gw9662 and Reperfusion-Injury

It is known that cerebral ischemia can cause brain inflammation and adiposome can serve as a depot of inflammatory mediators. In the study, the pro-inflammatory and pro-death role of adiposome in ischemic microglia and ischemic brain was newly investigated. The contribution of PPARγ to adiposome formation was also evaluated for the first time in ischemic microglia. Focal cerebral ischemia/reperfusion (I/R) animal model and the in vitro glucose-oxygen-serum deprivation (GOSD) cell model were both applied in the study. GOSD- or I/R-induced adiposome formation, inflammatory activity, cell death of microglia, and brain infarction were, respectively, determined, in the absence or presence of NS-398 (adiposome inhibitor) or GW9662 (PPARγ antagonist). GOSD-increased adiposome formation played a critical role in stimulating the inflammatory activity (production of TNF-α and IL-1β) and cell death of microglia. Similar results were also found in ischemic brain tissues. GOSD-induced PPARγ partially contributed to the increase of adiposomes and adiposome-mediated inflammatory responses of microglia. Blockade of adiposome formation with NS-398 or GW9662 significantly reduced not only the inflammatory activity and death rate of GOSD-treated microglia but also the brain infarct volume and motor function deficit of ischemic rats. The pathological role of microglia-derived adiposome in cerebral ischemia has been confirmed and attributed to its pro-inflammatory and/or pro-death effect upon ischemic brain cells and tissues. Adiposome and its upstream regulator PPARγ were therefore as potential targets for the treatment of ischemic stroke. Therapeutic values of NS-398 and GW9662 have been suggested.

Topics: Anilides; Animals; Animals, Newborn; Brain Ischemia; Cell Death; Culture Media, Serum-Free; Cyclooxygenase 2; Down-Regulation; Glucose; Inflammation; Interleukin-1beta; Lipid Droplets; Male; Microglia; Motor Activity; Neurons; Nitrobenzenes; Oxygen; PPAR gamma; Rats, Sprague-Dawley; Reperfusion Injury; Stroke; Sulfonamides; Tumor Necrosis Factor-alpha

PPAR-gamma (γ) is highly expressed in macrophages and its activation affects their polarization. The effect of PPAR-γ activation on Kupffer cells (KCs) and liver ischemia-reperfusion injury (IRI) has not yet been evaluated. We investigated the effect of PPAR-γ activation on KC-polarization and IRI.. Seventy percent (70%) liver ischemia was induced for 60mins. PPAR-γ-agonist or vehicle was administrated before reperfusion. PPAR-γ-antagonist was used to block PPAR-γ activation. Liver injury, necrosis, and apoptosis were assessed post-reperfusion. Flow-cytometry determined KC-phenotypes (pro-inflammatory Nitric Oxide +, anti-inflammatory CD206+ and anti-inflammatory IL-10+).. Liver injury assessed by serum AST was significantly decreased in PPAR-γ-agonist versus control group at all time points post reperfusion (1hr: 3092±105 vs 4469±551; p = 0.042; 6hr: 7041±1160 vs 12193±1143; p = 0.015; 12hr: 5746±328 vs 8608±1259; p = 0.049). Furthermore, liver apoptosis measured by TUNEL-staining was significantly reduced in PPAR-γ-agonist versus control group post reperfusion (1hr:2.46±0.49 vs 6.90±0.85%;p = 0.001; 6hr:26.40±2.93 vs 50.13±8.29%; p = 0.048). H&E staining demonstrated less necrosis in PPAR-γ-agonist versus control group (24hr:26.66±4.78 vs 45.62±4.57%; p = 0.032). The percentage of pro-inflammatory NO+ KCs was significantly lower at all post reperfusion time points in the PPAR-γ-agonist versus control group (1hr:28.49±4.99 vs 53.54±9.15%; p = 0.040; 6hr:5.51±0.54 vs 31.12±9.58%; p = 0.009; 24hr:4.15±1.50 vs 17.10±4.77%; p = 0.043). In contrast, percentage of anti-inflammatory CD206+ KCs was significantly higher in PPAR-γ-agonist versus control group prior to IRI (8.62±0.96 vs 4.88 ±0.50%; p = 0.04). Administration of PPAR-γ-antagonist reversed the beneficial effects on AST, apoptosis, and pro-inflammatory NO+ KCs.. PPAR-γ activation reduces IRI and decreases the pro-inflammatory NO+ Kupffer cells. PPAR-γ activation can become an important tool to improve outcomes in liver surgery through decreasing the pro-inflammatory phenotype of KCs and IRI.

Topics: Alanine Transaminase; Anilides; Animals; Apoptosis; Aspartate Aminotransferases; Cell Polarity; Cytokines; Disease Models, Animal; Kupffer Cells; Lectins, C-Type; Liver; Male; Mannose Receptor; Mannose-Binding Lectins; Mice; Mice, Inbred C57BL; Necrosis; Nitric Oxide; PPAR gamma; Receptors, Cell Surface; Reperfusion Injury; Rosiglitazone; Thiazolidinediones

Nitroalkene derivatives of oleic acid (OA-NO2) serve as high-affinity ligand for PPAR-γ, which regulates apoptosis, oxidation and inflammation and plays a central role in ischemia-reperfusion injury. In the present study, we elucidated the protective mechanisms of OA-NO2 against renal ischemia-reperfusion injury.. HK-2 cells were subjected to oxygen and glucose deprivation followed by re-oxygenation (OGD/R) to mimic renal ischemia-reperfusion injury. Cell apoptosis was analyzed by flow cytometry. Bax mitochondrial translocation, cytochrome c and apoptosis-inducing factor (AIF) cytosolic leakage and Akt/Gsk 3β phosphorylation were evaluated by Western blotting. Bax activation was visualized by immunocytochemistry. GW9662 and siRNA transfection were employed to examine the involvement of PPAR-γ.. OGD/R injury promoted mitochondrial translocation and activation of Bax, leakage of cytochrome c and AIF, subsequent caspase-3 activation, and eventually cell apoptosis. Pre-incubation with OA-NO2 (1.25 µM, 45min) inhibited Bax activation and blocked apoptotic cascade, while the protective effects were negated by GW9662 or PPAR-γ siRNA. Moreover, OA-NO2 restored Akt and Gsk 3β phosphorylation in a PPAR-γ-dependent way.. These findings suggest that OA-NO2 attenuates OGD/R-induced apoptosis by inhibiting Bax translocation and activation and the subsequent mitochondria-dependent apoptotic cascade in a PPAR-γ dependent manner.

Topics: Anilides; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Caspase 3; Cell Hypoxia; Cytochromes c; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Kidney; Mitochondria; Oleic Acid; PPAR gamma; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; Signal Transduction

Injurious inflammatory response is critical to the development of lung ischemia/reperfusion injury (LIRI). The cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs), which exert an anti-inflammatory effect on the cardiovascular system. We therefore cytochrome hypothesized that CYP2J2 overexpression and pretreatment with exogenous EETs may have the potential to reduce LIRI.. A rat model was used to mimic the condition of LIRI by clamping the left pulmonary hilum for 60 minutes, followed by reperfusion for 2 hours. Moreover, we developed a cell model using human pulmonary artery endothelial cells by anoxia for 8 hours, followed by reoxygenation for 16 hours to determine the anti-inflammatory effect and mechanism of CYP2J2 overexpression and exogenous 11,12-EET.. Lung ischemia/reperfusion increased lung wet/dry and lung weight/body weight ratios, protein concentration in bronchoalveolar lavage fluid and concentrations of pro-inflammatory, including mediators in serum IL-1β, IL-8, TNF-α, sP- and sE-selectin, and decreased concentration of anti-inflammatory mediator IL-10. Ischemia/reperfusion also leaded to pulmonary edema and inflammation under light microscopy. Furthermore, activation of NF-κB p65 and degradation of IκBα were remarkably increased in ischemia/reperfusion lung tissues. While CYP2J2 overexpression significantly inhibited the above effects (p<0.05). In vitro data further confirmed the anti-inflammatory effect of CYP2J2 overexpression and 11,12-EET, an effect that may probably be mediated by PPARγ activation.. CYP2J2 overexpression and administration of exogenous EETs can protect against LIRI via anti-inflammatory effects. This can be a novel potential strategy for prevention and treatment of LIRI.

Topics: 8,11,14-Eicosatrienoic Acid; Anilides; Animals; Anti-Inflammatory Agents; Cell Hypoxia; Cell Line; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Cytokines; Down-Regulation; E-Selectin; Humans; I-kappa B Proteins; Inflammation Mediators; Intercellular Adhesion Molecule-1; Lung; Male; NF-KappaB Inhibitor alpha; P-Selectin; PPAR gamma; Rats; Rats, Transgenic; Rats, Wistar; Reperfusion Injury; Transcription Factor RelA

Improper macrophage activation is pathogenically linked to various metabolic, inflammatory, and immune disorders. Therefore, regulatory proteins controlling macrophage activation have emerged as important new therapeutic targets. We recently demonstrated that netrin-1 regulates inflammation and infiltration of monocytes and ameliorates ischemia-reperfusion-induced kidney injury. However, it was not known whether netrin-1 regulates the phenotype of macrophages and the signaling mechanism through which it might do this. In this study, we report novel mechanisms underlying netrin-1's effects on macrophages using in vivo and in vitro studies. Overexpression of netrin-1 in spleen and kidney of transgenic mice increased expression of arginase-1, IL-4, and IL-13 and decreased expression of COX-2, indicating a phenotypic switch in macrophage polarization toward an M2-like phenotype. Moreover, flow cytometry analysis showed a significant increase in mannose receptor-positive macrophages in spleen compared with wild type. In vitro, netrin-1 induced the expression of M2 marker expression in bone marrow-derived macrophages, peritoneal macrophages, and RAW264.7 cells, and suppressed IFNγ-induced M1 polarization and production of inflammatory mediators. Adoptive transfer of netrin-1-treated macrophages suppressed inflammation and kidney injury against ischemia-reperfusion. Netrin-1 activated PPAR pathways and inhibition of PPAR activation abolished netrin-1-induced M2 polarization and suppression of cytokine production. Consistent with in vitro studies, administration of PPAR antagonist to mice abolished the netrin-1 protective effects against ischemia-reperfusion injury of the kidney. These findings illustrate that netrin-1 regulates macrophage polarization through PPAR pathways and confers anti-inflammatory actions in inflammed kidney tissue.

Topics: Anilides; Aniline Compounds; Animals; Inflammation; Interferon-gamma; Kidney; Kidney Diseases; Macrophage Activation; Macrophages, Peritoneal; Male; Maleimides; Mice; Mice, Transgenic; Nerve Growth Factors; Netrin-1; PPAR gamma; PPAR-beta; Reperfusion Injury; Spleen; Tumor Suppressor Proteins

PPAR-γ has been reported to be a protective regulator in ischaemia/reperfusion (I/R) injury. The receptor for advanced glycation end-products (RAGE) plays a major role in the innate immune response, and its expression is associated with PPAR-γ activation. Several angiotensin receptor blockers possess partial agonist activities towards PPAR-γ. Therefore, this study investigated the action of losartan, particularly with regard to PPAR-γ activation and RAGE signalling pathways during hepatic I/R.. Mice were subjected to 60 min of ischaemia followed by 6 h of reperfusion. Losartan (0.1, 1, 3 and 10 mg · kg⁻¹) was administered 1 h prior to ischaemia and immediately before reperfusion. GW9662, a PPAR-γ antagonist, was administered 30 min prior to first pretreatment with losartan.. Losartan enhanced the DNA-binding activity of PPAR-γ in I/R. Losartan attenuated the increased serum alanine aminotransferase activity, TNF-α and IL-6 levels, and nuclear concentrations of NF-κB in I/R. GW9662 reversed these beneficial effects. Losartan caused a decrease in apoptosis as assessed by TUNEL assay, in release of cytochrome c and in cleavage of caspase-3, and these effects were abolished by GW9662 administration. Losartan attenuated not only I/R-induced RAGE overexpression, but also its downstream early growth response protein-1-dependent macrophage inflammatory protein 2 level; phosphorylation of p38, ERK and JNK; and subsequent c-Jun phosphorylation. GW9662 reversed these effects of losartan administration.. Our findings suggest that losartan ameliorates I/R-induced liver damage through PPAR-γ activation and down-regulation of the RAGE signalling pathway.

Topics: Angiotensin II Type 1 Receptor Blockers; Anilides; Animals; Apoptosis; Cell Nucleus; Cytokines; Dose-Response Relationship, Drug; Down-Regulation; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Gene Silencing; Liver; Losartan; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; PPAR gamma; Protective Agents; Protein Transport; Random Allocation; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Reperfusion Injury

Accumulating evidences have demonstrated the beneficial actions of peroxisome proliferator-activated receptor gamma (PPAR gamma) in a variety of animal stroke models. Following middle cerebral artery occlusion (60 min) and 2-24 hr reperfusion in rats, we observed cerebral ischemia/reperfusion (I/R) induced up-regulation of PPAR gamma protein expression and translocation from the cytoplasm into the nucleus in a time-dependent manner. We also found that PPAR gamma agonist rosiglitazone enhanced whereas PPAR gamma antagonist GW9662 inhibited the alteration of PPAR gamma stimulated by I/R, suggesting that the changes of PPAR gamma may result from the activation by endogenous ligands. Moreover, the link between the 12/15-lipoxygenase and the production of activating ligands for PPAR gamma has been proved in various tissues. However, the relation of them in brain tissue has not been identified. We demonstrated that the I/R-induced PPAR gamma alteration was reversed by baicalein, the specific inhibitor of 12/15-lipoxygenase. Baicalein treatment significantly inhibited the up-regulation of PPAR gamma expression and, furthermore, suppressed PPAR gamma nuclear accumulation as well as maintained PPAR gamma cytoplasmic retention. Together, these results suggest that I/R induces both PPAR gamma expression and translocation, probably through the activation by endogenous ligands in a 12/15-lipoxygenase inhibitor-sensitive way.

Topics: Anilides; Animals; Antioxidants; Cell Nucleus; Cerebral Cortex; Cytosol; Disease Models, Animal; Flavanones; Hypoglycemic Agents; Infarction, Middle Cerebral Artery; Male; PPAR gamma; Protein Transport; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rosiglitazone; Thiazolidinediones; Time Factors; Up-Regulation

To determine the involvement of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in cytoprotection, we subjected N2-A cells to oxygen-glucose deprivation followed by reoxygenation (H-R). Following H-R insults, H(2)O(2) production was increased while cell viability declined, which was accompanied by loss of mitochondrial membrane potential (MMP), cytochrome c release, caspases 9 and 3 activation, poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis. Rosiglitazone up to 5 microM protected cell viability, normalized MMP, and prevented apoptotic signals. The protective effect of rosiglitazone was abrogated by GW9662, a PPAR-gamma antagonist, or a specific PPAR-gamma small interference RNA (siRNA) but not a control scRNA. PPAR-gamma overexpression alone was effective in maintaining MMP and preventing apoptosis and its protective effect was also abrogated by PPAR-gamma siRNA or GW9662. To elucidate the mechanism by which PPAR-gamma protects MMP and prevents apoptosis, we analyzed Bcl-2, Bcl-xl, and phosphorylated Bad (p-Bad). H-R suppressed them. Rosiglitazone or PPAR-gamma overexpression restored them via PPAR-gamma. Rosiglitazone or PPAR-gamma overexpression preserved phosphorylated Akt and 3-phosphoinositide-dependent kinase-1 (PDK-1) in a PPAR-gamma dependent manner. These results indicate that ligand-activated PPAR-gamma protects N2-A cells against H-R damage by enhancing Bcl-2/Bcl-xl and maintaining p-Bad via preservation of p-Akt.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Anilides; Animals; Apoptosis; bcl-Associated Death Protein; bcl-X Protein; Caspase 3; Caspase 9; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytoprotection; Dose-Response Relationship, Drug; Glucose; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Mice; Mitochondria; Neuroblastoma; Phosphorylation; Poly(ADP-ribose) Polymerases; PPAR gamma; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; RNA Interference; RNA, Small Interfering; Rosiglitazone; Thiazolidinediones; Time Factors; Transfection; Up-Regulation

Thiadiazolidinones (TDZDs) are small molecules that inhibit glycogen synthase kinase 3-beta (GSK3-beta) activity in a non competitive manner to ATP. NP00111, a new TDZD, besides causing inhibition of GSK-3beta, has also shown to be an agonist of PPARgamma . Since phosphorylation and consequent inhibition of GSK-3beta by PI-3K/Akt and agonism of PPARgamma have shown to afford neuroprotection in several in vitro and in vivo models, we have studied the potential neuroprotective effect of NP00111 in an "in vitro" model of ischemia-reperfusion. NP00111, at the concentration of 10 microM, significantly protected adult rat hippocampal slices subjected to oxygen and glucose deprivation (OGD) for 1 h followed by 3 h re-oxygenation, measured as lactic dehydrogenase (LDH) released to the extracellular media. The protective effects of NP00111 were more pronounced during the re-oxygenation period in comparison to the OGD period. Other GSK-3beta inhibitors like lithium or AR-A014418 did not afford protection in this model. However, the PPARgamma agonist rosiglitazone was protective at 3 microM. Protection afforded by NP00111 and rosiglitazone were prevented by the PPARgamma antagonist GW9662, suggesting that both NP00111 and rosiglitazone were preventing cell death caused by oxygen-glucose deprivation via activation of PPARgamma. NP00111 increased by two fold phosphorylation of ERK1/2 and its protective effects were lost when the hippocampal slices were co-incubated with the mitogen-activated protein kinase (MAPK) inhibitor PD98059. In conclusion, the novel TDZD NP00111 was protective against OGD in rat hippocampal slices by a mechanism related to phosphorylation of ERK1/2 via activation of PPARgamma.

Topics: Anilides; Animals; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Glucose; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Hypoxia-Ischemia, Brain; L-Lactate Dehydrogenase; Male; MAP Kinase Signaling System; Neuroprotective Agents; Organ Culture Techniques; Phosphorylation; PPAR gamma; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rosiglitazone; Thiadiazoles; Thiazolidinediones

To observe the effects of peroxisome proliferation activated receptor (PPAR)-gamma on neuronal cell death induced by hypoxia/reoxygenation and ischemia/reperfusion.. Cortical neural cells of fetal SD rats were cultured for 12 days and exposed to hypoxia/reoxygenation so as to establish a hypoxia/reoxygenation model. Another primary fetal rat cortical neuronal cells were pre-treated with different concentrations of GW9662, antagonist of PPAR-gamma, then underwent hypoxia for 3 hours, re-oxygenated for 21 hours. MTT was added one hour after to measure the cell viability. Eleven male SD rats underwent right middle cerebral artery occlusion (MCAO) using suture and reperfusion. Eleven rats underwent sham operation. after the rats were killed and their brains were taken out. Nucleoprotein was extracted from the cultured primary cortical cells and the cerebral cortexes of the rats and co-cultured with [gamma-(32)P]-labeled PPAR-gamma probe, EMSA to detect the PPAR-gamma binding activity.. The PPAR-gamma activity of the cultured fetal rat cortical neurons that underwent hypoxia/reoxygenation significantly increased: It began to increase 1 hour after hypoxia and peaked in the 3rd hour of hypoxia, when the neurons underwent hypoxia for 3 hours and were reoxygenated for 2 hours, the binding activity still remained at a high level, and basically returned to the level of the untreated group 8 hours after reoxygenation. Data were quantified with control group as 100, 3 h of hypoxia was 160.3, and 2, 4, 8 h after reoxygenation were 157.5, 136.6, 103.3 separately. One hour after reperfusion the PPAR-gamma binding activity of the cortical cells at the ischemic side of the rats began to increase and peaked at the 4th hour, significantly higher than those of the cortical cells at the opposite side and of the sham operation group (both P < 0.01) then remained at a high level for the following 24 hours. The survival rate of the cultured neurons that underwent hypoxia for 3 hours and reoxygenation for 21 hours was significantly lower than that of the untreated neurons. Data were quantified with sham surgery group as 100, the side of MCAO and the contra side in surgery group were 144.8 and 102.6 separately. The survival rate of the neurons that were pretreated with GW9662 and then underwent hypoxia/reoxygenation was significantly higher than that of those without pretreatment (P < 0.01) with the peak protection effect of GW9662 at the concentration of 2.5 - 10 micromol/L. Data were quantified with control group as 100, hypoxia/reoxygenation group was 184, GW9662 group was 105. The PPAR-gamma binding activity of the primary cortical neurons pretreated with 5 micromol/L GW9662 for 30 minutes and than exposed to hypoxia for 3 hours and reoxygenation for 2 hours was significantly lower than that of the only hypoxia/reoxygenation group (P < 0.01).. PPAR-gamma is involved in the pathogenesis of neuron death induced by hypoxia/ischemia and may become a new target of treatment of ischemic stroke.

Topics: Anilides; Animals; Cell Death; Cell Hypoxia; Cells, Cultured; Cerebral Cortex; Female; Fetus; Hypoxia-Ischemia, Brain; Male; Neurons; PPAR gamma; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury

We have recently reported that pretreatment of rats with endotoxin (lipopolysaccharide, LPS) and selective agonists of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) protect the kidney against ischemia/reperfusion (I/R) injury. Here we investigate the hypothesis that the renoprotective effects of LPS may be due to an enhanced formation of endogenous ligands of PPARgamma, rather than an up-regulation of PPARgamma expression.. Rats were pretreated with LPS (1 mg/kg, IP, 24 hours prior to ischemia) in the absence (control) or presence of the selective PPARgamma antagonist GW9662 (1 mg/kg, IP, 24 and 12 hours prior to ischemia). Twenty-four hours after injection of LPS, rats were subjected to 60 minutes of bilateral renal ischemia, followed by 6 hours of reperfusion. Serum and urinary indicators of renal injury and dysfunction were measured, specifically serum creatinine, aspartate aminotransferase, and gamma-glutamyl-transferase, creatinine clearance, urine flow, and fractional excretion of sodium. Kidney PPARgamma1 mRNA levels were determined by reverse transcriptase-polymerase chain reaction.. Pretreatment with LPS significantly attenuated all markers of renal injury and dysfunction caused by I/R. Most notably, GW9662 abolished the protective effects of LPS. Additionally, I/R caused an up-regulation of kidney PPARgamma1 mRNA levels compared to sham animals, which were unchanged in rats pretreated with LPS.. We document here for the first time that endogenous ligands of PPARgamma may contribute to the protection against renal I/R injury afforded by LPS pretreatment in the rat.

Topics: Anilides; Animals; Drug Interactions; Kidney Diseases; Lipopolysaccharides; Male; PPAR gamma; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger