gw9662 and Sepsis

The present study aims to reveal the molecular mechanism of peroxisome proliferator-activated receptor γ (PPARγ) on sepsis-induced acute lung injury (ALI). To do that, the rat injury model was established using cecal ligation and perforation (CLP) method, followed by different treatments, and the rats were divided into Sham group, CLP group, CLP + rosiglitazone (PPARγ agonist) group, CLP + GW9662 (PPARγ inhibitor) group, CLP + bpV (phosphatase and tensin homolog (PTEN) inhibitor) group, CLP + GW9662 + bpV group. Compared with Sham group, the mRNA and protein expression levels of PPARγ were down-regulated, the inflammation levels were elevated, and the apoptosis was increased in CLP group. After treatment with rosiglitazone, the protein expression level of PPARγ was significantly up-regulated, the phosphorylation level of PTEN/β-catenin pathway was decreased, the PTEN/β-catenin pathway was inhibited, the lung injury, inflammation and apoptosis were reduced. The opposite effect was observed after treatment with GW9662. Besides, bpV inhibited PTEN/β-catenin pathway, and relieved the lung tissue injury. The overexpression of PPARγ reduced inflammatory response and inhibited apoptosis in sepsis-induced ALI. Furthermore, PPARγ relieved the sepsis-induced ALI by inhibiting the PTEN/β-catenin pathway.

Topics: Acute Lung Injury; Anilides; Animals; Apoptosis; beta Catenin; Disease Models, Animal; Lung; Male; Phosphorylation; Pneumonia; PPAR gamma; PTEN Phosphohydrolase; Pulmonary Edema; Rats, Sprague-Dawley; Rosiglitazone; Sepsis; Signal Transduction

Previous reports have demonstrated that the newly identified lipid mediator protectin DX (PDX) could effectively attenuate multiple organ injuries in sepsis. The aim of our study was to clarify whether PDX could improve acute lung injury (ALI) induced by sepsis and elucidate the relevant potential mechanism. After inducing sepsis by the cecal ligation and puncture approach, mice were treated with a high or low dose of PDX. Pathological changes in the pulmonary tissue were analyzed by hematoxylin-eosin staining, and lung injury score was evaluated. Lung permeability and edema were assessed by lung wet/dry ratio, and protein and cellular load of the bronchoalveolar lavage fluid (BALF). Inflammatory cytokine levels in BALF were measured by ELISA and the expression of PPARγ in the lung tissue was analyzed by immunoblotting. The results suggested that PDX could diminish the inflammatory response in lung tissue after sepsis by upregulating PPARγ and inhibiting the phosphorylation and activation of NF-κB p65. PDX treatment lowered the levels of pro-inflammation cytokines IL-1β, IL-6, TNF-α, and MCP-1, and the levels of anti-inflammatory cytokine IL-10 was increased in the BALF. It also improved lung permeability and reduced lung injury. Furthermore, the protective effect of PDX on lung tissue could be reversed by GW9662, a specific PPAR-γ antagonist. Taken together, our study indicated that PDX could ameliorate the inflammatory response in ALI by activating the PPARγ/NF-κB pathway in a mouse model of sepsis.

Topics: Acute Lung Injury; Anilides; Animals; Anti-Inflammatory Agents; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Docosahexaenoic Acids; Humans; Inflammation; Inflammation Mediators; Lung; Male; Mice; PPAR gamma; Sepsis; Signal Transduction; Transcription Factor RelA

Sepsis is a life-threatening disease. Inflammation is a major concomitant symptom of sepsis Chrysophanol, an anthraquinone derivative isolated from the rhizomes of rheumpalmatum, has been reported to have a protective effect against lipopolysaccharide(LPS)-induced inflammation. However, the underlying molecular mechanisms are not well understood. The aim of this study was to explore the effect and mechanism of chrysophanol on lipopolysaccharide (LPS)-induced anti-inflammatory effect of RAW264.7 cells and its involved potential mechanism. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and inducible nitric oxide synthase (iNOS), nuclear factor kappa B (NF-κB) and PPAR-γ were measured by qRT-PCR and western blotting, the production of TNF-α, IL-1β was evaluated by ELISA. Then, the phosphorylation of NF-κB p65 was also detected by western blotting. And NF-κB p65 promoter activity was analyzed by the Dual-Luciferase reporter assay system as well. Meanwhile, PPAR-γ inhibitor GW9662 was performed to knockdown PPAR-γ expression in cells. Our data revealed that LPS induced the up-regulation of TNF-α, IL-1β, iNOS and NF-κB p65, the down-regulation of PPAR-γ were substantially suppressed by chrysophanol in RAW264.7 cells. Furthermore, our data also figured out that these effects of chrysophanol were largely abrogated by PPAR-γ inhibitor GW9662. Taken together, our results indicated that LPS-induced inflammation was potently compromised by chrysophanol very likely through the PPAR-γ-dependent inactivation of NF-κB in RAW264.7 cells.

Topics: Anilides; Animals; Anthraquinones; Anti-Inflammatory Agents; Interleukin-1beta; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; Nitric Oxide Synthase Type II; PPAR gamma; RAW 264.7 Cells; Rheum; Rhizome; Sepsis; Signal Transduction; Tumor Necrosis Factor-alpha

Dysregulation of the inflammatory response against infection contributes to mortality in sepsis. Inflammation provides critical host defense, but it can cause tissue damage, multiple organ failure, and death. Because the nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) exhibits therapeutic potential, we characterized the role of PPARγ in sepsis. We analyzed severity of clinical signs, survival rates, cytokine production, leukocyte influx, and bacterial clearance in a cecal ligation and puncture (CLP) model of sepsis in Swiss mice. The PPARγ agonist rosiglitazone treatment improved clinical status and mortality, while increasing IL-10 production and decreasing TNF-α and IL-6 levels, and peritoneal neutrophil accumulation 24 h after CLP. We noted increased bacterial killing in rosiglitazone treated mice, correlated with increased generation of reactive oxygen species. Polymorphonuclear leukocytes (PMN) incubated with LPS or Escherichia coli and rosiglitazone increased peritoneal neutrophil extracellular trap (NET)-mediated bacterial killing, an effect reversed by the PPARγ antagonist (GW 9662) treatment. Rosiglitazone also enhanced the release of histones by PMN, a surrogate marker of NET formation, effect abolished by GW 9662. Rosiglitazone modulated the inflammatory response and increased bacterial clearance through PPARγ activation and NET formation, combining immunomodulatory and host-dependent anti-bacterial effects and, therefore, warrants further study as a potential therapeutic agent in sepsis.

Topics: Anilides; Animals; Disease Models, Animal; Escherichia coli; Extracellular Traps; Male; Mice; Neutrophils; PPAR gamma; Rosiglitazone; Sepsis; Signal Transduction; Thiazolidinediones

To determine the role of activated status of peroxisome proliferator-activated receptorγ/nuclear factor-ΚB (PPAR-γ/NF-ΚB ) in coagulation disorders induced by sepsis.. Forty male Sprague-Dawley (SD) rats were randomly divided into four groups, n=10 in each group: control group, lipopolysaccharide (LPS) challenged group, rosiglitazone (ROSI, selective agonist of PPAR-γ) pretreatment group, and GW9662 (PPAR-γ antagonist) pretreatment group. The sepsis model was reproduced by injection of 6 mg/kg LPS via sublingual vein, and the rats in control group were injected with 2 mL/kg normal saline. The rats in ROSI pretreatment group were given 0.3 mg/kg ROSI by sublingual venous injection followed by injection of LPS 30 minutes later; and in GW9662 pretreatment group rats were given 0.3 mg/kg GW9662 by sublingual venous injection followed by 0.3 mg/kg ROSI 15 minutes later, followed by injection of LPS 30 minutes later. Blood was collected at 4 hours after LPS administration, and the expressions of PPAR-γ and NF-ΚBp65 in peripheral blood mononuclear cell (PBMC) were determined with immunocytocheminal technique and graph analysis. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and D-dimer were determined simultaneously.. (1) PPAR-γ/NF-ΚB pathway: the expressions of PPAR-γ and NF-ΚBp65 were lowered in control group, and they were expressed in cytoplasm. In LPS challenged group the expression of PPAR-γ (gray value) was slightly increased but with no significant difference as compared with control group (111.01±4.06 vs. 98.46±5.99, P>0.05). In ROSI pretreatment group the expression of PPAR-γ (gray value) was significantly higher than that in LPS challenged group (214.38±5.79 vs. 111.01±4.06, P<0.01), with dislocation into nuclei. In GW9662 pretreatment group the expression of PPAR-γ (gray value) was lowered but without significant difference compared with that of control group (44.21±2.64 vs. 98.46±5.99, P>0.05). In LPS challenged group the expression of NF-ΚBp65 (gray value) was significantly higher than that in control group (249.48±6.86 vs. 105.81±10.19, P<0.01), and it was translocated into the nuclei. In ROSI pretreatment group the expression of NF-ΚBp65 (gray value) was significantly lower than that in LPS challenged group (102.47±8.05 vs. 249.48±6.86, P<0.01), and it lied in cytoplasm. In GW9662 pretreatment group the expression of NF-ΚBp65 (gray value) showed no significant difference as compared with that of LPS challenged group (214.84±7.91 vs. 249.48±6.86, P>0.05). (2) Coagulation: compared with control group, PT and APTT were significantly prolonged, FIB was significantly decreased, and D-dimer was significantly increased in LPS challenged group [PT (s): 18.32±2.03 vs. 12.22±1.38, APTT (s): 40.05±2.72 vs. 26.64±2.73, FIB (g/L): 1.65±0.51 vs. 3.60±0.37, D-dimer (mg/L): 2.58±0.73 vs. 0.37±0.06, all P<0.01]. Compared with LPS challenged group, APTT and PT were significantly shortened, FIB was significantly increased, and D-dimer was significantly lowered in ROSI pretreatment group [PT (s): 13.93±1.67 vs. 18.32±2.03, APTT (s): 30.29±0.86 vs. 40.05±2.72, FIB (g/L): 3.18±0.69 vs 1.65±0.51, D-dimer (mg/L): 0.40±0.12 vs. 2.58±0.73, all P<0.01]. All parameters in GW9662 pretreatment group showed no significant difference as compared with those of LPS challenged group.. PPAR-γ agonist ROSI may ameliorate coagulation disorders in septic rats. PPAR-γ/NF-ΚB transduction pathway plays an important role in septic coagulopathy.

Topics: Anilides; Animals; Blood Coagulation; Blood Coagulation Tests; Fibrin Fibrinogen Degradation Products; Leukocytes, Mononuclear; Lipopolysaccharides; Male; NF-kappa B; PPAR gamma; Rats; Rats, Sprague-Dawley; Rosiglitazone; Sepsis; Signal Transduction; Thiazolidinediones

To explore the effects of rosiglitazone, a synthetic ligand of proliferator-activated receptor-γ (PPAR-γ) on vascular endothelial injuries in septic rats.. A total of 40 male Sprague-Dawley rats were randomly divided into 4 groups of vehicle control, lipopolysaccharide (LPS), pretreatment of rosiglitazone (ROSI) and pretreatment of PPAR-γ antagonist 2-chloro-5-nitroaniline (GW9662) (n=10 each). At 4 hours post-intervention, blood samples were collected to detect the expression of PPAR-γ by immunocytochemistry and image analysis. And the following parameters of vascular endothelial injury were measured: Vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), angiopoietin-2 (Ang-2), thrombomodulin (TM), anti-thrombin III (AT-III), tissue factor (TF), von Willebrand factor (vWF) and circulating endothelial cell (CEC).. ① In ROSI group, the expression of PPAR-γ was significantly higher than that in LPS group (P<0.01). In GW9662 group, the expression of PPAR-γ had no significant difference compared to vehicle control group (P>0.05). ② The serum concentrations of VCAM-1, ICAM-1, Ang-2, TM, AT-III, TF and vWF were significantly higher in LPS group than those in vehicle control group (P<0.01). The concentrations of these parameters in ROSI group were significantly lower than those in LPS group (P<0.01). In GW9662 group, the concentrations of these parameters had no significant difference compared with LPS group (P>0.05). ③ The numbers of CEC were significantly higher in LPS group than those in vehicle control group (P<0.01). And the numbers of CEC were significantly lower in ROSI group than those in LPS group (P<0.01). In GW9662 group, the numbers of CEC had no significant difference compared with LPS group (P>0.05).. Proliferator-activated receptor-γ agonist improves sepsis-induced vascular endothelial injury. And its mechanism may be through stabilizing vascular endothelial cell for improving serious inflammatory reaction and blood coagulation dysfunction.

Topics: Angiopoietin-2; Anilides; Animals; Endothelium, Vascular; Intercellular Adhesion Molecule-1; Lipopolysaccharides; Male; PPAR gamma; Rats; Rats, Sprague-Dawley; Rosiglitazone; Sepsis; Thiazolidinediones; Thromboplastin; Vascular Cell Adhesion Molecule-1

Despite intensive research, sepsis displays the most prevalent cause of death on intensive care units. The hallmark of sepsis is an overshooting T-cell death that reduces host defense mechanisms and that is associated with poor patient survival. Previous in vitro studies revealed that the expression of the transcription factor peroxisome proliferator-activated receptor (PPAR) γ was increased in isolated T cells of patients with sepsis.. We determined the importance of targeting PPARγ for sepsis treatment and underlying molecular mechanisms for T-cell apoptosis in vivo.. To mimic human systemic inflammation and septic conditions, we used a nonlethal endotoxemia and a lethal cecum ligation and puncture polymicrobial sepsis model.. PPARγ inhibition in T cells with either the PPARγ antagonist GW9662 or a newly generated T cell-specific PPARγ knockout (Tc-PPARγ(-/-)) mice provided a survival advantage during polymicrobial sepsis in mice, which correlated with abrogated T-cell depletion in both in vivo models. Pathway analysis revealed increased antiapoptotic IL-2 and Bcl-2 expression, and activated prosurvival PI3K/Akt signaling under PPARγ-deficient conditions. In line, neutralizing IL-2 in Tc-PPARγ(-/-) mice resulted in T-cell apoptosis and increased mortality.. Our results provide evidence for a pivotal involvement of PPARγ in T-cell depletion by activating two important apoptosis pathways, and subsequently provoking the breakdown of defense mechanisms during systemic inflammation and sepsis.

Topics: Anilides; Animals; Apoptosis; Interleukin-2; Mice; Mice, Knockout; NFATC Transcription Factors; Peritonitis; Phosphatidylinositol 3-Kinases; PPAR gamma; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Sepsis; Signal Transduction; Survival Rate; T-Lymphocytes

Neutrophils (polymorphonuclear leukocytes [PMNs]) are critical to the immune response, including clearance of infectious pathogens. Sepsis is associated with impaired PMN function, including chemotaxis. PMNs express peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a ligand-activated nuclear transcription factor involved in immune and inflammatory regulation. The role of PPAR-gamma in PMN responses, however, is not well characterized. We report that freshly isolated human PMNs constitutively express PPAR-gamma, which is up-regulated by the sepsis-induced cytokines TNF-alpha and IL-4. PMN chemotactic responses to formylmethionyl-leucyl-phenylalanine (fMLP) and IL-8 were dose-dependently inhibited by treatment with the PPAR-gamma ligands troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and by transfection of PMN-like HL-60 cells with a constitutively active PPAR-gamma construct. Inhibition of chemotaxis by PPAR-gamma ligands correlated with decreases in extracellular signal-regulated kinase-1 and -2 activation, actin polymerization, and adherence to a fibrinogen substrate. Furthermore, PMN expression of PPAR-gamma was increased in sepsis patients and mice with either of 2 models of sepsis. Finally, treatment with the PPAR-gamma antagonist GW9662 significantly reversed the inhibition of PMN chemotaxis and increased peritoneal PMN recruitment in murine sepsis. This study indicates that PPAR-gamma activation is involved in PMN chemotactic responses in vitro and may play a role in the migration of these cells in vivo.

Topics: Actins; Anilides; Animals; Antineoplastic Agents; Cell Adhesion; Chemotaxis; Chromans; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fibrinogen; HL-60 Cells; Humans; Inflammation; Interleukin-4; Interleukin-8; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; N-Formylmethionine Leucyl-Phenylalanine; PPAR gamma; Prostaglandin D2; Sepsis; Thiazolidinediones; Troglitazone; Tumor Necrosis Factor-alpha; Up-Regulation

Although phytochemical curcumin has been shown to possess anti-inflammatory properties, it remains unknown whether this agent has any beneficial effects in sepsis. The purpose of this study was to demonstrate whether curcumin protects septic animals and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR)-gamma, an anti-inflammatory nuclear receptor, plays any role.. Prospective, controlled, and randomized animal study.. A research institute laboratory.. Male Sprague-Dawley rats.. A bolus injection of 0.2 micromol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 micromol/day) for 3 days via a primed 2-mL mini-pump. The rats were then subjected to sepsis by cecal ligation and puncture (CLP).. Serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase), lactate, albumin, and tumor necrosis factor (TNF)-alpha were measured at 20 hrs after CLP (i.e., late stage of sepsis). In addition, a 10-day survival curve was conducted following CLP and cecal excision with or without curcumin treatment. Furthermore, macrophages cell line RAW 264.7 cells were treated with curcumin followed by stimulation with endotoxin. TNF-alpha and PPAR-gamma expression were then measured. The results indicate that intravenous administration of curcumin before the onset of sepsis attenuated tissue injury, reduced mortality, and decreased the expression of TNF-alpha in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-gamma in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-gamma antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-alpha expression and markedly up-regulated PPAR-gamma expression without affecting cell viability. Curcumin also prevented morphologic alterations in macrophages induced by endotoxin.. The protective effect of curcumin makes it or its analogues strong candidates as a novel therapy for sepsis. The beneficial effect of curcumin appears to be mediated by up-regulation of nuclear receptor PPAR-gamma.

Topics: Anilides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Survival; Curcumin; Down-Regulation; Endotoxins; Lactates; Liver; Macrophages; Male; PPAR gamma; Prospective Studies; Random Allocation; Rats; Rats, Sprague-Dawley; Sepsis; Serum Albumin; Transaminases; Tumor Necrosis Factor-alpha; Up-Regulation