Protective Effects And Mechanisms Of Peroxisome Proliferator-Activated Receptor γ On Endotoxin-induced Acute Lung Injury

Part IThe change and significance of the lung expression of peroxisome proliferator-activated receptor γ in rats with endotoxin-inducedacute lung injuryObjective To investigate the time course profile of the lung expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and to explore its functional significance in rats with endotoxin-induced acute lung injury. Methods Thirty male Wistar rats were randomly assigned to one of the following five groups: saline control group and different lipopolysaccaride (LPS) groups (2 h, 4 h, 6 h and 8 h after LPS 6 mg/kg i.v.). At predefined time points, blood samples were collected to measure arterial oxygen pressure (PaO₂) and lungs were removed for evaluation of histopathological changes and determination of wet-to-dry weight (W/D) ratio, myeloperoxidase (MPO) activity and tumor necrosis factor (TNF)-a level. Nuclear expressions of nuclear factor (NF)-κB p65 and PPAR-γ in lung tissues were also examined in each group. Results LPS treatment resulted in a progressive decrease in PaO₂, reaching the minimum at 8 h after LPS injection. Administration of LPS also induced a time-dependent increase in the magnitude of morphological lung damages and the levels of W/D ratio and MPO activity in the lung, peaking at 8 h after LPS injection. In addition, LPS also caused a marked increase in pulmonary TNF-α, which was paralleled by the magnitude of NF-κB activation in overall trends. RT-PCR analysis of lung tissue samples showed that LPS treatment had no effect on either PPAR-γ1 or PPAR-γ mRNA levels at all time points. However, Western blot analysis showed that nuclear PPAR-γ protein decreased beginning 2 h after LPS treatment, and progressively further decreased for up to 8 h after LPS treatment. Immunohistochemistry revealed a reduction of PPAR-γ expression in alveolar epithelial cells and macrophages in lungs from LPS-treated rats. Conclusion Our data suggest that decreased expression of PPAR-γ protein in lung may be implicated in the pathogenesis of LPS-induced acute lung injury.Part IIProtective effects and mechanisms of rosiglitazone, an agonist ofperoxisome proliferator-activated receptor γ, onendotoxin-induced acute lung injuryStudy 1 Protective effects of pretreatment with rosiglitazone on endotoxin-induced acute lung injury in ratObjective To investigate the effects of rosiglitazone (ROSI), a potent agonist of peroxisome proliferator-activated receptor γ (PPAR-γ ), on acute lung injury (ALI) induced by endotoxin in rats. Methods Thirty-six male Wistar rats were randomly assigned to one of six groups (n = 6 per group): control group received 10% dimethyl sulfoxide (DMSO) 30 min prior to the administration of saline; ROSI group was identical to the control group except that ROSI (0.3 mg/kg i.v.) was administered instead of DMSO; GW9662 group was identical to the control group except that GW9662 (0.3 mg/kg i.v.) was administered instead of DMSO; endotoxin group received 10% DMSO 30 min before treatment with lipopolysaccaride (LPS) 6 mg/kg i.v.; ROSI-LPS group was identical to the LPS group but received ROSI (0.3 mg/kg, i.v.) instead of DMSO; GW9662-ROSI-LPS group was identical to the ROSI-LPS group except that GW9662 0.3 mg/kg was given 20 min before ROSI. Four hours after LPS injection, rats were sacrificed and the lungs were removed for evaluation of histological injury and determination of wet/dry lung weight (W/D) ratio and myeloperoxidase (MPO) activity as well as malondialdehyde (MDA) and nitric oxide (NO) contents. Inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation were also studied in the lung tissues. Results LPS induced marked lung histological injury and a significant increase in W/D ratio and MPO activity as well as MDA and NO levels in the lung. All of these changes were significantly attenuated by pretreatment with ROSI. ROSI pretreatment also markedly suppressed LPS-induced expression of iNOS messenger RNA and protein in the lung, as demonstrated by reverse transcription-polymerase chain reaction or Western blot analysis. Immunohisto-chemical analysis revealed that ROSI inhibited the formation of nitrotyrosine, a marker for peroxynitrite reactivity, in the lung tissue. In addition, the specific PPAR-γ antagonist GW9662 abolished the effects of ROSI. Conclusion This study provides evidence, for the first time, that the PPAR-γ agonist ROSI has protective effect on endotoxin-induced ALI in rats. The mechanism of the salutary effect of ROSI is dependent on the activation of PPAR-γ.Study 2 Effect of rosiglitazone on pulmonary inflammation in rats with endotoxin-induced acute lung injury and possible mechanismsObjective To investigate the effect of rosiglitazone (ROSI), a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, on acute pulmonary inflammation and nuclear factor-κB (NF-κB) activation in rats with endotoxin-induced acute lung injury. Methods Thirty-six male Wistar rats were randomly assigned to one of six groups (n = 6 per group): control group received 10% dimethyl sulfoxide (DMSO) 30 min prior to the administration of saline; ROSI group was identical to the control group except that ROSI (0.3 mg/kg i.v.) was administered instead of DMSO; GW9662 group was identical to the control group except that GW9662 (0.3 mg/kg i.v.) was administered instead of DMSO; endotoxin group received 10% DMSO 30 min before treatment with lipopolysaccaride (LPS) 6 mg/kg i.v.; ROSI-LPS group was identical to the LPS group but received ROSI (0.3 mg/kg, i.v.) instead of DMSO; GW9662-ROSI-LPS group was identical to the ROSI-LPS group except that GW9662 0.3 mg/kg was given 20 min before ROSI. Four hours after LPS injection, the concentrations of tumor necrosis factor-α (TNF-α) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured in rat lung tissues. Expression of intercellular adhesion molecule-1 (ICAM-1), nuclear factor (NF)-κB p65 and PPAR-γ were also examined by immunohistochemistry or Western blot analysis. Results ROSI pretreatment significantly attenuated the increases in the lung levels of TNF-α and CINC-1 as well as expression of ICAM-1 following LPS challenge. The specific PPAR-γ antagonist GW9662 abolished the effect of ROSI. In addition, ROSI also inhibited the activation of NF-κB and up-regulated the expression of PPAR-γ protein. Conclusion PPAR-γ agonist ROSI could reduce LPS-induced pulmonary inflammation. The mechanisms underlying the antiinflammatory effect of ROSI involve inhibition of NF-κB activation and upregulation of PPAR-γ expression in the lung.Part IIIProtective effects and mechanisms of 15-deoxy-Δ^12,14-prostaglandin J₂, anagonist of peroxisome proliferator-activated receptor γ, onendotoxin-induced acute lung injuryStudy 1 Protective effects of pretreatment with 15-deoxy-Δ^12,14-prostaglandin J₂ onendotoxin-induced acute lung injury in ratObjective To investigate the effects of 15-deoxy-Δ^12,14-prostaglandin J₂ (15-PGJ₂), a nature ligand of PPAR-γ, on acute lung injury (ALI) induced by endotoxin in rats. Methods Thirty-six male Wistar rats were randomly assigned to one of six groups (n = 6 per group): control group received 10% dimethyl sulfoxide (DMSO) 30 min prior to the administration of saline; 15d-PGJ2 group was identical to the control group except that 15d-PGJ₂ (0.3 mg/kg i.v.) was administered instead of DMSO; GW9662 (GW) group was identical to the control group except that GW9662 (0.3 mg/kg i.v.) was administered instead of DMSO; endotoxin group received 10% DMSO 30 min before treatment with lipopolysaccaride (LPS) 6 mg/kg i.v.; LPS + 15d-PGJ₂ group was identical to the LPS group but received 15d-PGJ₂ (0.3 mg/kg, i.v.) instead of DMSO; LPS + 15d-PGJ₂+ GW group was identical to the LPS + 15d-PGJ₂ group except that GW9662 0.3 mg/kg was given 20 min before 15d-PGJ₂. Four hours after LPS injection, rats were sacrificed and the lungs were removed for evaluation of histological injury and determination of wet/dry lung weight (W/D) ratio, myeloperoxidase (MPO) activity, malondialdehyde (MDA), and nitric oxide (NO) concentrations. Inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation were also studied in the lung tissues. Results Pretreatment with 15d-PGJ2 significantly ameliorated LPS-induced lung histological injury and attenuated the increases in W/D ratio and MPO activity as well as MDA and NO levels in the lung tissue. In addition, 15d-PGJ2 also markedly suppressed LPS-induced expression of iNOS messenger RNA and protein in the lung, as demonstrated by reverse transcription-polymerase chain reaction or Western blot analysis. Immunohistochemical analysis revealed that 15d-PGJ2 inhibited the formation of nitrotyrosine, a marker for peroxynitrite reactivity, in the lung tissue. In addition, the specific PPAR-γ antagonist GW9662 could partly antagonize the effects of 15d-PGJ2. Conclusion This study provides evidence that the PPAR-γ agonist 15d-PGJ₂ has protective effect on endotoxin-induced ALI in rats. The mechanism of the salutary effect of 15d-PGJ2 is, at lest in part, dependent on the activation of PPAR-γ .Study 2 Effect of 15-deoxy-Δ^12,14-prostaglandin J₂ on pulmonary inflammation in rats with endotoxin-induced acute lung injury and possible MechanismsObjective The present study was undertaken to investigate the effect of 15-deoxy-Δ^12,14-prostaglandin J₂ (I5-PGJ₂), on acute pulmonary inflammation in rats with endotoxin-induced acute lung injury and to explore its possible mechanisms. Methods Thirty-six male Wistar rats were randomly assigned to one of six groups (n = 6 per group): control group received 10% dimethyl sulfoxide (DMSO) 30 min prior to the administration of saline; 15d-PGJ2 group was identical to the control group except that 15d-PGJ₂ (0.3 mg/kg i.v.) was administered instead of DMSO; GW9662 (GW) group was identical to the control group except that GW9662 (0.3 mg/kg i.v.) was administered instead of DMSO; endotoxin group received 10% DMSO 30 min before treatment with LPS 6 mg/kg i.v.; LPS + 15d-PGJ₂ group was identical to the LPS group but received 15d-PGJ₂ (0.3 mg/kg, i.v,) instead of DMSO; LPS + 15d-PGJ₂+ GW group was identical to the LPS + 15d-PGJ₂ group except that GW9662 0.3 mg/kg was given 20 min before 15d-PGJ₂. Results 15d-PGJ₂ pretreatment significantly attenuated the increases in the lung levels of TNF-α and CINC-1 as well as expression of ICAM-1 following LPS challenge. The specific PPAR-γ antagonist GW9662 could partly antagonize the effect of 15d-PGJ₂. In addition, 15d-PGJ₂ also inhibited the nuclear localization of nuclear factor-κB (NF-κB) and up-regulated the expression of PPAR-γ and HO-1 protein. Conclusions These data suggest that PPAR-γ agonist 15d-PGJ₂ can reduce LPS-induced pulmonary inflammation. The mechanisms underlying the antiinflammatory effect of 15d-PGJ₂ involve activation of PPAR-γ , inhibition of NF-κB activation and upregulation of HO-1 protein expression in the lung.Part IVEffects of PPAR-γ agonists on nitric oxide production and nuclearfactor-κB activation in lipopolysaccharide-stimulated ratalveolar macrophagesObjective To investigate the effects of PPAR-γ agonists, rosiglitazone and 15-deoxy-Δ^12,14-prostaglandin J₂ (15-PGJ₂), on the production of nitric oxide (NO) and activation of nuclear factor (NF)-kB induced by LPS in rat alveolar macrophages (AMs). Methods Pulmonary AMs isolated from normal Wistar rats were used. The cells were pretreated with the vehicle or increasing concentrations (0.5 to 10 μM) of each PPAR-γ agonist for 30 min and then incubated with LPS (10 ng/ml) for 24 h. Culture supernatants were collected for NO quantification. In parallel studies, rat AMs were pretreated for 30 min with either rosiglitazone (10 μM) or 15-PGJ₂(10 μM) followed by stimulation with LPS (10 μg/ml) for 1 h or 24 h. Then, activation of NF-κB and the protein expression of inducible nitric oxide synthase (iNOS) were examined by immunocytochemistry or Western blot analysis, respectively. Results In rat AMs, 10 μg/ml LPS treatment significantly increased NO production, iNOS protein expression and NF-κB activation. Both rosiglitazone and 15d-PGJ2 (0.5 to 10 μM) pretreatment caused a dose-dependent suppression of the production of NO induced by LPS. At 10 μM concentrations, rosiglitazone and 15d-PGJ2 also inhibited the protein expression of iNOS in response to LPS. Immunocytochemical staining revealed that both rosiglitazone and 15d-PGJ2 (10 μM) significantly attenuated LPS-induced NF-κB activation in AMs. Conclusion These results show that PPAR-γ agonists suppress NO production by inhibiting iNOS expression in LPS-activated AMs, which may be associated with the inhibition of the activation of NF-κB following LPS stimulation.