cytochrome-c-t and Inflammation

Both mitochondrial dysfunction and neuroinflammation are implicated in neurodegeneration and neurodegenerative diseases. Accumulating evidence shows multiple links between mitochondrial dysfunction and neuroinflammation. Mitochondrial-derived damage-associated molecular patterns (DAMPs) are recognized by immune receptors of microglia and aggravate neuroinflammation. On the other hand, inflammatory factors released by activated glial cells trigger an intracellular cascade, which regulates mitochondrial metabolism and function. The crosstalk between mitochondrial dysfunction and neuroinflammatory activation is a complex and dynamic process. There is strong evidence that mitochondrial dysfunction precedes neuroinflammation during the progression of diseases. Thus, an in-depth understanding of the specific molecular mechanisms associated with mitochondrial dysfunction and the progression of neuroinflammation in neurodegenerative diseases may contribute to the identification of new targets for the treatment of diseases. In this review, we describe in detail the DAMPs that induce or aggravate neuroinflammation in neurodegenerative diseases including mtDNA, mitochondrial unfolded protein response (mtUPR), mitochondrial reactive oxygen species (mtROS), adenosine triphosphate (ATP), transcription factor A mitochondria (TFAM), cardiolipin, cytochrome c, mitochondrial Ca

Topics: Adenosine Triphosphate; Alarmins; Cardiolipins; Cytochromes c; DNA, Mitochondrial; Humans; Inflammation; Iron; Mitochondria; Neurodegenerative Diseases; Neuroinflammatory Diseases; Reactive Oxygen Species; Transcription Factors

Mitochondrial dysfunction has been established as a common feature of neurodegenerative disorders that contributes to disease pathology by causing impaired cellular energy production. Mitochondrial molecules released into the extracellular space following neuronal damage or death may also play a role in these diseases by acting as signaling molecules called damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs have been shown to initiate proinflammatory immune responses from nonneuronal glial cells, including microglia and astrocytes; thereby, they have the potential to contribute to the chronic neuroinflammation present in these disorders accelerating the degeneration of neurons. In this review, we highlight the mitochondrial DAMPs cytochrome

Topics: Animals; Cytochromes c; DNA-Binding Proteins; Humans; Inflammation; Mitochondria; Mitochondrial Proteins; Neurodegenerative Diseases; Transcription Factors

Parthenolide, a naturally occurring sesquiterpene lactone derived from feverfew (Tanacetum parthenium), exhibits exceptional anti-cancer and anti-inflammatory properties, making it a prominent candidate for further studies and drug development. In this review, we briefly investigate molecular events and cell-specific activities of this chemical in relation to cytochrome c, nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), signal transduction and activation of transcription (STAT), reactive oxygen species (ROS), TCP, HDACs, microtubules, and inflammasomes. This paper reports that parthenolide shows strong NF-κB- and STAT-inhibition-mediated transcriptional suppression of pro-apoptotic genes. This compound acts both at the transcriptional level and by direct inhibition of associated kinases (IKK-β). Similarly, this review discusses parthenolide-induced ROS-mediated apoptosis of tumor cells via the intrinsic apoptotic signaling pathway. The unique ability of this compound to not harm normal cells but at the same time induce sensitization to extrinsic as well as intrinsic apoptosis signaling in cancer cells provides an important, novel therapeutic strategy for treatment of cancer and inflammation-related disorders.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Cytochromes c; Humans; I-kappa B Kinase; Inflammation; Neoplasms; NF-kappa B; Plant Extracts; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction; STAT Transcription Factors; Tanacetum parthenium; Transcription, Genetic

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Cell Respiration; Cytochromes c; Electron Transport Complex IV; Humans; Inflammation; Mitochondria; Models, Biological; Phosphorylation; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction

The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Respiration; Cyclic AMP; Cytochromes c; Electron Transport Complex IV; Humans; Inflammation; Membrane Potential, Mitochondrial; Molecular Sequence Data; Neoplasms; Phosphorylation; Protein Kinase C; Reactive Oxygen Species; Signal Transduction

Mcl-1 is a Bcl-2 family protein which can act as an apical molecule in apoptosis control, promoting cell survival by interfering at an early stage in a cascade of events leading to release of cytochrome c from mitochondria. Mcl-1 has a short half life and is a highly regulated protein, induced by a wide range of survival signals and also rapidly down regulated during apoptosis. Mcl-1 can also readily be cleaved by caspases during apoptosis to produce a cell death promoting molecule. The multiple levels of control of Mcl-1 expression suggest that Mcl-1 plays a critical role in controlling life and death decisions in response to rapidly changing environmental cues and Mcl-1 is required for embryonic development and the function of the immune system. Expression of Mcl-1 may be useful in informing decision making in the treatment of various cancers, and countering Mcl-1 function may be an attractive therapeutic strategy in malignancy, inflammatory conditions and infectious disease where Mcl-1 may play a major role in suppressing apoptosis.

Topics: Animals; Apoptosis; Caspases; Cell Survival; Communicable Diseases; Cytochromes c; Drug Delivery Systems; Embryonic Development; Humans; Inflammation; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Apoptotic cell death is an active process mediated by various signaling pathways, which include the caspase cascade and the stress-activated protein kinase pathways. The caspase cascade is activated by two distinct routes: one from cell surface and the other from mitochondria. Activation of the route from cell surface requires the cellular components that include membrane receptors, adaptor proteins such as TRADD and FADD, and caspase-8, while activation of the other from mitochondria requires Apaf-1, caspase-9, and cytosolic cytochrome c. On the other hand, persistent stimulation of the stress-activated protein kinase pathway is also shown to mediate apoptosis in many cell types. Gene-targeting studies with jnk- or jip-null mice, in particular, strongly suggest that this signaling pathway plays a pivotal role in the cellular machinery for apoptosis.

Topics: Animals; Apoptosis; Arabidopsis Proteins; Caspase 9; Caspases; Cell Membrane; Cytochromes c; Cytosol; Enzyme Activation; Fatty Acid Desaturases; Humans; Inflammation; Mice; Mitogen-Activated Protein Kinases; Models, Biological; Signal Transduction; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

To establish whether plasma cytochrome c is detectable in patients undergoing cardiac surgery, whether cytochrome c levels are associated with lactate/inflammatory markers/cellular oxygen consumption, and whether cytochrome c levels are associated with clinical outcomes.. This was an observational sub-study of a randomized trial comparing thiamine to placebo in patients undergoing coronary artery bypass grafting. Patients had blood drawn before, after, and again 6h after surgery. Cytochrome c, inflammatory markers, and cellular oxygen consumption were measured.. 64 patients were included. Cytochrome c was detectable in 63 (98%) patients at baseline with a median cytochrome c level of 0.18ng/mL (quartiles: 0.13, 0.55). There was no difference from baseline level to post-surgical level (0.19ng/mL [0.09, 0.51], p=0.36) or between post-surgical level and 6-hour post-surgical level (0.17ng/mL [0.10, 0.57], p=0.61). There was no difference between the thiamine and placebo groups' change in cytochrome c levels from baseline to after surgery (p=0.22). Cytochrome c levels were not associated with lactate, inflammatory markers, cellular oxygen consumption, or clinical outcomes.. Cytochrome c levels did not increase after cardiac surgery and was not associated with the degree of inflammation or clinical outcomes.

Topics: Aged; Biomarkers; Cardiopulmonary Bypass; Coronary Artery Bypass; Cytochromes c; Double-Blind Method; Endothelium, Vascular; Female; Humans; Inflammation; Lactic Acid; Male; Oxygen Consumption; Thiamine; Vitamin B Complex

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Beclin-1; Cardiotoxicity; Caspase 3; Caspase 9; Cyclooxygenase 2; Cytochromes c; Inflammation; Interleukin-2; Male; Mice; Mice, Inbred C57BL; Nickel; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Thioredoxin-Disulfide Reductase; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Background: The kidney is the most common extrapulmonary organ injured in sepsis. The current study examines the ability of aerosolized nanochemically modified tetracycline 3 (nCMT-3), a pleiotropic anti-inflammatory agent, to attenuate acute kidney injury (AKI) caused by intratracheal LPS. Methods: C57BL/6 mice received aerosolized intratracheal nCMT-3 (1 mg/kg) or saline, followed by intratracheal LPS (2.5 mg/kg) to induce acute lung injury-induced AKI. Tissues were harvested at 24 h. The effects of nCMT-3 and LPS on AKI were assessed by plasma/tissue levels of serum urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, kidney injury molecule 1, and renal histology. Renal matrix metalloproteinase (MMP) level/activity, cytochrome C, Bax, Bcl-2, caspase-3, p38 mitogen-activated protein kinase activation, NLRP3, and caspase-1 were also measured. Apoptotic cells in kidney were determined by TUNEL assay. Renal levels of IL-1β and IL-6 were measured to assess inflammation. Results: Acute lung injury-induced AKI was characterized by increased plasma blood urea nitrogen, creatinine, injury biomarkers (neutrophil gelatinase-associated lipocalin, kidney injury molecule 1), and histologic evidence of renal injury. Lipopolysaccharide-treated mice demonstrated renal injury with increased levels of inflammatory cytokines (IL-1β, IL-6), active MMP-2 and MMP-9, proapoptotic proteins (cytochrome C, Bax/Bcl-2 ratio, cleaved caspase-3), apoptotic cells, inflammasome activation (NLRP3, caspase-1), and p38 signaling. Intratracheal nCMT-3 significantly attenuated all the measured markers of renal injury, inflammation, and apoptosis. Conclusions: Pretreatment with aerosolized nCMT-3 attenuates LPS-induced AKI by inhibiting renal NLRP3 inflammasome activation, renal inflammation, and apoptosis.

Topics: Acute Kidney Injury; Acute Lung Injury; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 1; Caspase 3; Creatinine; Cytochromes c; Inflammasomes; Inflammation; Interleukin-6; Lipocalin-2; Lipopolysaccharides; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Proto-Oncogene Proteins c-bcl-2; Sepsis; Tetracyclines

Thymoquinone (TQ) is the main active constituent of Nigella sativa. The present study aimed to investigate the effect of TQ on apoptotic parameters and MMP-9 expression in isoproterenol (ISP)-induced myocardial infarction (MI). TQ was given once daily for 7 days at doses of 10 and 20 mg/kg orally with ISP (86 mg/kg; s.c.) administered on the sixth and seventh days. TQ pre-treatment protected against ISP-induced MI as approved by normalisation of electrocardiogram (ECG) and b (CK)-MB, minimal histopathological changes, and reduction of the infarction size. Effects of TQ could be supported by its antioxidant activity, evidenced by the increase of cardiac reduced glutathione and total serum antioxidant capacity, and the inhibition of ISO-induced lipid peroxidation. TQ anti-inflammatory activity was associated with reduced expression of NF-κB and TNF-α. TQ ameliorated cardiomyocytes

Topics: Animals; Apoptosis; Benzoquinones; Biomarkers; Cytochromes c; Gene Expression Regulation, Enzymologic; Inflammation; Isoproterenol; Matrix Metalloproteinase 9; Myocardial Infarction; Random Allocation; Rats; Rats, Wistar

Topics: Animals; Cytochromes c; Disease Models, Animal; Fibrosis; Inflammation; Lung; Mice; Silicon Dioxide; Silicosis; Spectrum Analysis, Raman

Excessive fluoride intake has been reported to cause toxicities to brain, thyroid, kidney, liver and testis tissues. Hesperidin (HSP) is an antioxidant that possesses anti-allergenic, anti-carcinogenic, anti-oxidant and anti-inflammatory activities. Presently, the studies focusing on the toxic effects of sodium fluoride (NaF) on heart tissue at biochemical and molecular level are limited. This study was designed to evaluate the ameliorative effects of HSP on toxicity of NaF on the heart of rats in vivo by observing the alterations in oxidative injury markers (MDA, SOD, CAT, GPX and GSH), pro-inflammatory markers (NF-κB, IL-1β, TNF-α), expressions of apoptotic genes (caspase-3, -6, -9, Bax, Bcl-2, p53, cytochrome c), levels of autophagic markers (Beclin 1, LC3A, LC3B), expression levels of PI3K/Akt/mTOR and cardiac markers. HSP treatment attenuated the NaF-induced heart tissue injury by increasing activities of SOD, CAT and GPx and levels of GSH, and suppressing lipid peroxidation. In addition, HSP reversed the changes in expression of apoptotic (caspase-3, -6, -9, Bax, Bcl-2, p53, cytochrome c), levels of autophagic and inflammatory parameters (Beclin 1, LC3A, LC3B, NF-κB, IL-1β, TNF-α), in the NaF-induced cardiotoxicity. HSP also modulated the gene expression levels of PI3K/Akt/mTOR signaling pathway and levels of cardiac markers (LDH, CK-MB). Overall, these findings reveal that HSP treatment can be used for the treatment of NaF-induced cardiotoxicity.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Beclin-1; Cardiotoxicity; Caspase 3; Cytochromes c; Heart Diseases; Hesperidin; Inflammation; NF-kappa B; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Sodium Fluoride; Superoxide Dismutase; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Endothelial dysfunction is a driving force during the development and progression of cardiovascular complications in diabetes. Targeting endothelial injury may be an attractive avenue for the management of diabetic vascular disorders. Chicoric acid is reported to confer antioxidant and anti-inflammatory properties in various diseases including diabetes. However, the role and mechanism of chicoric acid in hyperglycemia-induced endothelial damage are not well understood.. In the present study, human umbilical vein endothelial cells (HUVECs) were incubated with high glucose/high fat (HG + HF) to induce endothelial cell injury.. We found that exposure of HUVECs to HG + HF medium promoted the release of cytochrome c (cytc) from mitochondrion into the cytoplasm, stimulated the cleavage of caspase-3 and poly ADP-ribose-polymerase (PARP), then inducing cell apoptosis, the effects that were prevented by administration of chicoric acid. Besides, we found that chicoric acid diminished HG + HF-induced phosphorylation and degradation of IκBα, and subsequent p65 NFκB nuclear translocation, thereby contributing to its anti-inflammatory effects in HUVECs. We also confirmed that chicoric acid mitigated oxidative/nitrative stresses under HG + HF conditions. Studies aimed at exploring the underlying mechanisms found that chicoric acid activated the AMP-activated protein kinase (AMPK) signaling pathway to attenuate HG + HF-triggered injury in HUVECs as AMPK inhibitor Compound C or silencing of AMPKα1 abolished the beneficial effects of chicoric acid in HUVECs.. Collectively, chicoric acid is likely protected against diabetes-induced endothelial dysfunction by activation of the AMPK signaling pathway. Chicoric acid could be a novel candidate for the treatment of the diabetes-associated vascular endothelial injury.

Topics: AMP-Activated Protein Kinases; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Caffeic Acids; Cell Survival; Cytochromes c; Cytoplasm; Endothelium, Vascular; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Inflammation; L-Lactate Dehydrogenase; Mitochondria; Nitrosative Stress; Oxidative Stress; Poly(ADP-ribose) Polymerases; RNA, Small Interfering; Succinates

Exposure to high oxygen concentrations leads to generation of excessive reactive oxygen species, causing cellular injury and multiple organ dysfunctions and is associated with a high mortality rate. Clusterin (CLU) is a heterodimeric glycoprotein that mediates several intracellular signaling pathways, including cell death and inflammation. However, the role of CLU in the pathogenesis of hyperoxic acute lung injury (HALI) is unknown. Wild-type (WT) and CLU-deficient mice and cultured human airway epithelial cells were used. Changes in cell death- and inflammation-related molecules with or without hyperoxia exposure in cells and animals were determined. Hyperoxia induced an increase in CLU expression in mouse lungs and human airway epithelial cells. Mice lacking CLU had increased HALI and mortality rate compared with WT mice. In vitro, CLU-disrupted cells showed enhanced release of cytochrome c, Bax translocation, cell death and inflammatory cytokine expression. However, treatment with recombinant CLU attenuated hyperoxia-induced apoptosis. Moreover, the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses revealed metabolic pathways, hematopoietic cell lineage, response to stress and localization and regulation of immune system that were differentially regulated between WT and CLU

Topics: Acute Lung Injury; Animals; Apoptosis; bcl-2-Associated X Protein; Clusterin; Cytochromes c; Epithelial Cells; Gene Expression Profiling; Gene Expression Regulation; Humans; Hyperoxia; Inflammation; Lung; Male; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mice, Knockout; Microarray Analysis; RNA, Messenger

Pulmonary arterial hypertension (PH) is a progressive disease with limited therapeutic options, ultimately leading to right heart failure and death. Recent findings indicate the role of the Warburg effect (aerobic glycolysis) in the development of PH. However, the effect of the glycolysis inhibitor 3-bromopyruvate (3-BrPA) on the pathogenesis of PH has not been well investigated. This study aimed to determine whether 3-BrPA inhibits PH and its possible mechanism.. PH was induced in adult Sprague-Dawley rats by a single intraperitoneal injection of monocrotaline (MCT). 3-BrPA, or phosphate-buffered saline (PBS) was administered via intraperitoneal injection every other day from the first day of MCT-injection to 4 weeks of follow-up, and indices such as right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), pulmonary arteriolar remodeling indicated by percent media thickness (% MT), lactate levels and glucose consumption, were evaluated. Pulmonary arteriolar remodeling and right ventricular hypertrophy were observed in hematoxylin-eosin-stained lung sections. Western blotting, immunohistochemistry, and/or immunofluorescence analyses were used to measure the expression of relevant proteins. A cytochrome C release apoptosis assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining were used to measure cell apoptosis.. MCT-induced PH showed a significant increase in glucose consumption (0 vs. 4 weeks: 0.87 ± 0.23 vs. 2.94 ± 0.47, P = 0.0042) and lactate production (0 vs. 4 weeks: 4.19 ± 0.34 vs. 8.06 ± 0.67, P = 0.0004). Treatment with 3-BrPA resulted in a concomitant reduction in glucose consumption (1.10 ± 0.35 vs. 3.25 ± 0.47, P = 0.0063), lactate production (5.09 ± 0.55 vs. 8.06 ± 0.67, P = 0.0065), MCT-induced increase in RVSP (39.70 ± 2.94 vs. 58.85 ± 2.32, P = 0.0004), pulmonary vascular remodeling (% MT, 43.45% ± 1.41% vs. 63.66% ± 1.78%, P < 0.0001), and right ventricular hypertrophy (RVHI, 38.57% ± 2.69% vs. 62.61% ± 1.57%, P < 0.0001) when compared with those of the PBS-treated group. 3-BrPA, a hexokinase 2 inhibitor, exerted its beneficial effect on PH by decreasing aerobic glycolysis and was also associated with inhibiting the expression of glucose transporter protein-1, inducing apoptosis, and suppressing inflammation.. 3-BrPA might have a potential beneficial effect on the PH treatment.

Topics: Animals; Apoptosis; Blood Pressure; Blotting, Western; Cytochromes c; Fluorescent Antibody Technique; Glycolysis; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Male; Monocrotaline; Pulmonary Arterial Hypertension; Pyruvates; Rats; Rats, Sprague-Dawley

Triple-negative breast cancer (TNBC), defined as loss of estrogen, progesterone, and Her2 receptors, is a subtype of highly aggressive breast cancer with worse prognosis and poor survival rate. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine aberrantly expressed in many solid tumors and known to promote tumor progression and metastasis. However, its role in TNBC progression and metastasis is unexplored. Here we have shown that in TNBC patients, MIF expression was significantly enriched in the tumor compared to adjacent normal tissue. Using publically available patient datasets, we showed that MIF overexpression correlates with worse survival in TNBC compared to other hormonal status. Orthotopic implantation of TNBC cells into MIF knockout mice showed reduced tumor growth compared to wild-type mice. In addition, we have shown that MIF downregulation inhibits TNBC growth and progression in a syngeneic mouse model. We further showed that CPSI-1306, a small-molecule MIF inhibitor, inhibits the growth of TNBC cells in vitro. Mechanistic studies revealed that CPSI-1306 induces intrinsic apoptosis by alteration in mitochondrial membrane potential, cytochrome c (Cyt c) release, and activation of different caspases. In addition, CPSI-1306 inhibits the activation of cell survival and proliferation-related molecules. CPSI-1306 treatment also reduced the tumor growth and metastasis in orthotopic mouse models of mammary carcinoma. CPSI-1306 treatment of tumor-bearing mice significantly inhibited TNBC growth and pulmonary metastasis in a dose-dependent manner. Histological analysis of xenograft tumors revealed a higher number of apoptotic cells in CPSI-1306-treated tumors compared to vehicle controls. Our studies, for the first time, show that MIF overexpression in TNBC enhances growth and metastasis. Taken together, our results indicate that using small molecular weight MIF inhibitors could be a promising strategy to inhibit TNBC progression and metastasis.

Topics: Animals; Apoptosis; Caspases; Cell Movement; Cell Survival; Cytochromes c; Disease Progression; Enzyme Activation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Inflammation; Intramolecular Oxidoreductases; Isoxazoles; Macrophage Migration-Inhibitory Factors; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Morpholines; Neoplasm Metastasis; Neoplasm Transplantation; Treatment Outcome; Triple Negative Breast Neoplasms; Wound Healing

Increasing evidence suggests that apoptosis of tubular cells and renal inflammation mainly determine the outcome of sepsis-associated acute kidney injury (AKI). The study aim was to investigate the molecular mechanism involved in the renoprotective effects of simvastatin in endotoxin (lipopolysaccharide, LSP)-induced AKI. A sepsis model was established by intraperitoneal injection of a single non-lethal LPS dose after short-term simvastatin pretreatment. The severity of the inflammatory injury was expressed as renal damage scores (RDS). Apoptosis of tubular cells was detected by Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL assay) (apoptotic DNA fragmentation, expressed as an apoptotic index, AI) and immunohistochemical staining for cleaved caspase-3, cytochrome C, and anti-apoptotic Bcl-xL and survivin. We found that endotoxin induced severe renal inflammatory injury (RDS = 3.58 ± 0.50), whereas simvastatin dose-dependently prevented structural changes induced by LPS. Furthermore, simvastatin 40 mg/kg most profoundly attenuated tubular apoptosis, determined as a decrease of cytochrome C, caspase-3 expression, and AIs (

Topics: Acute Kidney Injury; Animals; Apoptosis; bcl-X Protein; Cell Survival; Cytochromes c; Endotoxins; Epithelial Cells; Humans; Inflammation; Kidney; Kidney Tubules; Lipopolysaccharides; Rats; Simvastatin

Cytochrome c (Cyt c), a heme-containing mitochondrial protein, has a critical function in both respiration and apoptosis. Consistent with these vital functions, somatic Cyt c mouse knockout is embryonic lethal. In order to investigate the sensitivity of postnatal neurons to Cyt c depletion, we developed a neuron-specific conditional knockout model. Neuron-specific Cyt c KO mouse (nCytc

Topics: Animals; Apoptosis; Cytochromes c; Electron Transport Complex IV; Gene Deletion; Inflammation; Locomotion; Mice, Knockout; Mitochondria; Nerve Degeneration; Neurons; Oxidative Phosphorylation; Oxidative Stress; Phenotype; Prosencephalon

Current predictive model is not developed by inflammation-related genes to evaluate clinical outcome of breast cancer patients.. With mRNA expression profiling, we identified 3 mRNAs with significant expression between 15 normal samples and 669 breast cancer patients. Using 7 cell lines and 150 paraffin-embedded specimens, we verified the expression pattern by bio-experiments. Then, we constructed a three-mRNA model by Cox regression method and approved its predictive accuracy in both training set (n = 1095) and 4 testing sets (n = 703).. We developed a three-mRNA (TBX21, TGIF2, and CYCS) model to stratify patients into high- and low-risk subgroup with significantly different prognosis. In training set, 5-year OS rate was 84.5% (78.8%-90.5%) vs 73.1% (65.9%-81.2%) for the low- and high-risk group (HR = 1.573 (1.090-2.271); P = 0.016). The predictive value was similar in four independent testing sets (HR>1.600; P < 0.05). This model could assess survival independently with better predictive power compared with single clinicopathological risk factors and any of the three mRNAs. Patients with both low-risk values and any poor prognostic factors had more favorable survival from nonmetastatic status (HR = 1.740 (1.028-2.945), P = 0.039). We established two nomograms for clinical application that integrated this model and another three significant risk factors to forecast survival rates precisely in patients with or without metastasis.. This model is a dependable tool to predict the disease recurrence precisely and could improve the predictive accuracy of survival probability for breast cancer patients with or without metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Inflammation; Male; Middle Aged; Prognosis; Repressor Proteins; RNA, Messenger; T-Box Domain Proteins

Neuroinflammation has been acknowledged as a primary factor contributing to the pathogenesis of neurodegenerative disease. However, the molecular mechanism underlying inflammation stress-mediated neuronal dysfunction is not fully understood. The aim of our study was to explore the influence of mammalian STE20-like kinase 1 (Mst1) in neuroinflammation using TNFα and CATH.a cells in vitro. The results of our study demonstrated that the expression of Mst1 was dose-dependently increased after TNFα treatment. Interestingly, knockdown of Mst1 using siRNA transfection significantly repressed TNFα-induced neuronal death. We also found that TNFα treatment was associated with mitochondrial stress, including mitochondrial ROS overloading, mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential reduction, and mitochondrial pro-apoptotic factor release. Interestingly, loss of Mst1 attenuated TNFα-triggered mitochondrial stress and sustained mitochondrial function in CATH.a cells. We found that Mst1 modulated mitochondrial homeostasis and cell viability via the JNK pathway in a TNFα-induced inflammatory environment. Inhibition of the JNK pathway abolished TNFα-mediated CATH.a cell death and mitochondrial malfunction, similar to the results obtained via silencing of Mst1. Taken together, our results indicate that inflammation-mediated neuronal dysfunction is implicated in Mst1 upregulation, which promotes mitochondrial stress and neuronal death by activating the JNK pathway. Accordingly, our study identifies the Mst1-JNK-mitochondria axis as a novel signaling pathway involved in neuroinflammation.

Topics: Animals; Anthracenes; Apoptosis; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Gene Knockdown Techniques; Inflammation; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mitochondria; Neurodegenerative Diseases; Oxidative Stress; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Chronic activation of glial cells contributes to neurodegenerative diseases. Cytochrome c (CytC) is a soluble mitochondrial protein that can act as a damage-associated molecular pattern (DAMP) when released into the extracellular space from damaged cells. CytC causes immune activation of microglia in a toll-like receptor (TLR) 4-dependent manner. The effects of extracellular CytC on astrocytes are unknown. Astrocytes, which are the most abundant glial cell type in the brain, express TLR 4 and secrete inflammatory mediators; therefore, we hypothesized that extracellular CytC can interact with the TLR 4 of astrocytes inducing their release of inflammatory molecules and cytotoxins.. Experiments were conducted using primary human astrocytes, U118 MG human astrocytic cells, BV-2 murine microglia, and SH-SY5Y human neuronal cells.. Extracellularly applied CytC increased the secretion of interleukin (IL)-1β, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-12 p70 by cultured primary human astrocytes. Anti-TLR 4 antibodies blocked the CytC-induced secretion of IL-1β and GM-CSF by astrocytes. Supernatants from CytC-activated astrocytes were toxic to human SH-SY5Y neuronal cells. We also demonstrated CytC release from damaged glial cells by measuring CytC in the supernatants of BV-2 microglia after their exposure to cytotoxic concentrations of staurosporine, amyloid-β peptides (Aβ42) and tumor necrosis factor-α.. CytC can be released into the extracellular space from damaged glial cells causing immune activation of astrocytes in a TLR 4-dependent manner.. Astrocyte activation by CytC may contribute to neuroinflammation and neuronal death in neurodegenerative diseases. Astrocyte TLR 4 could be a potential therapeutic target in these diseases.

Topics: Astrocytes; Cell Line, Tumor; Cytochromes c; Humans; Inflammation; Interleukin-12; Interleukin-1beta; Toll-Like Receptor 4

Peroxiredoxin 2 (Prdx2) is a ubiquitous antioxidant enzyme in mammalian brain. Although a protective role of Prdx2 has been established in cerebral ischemia and several neurodegenerative diseases, its contribution against iron-induced neurocytotoxicity still remains to be determined. Accordingly, in this study, we aimed to investigate the effects of Prdx2 on iron-induced cytotoxicity using an in vitro model in which PC12 cells are exposed to ferrous sulfate (FS). The FS treatment increased Prdx2 expression, and promoted lactate dehydrogenase (LDH) release and cell apoptosis in PC12 cells, accompanied by the increase in the Bax/Bcl2 ratio, cytochrome c release, and caspase-3 cleavage. FS exposure also increased the malondialdehyde content (lipid peroxidation), 3'-nitrotyrosine expression (protein nitration), γ-H2A.X formation (DNA oxidation), and promoted nuclear factor kappa B nuclear translocation, cyclooxygenase-2 expression, and release of tumor necrosis factor-α and interleukin-1β. Lentivirus-mediated Prdx2 knockdown intensified the FS-induced LDH release and cell apoptosis by aggravating the oxidative and inflammatory damage. In conclusion, our findings demonstrated that Prdx2 played a vital role in the protection against iron-induced cytotoxicity in PC12 cells.

Topics: Animals; Antioxidants; Cytochromes c; Inflammation; Lipid Peroxidation; Malondialdehyde; Membrane Potential, Mitochondrial; Neuroprotective Agents; Oxidative Stress; PC12 Cells; Peroxiredoxins; Rats; Reactive Oxygen Species

Exposure to oxidative stress will lead to the progression of retinal degenerative diseases, and unfortunately the exact mechanisms have not been fully understood. In this study, the protective effects of (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (TIM) against the lipoteichoic acid (LTA)-induced cell damage in mouse photoreceptor-derived 661W cells were investigated.. 661W cells were pre-treated with TIM at different concentrations (0.1-2.5 μM) before exposure to LTA. The oxidative stress and inflammatory response were detected in 661W cells.. Pre-treatment of 661W cells with TIM (0.1-2.5 μM) for 4 h significantly decreased the LTA-induced toxicity. Meanwhile, pre-treatment with TIM could attenuate the imbalance state of redox in 661W cells by decreasing the levels of intracellular ROS and MDA, as well as enhancing the SOD activity and the level of GSH, through increasing the protein expression of Nrf2. Moreover, TIM pre-treatment decreased pro-inflammatory factors IL-1β, IL-12 and TNFα, through inhibiting the nuclear factor kappa B. Pre-treatment with TIM also suppressed Egr1, Fosl1, and Lox12 gene expression.. These results suggested that TIM may exert its protective effects against LTA-induced toxicity in 661W cells, through counteracting the oxidative stress and inhibiting inflammatory response. Our findings provided the scientific rational to develop TIM in the treatment of oxidative stress-induced photoreceptor cell damage.

Topics: Animals; Anti-Inflammatory Agents; Caspases; Cell Line; Cell Survival; Chromans; Cytochromes c; Cytokines; Gene Expression; Inflammation; Lipopolysaccharides; Membrane Potential, Mitochondrial; Mice; Neuroprotective Agents; Oxidation-Reduction; Oxidative Stress; Photoreceptor Cells, Vertebrate; Teichoic Acids

Rutin, a natural flavonoid, possess beneficial health effects. However, its renoprotective effect against carbon tetrachloride (CCl

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calpain; Carbon Tetrachloride; Caspase 3; Ceramides; Cytochromes c; Inflammation; Kidney; Male; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; Oxidative Stress; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Rutin; Tumor Suppressor Protein p53

Topics: Apoptosis; bcl-2-Associated X Protein; Cytochromes c; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia; Inflammation; Lysophospholipids; Reactive Oxygen Species; Sphingosine

Hind limb ischemia-reperfusion injury is an important pathology in vascular surgery. Reactive oxygen species are thought to be involved in the pathogenesis of hind limb ischemia-reperfusion injury. SS-31, which belongs to a family of mitochondrion-targeted peptide antioxidants, was shown to reduce mitochondrial reactive oxygen species production. In this study, we investigated whether the treatment of SS-31 could protect hind limb from ischemia-reperfusion injury in a mouse model. The results showed that SS-31 treatment either before or after ischemia exhibited similar protective effects. Histopathologically, SS-31 treatment prevented the IR-induced histological deterioration compared with the corresponding vehicle control. SS-31 treatment diminished oxidative stress revealed by the reduced malondialdehyde level and increased activities and protein levels of Sod and catalase. Cellular ATP contents and mitochondrial membrane potential increased and the level of cytosolic cytC was decreased after SS-31 treatment in this IR model, demonstrating that mitochondria were protected. The IR-induced increase of levels of inflammatory factors, such as Tnf-α and Il-1β, was prevented by SS-31 treatment. In agreement with the reduced cytosolic cytC, cleaved-caspase 3 was kept at a very low level after SS-31 treatment. Overall, the effect of SS-31 treatment before ischemia is mildly more effective than that after ischemia. In conclusion, our results demonstrate that SS-31 confers a protective effect in the mouse model of hind limb ischemia-reperfusion injury preventatively and therapeutically.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Caspase 3; Catalase; Cytochromes c; Disease Models, Animal; Hindlimb; Inflammation; Inflammation Mediators; Interleukin-1beta; Male; Malondialdehyde; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria, Muscle; Muscle, Skeletal; Oligopeptides; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Notwithstanding uncertain pathogenesis of inflammatory bowel disease (IBD), deregulation of adaptive immunity is paramount for the development of inflammation. Essential role in the resolution of inflammation is played by apoptosis, deregulated in lymphocytes isolated from inflamed intestine. Despite IBD being a systemic disease, little is known about apoptosis of peripheral lymphocytes. The concentrations of Bcl-2, cytochrome c, p53, and caspase-9 were determined (ELISA) in lymphocyte-enriched fractions of peripheral blood mononuclear cells (LE-PBMCs) from 64 individuals (42 with IBD) and related to IBD phenotype and activity, treatment, and inflammatory and hematological indices. The diagnostic potential of evaluated markers was determined as well. All evaluated molecules were significantly lower in IBD patients, of which cytochrome c and p53 were significantly lower exclusively in patients with Crohn's disease (CD) and cytochrome c differed significantly between CD and ulcerative colitis (UC). Caspase 9 was significantly lower in active IBD and Bcl-2 in active UC whereas cytochrome c was higher in active CD. Treatment with corticosteroids affected the concentrations of cytochrome c and p53. Both positively correlated with hsCRP and the concentrations of all markers were interrelated. As IBD markers, Bcl-2 and caspase-9 displayed good accuracy and, as a panel of markers with cytochrome c, their accuracy was excellent (92%). As CD markers Bcl-2, cytochrome c, and p53 displayed fair accuracy but combined determination of Bcl-2 and cytochrome c improved the accuracy to 85%. Taken together, our results imply diminished intrinsic apoptotic capacity of LE-PBMCs in IBD but an upregulation of proapoptotic features parallel to increasing severity of inflammation. Observed abnormalities in intrinsic pathway of apoptosis are more pronounced in CD. Upon positive validation on a larger set of patients, combined quantification of Bcl-2 and cytochrome c might be considered as an adjunct in differential diagnosis of UC and CD of colon and rectum.

Topics: Adaptive Immunity; Adult; Apoptosis; Caspase 9; Colitis, Ulcerative; Colon; Crohn Disease; Cytochromes c; Female; Humans; Inflammation; Inflammatory Bowel Diseases; Leukocytes, Mononuclear; Lymphocytes; Male; Proto-Oncogene Proteins c-bcl-2; Rectum; Tumor Suppressor Protein p53

Burns are associated with activation of the innate immunity that can contribute to complications. Damage-associated molecular patterns (DAMPs) released after tissue injury play a critical role in the activation of the innate immunity, which appears to be mediated via toll-like receptors (TLRs). Previous findings have shown that TLRs and TLR-mediated responses are up-regulated after burn. Nonetheless, it is unclear what impact burn has on circulating levels of DAMPs. To study this, male C57BL/6 mice were subjected to a major burn or sham procedure. Three hours to 7days thereafter, plasma was collected and assayed for the representative DAMPs (i.e., HMGB1, cytochrome C, DNA and S100A) and extracellular cleavage products (fibronectin and hyaluronan). HMGB1, cytochrome C, fibronectin and hyaluronan levels were elevated in a time-dependent manner after burn as compared to sham levels. A significant elevation in TNF-α, IL-6 and IL-10 cytokine plasma levels was also found after burn. All cytokine levels were increased as early as 3h and remained elevated up to 24h. Circulating CD11b

Topics: Alarmins; Animals; Biomarkers; Burns; Cytochromes c; Disease Models, Animal; Fibronectins; HMGB1 Protein; Hyaluronic Acid; Inflammation; Interleukin-10; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Monocytes; Tumor Necrosis Factor-alpha

Allicin, a major component of garlic, is regarded as a cardioprotective agent and is associated with increased endothelial function.. The effects of allicin on lipopolysaccharide (LPS)-induced vascular oxidative stress and inflammation in cultured human umbilical vein endothelial cells (HUVECs) and the mechanisms underlying these effects were studied. The protective effects were measured using cell viability, a lactate dehydrogenase (LDH) assay and cell apoptosis as indicators, and the anti-oxidative activity was determined by measuring reactive oxygen species (ROS) generation, oxidative products and endogenous antioxidant enzyme activities. HUVEC mitochondrial function was assessed by determining mitochondrial membrane potential (MMP) collapse, cytochrome c production and mitochondrial ATP release. To investigate the potential underlying mechanisms, we also measured the expression of dynamic mitochondrial proteins using western blotting. Furthermore, we evaluated the Nrf2 antioxidant signaling pathway using an enzyme-linked immunosorbent assay (ELISA).. Our results demonstrated that allicin enhanced HUVEC proliferation, which was suppressed by LPS exposure, and LDH release. Allicin ameliorated LPS-induced apoptosis, suppressed ROS overproduction, reduced lipid peroxidation and decreased the endogenous antioxidant enzyme activities in HUVECs. These protective effects were associated with the inhibition of mitochondrial dysfunction as indicated by decreases in the MMP collapse, cytochrome c synthesis and mitochondrial ATP release. In addition, allicin attenuated the LPS-induced inflammatory responses, including endothelial cell adhesion and TNF-α and IL-8 production. Furthermore, allicin increased the expression of LXRα in a dose-dependent manner. Allicin-induced attenuation of inflammation was inhibited by LXRα siRNA treatment. Finally, allicin activated NF-E2-related factor 2 (Nrf2), which controls the defense against oxidative stress and inflammation.. Taken together, the present data suggest that allicin attenuated the LPS-induced vascular injury process, which may be closely related to the oxidative stress and inflammatory response in HUVECs. Allicin modulated Nrf2 activation and protected the cells against LPS-induced vascular injury. Our findings suggest that allicin attenuated the LPS-induced inflammatory response in blood vessels.

Topics: Apoptosis; Cell Adhesion; Cytochromes c; Disulfides; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Interleukin-8; Lipid Peroxidation; Lipopolysaccharides; Liver X Receptors; Malondialdehyde; Membrane Potential, Mitochondrial; Mitochondria; Neutrophils; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; RNA Interference; Sulfinic Acids; Tumor Necrosis Factor-alpha

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.

Topics: Acute-Phase Reaction; alpha-2-HS-Glycoprotein; Animals; Cattle; Chromatography, Liquid; Computational Biology; Cytochromes c; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Filtration; Haptoglobins; Hemopexin; Inflammation; Orosomucoid; Proteins; Proteolysis; Sepsis; Software; Sorption Detoxification; Swine; Tandem Mass Spectrometry

Clinical evidences showing zinc (Zn) accumulation in the post-mortem brain of Parkinson's disease (PD) patients and experimental studies on rodents chronically exposed to Zn suggested its role in PD. While oxidative stress is implicated in Zn-induced neurodegeneration, roles of inflammation and apoptosis in degeneration of the nigrostriatal dopaminergic neurons have yet been elusive. The present study investigated the contribution of the nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and B-cell lymphoma 2 (Bcl-2) family proteins in Zn-induced Parkinsonism. Male Wistar rats were treated with/without zinc sulfate (Zn; 20 mg/kg, intraperitoneally), twice a week, for 2-12 weeks. In a few sets, animals were treated intraperitoneally with a NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100 mg/kg), a TNF-α inhibitor, pentoxyfylline (PTX; 50 mg/kg), and an anti-inflammatory agent, dexamethasone (DEX; 5 mg/kg), prior to Zn exposure along with respective controls. Zn caused neurobehavioral impairments and reduction in dopamine and its metabolites, tyrosine hydroxylase (TH)-positive neurons, catalase activity, and expression of TH, Bcl-2, and NOXA. On the contrary, Zn augmented lipid peroxidation, activity of superoxide dismutase, expression of TNF-α, IL-1β, Bcl-xl, and p53-upregulated modulator of apoptosis (PUMA), and translocation of NF-κB and Bax from the cytosol to the nucleus and mitochondria, respectively, with concomitant increase in the mitochondrial cytochrome c release and activation of procaspase-3 and -9. Pre-treatment with PTX, DEX, or PDTC invariably ameliorated Zn-induced changes in behavioral and neurodegenerative indexes, inflammatory mediators, and apoptosis. Results demonstrate that inflammation regulates Bax expression that subsequently contributes to the nigrostriatal dopaminergic neurodegeneration.

Topics: Animals; Antigens, Nuclear; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Behavior, Animal; Cytochromes c; Dopamine; Dopaminergic Neurons; Gene Expression Regulation; Inflammation; Interleukin-1beta; Lipid Peroxidation; Male; Metabolome; Mitochondria; Neostriatum; Nerve Degeneration; Nerve Tissue Proteins; NF-kappa B; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats, Wistar; RNA, Messenger; Serotonin; Substantia Nigra; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase; Zinc

Recent evidence suggests that nerve growth factor IB (Nur77) and nuclear receptor related1 (Nurr1) are differentially involved in dopaminergic neurodegeneration. Since memantine has shown clinically relevant efficacy in Parkinson's disease (PD) and displayed a potent protective effect on dopaminergic neurons in experimental PD models, we asked if it exerts its neuroprotection by regulating Nur77 and Nurr1 signaling. We adopted a well-established in vitro PD model, 6-hydroxydopamine (OHDA)-lesioned PC12 cells, to test our hypothesis. Different concentrations of memantine were incubated with 6-OHDA-lesioned PC12 cells, and Nur77/Nurr1 and their related signaling molecules were examined by Western blot and immunocytochemistry. Nur77-deficient PC12 cells were used to verify the influences of Nur77 on neurodegeneration and memantine-mediated neuroprotection. We found that memantine reversed Nur77 upregulation and restored Nurr1 downregulation in 6-OHDA-lesioned PC12 cells. 6-OHDA incubation caused Nur77 translocation from the nucleus to cytosol and induced co-localization of Cyt c/HSP60/Nur77 in the cytosol. Memantine strongly reduced the sub-cellular translocations of Nur77/Cyt c/HSP60 under 6-OHDA-induced oxidative condition. Knockdown of Nur77 enhanced the viability of PC12 cells exposed to 6-OHDA, while memantine-induced neuroprotection was much less in the cells with Nur77 knockdown than in those without it. We conclude that Nur77 plays a crucial role in modulating mitochondrial impairment and contributes to neurodegeneration under the experimental PD condition. Memantine effectively suppresses such Nur77-mediated neurodegeneration and promotes survival signaling through post-translational modification of Nurr1. Nur77 and Nurr1 present a contra-directionally coupling interaction in memantine-mediated neuroprotection.

Topics: Animals; Cell Survival; Cytochromes c; Dopamine Plasma Membrane Transport Proteins; Gene Knockdown Techniques; Glutamic Acid; Inflammation; Interleukin-6; L-Lactate Dehydrogenase; MAP Kinase Signaling System; Memantine; Mitochondria; Nerve Degeneration; Neuroprotection; Nuclear Receptor Subfamily 4, Group A, Member 1; Nuclear Receptor Subfamily 4, Group A, Member 2; Oxidopamine; PC12 Cells; Rats; Subcellular Fractions; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase

Endosulfan is one of the organochlorine pesticides. It has been associated with a wide range of adverse health effects. However, it is unknown whether endosulfan causes endothelial dysfunction. In the present study, we investigated the effects of endosulfan on human vascular endothelial cells. We exposed human umbilical vein endothelial cells (HUVEC-C) to varying concentrations of endosulfan for 48 h. The results showed that endosulfan lowered cell viability and inhibited cell proliferation in a dose-dependent manner. Flow cytometric analysis showed that endosulfan at 60 μM induced G1 cell cycle arrest, a response attributed to down-regulation of CDK6 and pRb dephosphorylation. We observed that endosulfan at 40 and 60 μM induced a considerable percentage of cells to undergo apoptosis, as detected by Annexin-V binding assays. Endosulfan reduced mitochondrial transmembrane potential, leading to the release of cytochrome c into the cytoplasm; meanwhile, endosulfan also inhibited the mRNA expression level of survivin, which resulted in the activation of caspase-3. These results indicated that the intrinsic mitochondria-mediated pathway was involved in apoptotic process. Exposure to endosulfan increased the secretion and mRNA expression levels of inflammation factors interleukin (IL)-6 and IL-8, suggesting that endosulfan could cause inflammation. Overall, these findings suggested that endosulfan is toxic to HUVEC-C cells, resulting in endothelial dysfunction. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1785-1795, 2016.

Topics: Apoptosis; Caspase 3; Cell Cycle; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase 6; Cytochromes c; Down-Regulation; Endosulfan; Enzyme Activation; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Interleukin-8; Mitochondria; Pesticides; Phosphorylation; Retinoblastoma Protein

Doxycycline (DOX) exhibits anti-inflammatory, anti-tumor, and pro-apoptotic activity and is being tested in clinical trials as a chemotherapeutic agent for several cancers, including colon cancer.. In the current study, the chemotherapeutic activity of doxycycline was tested in a rat model of colon carcinogenesis, induced by colon specific cancer promoter, 1,2, dimethylhydrazine (DMH) as well as study the effect of DOX-alone on a separate group of rats.. Doxycycline administration in DMH-treated rats (DMH-DOX) unexpectedly increased tumor multiplicity, stimulated progression of colonic tumor growth from adenomas to carcinomas and revealed metastasis in small intestine as determined by macroscopic and histopathological analysis. DOX-alone treatment showed markedly enhanced chronic inflammation and reactive hyperplasia, which was dependent upon the dose of doxycycline administered. Moreover, immunohistochemical analysis revealed evidence of inflammation and anti-apoptotic action of DOX by deregulation of various biomarkers.. These results suggest that doxycycline caused chronic inflammation in colon, small intestine injury, enhanced the efficacy of DMH in tumor progression and provided a mechanistic link between doxycycline-induced chronic inflammation and tumorigenesis. Ongoing studies thus may need to focus on the molecular mechanisms of doxycycline action, which lead to its inflammatory and tumorigenic effects.

Topics: 1,2-Dimethylhydrazine; Animals; Body Weight; Carcinogenesis; Caspase 3; Caspase 9; Cell Proliferation; Chronic Disease; Colonic Neoplasms; Cytochromes c; Down-Regulation; Doxycycline; Immunohistochemistry; Inflammation; Intestine, Small; Male; Matrix Metalloproteinase 9; Neoplasm Metastasis; NF-kappa B; Rats, Sprague-Dawley; Tumor Suppressor Protein p53; Up-Regulation; Vascular Endothelial Growth Factor A

Perfluorinated chemicals (PFCs) are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS) caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs). In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1) by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP) assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α) by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE) in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation.

Topics: Acetylation; Alkanesulfonic Acids; Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Carnitine; Cell Line; Cell Proliferation; Cytochromes c; Cytosol; Fluorocarbons; Genes, Reporter; Inflammation; Kidney Function Tests; Kidney Tubules; Male; Mice, Inbred BALB C; Oxidation-Reduction; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR gamma; Protective Agents; Protein Binding; Rats; Response Elements; Rosiglitazone; Signal Transduction; Sirtuin 1; Thiazolidinediones; Tumor Suppressor Protein p53

After focal brain injuries occur, in addition to the effects that are attributable to the primary site of damage, the resulting functional impairments depend highly on changes that occur in regions that are remote but functionally connected to the site of injury. Such effects are associated with apoptotic and inflammatory cascades and are considered to be important predictors of outcome. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique that is used to treat various central nervous system (CNS) pathologies and enhance functional recovery after brain damage.. This study examined the efficacy of rTMS in mitigating remote degeneration and inflammation and in improving functional recovery in a model of focal brain damage.. Rats that were undergoing hemicerebellectomy (HCb) were treated with an rTMS protocol for 7 days, and neuronal death indices, glial activation, and functional recovery were assessed.. rTMS significantly reduced neuronal death and glial activation in remote regions and improved functional recovery.. Our finding opens up a completely new scenario for exploiting the potential of rTMS as an anti-apoptotic and anti-inflammatory treatment.

Topics: Animals; Apoptosis; Brain Injuries; Calcium-Binding Proteins; Cytochromes c; Disease Models, Animal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Inflammation; Male; Microfilament Proteins; Neuroglia; Phosphopyruvate Hydratase; Rats; Rats, Wistar; Recovery of Function; RNA, Messenger; Transcranial Magnetic Stimulation

The presence of lead (Pb) in fetal brain may affect brain development-related proteins. We studied whether gestational/lactational Pb-exposure affects oxidative stress, proinflammatory response, apoptosis and levels of brain development/cognition-related proteins, including presynaptic synaptosome-associated protein-25 (SNAP-25), postsynaptic density protein-95 (PSD-95), brain-derived neurotropic factor (BDNF), tyrosine receptor-kinase protein B (TrkB) and vesicular acetylcholine transporter (VAChT) in the offspring. Female Wistar rats were randomly divided into control and Pb-exposed mother groups. The Pb-exposed rats received 0.1% (w/v) Pb acetate via drinking water during pregnancy and lactation. Milk and mammary glands were collected from lactating mothers to measure milk/mammary gland levels of lipid peroxide (LPO), as indicator of oxidative stress and proinflammatory TNF-α. Afterwards, the pups were sacrificed to determine brain levels of Pb, LPO, TNF-α, cytochrome C, SNAP-25, PSD-95, BDNF, TrkB and VAChT. The levels of LPO and TNF-α increased in the milk/mammary glands of the Pb-exposed mothers, concurrently with increases in the levels of Pb, LPO, TNF-α and cytochrome C and decreases in the levels of SNAP-25, PSD-95, BDNF, TrkB and VAChT in the brains of their offspring. Our results demonstrate that Pb-exposure during development reduces the brain levels of PSD-95 and SNAP-25 (synaptogenesis-markers), with concomitant upsurges of oxidative stress, TNF-α and apoptosis in the offspring. Furthermore, BDNF-TrkB proteins that comprehend memory-related brain cognitions and/or VAChT that comprises cholinergic-neuromotor activities might be impaired by Pb-exposure. These findings provide evidence of toxic effects of Pb on brain development, at least, partially by decreasing the levels of PSD-95, SNAP-25 and other cognition-related proteins.

Topics: Animals; Apoptosis; Brain; Brain-Derived Neurotrophic Factor; Cytochromes c; Cytokines; Female; Humans; Inflammation; Lead; Lipid Peroxidation; Male; Mammary Glands, Human; Membrane Proteins; Oxidative Stress; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Wistar; Up-Regulation

Objective To investigate the protective effects of epigallocatechin-3-gallate (EGCG), a major polyphenol source in green tea, against hepatic ischaemia-reperfusion injury in mice. Methods The partial hepatic ischaemia-reperfusion injury model was created by employing the hanging-weight method in C57BL/6 male mice. EGCG (50 mg/kg) was administered via an intraperitoneal injection 45 min before performing the reperfusion. A number of markers of inflammation, oxidative stress, apoptosis and liver injury were measured after the ischaemia-reperfusion injury had been induced. Results The treatment groups were: sham-operated (Sham, n = 10), hepatic ischaemia-reperfusion injury (IR, n = 10), and EGCG with ischaemia-reperfusion injury (EGCG-treated IR, n = 10). Hepatic ischaemia-reperfusion injury increased the levels of biochemical and histological markers of liver injury, increased the levels of malondialdehyde, reduced the glutathione/oxidized glutathione ratio, increased the levels of oxidative stress and lipid peroxidation markers, decreased B-cell lymphoma 2 levels, and increased the levels of Bax, cytochrome c, cleaved caspase-3, and cleaved caspase-9. Pretreatment with EGCG ameliorated all of these changes. Conclusion The antioxidant and antiapoptotic effects of EGCG protected against hepatic ischaemia-reperfusion injury in mice.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Catechin; Cytochromes c; Disease Models, Animal; Glutathione; Glutathione Disulfide; Inflammation; Injections, Intraperitoneal; Lipid Peroxidation; Liver; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury

Obacunone and obacunone glucoside (OG) are naturally occurring triterpenoids commonly found in citrus and other plants of the Rutaceae family. The current study reports the mechanism of cytotoxicity of citrus-derived obacunone and OG on human androgen-dependent prostate cancer LNCaP cells. Both limonoids exhibited time- and dose-dependent inhibition of cell proliferation, with more than 60% inhibition of cell viability at 100 μM, after 24 and 48 h. Analysis of fragmentation of DNA, activity of caspase-3, and cytosolic cytochrome-c in the cells treated with limonoids provided evidence for activation of programmed cell death by limonoids. Treatment of LNCaP cells with obacunone and OG resulted in dose-dependent changes in expression of proteins responsible for the induction of programmed cell death through the intrinsic pathway and down-regulation of Akt, a key molecule in cell signaling pathways. In addition, obacunone and OG also negatively regulated an inflammation-associated transcription factor, androgen receptor, and prostate-specific antigen, and activated proteins related to the cell cycle, confirming the ability of limonoids to induce cytotoxicity through multiple pathways. The results of this study provided, for the first time, an evidence of the cytotoxicity of obacunone and OG in androgen-dependent human prostate cancer cells.

Topics: Apoptosis; Benzoxepins; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Citrus paradisi; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Down-Regulation; Glucosides; Humans; Inflammation; Limonins; Male; Plant Extracts; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction; Triterpenes

IL-17, classified as an inflammatory cytokine, plays a key role in the activation of inflammatory processes involving neutrophils.. Twenty healthy voluntary blood donors were controlled in the study. The granulocyte suspensions were stimulated with rhIL-17 and fMLP. Expression of Bcl-xl, Smac/DIABLO, and Omi/HtrA2 in neutrophil lysates were assessed by Western blot. The level of cytochrome c and activity of caspase 9 was also assayed in these cells.. The results of existing research highlight the importance of rhIL-17 in reducing the survival of neutrophils via the mitochondria, depending on the Bcl-2 protein family. Our research has indicated that rhIL-17 regulates the mutual relationships between the proteins of that family. The proapoptotic effect observed in neutrophils affected by rhIL-17 is a result of a decreased expression of Bcl-xl. Consequently, the expression of apoptogenic proteins, including cytochrome c, Smac/DIABLO, and Omi/HtrA2, is elevated. Surprisingly, there have been no observations of the cytokine influencing the activity of caspase 9.. Results have shown for the first time that IL-17 has a direct effect on the decrease of Bcl-xl. In conclusion, the results of the research presented in this article confirm the dual action of IL-17, which, on the one hand, leads to an array of proinflammatory mechanisms regarding neutrophils and, on the other hand, reduces the survival of those cells via an immediate influence on the Bcl-2 family of proteins and apoptogenic factors.

Topics: Adult; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Blood Donors; Cell Survival; Cytochromes c; Granulocytes; Healthy Volunteers; High-Temperature Requirement A Serine Peptidase 2; Humans; Inflammation; Interleukin-17; Intracellular Signaling Peptides and Proteins; Middle Aged; Mitochondria; Mitochondrial Proteins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Serine Endopeptidases; Young Adult

The present results demonstrated that high glucose (G), salt (S), and cholesterol C (either alone or in combination), as mimicking extracellular changes in metabolic syndrome, damage cardiomyocyte-like H9c2 cells and reduce their viability in a time-dependent manner. However, the effects were greatest when cells were exposed to all three agents (GSC). The mRNA of glycoprotein (gp) 130 and WSX-1, both components of the interleukin (IL)-27 receptor, were present in H9c2 cells. Although mRNA expression was not affected by exogenous treatment with IL-27, the expression of gp130 mRNA (but not that of WSX-1 mRNA) was attenuated by GSC. Treatment of IL-27 to H9c2 cells increased activation of signal transducer and activator of transcription 3 (STAT3) and protected cells from GSC-induced cytochrome c release and cell damage. The protective effects of IL-27 were abrogated by the STAT3 inhibitor, stattic. The results of the present study clearly demonstrate that the STAT3 pathway triggered by anti-inflammatory IL-27 plays a role in protecting cardiomyocytes against GSC-mediated damage.

Topics: Cell Survival; Cholesterol; Cytochromes c; Gene Expression Regulation; Glucose; Glycoproteins; Humans; Inflammation; Interleukins; Metabolic Syndrome; Myocytes, Cardiac; Receptors, Interleukin; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor

Global or local ischemia contributes to the pathogenesis of acute kidney injury (AKI). Currently there are no specific therapies to prevent AKI. Potentiation of glycolytic metabolism and attenuation of mitochondrial respiration may decrease cell injury and reduce reactive oxygen species generation from the mitochondria. Meclizine, an over-the-counter anti-nausea and -dizziness drug, was identified in a 'nutrient-sensitized' chemical screen. Pretreatment with 100 mg/kg of meclizine, 17 h prior to ischemia protected mice from IRI. Serum creatinine levels at 24 h after IRI were 0.13 ± 0.06 mg/dl (sham, n = 3), 1.59 ± 0.10 mg/dl (vehicle, n = 8) and 0.89 ± 0.11 mg/dl (meclizine, n = 8). Kidney injury was significantly decreased in meclizine treated mice compared with vehicle group (p < 0.001). Protection was also seen when meclizine was administered 24 h prior to ischemia. Meclizine reduced inflammation, mitochondrial oxygen consumption, oxidative stress, mitochondrial fragmentation, and tubular injury. Meclizine preconditioned kidney tubular epithelial cells, exposed to blockade of glycolytic and oxidative metabolism with 2-deoxyglucose and NaCN, had reduced LDH and cytochrome c release. Meclizine upregulated glycolysis in glucose-containing media and reduced cellular ATP levels in galactose-containing media. Meclizine inhibited the Kennedy pathway and caused rapid accumulation of phosphoethanolamine. Phosphoethanolamine recapitulated meclizine-induced protection both in vitro and in vivo.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Cell Respiration; Cytochromes c; Deoxyglucose; Disease Models, Animal; Epithelial Cells; Ethanolamines; Galactose; Glycolysis; Humans; Inflammation; Ischemic Preconditioning; Kidney; Kidney Tubules; L-Lactate Dehydrogenase; LLC-PK1 Cells; Male; Meclizine; Mice, Inbred C57BL; Mitochondria; Protective Agents; Reperfusion Injury; Sodium Cyanide; Swine; Up-Regulation

Inflammation is common in hemodialysis (HD) patients. Mitochondrial damage-associated molecular patterns (DAMPs) are released during cell necrosis or apoptosis and induce inflammation. Cell apoptosis is increased in HD patients. The mitochondrial protein cytochrome c, as a marker of released mitochondrial DAMPs, and interleukin-6 (IL-6), as a marker of inflammation, were evaluated in HD patients.. Thirty-four HD patients and 20 controls were enrolled in the study. Serum cytochrome c and IL-6 were measured by means of enzyme-linked immunosorbent assay.. Compared to controls, cytochrome c was markedly increased in HD patients (1392.88 ± 905.24 pg/mL vs. 212.95 ± 91.71 pg/mL). IL-6 was also significantly increased in HD patients (50.32 ± 35.89 pg/mL vs. 14.27 ± 6.83 pg/mL). In HD patients serum IL-6 was positively related to serum cytochrome c (r = 0.458).. Both circulating cytochrome c and IL-6 are markedly increased in HD patients. Cytochrome c is positively related to IL-6.

Topics: Aged; Apoptosis; Biomarkers; Case-Control Studies; Cytochromes c; Female; Humans; Inflammation; Interleukin-6; Kidney Failure, Chronic; Male; Middle Aged; Mitochondrial Proteins; Renal Dialysis

Chronic inflammation promotes prostate cancer formation and progression. Furthermore, alterations in energy metabolism are a hallmark of prostate cancer cells. However, the actions of inflammatory factors on the energy metabolism of prostate epithelial cells have not been previously investigated. This is the first study to report on the effect of the inflammatory cytokine tumor necrosis factor alpha (TNFα) on the glycolytic and oxidative metabolism, and the mitochondrial function of widely used prostate epithelial cells.. Pre-malignant RWPE-1 and cancerous LNCaP and PC-3 cells were treated with low-dose TNFα. Glycolytic and oxidative metabolism was quantified by measuring extracellular acidification and oxygen consumption rates, respectively. ATP content and lactate export were measured by luminescence and fluorescence, respectively. Mitochondrial content and the expression of glucose transporter 1 (GLUT1), peroxisome proliferator-activated receptor co-activator 1 alpha (PGC-1α), and Cytochrome C were measured by flow cytometry.. Our data suggest that TNFα increases glycolysis, ATP production, and lactate export, while it reduces oxidative metabolism and mitochondrial function in prostate epithelial cells. The highly aggressive PC-3 cells tend to be less responsive to the actions of TNFα than the pre-malignant RWPE-1 and the non-aggressive LNCaP cells.. Cellular energetics, that is, glycolytic and oxidative metabolism is significantly influenced by low-level inflammation in prostate epithelial cells. In widely used prostate epithelial cell models, the micro-environmental inflammatory cytokine TNFα induces aerobic glycolysis while inhibiting oxidative metabolism. This supports the hypothesis that low-level inflammation can induce Warburg metabolism in prostate epithelial cells, which may promote cancer formation and progression.

Topics: Cell Line, Tumor; Cytochromes c; Disease Progression; Energy Metabolism; Epithelial Cells; Glucose Transporter Type 1; Glycolysis; Humans; Inflammation; Male; Mitochondria; Oxidative Stress; PPAR alpha; Precancerous Conditions; Prostate; Prostatic Neoplasms; Tumor Microenvironment; Tumor Necrosis Factor-alpha

Increasing evidence has established causative links between obesity, chronic inflammation and insulin resistance; the core pathophysiological feature in type 2 diabetes mellitus. This study was designed to examine whether the combination of L-cysteine and metformin would provide additional benefits in reducing oxidative stress, inflammation and insulin resistance in streptozotocin-induced type 2 diabetes in rats. Male Wistar rats were fed a high-fat diet (HFD) for 8 weeks to induce insulin resistance after which they were rendered diabetic with low-dose streptozotocin. Diabetic rats were treated with metformin (300 mg/kg/day), L-cysteine (300 mg/kg/day) and their combination along with HFD for another 2 weeks. Control rats were fed normal rat chow throughout the experiment. At the end of treatment, fasting blood glucose, fasting serum insulin, homeostasis model assessment-insulin resistance index (HOMA-IR) and serum free fatty acids (FFAs) were measured. Serum levels of the inflammatory markers; monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP) and nitrite/nitrate were also determined. The liver was isolated and used for determination of malondialdehyde (MDA), reduced glutathione (GSH), caspase-3 and cytochrome c levels. The hypoglycemic effect of the combination therapy exceeded that of metformin and L-cysteine monotherapies with more improvement in insulin resistance. All treated groups exhibited significant reductions in serum FFAs, oxidative stress and inflammatory parameters, caspase-3 and cytochrome c levels compared to untreated diabetic rats with the highest improvement observed in the combination group. In conclusion, the present results clearly suggest that L-cysteine can be strongly considered as an adjunct to metformin in management of type 2 diabetes.

Topics: Animals; Body Weight; C-Reactive Protein; Caspase 3; Chemokine CCL2; Cysteine; Cytochromes c; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Drug Interactions; Fatty Acids, Nonesterified; Glutathione; Inflammation; Insulin Resistance; Liver; Male; Malondialdehyde; Metformin; Nitrates; Nitrites; Oxidative Stress; Rats; Rats, Wistar

Quercetin, existing mostly in its glycoside form quercitrin, is the most widely distributed flavonoid in nature. It possesses various potential effects as an antioxidant, anti-inflammatory for cell damage of β-cells, however, studies on this topic are limited and controversial. In order to examine the effects of quercetin on type I diabetes mellitus, we investigated the role of quercetin/quercitrin in cytokine-induced β-cell injuries in RINm5F rat insulinoma cells. Cell viability, glucose-stimulated insulin secretion (GSIS), intracellular reactive oxygen species (ROS), nitric oxide (NO) and inflammation or apoptosis-associated protein expression were measured with or without quercetin/quercitrin treatment. We also compared the differences between the aglycone and the glycoside forms of quercetin, with the aim to shed some light on their structures and transportation into cells. The results showed that quercetin/quercitrin protected against cytokine-induced cell death, improved GSIS, and inhibited ROS as well as NO accumulation. These effects were associated with reduced expression of inducible nitric oxide synthases (iNOS) and inhibited translocation of nuclear factor-κB (NF-κB). Also, quercetin/quercitrin suppressed cytochrome c release from mitochondria and the following alteration of downstream proteins, suggesting that mitochondrial apoptosis was attenuated by quercetin treatment. In summary, quercetin and quercitrin are potential candidates to prevent β-cell death via the mitochondrial pathway and NF-κB signaling, and quercetin may be more efficacious than quercitrin as an anti-diabetic agent.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytokines; Glucose; Hypoglycemic Agents; Inflammation; Insulin; Insulin Secretion; Insulin-Secreting Cells; Insulinoma; Mitochondria; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Quercetin; Rats; Reactive Oxygen Species; Signal Transduction

Pro-inflammatory and pro-apoptotic mediators have been involved in the pathogenesis of neurodegenerative diseases. Tigecycline (Tig), a glycylcycline antibiotic and an analog of Minocycline, is shown to exert anti-inflammatory effects that are distinct from its anti-microbial activity. Its neuroprotective mechanism is unknown. In this study, we investigated the direct protective mechanisms of tigecycline against lipopolysaccharide (LPS)-induced Rat pheochromocytoma (PC12) cells. The results showed that tigecycline significantly attenuated the expression and the release of nuclear factor-kappa beta (NF-κB), tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β), as well as nitric oxide (NO) levels in LPS-induced PC12 cells. In addition, tigecycline dose-dependently decreased cytochrome c release and caspase-3 activity. This later finding corroborated the results of decreased pro-apoptotic Bad, and increased anti-apoptotic Bcl-2 protein expression thus, confirming a neuroprotective effect of the drug in differentiated PC12 cells induced with LPS. The findings of our study suggest new targets for tigecycline and support the potential for tigecycline to be investigated as a therapeutic agent for neurodegenerative disorders.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Caspase 3; Cell Survival; Cytochromes c; Inflammation; Inflammation Mediators; Interleukin-1beta; Lipopolysaccharides; Minocycline; Neurons; Neuroprotective Agents; NF-kappa B; Nitrites; PC12 Cells; Rats; Tigecycline; Tumor Necrosis Factor-alpha

6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinson's disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Caspase 3; Caspase 9; Corpus Striatum; Cytochromes c; Inflammation; JNK Mitogen-Activated Protein Kinases; Male; Mitochondria; Neurotoxicity Syndromes; NF-E2-Related Factor 2; NF-kappa B; Oxidopamine; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Wistar; Signal Transduction; Up-Regulation

Gastric ulcer is a multifaceted process that involves reactive oxygen species (ROS) generation, extracellular matrix degradation and mitochondrial damage. Mitochondria play a crucial role for homeostasis of ROS and cell survival. In our study, we investigated the efficacy and mechanism of polymeric nanocapsuled quercetin (NQC) over the free quercetin (QC) molecule in prevention of ethanol-induced gastric ulcer in rat. NQC possessed significantly higher efficacy (~20 fold) than free QC while preventing gastric ulcers. Our data show that prior administration of NQC and/or QC significantly blocked synthesis and secretion of matrix metalloproteinase (MMP)-9 as well as infiltration of inflammatory cells and oxidative damage in rat gastric tissues. As compared to free QC, NQC protected much better the mitochondrial integrity and size along with mitochondrial functions by controlling succinate dehydrogenase and NADH oxidase in rat gastric tissues. In addition, both free QC and NQC down regulated PARP-1 as well as apoptosis during protection against ethanol-induced gastric ulcer. Herein, the effect of NQC was greater than QC on expression of enzymes like cyclooxygenase and nitric oxidase synthase (NOS)-2. We conclude that NQC with greater bioavailability offers significantly higher potency in downregulating MMP-9 and NOS-2 as well as oxidative stress in blocking ethanol-induced gastric ulcer.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cytochromes c; Cytokines; Ethanol; Gastric Mucosa; Glutathione; Inflammation; Lactic Acid; Male; Matrix Metalloproteinase 9; Membrane Potential, Mitochondrial; Mitochondria; Nanoparticles; Nitric Oxide Synthase Type II; Particle Size; Peroxidase; Poly(ADP-ribose) Polymerases; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Quercetin; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stomach; Stomach Ulcer; Up-Regulation

We tested the hypothesis that obesity reduced circulating number of endothelial progenitor cells (EPCs), angiogenic ability, and blood flow in ischemic tissue that could be reversed after obesity control.. 8-week-old C57BL/6J mice (n=27) were equally divided into group 1 (fed with 22-week control diet), group 2 (22-week high fat diet), and group 3 (14-week high fat diet, followed by 8-week control diet). Critical limb ischemia (CLI) was induced at week 20 in groups 2 and 3. The animals were sacrificed at the end of 22 weeks.. Heart weight, body weight, abdominal fat weight, serum total cholesterol level, and fasting blood sugar were highest in group 2 (all p<0.001). The numbers of circulating EPCs (C-kit/CD31+, Sca-1/KDR + and CXCR4/CD34+) were lower in groups 1 and 2 than in group 3 at 18 h after CLI induction (p<0.03). The numbers of differentiated EPCs (C-kit/CD31+, CXCR4/CD34+ and CD133+) from adipose tissue after 14-day cultivation were also lowest in group 2 (p<0.001). Protein expressions of VCAM-1, oxidative index, Smad3, and TGF-β were higher, whereas the Smad1/5 and BMP-2, mitochondrial cytochrome-C SDF-1α and CXCR4 were lower in group 2 than in groups 1 and 3 (all p<0.02). Immunofluorescent staining of CD31+ and vWF + cells, the number of small vessel (<15 μm), and blood flow through Laser Doppler scanning of ischemic area were lower in group 2 compared to groups 1 and 3 on day 14 after CLI induction (all p<0.001).. Obesity suppressed abilities of angiogenesis and recovery from CLI that were reversed by obesity control.

Topics: Adipose Tissue; Animals; Biomarkers; Cell Movement; Cytochromes c; Cytosol; Endothelial Cells; Fibrosis; Fluorescent Antibody Technique; Hindlimb; Inflammation; Ischemia; Laser-Doppler Flowmetry; Male; Mice; Mitochondria; Neovascularization, Physiologic; Obesity; Oxidative Stress; Regional Blood Flow; Stem Cells

Paeoniflorin (PF), the principal component of Paeoniae Radix prescribed in traditional Chinese medicine, has been reported to exhibit many pharmacological effects including protection against ischemic injury. However, the mechanisms underlying the protective effects of PF on cerebral ischemia are still under investigation. The present study showed that PF treatment for 14 days could significantly inhibit transient middle cerebral artery occlusion (MCAO)-induced over-activation of astrocytes and microglia, and prevented up-regulations of pro-inflamamtory mediators (TNFα, IL-1β, iNOS, COX(2) and 5-LOX) in plasma and brain. Further study demonstrated that chronic treatment with PF suppressed the activations of JNK and p38 MAPK, but enhanced ERK activation. And PF could reverse ischemia-induced activation of NF-κB signaling pathway. Moreover, our in vitro study revealed that PF treatment protected against TNFα-induced cell apoptosis and neuronal loss. Taken together, the present study demonstrates that PF produces a delayed protection in the ischemia-injured rats via inhibiting MAPKs/NF-κB mediated peripheral and cerebral inflammatory response. Our study reveals that PF might be a potential neuroprotective agent for stroke.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Astrocytes; bcl-2-Associated X Protein; Benzoates; Brain; Brain Ischemia; Bridged-Ring Compounds; Cerebral Infarction; Cyclooxygenase 2; Cytochromes c; Disease Models, Animal; Gene Expression Regulation; Glucosides; Hippocampus; Inflammation; Interleukin-1beta; Lipoxygenase; Male; Microglia; Mitogen-Activated Protein Kinases; Monoterpenes; Neurons; NF-kappa B; Nitric Oxide Synthase Type II; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Tumor Necrosis Factor-alpha

Apoptosis and inflammatory/oxidative stress have been associated with hyperglycemia in human peritoneal mesothelial cells (HPMCs) and other cell types. We and others have highlighted the role of early products of non-enzymatic protein glycation in inducing proinflammatory conditions and increasing apoptotic rates in HPMCs. Loss of HPMCs seems to be a hallmark of complications associated with peritoneal membrane dysfunction. The aim of this work is to elucidate the mechanisms by which Amadori adducts may act upon HPMC apoptosis.. HPMCs isolated from different patients were exposed to different Amadori adducts, i.e. highly glycated hemoglobin (10 nM) and glycated bovine serum albumin (250 μg/ml), to study cell death and several proapoptotic markers by different experimental approaches.. Amadori adducts, but not their respective controls, impaired cell proliferation and cell viability by means of apoptosis in a time-dependent manner. They regulated the intrinsic mitochondrial cell death signaling pathway and modulated activation of caspases, Bax, iNOS, p53, NF-κB, and mitogen-activated protein kinases (p38 and JNK) through different reactive oxygen and nitrosative species.. Our data strongly support the idea that long-term hyperglycemia could act as an inducer of apoptosis in HPMCs through Amadori adducts, involving different oxidative and nitrosative reactive species.

Topics: Animals; Apoptosis; Cattle; Cell Death; Cytochromes c; Epithelium; Glycolipids; Humans; Hyperglycemia; Inflammation; L-Lactate Dehydrogenase; MAP Kinase Signaling System; Nitrogen; Oxidative Stress; Phosphatidylethanolamines; Proto-Oncogene Proteins c-jun; Signal Transduction

Nerve growth factor (NGF) antagonism has long been proposed as a chronic pain treatment. In 2010, the FDA suspended clinical trials using tanezumab, a humanized monoclonal anti-NGF antibody, to treat osteoarthritis due to worsening joint damage in 16 patients. Increased physical activity in the absence of acute pain which normally prevents self-harm was purported as a potential cause. Such an adverse effect is consistent with an extension of tanezumab's primary mechanism of action by decreasing pain sensitivity below baseline levels. In animal inflammatory pain models, NGF antagonism decreases intraepidermal nerve fiber (IENF) density and attenuates increases in expression of nociception-related proteins, such as calcitonin gene-related peptide (CGRP) and substance P (SP). Little is known of the effects of NGF antagonism in noninflamed animals and the hypoalgesia that ensues. In the current study, we immunized rats with NGF or cytochrome C (cytC) and examined (1) nocifensive behaviors with thermal latencies, mechanical thresholds, the hot plate test, and the tail flick test, (2) IENF density, and (3) expression of CGRP, SP, voltage-gated sodium channel 1.8 (Nav1.8), and glutaminase in subpopulations of dorsal root ganglion (DRG) neurons separated by size and isolectin B4 (IB4) labeling. Rats with high anti-NGF titers had delayed responses on the hot plate test but no other behavioral abnormalities. Delayed hot plate responses correlated with lower IENF density. CGRP and SP expression was decreased principally in medium (400-800 μm(2)) and small neurons (<400 μm(2)), respectively, regardless of IB4 labeling. Expression of Nav1.8 was only decreased in small and medium IB4 negative neurons. NGF immunization appears to result in a more profound antagonism of NGF than tanezumab therapy, but we hypothesize that decreases in IENF density and nociception-related protein expression are potential mechanisms for tanezumab-induced hypoalgesia.

Topics: Age Factors; Animals; Antibodies, Monoclonal, Humanized; Calcitonin Gene-Related Peptide; Cytochromes c; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Ganglia, Spinal; Glycoproteins; Immunoglobulins; Inflammation; Male; NAV1.8 Voltage-Gated Sodium Channel; Nerve Growth Factor; Nerve Tissue Proteins; Pain; Pain Threshold; Rats; Rats, Sprague-Dawley; Sodium Channels; Substance P

This study tested the superiority of combined cyclosporine A (CsA)-erythropoietin (EPO) therapy compared with either one in limiting brain infarction area (BIA) and preserving neurological function in rat after ischemic stroke (IS).. Fifty adult-male SD rats were equally divided into sham control (group 1), IS plus intra-peritoneal physiological saline (at 0.5/24/48 h after IS) (group 2), IS plus CsA (20.0 mg/kg at 0.5/24h, intra-peritoneal) (group 3), IS plus EPO (5,000IU/kg at 0.5/24/48h, subcutaneous) (group 4), combined CsA and EPO (same route and dosage as groups 3 and 4) treatment (group 5) after occlusion of distal left internal carotid artery.. BIA on day 21 after acute IS was higher in group 2 than in other groups and lowest in group 5 (all p < 0.01). The sensorimotor functional test showed higher frequency of left turning in group 2 than in other groups and lowest in group 5 (all p < 0.05). mRNA and protein expressions of apoptotic markers and number of apoptotic nuclei on TUNEL were higher in group 2 than in other groups and lowest in group 1 and 5, whereas the anti-apoptotic markers exhibited an opposite trend (all p < 0.05). The expressions of inflammatory and oxidized protein were higher in group 2 than in other groups and lowest in group 1 and 5, whereas anti-inflammatory markers showed reversed changes in group 1 and other groups (all p < 0.05). The number of aquaporin-4+ and glial fibrillary acid protein+ stained cells were higher in group 2 as compared to other groups and lowest in groups 1 and 5 (all p < 0.01).. combined treatment with CsA and EPO was superior to either one alone in protecting rat brain from ischemic damage after IS.

Topics: Animals; Apoptosis; Aquaporin 4; Brain Infarction; Cell Nucleus; Cyclosporine; Cytochromes c; Drug Therapy, Combination; Erythropoietin; Gene Expression Regulation; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Inflammation; Male; Oxidative Stress; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Rats; Rats, Sprague-Dawley; Recovery of Function; RNA-Binding Proteins; RNA, Messenger; Stroke; Transcription Factors

Pro-inflammatory cytokine-mediated pancreatic β-cell dysfunction is a key pathological event in type 1 diabetes mellitus. There are few studies about the protection of epigallocatechin-3-gallate (EGCG) against pro-inflammatory cytokine-induced β-cell apoptosis. To examine the direct effects of EGCG on β-cells, insulin-producing RINm5F cells were exposed to a combination of recombinant interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ), with or without EGCG pretreatment for 24h. Cell death was monitored by the MTT assay. Glucose-stimulated insulin release was measured using radio immunoassay. Intracellular reactive oxygen species was examined with dichlorofluorescein (DCF) fluorescence by flow cytometry. To evaluate RINm5F cells mitochondrial function, change in mitochondrial membrane potential, intracellular ATP levels, and nitric oxide was assessed. The expression of cytochrome c, Bax, Bcl-2, and iNOS proteins was measured by western blotting. In the present study, EGCG pretreatment protected against cytokines inducing cell death and restored glucose stimulated-insulin secretion in RINm5F cells. EGCG reduced the cytokine-induced generation of reactive oxygen species, the loss of mitochondrial membrane potential (Δψm), the release of cytochrome c from the mitochondria, and translocation of Bax protein to the mitochondria from the cytosol. EGCG pretreatment prevented cytokine-induced iNOS overexpression and NO generation. In summary, pro-inflammatory cytokines lead to a reduction of glucose-induced insulin secretion, mitochondrial activity and viability in RINm5F cells. The pro-inflammatory cytokine-induced effects can be prevented by EGCG pretreatment via the mitochondrial pathway.

Topics: Adenosine Triphosphate; bcl-2-Associated X Protein; Catechin; Cell Death; Cell Line; Cytochromes c; Cytokines; Down-Regulation; Gene Expression Regulation, Enzymologic; Inflammation; Insulin; Insulin Secretion; Insulin-Secreting Cells; Membrane Potential, Mitochondrial; Mitochondria; Nitric Oxide; Nitric Oxide Synthase Type II; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFalpha). TNFalpha has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFalpha. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 microg of antihuman TNFalpha blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFalpha treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFalpha production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.

Topics: Animals; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Cell Death; Chagas Disease; Cytochromes c; Humans; In Situ Nick-End Labeling; Inflammation; Liver; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor, Type I; Trypanosoma cruzi; Tumor Necrosis Factor-alpha

Our laboratories have previously identified the alpha7 nAChR-JAK2 pathway as playing a central role in nicotine-induced neuroprotection. We have also reported that the angiotensin II (Ang II) AT(2) receptor induced activation of SHP-1 induces the tyrosine dephosphorylation of JAK2 that results in a complete neutralization of the alpha7 nAChR-JAK2 pro-survival cascade. In this study, we investigated the effects of inhibiting the alpha7 nAChR-JAK2 pro-survival cascade on the nicotine-induced production of the survival factor Bcl-2 and the transcriptional activation of NF-kappaB, AP-1, STAT1, STAT3, and STAT5. We report that nicotine induced the production of Bcl-2 and increased the transcriptional activation of NF-kappaB, AP-1, STAT1, and STAT3, and with the exception of AP-1, the other transcription factors (NF-kappaB, STAT1, and STAT3) were significantly reduced by JAK2 inhibition. We also demonstrate that, via transfection of either Bcl-2 antisense or NF-kappaB, STAT1 and STAT3 transcription factor decoys oligodeoxyribonucleotides into PC12 cells, nicotine induces its neuroprotection in PC12 cells via activation of the alpha7 nAChR-JAK2-(NF-kappaB; STAT3)-Bcl-2 pro-survival pathway. Finally, the neuroprotective nicotine-induced production of Bcl-2 appears to fully counteract the Abeta (1-42)-induced apoptosis of PC12 cells by blocking Abeta (1-42)-induced mitochondrial release of cytosolic cytochrome C.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Amyloid beta-Peptides; Animals; Apoptosis; Cytochromes c; Enzyme Activation; Gene Expression; Inflammation; Janus Kinase 2; Mitochondria; Neuroprotective Agents; NF-kappa B; Nicotine; PC12 Cells; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Rats; Receptors, Nicotinic; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Transcription Factor AP-1; Tyrphostins

In genetically susceptible humans and/or experimental animals, ions of heavy metals, Hg (II), Au (III), and Ag (I) have been shown to strongly induce autoimmunity, in which mast cells have been implicated to play a role. Here, we demonstrate that Ag (I) application results in mast cell death through a unique Ca(2+)- and mitochondria-dependent pathway. As cellular susceptibilities to Ag (I) cytotoxicity varied considerably, we analyzed the cell death pathway in the low and high responding cells. In the low responding cells, long application (e.g., 20 h) of Ag (I) at concentrations (>or=30 microM) induced cell death, which was accompanied by mitochondrial membrane depolarization, cyt c release, and caspase-3/7 activation but was not prevented by selective inhibitors of caspase-3/7 and the mitochondrial permeability transition. The cell death was preceded by elevations in the cytoplasmic and mitochondrial Ca(2+) levels, and Ca(2+) responses and cell death were prevented by thiol reagents, including DTT, N-acetylcysteine, and reduced glutathione monoethyl ester. In the high responding cells, Ag (I) evoked considerable cell death by necrosis within 1 h, without inducing caspase activation, and this cell death was reduced significantly by depleting extracellular but not intracellular Ca(2+). Moreover, Ag (I) strongly induced Ca(2+)-dependent CL oxidation and intracellular ATP depletion, both of which were blocked by thiol reagents. These results suggest that Ag (I) activates thiol-dependent Ca(2+) channels, thereby promoting Ca(2+)-dependent CL oxidation, cyt c release, and ATP depletion. This necrotic cell death may play roles in Ag-induced inflammation and autoimmune disorders.

Topics: Adenosine Triphosphate; Animals; Autoimmunity; Calcium; Calcium Channels; Cardiolipins; Cytochromes c; Inflammation; Mast Cells; Mice; Mice, Inbred C57BL; Mitochondria; Necrosis; Oxidation-Reduction; Silver

Mice with a keratinocyte-specific deletion of Tak1 exhibit severe skin inflammation due to hypersensitivity to tumor necrosis factor (TNF) killing. Here we have examined the mechanisms underlying this hypersensitivity. We found that TAK1 deficiency up-regulates reactive oxygen species (ROS) resulting in cell death upon TNF or oxidative stress challenge. Because blockade of NF-kappaB did not increase ROS or did not sensitize cells to oxidative stress in keratinocytes TAK1 regulates ROS mainly through the mechanisms other than those mediated by NF-kappaB. We found that c-Jun was decreased in TAK1-deficient keratinocytes and that ectopic expression of c-Jun could partially inhibit TNF-induced increase of ROS and cell death. Finally, we show that, in an in vivo setting, the antioxidant treatment could reduce an inflammatory condition in keratinocyte-specific Tak1 deletion mice. Thus, TAK1 regulates ROS partially through c-Jun, which is important for preventing ROS-induced skin inflammation.

Topics: Animals; Antioxidants; Cytochromes c; Gene Expression Regulation; Inflammation; Keratinocytes; MAP Kinase Kinase Kinases; Mice; Mice, Transgenic; Models, Biological; NF-kappa B; Oxidative Stress; Proto-Oncogene Proteins c-jun; Reactive Oxygen Species; Skin

The purposes of this study were to investigate the effects of strenuous exercise on apoptosis of the gastrocnemius and soleus muscle fibers and clarify the role of oxidative metabolism in the strenuous exercise-induced apoptosis. The experiment was designed with 49 (n = 49) male, 24-week-old, L. Wistar albino rats. Strenuous exercise model was applied to 42 (n = 42) rats and seven (n = 7) rats served as rested controls. All rats were randomly assigned to one of the following groups (n = 7): rested control (C), immediately after exercise (0 h) and 3, 6, 12, 24, and 48 h after exercise. Apoptotic nuclei were shown by single stranded DNA (ssDNA) determination. Oxidative damage in mitochondrial fractions of the muscle tissues was evaluated by malondialdehyde (MDA) levels and reduced/oxidized glutathione (GSH/GSSG) ratios. Caspase-9, -8 and -3 activities and the level of cytochrome c (Cyt c) were measured in the cytosolic fractions of muscle tissues to follow mitochondrial-dependent (intrinsic) or ligand-mediated death receptor (extrinsic) pathways of apoptosis. Plasma interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) levels were also determined. Based on our results, apoptosis is significantly triggered in muscle fibers by strenuous exercise (P < 0.05). Apoptosis in the soleus muscle tissues mostly depends on the intrinsic pathway and may be triggered by increased oxidative stress. In contrast, extrinsic pathway of apoptosis was predominant in the gastrocnemius muscle and increases of TNF-alpha and IL-6 may play a significant role.

Topics: Animals; Apoptosis; Biomarkers; Caspase 3; Caspase 8; Caspase 9; Cytochromes c; Cytosol; DNA, Single-Stranded; Glutathione; Inflammation; Interleukin-6; Lipid Peroxidation; Male; Malondialdehyde; Mitochondria, Muscle; Muscle Fibers, Skeletal; Muscle, Skeletal; Oxidative Stress; Physical Exertion; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

Imiquimod is an immune response modifier currently used as a topical treatment of genital warts, basal cell carcinoma, cutaneous metastasis of malignant melanoma, and vascular tumors. We developed more efficient killers from the same family of compounds that can induce apoptosis without the prominent pro-inflammatory response associated with imiquimod. Among these new products, tk;4EAPB0203, a member of the imidazo[1,2-a]quinoxalines, exhibits an important cytotoxic activity in vitro. HTLV-I-associated adult T-cell leukemia (ATL) and HTLV-I-negative peripheral T-cell lymphomas are associated with poor prognosis. Using potentially achievable concentrations of EAPB0203, we demonstrate inhibition of cell proliferation, G2/M cell- cycle arrest, and induction of apoptosis in HTLV-I-transformed and HTLV-I-negative malignant T cells and fresh ATL cells, whereas normal resting or activated T lymphocytes were resistant. EAPB0203 treatment significantly down-regulated the antiapoptotic proteins c-IAP-1 and Bcl-XL and resulted in a significant loss of mitochondrial membrane potential, cytoplasmic release of cytochrome c, and caspase-dependent apoptosis. Moreover, in HTLV-I-transformed cells only, EAPB0203 treatment stabilized p21 and p53 proteins but had no effect on NF-kappaB activation. These results support a potential therapeutic role for EAPB0203 in ATL and HTLV-I-negative T-cell lymphomas, either as a systemic or topical therapy for skin lesions.

Topics: Aminoquinolines; Antineoplastic Agents; Apoptosis; bcl-X Protein; Caspases; Cell Division; Cytochromes c; G2 Phase; Human T-lymphotropic virus 1; Humans; Imiquimod; Inflammation; Inhibitor of Apoptosis Proteins; Jurkat Cells; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Activation; Lymphoma, T-Cell; Membrane Potential, Mitochondrial; NF-kappa B; Quinoxalines; Skin Neoplasms; Tumor Suppressor Protein p53

In the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species-dependent manner. Under inflammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identified a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D.

Topics: Animals; Apoptosis; Caspase 8; Cathepsin B; Cathepsin D; Cytochromes c; Enzyme Activation; Humans; Immunity, Innate; In Vitro Techniques; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Reactive Oxygen Species; Shock, Septic

Studies in sepsis suggest that mitochondria mediate multiple organ dysfunction, including cardiac failure; however, the underlying molecular mechanisms remain elusive. This study examined changes in mitochondrial membrane integrity, antioxidant activities, and oxidative stress in the heart after infectious challenge (intratracheal Streptococcus pneumoniae, 4 x 10(6) colony-forming units). Inflammation responses also were examined.. Cardiac tissues were harvested from Sprague-Dawley rats 4, 8, 12, and 24 h after bacterial challenge (or intratracheal vehicle for sham-treated animals) and homogenized, followed by preparation of subcellular fractions (mitochondrial, cytosol, and nuclei) or whole-tissue lysate. We examined mitochondrial outer membrane damage and cytochrome C translocation to evaluate mitochondrial integrity, mitochondrial lipid and protein oxidation to assess oxidative stress, and mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities to estimate antioxidant defense. In addition, we measured nuclear factor-kappa B (NF-kappaB) activation in myocardium and cytokine production to investigate inflammatory responses to septic challenge.. Oxidation of mitochondrial protein and lipid was evident 4 h through 24 h after bacterial challenge. Mitochondrial outer membrane damage and cytochrome C release were accompanied by down-regulation of mitochondrial SOD and GPx activity. After bacterial challenge, systemic and myocardial cytokine production increased progressively, and NF-kappaB was activated gradually.. Sepsis impaired cardiac mitochondria by damaging membrane integrity, increasing oxidative stress, and altering defenses against reactive oxygen species. These alterations occur earlier than or simultaneously with inflammatory responses in myocardium after infectious challenge, suggesting that mitochondria play a role in modulating inflammation in sepsis.

Topics: Animals; Cytochromes c; Cytokines; Disease Models, Animal; Glutathione Peroxidase; Inflammation; Male; Membrane Lipids; Mitochondria, Heart; Mitochondrial Membranes; Mitochondrial Proteins; Myocardium; NF-kappa B; Oxidation-Reduction; Oxidative Stress; Pneumococcal Infections; Rats; Rats, Sprague-Dawley; Sepsis; Shock; Streptococcus pneumoniae; Superoxide Dismutase

This issue's recently published papers concentrates on early goal directed therapy, starting with new data from the original study through to new studies that may have a major bearing on the treatment of septic shock in years to come. A timely reminder about talking, walking and teaching clinical medicine completes the roundup.

Topics: Animals; Critical Care; Cytochromes c; Disease Models, Animal; Education, Medical; Humans; Hydrogen; Hypoxia; Inflammation; Mice; Mitochondria; Rats; Sepsis; Time Factors; Vasoconstrictor Agents

This study examined the effects of antioxidant vitamins on several aspects of sepsis-related myocardial signaling cascades. Sprague-Dawley rats were divided into four groups: group 1, vehicle-treated shams; group 2, sham-operated rats given antioxidant vitamins (vitamin C, 24 mg/kg; vitamin E, 20 U/kg; vitamin A, 417 U/kg; and zinc, 3.7 ng/kg) by oral gavage in 0.5 ml water twice daily for 3 days and no septic challenge (vitamin-treated, sham-operated rats); group 3, intratracheal delivery of Streptococcus pneumoniae, 4 x 10(6) colony forming units in a volume of 0.3 ml phosphate buffer solution; group 4, S. pneumonia challenge as described for group 3 plus antioxidant vitamins (as described for group 2). Hearts collected 24 h after septic challenge were used to examine several aspects of cell signaling and ventricular function. As a result, when compared with sham-operated rats, sepsis in the absence of antioxidant therapy promoted NF-kappaB activation, increased mitochondrial cytochrome c release, increased myocyte cytokine secretion, increased caspase activation, and impaired left ventricular function. Antioxidant vitamin therapy plus septic challenge prevented NF-kappaB activation, reduced mitochondrial cytochrome c release, decreased caspase activity, abrogated cardiomyocyte secretion of inflammatory cytokines, and improved myocardial contractile function. In conclusion, antioxidant vitamin therapy abrogated myocardial inflammatory cytokine signaling and attenuated sepsis-related contractile dysfunction, suggesting that antioxidant vitamin therapy may be a potential approach to treat injury and disease states characterized by myocardial dysfunction.

Topics: Animals; Antioxidants; Apoptosis; Calcium; Cardiomyopathies; Caspases; Cytochromes c; Cytokines; Inflammation; Male; Myocardial Contraction; Myocardium; Myocytes, Cardiac; NF-kappa B; Pneumococcal Infections; Rats; Rats, Sprague-Dawley; Sepsis; Signal Transduction; Sodium; Streptococcus pneumoniae; Ventricular Function, Left; Vitamins

Although great achievements have been made in elucidating the molecular mechanisms contributing to acute myocardial ischemia/reperfusion (I/R) injury, an effective pharmacological therapy to protect cardiac tissues from serious damage associated with acute myocardial infarction, coronary arterial bypass grafting surgery, or acute coronary syndromes has not been developed. We examined the in vivo cardioprotective effects of caffeic acid phenethyl ester (CAPE), a natural product with potent anti-inflammatory, antitumor, and antioxidant activities. CAPE was systemically delivered to rabbits either 60 min before or 30 min after surgically inducing I/R injury. Infarct dimensions in the area at risk were reduced by >2-fold (P < 0.01) with CAPE treatment at either period. Accordingly, serum levels of normally cytosolic enzymes lactate dehydrogenase, creatine kinase (CK), MB isoenzyme of CK, and cardiac-specific troponin I were markedly reduced in both CAPE treatment groups (P < 0.05) compared with the vehicle-treated control group. CAPE-treated tissues displayed significantly less cell death (P < 0.05), which was in part due to inhibition of p38 mitogen-activated protein kinase activation and reduced DNA fragmentation often associated with caspase 3 activation (P < 0.05). In addition, CAPE directly blocked calcium-induced cytochrome c release from mitochondria. Finally, the levels of inflammatory proteins IL-1beta and TNF-alpha expressed in the area at risk were significantly reduced with CAPE treatment (P < 0.05). These data demonstrate that CAPE has potent cardioprotective effects against I/R injury, which are mediated, at least in part, by the inhibition of inflammatory and cell death responses. Importantly, protection is conferred when CAPE is systemically administered after the onset of ischemia, thus demonstrating potential efficacy in the clinical scenario.

Topics: Acute Disease; Animals; Apoptosis; Blotting, Western; Caffeic Acids; Cardiotonic Agents; Caspases; Creatine Kinase; Cytochromes c; In Situ Nick-End Labeling; Inflammation; Interleukin-1; L-Lactate Dehydrogenase; Male; Mitochondria, Heart; Myocardial Reperfusion Injury; p38 Mitogen-Activated Protein Kinases; Phenylethyl Alcohol; Rabbits; Rats; Rats, Sprague-Dawley; Troponin I; Tumor Necrosis Factor-alpha

Volatile anesthetics attenuate apoptosis. The underlying mechanisms remain undefined. The authors tested whether isoflurane reduces apoptosis in cardiomyocytes subjected to oxidative or inflammatory stress by enhancing Akt and B-cell lymphoma-2 (Bcl-2).. Adult and neonatal rat ventricular myocytes and atrial HL-1 myocytes were exposed to hypoxia, hydrogen peroxide, or neutrophils with or without isoflurane pretreatment. The authors assessed cell damage and investigated apoptosis using mitochondrial cytochrome c release, caspase activity, and TUNEL assay. They determined expression of phospho-Akt and Bcl-2 and tested their involvement by blocking phospho-Akt with wortmannin and Bcl-2 with HA14-1.. Isoflurane significantly reduced the cell damage and apoptosis induced by hypoxia, H2O2, and neutrophils. Isoflurane reduced hypoxia-induced mitochondrial cytochrome c release in HL-1 cells by 45 +/- 12% and caspase activity by 28 +/- 4%; in neonatal cells, it reduced caspase activity by 43 +/- 5% and TUNEL-positive cells by 50 +/- 2%. Isoflurane attenuated H2O2-induced caspase activity in HL-1 cells by 48 +/- 16% and TUNEL-positive cells by 78 +/- 3%; in neonatal cells, it reduced caspase activity by 30 +/- 3% and TUNEL-positive cells by 32 +/- 7%. In adult cardiomyocytes exposed to neutrophils, isoflurane decreased both mitochondrial cytochrome c and caspase activity by 47 +/- 3% and TUNEL-positive cells by 25 +/- 4%. Isoflurane enhanced phospho-Akt and Bcl-2 expression. Wortmannin and HA14-1 prevented the action of isoflurane (53 +/- 8% and 54 +/- 7% apoptotic cells vs. 18 +/- 1% without blockers).. Isoflurane protects cardiomyocytes against apoptosis induced by hypoxia, H2O2, or activated neutrophils through Akt activation and increased Bcl-2 expression. This suggests that a reduction in apoptosis contributes to the cardioprotective effects of isoflurane.

Topics: Anesthetics, Inhalation; Animals; Animals, Newborn; Apoptosis; Biotransformation; Caspases; Cell Line; Cell Separation; Cytochromes c; Dogs; Genes, bcl-2; Hydrogen Peroxide; Hypoxia; In Situ Nick-End Labeling; Inflammation; Isoflurane; L-Lactate Dehydrogenase; Male; Myocytes, Cardiac; Neutrophil Activation; Neutrophils; Oxidation-Reduction; Oxidative Stress; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Tetrazolium Salts; Thiazoles

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Topics: Adenosine Triphosphate; Animals; Body Weight; Carcinogenicity Tests; Carcinogens; Cell Proliferation; Cytochromes c; DNA Adducts; Genes, ras; Hydroxyl Radical; Hyperplasia; Inflammation; Leukocytes; Luminescent Measurements; Mice; Mice, Inbred SENCAR; Oxidation-Reduction; Oxidative Stress; Peroxidase; Salicylates; Skin; Skin Neoplasms; Smoke; Superoxides

Tumor necrosis factor-alpha (TNF-a) is produced by alveolar macrophages (AM) in response to bleomycin (BLM) exposure. This cytokine has been linked to BLM-induced pulmonary inflammation, an early drug effect, and to lung fibrosis, the ultimate toxic effect of BLM. The present study was carried out to study the time dependence of apoptotic signaling pathways and the potential roles of TNF receptors in BLM-induced AM apoptosis. Male Sprague-Dawley rats were exposed to saline or BLM (1 mg/kg) by intratracheal instillation. At 1, 3, or 7 d postexposure, AM were isolated by bronchoalveolar (BAL) lavage and evaluated for apoptosis by ELISA. The release of cytochrome c from mitochrondria, the activation of caspase-3, -8, and -9, the cleavage of nuclear poly(ADP-ribose) polymerase (PARP), and the expression of TNF receptors (TNF-R1/p55 and TNF-R2/p75), TNF-R-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis 1 (c-IAP1) were determined by immunoblotting. The results showed that BLM exposure induced AM apoptosis, with the highest apoptotic effect occurring at 1 d after exposure and gradually decreasing at 3 and 7 d postexposure, but still remaining significantly above the control level. The maximal translocation of cytochromec from mitochondria into the cytosol was observed at 1 d postexposure, whereas the activation of caspase-9 and caspase-3 and caspase-3-dependent cleavage of PARP was found to reach a peak level at 3 d postexposure. BLM exposure had no marked effect on AM expression of TNF-R1 or caspase-8 activation, but significantly increased the expression of TNF-R2 that was accompanied by a rise in c-IAP1 and a decrease in TRAF2. This induction of TNF-R2 by BLM was significant on d 1 and increased with greater exposure time. In vitro studies showed that pretreatment of naive AM with a TNF-R2 antibody significantly inhibited BLM-induced caspase-3 activity and apoptosis. These results suggest that BLM-induced apoptosis involves multiple pathways in a time-dependent manner. Since maximal BLM-induced AM apoptosis (1 d postexposure) preceded maximal changes in caspase-9 and -3 (3 d postexposure), it is possible that a caspase-independent mechanism is involved in this initial response. These results indicate that the sustained expression of TNF-R2 in AM by BLM exposure may sensitize these cells to TNF-a-mediated toxicity.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, CD; Apoptosis; Bleomycin; Bronchoalveolar Lavage Fluid; Caspase 3; Caspase 8; Caspase 9; Caspases; Cytochromes c; Drug Evaluation, Preclinical; Environmental Exposure; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Inflammation; Instillation, Drug; Macrophages, Alveolar; Male; Poly(ADP-ribose) Polymerases; Proteins; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Time Factors; TNF Receptor-Associated Factor 2; Translocation, Genetic

The local anesthetic richlocaine decreased the area of necrosis in the skin flap under conditions of reduced blood flow by 29.5%. Improved survival of skin flap after richlocaine treatment alleviated endogenous intoxication, reduced secondary inflammatory reaction, improved liver function, and normalized the ratio between vasoconstricting and vasodilating prostaglandins. This effect was most pronounced after combination therapy with richlocaine and direct-action antihypoxant energostim.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Cell Survival; Cytochromes c; Drug Combinations; Endotoxemia; Erythrocytes; Histamine; Hydroxyproline; Hypoxia; Inflammation; Inosine; Keratinocytes; Lactates; Male; NAD; Necrosis; Piperidines; Rats; Regional Blood Flow; Serotonin; Skin; Surgical Flaps; Time Factors; Vasodilator Agents

We studied morphological characteristics of the regenerative process in gingival tissues during therapy of chronic periodontitis. Energostim stimulated macrophageal reaction and promoted neoangiogenesis in the inflammatory infiltrate. It was not observed after traditional drug therapy. Energostim promoted vascularization in regenerating tissues, normalized the structure at a greater area of the lamina propria of the gingiva, and prevented fibrous and sclerotic changes. The volume of histiolymphocytic infiltrates in regenerating gingival tissues decreased after application of intradental splints.

Topics: Capillaries; Chronic Disease; Cytochromes c; Drug Combinations; Epithelium; Fibroblasts; Gingiva; Gingivitis; Humans; Inflammation; Inosine; Macrophages; Mucous Membrane; NAD; Periodontitis; Splints; Time Factors; Wound Healing