cytochrome-c-t and Acute-Lung-Injury

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

Disruption of the alveolar−endothelial barrier caused by inflammation leads to the progression of septic acute lung injury (ALI). In the present study, we investigated the beneficial effects of simvastatin on the endotoxin lipopolysaccharide (LPS)-induced ALI and its related mechanisms. A model of ALI was induced within experimental sepsis developed by intraperitoneal injection of a single non-lethal LPS dose after short-term simvastatin pretreatment (10−40 mg/kg orally). The severity of the lung tissue inflammatory injury was expressed as pulmonary damage scores (PDS). Alveolar epithelial cell apoptosis was confirmed by TUNEL assay (DNA fragmentation) and expressed as an apoptotic index (AI), and immunohistochemically for cleaved caspase-3, cytochrome C, and anti-apoptotic Bcl-xL, an inhibitor of apoptosis, survivin, and transcriptional factor, NF-kB/p65. Severe inflammatory injury of pulmonary parenchyma (PDS 3.33 ± 0.48) was developed after the LPS challenge, whereas simvastatin significantly and dose-dependently protected lung histology after LPS (p < 0.01). Simvastatin in a dose of 40 mg/kg showed the most significant effects in amelioration alveolar epithelial cells apoptosis, demonstrating this as a marked decrease of AI (p < 0.01 vs. LPS), cytochrome C, and cleaved caspase-3 expression. Furthermore, simvastatin significantly enhanced the expression of Bcl-xL and survivin. Finally, the expression of survivin and its regulator NF-kB/p65 in the alveolar epithelium was in strong positive correlation across the groups. Simvastatin could play a protective role against LPS-induced ALI and apoptosis of the alveolar−endothelial barrier. Taken together, these effects were seemingly mediated by inhibition of caspase 3 and cytochrome C, a finding that might be associated with the up-regulation of cell-survival survivin/NF-kB/p65 pathway and Bcl-xL.

Topics: Acute Lung Injury; Alveolar Epithelial Cells; Apoptosis; Caspase 3; Cytochromes c; Endotoxins; Humans; Lipopolysaccharides; Lung; NF-kappa B; Simvastatin; Survivin; Up-Regulation

Our previous study found that Lianhuaqingwen reduces lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice by suppressing p53-mediated apoptosis. To identify the type of lung cells affected by Lianhuaqingwen, we conducted a cell experiment.. C57/B6 mice and A549 cells were administered Lianhuaqingwen and LPS. A549 cells were transfected with p53 siRNA to inhibit p53. The degree of ALI in mice was validated by haematoxylin and eosin staining as well as measurement of IL-1β and MCP-1 levels. In A549 cells, Cell Counting Kit-8 (CCK-8), DHE and TUNEL assays were used to assess cell viability, reactive oxygen species (ROS) production and apoptosis, respectively. Western blot analysis was used to evaluate the protein expression of p53, Bcl-2, Bax, caspase-9 and caspase-3. Co-immunofluorescence was used to detect cytochrome C distribution.. Lianhuaqingwen alleviated LPS-induced ALI in vivo. Lianhuaqingwen at 300 μg/ml increased cell viability, lowered ROS production and reduced apoptotic cells in vitro. Lianhuaqingwen enhanced Bcl-2 expression and reduced Bax, caspase-9 and caspase-3 expression as well as blocked cytochrome C release under LPS stimulation. Treatment with a combination of Lianhuaqingwen and p53 siRNA was more effective than treatment with Lianhuaqingwen alone.. Lianhuaqingwen inhibits p53-mediated apoptosis in alveolar epithelial cells, thereby preventing LPS-induced ALI.

Topics: Acute Lung Injury; Alveolar Epithelial Cells; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cytochromes c; Drugs, Chinese Herbal; Lipopolysaccharides; Mice; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Small Interfering; Tumor Suppressor Protein p53

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

Recent evidence has shown that microRNAs (miRs) are involved in endothelial dysfunction and vascular injury in lung-related diseases. However, the potential role of miR-34a in the regulation of pulmonary endothelial dysfunction, vascular injury, and endothelial cells (ECs) apoptosis in acute lung injury (ALI)/acute lung respiratory distress syndrome is largely unknown. Here, we show that miR-34a-5p was upregulated in whole lungs, isolated ECs from lungs, and ECs stimulated with various insults (LPS and hyperoxia). Overexpression of miR-34a-5p in ECs exacerbated endothelial dysfunction, inflammation, and vascular injury, whereas the suppression of miR-34a-5p expression in ECs and miR-34a-null mutant mice showed protection against LPS- and hyperoxia-induced ALI. Furthermore, we observed that miR-34a-mediated endothelial dysfunction is associated with decreased miR-34a direct-target protein, sirtuin-1, and increased p53 expression in whole lungs and ECs. Mechanistically, we show that miR-34a leads to translocation of p53 and Bax to the mitochondrial compartment with disruption of mitochondrial membrane potential to release cytochrome C into the cytosol, initiating a cascade of mitochondrial-mediated apoptosis in lungs. Collectively, these data show that downregulating miR-34a expression or modulating its target proteins may improve endothelial dysfunction and attenuate ALI.

Topics: Acute Lung Injury; Animals; Apoptosis; bcl-2-Associated X Protein; Cytochromes c; Cytosol; Disease Models, Animal; Endothelial Cells; Lipopolysaccharides; Lung; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Mitochondria; Sirtuin 1; Tumor Suppressor Protein p53

Losartan is a selective antagonist of AngⅠ type (AT1) receptor of Angiotensin Ⅱ (Ang Ⅱ), which is widely used as a clinical medicine for the hypertension. Recent studies have shown that losartan was shown to protect from acute lung injury (ALI). However, the underlying mechanism remains unclear. The aim of this research was to clarify whether Ang Ⅱ participated in the inflammatory response of ALI induced by seawater inhalation, and whether losartan had the protective effects on ALI by blocking the combination of Ang Ⅱ and AT1 receptor. In the current study, the severity of lung injury and the inflammatory reactions during seawater drowning induced ALI were assessed. Besides, we also detected the activation of relative pathways such as NF-κB, JAK2/STATs and apoptosis. The results showed that seawater inhalation could up-regulate the expression of Ang Ⅱ and AT1. While pretreatment of losartan (especially 15 mg/kg and 30 mg/kg) alleviated lung injury by inhibiting Ang-Ⅱ and AT1 receptor combination and in turn decreased the expression of p-NF-κB and activation of JAK2/STATs pathway. We also confirmed that losartan could reduce the apoptotic ratio of cells in the lung by modulating the phosphorylation of JNK and leak of cytochrome C to cytosol. Taken together, these findings demonstrate that losartan might have a therapeutic potential as an anti-inflammatory agent for treating SWI-ALI.

Topics: Acute Lung Injury; Angiotensin II Type 1 Receptor Blockers; Animals; Anti-Inflammatory Agents; Apoptosis; Cytochromes c; Janus Kinase 2; Losartan; Male; NF-kappa B; Phosphorylation; Rats; Rats, Sprague-Dawley; Seawater; STAT Transcription Factors

Acute lung injury (ALI) is a serious disease with high incidence in ICU, and impaired mitochondria function plays a significant role in ALI. In this study, we examined the possible roles of exogenous hydrogen sulfide (H2S) in lung mitochondria regulation in ALI rats.. The rat ALI model was induced by an intra-tongue vein Lipopolysaccharide (LPS) injection. We used sodium hydrosulphide (NaHS) as the H2S donor. We randomly divided 40 Sprague-Dawley rats into five groups: control, LPS injury, LPS + low-dose NaHS (0.78 mg • kg(-1)), LPS + middle-dose NaHS (1.56 mg • kg(-1)), and LPS + high-dose NaHS (3.12 mg • kg(-1)). Rats were killed 3 h after NaHS administration. We calculated a semi-quantitative histological index of lung injury assessments and measured the lung wet-to-dry weight ratio. We further analyzed serum for interleukin-1β levels using enzyme-linked immunosorbent assays. We observed lung mitochondria ultrastructures with an electron microscope. We examined oxidative stress markers in lung mitochondria and the mitochondrial swelling and activity. We analyzed lung mitochondria and cytosol Cyt-c protein expression using Western blotting.. Compared to the control group, the quantitative assessment score index, wet-to-dry weight ratios, and interleukin-1β content in the LPS injury group were significantly increased and the mitochondrial ultrastructure damaged. Furthermore, mitochondrial activity, adenosine triphosphatease, superoxide dismutase, glutathione peroxidase, and mitochondrial Cyt-c protein expression were significantly decreased, and malondialdehyde content, mitochondrial swelling, and cytosol Cyt-c protein expression were significantly increased in the LPS injury group compared to the control group. These effects were lessened by NaHS.. Exogenous H2S provided a protective effect against ALI by decreasing the mitochondrial lipid peroxidation level and protecting the cell structure in the LPS-induced rat models. Its regulatory effect on lung mitochondria is positively correlated with the dosage.

Topics: Acute Lung Injury; Animals; Cytochromes c; Cytosol; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Interleukin-1beta; Lipid Peroxidation; Lipopolysaccharides; Lung; Male; Malondialdehyde; Mitochondria; Rats; Rats, Sprague-Dawley; Sulfides; Superoxide Dismutase

Normal hepatocytes do not express endogenous uncoupling protein 2 (UCP2) in adult liver, although Kupffer cells do, and it is strikingly induced in hepatocytes in steatotic liver and obese conditions. However, the direct link of UCP2 with the pathogenic development of liver diseases and liver injury remains elusive. Here we report that targeted expression of UCP2 to mouse liver increases susceptibility to acute liver injury induced by lipopolysaccharide (LPS) and galactosamine (GalN). UCP2 appears to enhance proton leak, leading to mild uncoupling in a guanosine diphosphate-repressible manner. Indeed, mitochondria from the genetically manipulated mouse liver have increased state 4 respiration, lower respiratory control ratio, and reduced adenosine triphosphate (ATP) levels, which altered mitochondrial physiology. To address the underlying mechanism of how UCP2 and the reduced energy coupling efficiency enhance cell death in mouse liver, we show that the reduced ATP levels lead to activation of 5'AMP-activated protein kinase (AMPK) and its downstream effector, c-Jun N-terminal kinase; thus, the increased sensitivity toward LPS/GalN-induces apoptosis. Importantly, we show that inhibition of UCP2 activity by its pharmacological inhibitor genipin prevents LPS/GalN-induced ATP reduction, AMPK activation, and apoptosis. Also, inhibition of ATP production by oligomycin promotes LPS/GalN-induced cell death both in vivo and in vitro.. Our results clearly show that targeted expression of UCP2 in liver may result in compromised mitochondrial physiology that contributes to enhanced cell death and suggests a potential role of UCP2 in the development of liver diseases.

Topics: Acute Lung Injury; Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Apoptosis; Caspase 3; Cytochromes c; Disease Models, Animal; Female; Galactosamine; Genetic Predisposition to Disease; Hepatocytes; Ion Channels; Lipopolysaccharides; Liver; Male; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Liver; Mitochondrial Proteins; Uncoupling Protein 2

The present study investigated whether the pathophysiological changes induced by burn and smoke inhalation are modulated by parenteral administration of Na(2)S, a H(2)S donor.. The study used a total of 16 chronically instrumented, adult female sheep. Na(2)S was administered 1 h post injury, as a bolus injection at a dose of 0.5 mg.kg(-1) and subsequently, as a continuous infusion at a rate of 0.2 mg.kg(-1).h(-1) for 24 h. Cardiopulmonary variables (mean arterial and pulmonary arterial blood pressure, cardiac output, ventricular stroke work index, vascular resistance) and arterial and mixed venous blood gases were measured. Lung wet-to-dry ratio and myeloperoxidase content and protein oxidation and nitration were also measured. In addition, lung inducible nitric oxide synthase expression and cytochrome c were measured in lung homogenates via Western blotting and enzyme-linked immunosorbent assay (elisa) respectively.. The H(2)S donor decreased mortality during the 96 h experimental period, improved pulmonary gas exchange and lowered further increase in inspiratory pressure and fluid accumulation associated with burn- and smoke-induced acute lung injury. Further, the H(2)S donor treatment reduced the presence of protein oxidation and 3-nitrotyrosine formation following burn and smoke inhalation injury.. Parenteral administration of the H(2)S donor ameliorated the pulmonary pathophysiological changes associated with burn- and smoke-induced acute lung injury. Based on the effect of H(2)S observed in this clinically relevant model of disease, we propose that treatment with H(2)S or its donors may represent a potential therapeutic strategy in managing patients with acute lung injury.

Topics: Acute Lung Injury; Animals; Blotting, Western; Burns; Cytochromes c; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Hydrogen Sulfide; Nitric Oxide Synthase Type II; Sheep; Smoke Inhalation Injury; Sulfides