mitotempo and Inflammation

Excess mitochondrial reactive oxygen species (mROS) in sepsis is associated with organ failure, in part by generating inflammation through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. We determined the impact of a mitochondrial-targeted antioxidant (MitoTEMPO) on mitochondrial dysfunction in renal proximal tubular epithelial cells, peritoneal immune cell function ex vivo, and organ dysfunction in a rat model of sepsis.. The effects of MitoTEMPO were assessed ex vivo using adenosine triphosphate and lipopolysaccharide-stimulated rat peritoneal immune cells and fresh rat kidney slices exposed to serum from septic rats. We assessed mROS production and phagocytotic capacity (flow cytometry), mitochondrial functionality (multiphoton imaging, respirometry), and NLRP3 inflammasome activation in cell culture. The effect of MitoTEMPO on organ dysfunction was evaluated in a rat model of faecal peritonitis.. MitoTEMPO decreased septic serum-induced mROS (P<0.001) and maintained normal reduced nicotinamide adenine dinucleotide redox state (P=0.02) and mitochondrial membrane potential (P<0.001) in renal proximal tubular epithelial cells ex vivo. In lipopolysaccharide-stimulated peritoneal immune cells, MitoTEMPO abrogated the increase in mROS (P=0.006) and interleukin-1β (IL-1β) (P=0.03) without affecting non-mitochondrial oxygen consumption or the phagocytotic-induced respiratory burst (P>0.05). In vivo, compared with untreated septic animals, MitoTEMPO reduced systemic IL-1β (P=0.01), reduced renal oxidative stress as determined by urine isoprostane levels (P=0.04), and ameliorated renal dysfunction (reduced serum urea (P<0.001) and creatinine (P=0.05).. Reduction of mROS by a mitochondria-targeted antioxidant reduced IL-1β, and protected mitochondrial, cellular, and organ functionality after septic insults.

Topics: Animals; Antioxidants; Disease Models, Animal; Inflammasomes; Inflammation; Interleukin-1beta; Kidney Diseases; Male; Membrane Potential, Mitochondrial; Mitochondria; Organophosphorus Compounds; Oxidative Stress; Peritonitis; Piperidines; Rats; Rats, Wistar; Reactive Oxygen Species; Sepsis

MitoTEMPO, a mitochondrial antioxidant, has protective effects on liver-related diseases. However, the role of MitoTEMPO on nonalcoholic fatty liver disease (NAFLD) and its possible mechanisms are largely unknown. Here, we investigated the effects of MitoTEMPO on NAFLD using high fat diet- (HFD-) induced obese mice as animal models. MitoTEMPO was intraperitoneally injected into HFD mice. Liver morphological changes were observed by H&E and Oil Red O staining, and the frequency of MDSCs in peripheral blood was analyzed by flow cytometry. Moreover, real-time quantitative PCR, western blot, and immunohistochemistry were conducted to detect the mRNA and protein expressions in the liver tissues. The results showed that the hepatic steatosis in liver tissues of HFD mice injected with MitoTEMPO was significantly ameliorated. Additionally, MitoTEMPO reduced the frequency of CD11b

Topics: Animals; Gene Expression Regulation; Inflammation; Liver; Male; Mice; Myeloid-Derived Suppressor Cells; Non-alcoholic Fatty Liver Disease; Organophosphorus Compounds; Piperidines

Ischemia/reperfusion (I/R) is a critical risk factor for acute kidney injury (AKI). Recent studies provided evidence that tubular epithelial cells (TEC)-associated inflammation aggravates kidney injury and impairs tissue repair after I/R injury. Here we demonstrated that the Nod-like receptor protein 3 (NLRP3) inflammasome is activated by mitochondrial reactive oxygen species (mROS) during I/R injury via direct interactions between the inflammasome and thioredoxin-interacting protein (TXNIP). Firstly, we found that NLRP3 inflammasome activation was induced by I/R injury, peaking at day 3 after reperfusion. Consistent with this observation, NLRP3 deletion significantly attenuated I/R-induced kidney damage and markers of inflammasome activation. Then, we observed mitochondrial dysfunction, characterized by ultrastructural changes and cytochrome C (Cyt c) redistribution. Mitochondria-targeted antioxidant MitoTEMPO prevented mROS overproduction and the decline in mitochondrial membrane potential (MMP) in vitro. MitoTEMPO treatment also inhibited NLRP3 inflammasome activation and co-localization of NLRP3 and TXNIP after simulated ischemia/reperfusion (SI/R) injury. Finally, we transfected HK-2 cells with TXNIP siRNA to explore the role of TXNIP in mROS-induced NLRP3 inflammasome activation. We found that TXNIP siRNA significantly inhibited NLRP3 inflammasome activation. These results demonstrate that NLRP3 inflammasome is activated through the mROS-TXNIP-NLRP3 pathway and provide a potential therapeutic target in ischemic AKI.

Topics: Acute Kidney Injury; Animals; Antioxidants; Carrier Proteins; Cell Proliferation; Cells, Cultured; Inflammasomes; Inflammation; Interleukin-1beta; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; NLR Family, Pyrin Domain-Containing 3 Protein; Organophosphorus Compounds; Piperidines; Reactive Oxygen Species; Reperfusion Injury; Thioredoxins

Psychological stress, depression and anxiety lead to multiple organ dysfunctions wherein stress-related mucosal disease (SRMD) is common to people experiencing stress and also occur as a side effect in patients admitted to intensive care units; however the underlying molecular aetiology is still obscure. We report that in rat-SRMD model, cold restraint-stress severely damaged gut mitochondrial functions to generate superoxide anion (O

Topics: Adenosine Triphosphate; Animals; Antipsychotic Agents; bcl-2-Associated X Protein; Cold Temperature; Corticosterone; Electron Transport Complex I; Gastric Mucosa; Gene Expression Regulation; Immobilization; Inflammation; Membrane Transport Proteins; Mifepristone; Mitochondria; Mitochondrial Dynamics; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Mitophagy; NF-kappa B; Organophosphorus Compounds; Oxidative Stress; Piperidines; Protein Kinases; Rats, Sprague-Dawley; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Glucocorticoid; Stomach; Stress, Psychological; Superoxides; Ubiquitin-Protein Ligases

Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.

Topics: Acetylcysteine; Adipocytes; Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cells, Cultured; Culture Media, Conditioned; Cytokines; Dual-Specificity Phosphatases; Endoplasmic Reticulum Stress; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Inflammation Mediators; Macrophages; Mice, Inbred C57BL; Mitochondria; Mitogen-Activated Protein Kinase Phosphatases; Mitogen-Activated Protein Kinases; NF-kappa B; Organophosphorus Compounds; Oxygen; Palmitates; Phosphorylation; Piperidines; Reactive Oxygen Species; Signal Transduction

Increasing evidences suggest that, apart from activation of guanylyl cyclase, intracellular nitric oxide (NO) signaling is associated with an interaction between NO and reactive oxygen species (ROS) to modulate physiological or pathophysiological processes. The aim of this study was to understand the contribution of mitochondrial ROS (mtROS) to NO-mediated signaling in hepatocytes on inflammation.. In rats treated with lipopolysaccharide (LPS), mitochondria-targeted antioxidants (mtAOX) (mitoTEMPO and SkQ1) reduced inducible nitric oxide synthase (iNOS) gene expression in liver, NO levels in blood and plasma, and markers of organ damage (lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase). In cultured hepatocytes, treated with inflammatory mediators, generated ex vivo by incubation of white blood cells with LPS, we observed an increase in NO and mtROS levels. l-NG-monomethyl arginine citrate, a NOS inhibitor, decreased both NO and mtROS levels. mtAOX reduced mtROS, cytoplasmic ROS levels, and expression of iNOS and interleukin (IL)-6. These data suggest that NO, generated by iNOS, elevates mtROS, which, in turn, diffuse into the cytoplasm and upregulate iNOS and IL-6.. Here, for the first time, we show that intracellular signaling pathways mediated by NO and ROS are linked to each other via mtROS and form an iNOS-mtROS feed-forward loop which aggravates liver failure on acute inflammation.. Our results provide a mechanistic explanation of how NO and mtROS cooperate to conduct inflammatory intracellular signals. We anticipate our results to be the missing mechanistic link between acute systemic inflammation and liver failure.

Topics: Animals; Antioxidants; Biomarkers; Cell Line; Hepatocytes; Inflammation; Lipopolysaccharides; Liver; Male; Mitochondria; Nitric Oxide; Nitric Oxide Synthase Type II; Organophosphorus Compounds; Piperidines; Plastoquinone; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction

Activation of microglia cells in the brain contributes to neurodegenerative processes promoted by many neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO). Reactive oxygen species (ROS) actively affect microglia-associated neurodegenerative diseases through their role as pro-inflammatory molecules and modulators of pro-inflammatory processes. Although the ROS which involved in microglia activation are thought to be generated primarily by NADPH oxidase (NOX) and involved in the immune response, mitochondrial ROS have also been proposed as important regulators of the inflammatory response in the innate immune system. However, the role of mitochondrial ROS in microglial activation has yet to be fully elucidated. In this study, we demonstrate that inhibition of mitochondrial ROS by treatment with Mito-TEMPO effectively suppressed the level of mitochondrial and intracellular ROS. Mito-TEMPO treatment also significantly prevented LPS-induced increase in the TNF-α, IL-1β, IL-6, iNOS and Cox-2 in BV-2 and primary microglia cells. Furthermore, LPS-induced suppression of mitochondrial ROS generation not only affected LPS-stimulated activation of MAPKs, including ERK, JNK, and p38, but also regulated IκB activation and NF-κB nuclear localization. These results indicate that mitochondria constitute a major source of ROS generation in LPS-mediated activated microglia cells. Additionally, suppression of LPS-induced mitochondrial ROS plays a role in modulating the production of pro-inflammatory mediators by preventing MAPK and NF-κB activation in microglia cells. Our findings suggest that a potential strategy in the development of therapy for inflammation-associated degenerative neurological diseases involves targeting the regulation of mitochondrial ROS in microglial cells.

Topics: Animals; Cell Line; Extracellular Signal-Regulated MAP Kinases; Inflammation; Lipopolysaccharides; MAP Kinase Kinase 4; Mice; Microglia; Mitochondria; Mitogen-Activated Protein Kinases; NF-kappa B; Organophosphorus Compounds; p38 Mitogen-Activated Protein Kinases; Piperidines; Reactive Oxygen Species; Signal Transduction

The NLRP3 (NALP3, cryopyrin) inflammasome, a key component of the innate immune system, facilitates caspase-1 and interleukin (IL)-1β processing, which amplifies the inflammatory response. Here, we investigated whether NLRP3 knockdown decreases neutrophil infiltration, reduces brain edema, and improves neurological function in an intracerebral hemorrhage (ICH) mouse model. We also determined whether mitochondrial reactive oxygen species (ROS) governed by mitochondrial permeability transition pores (mPTPs) would trigger NLRP3 inflammasome activation following ICH.. ICH was induced by injecting autologous arterial blood (30μl) into a mouse brain. NLRP3 small interfering RNAs were administered 24 hours before ICH. A mPTP inhibitor (TRO-19622) or a specific mitochondria ROS scavenger (Mito-TEMPO) was coinjected with the blood. In naive animals, rotenone, which is a respiration chain complex I inhibitor, was applied to induce mitochondrial ROS production, and followed by TRO-19622 or Mito-TEMPO treatment. Neurological deficits, brain edema, enzyme-linked immunosorbent assay, Western blot, in vivo chemical cross-linking, ROS assay, and immunofluorescence were evaluated.. ICH activated the NLRP3 inflammasome. NLRP3 knockdown reduced brain edema and decreased myeloperoxidase (MPO) levels at 24 hours, and improved neurological functions from 24 to 72 hours following ICH. TRO-19622 or Mito-TEMPO reduced ROS, NLRP3 inflammasome components, and MPO levels following ICH. In naive animals, rotenone administration induced mPTP formation, ROS generation, and NLRP3 inflammasome activation, which were then reduced by TRO-19622 or Mito-TEMPO.. The NLRP3 inflammasome amplified the inflammatory response by releasing IL-1β and promoting neutrophil infiltration following ICH. Mitochondria ROS may be a major trigger of NLRP3 inflammasome activation. The results of our study suggest that the inhibition of the NLRP3 inflammasome may effectively reduce the inflammatory response following ICH.ANN NEUROL 2014;75:209-219.

Topics: Animals; Antioxidants; Brain Edema; Carrier Proteins; Cerebral Hemorrhage; Cholestenones; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Hematoma; Inflammation; Injections, Intraventricular; Male; Mice; Neutrophil Infiltration; NLR Family, Pyrin Domain-Containing 3 Protein; Organophosphorus Compounds; Piperidines; RNA, Small Interfering