The Anti-inflammatory Effects And Mechanisms Of Peroxisome Proliferators-activated Receptor γ In Vitro

BackgroudAcute lung injury (ALI) is a common disorder associated with significant morbidity and mortality. Severe hypoxemia and high mortality are popular in clinic. The pathological features mainly include diffuse injury of alveolar epithelial and pulmonary vascular endothelial cells. Previous studies have indicated that ALI is an excessive inflammatory response for the imbalance between the inflammation mediators. Because there are so many mediators and the interaction of these agents is complex, true insight into the process is still unclear and current therapy is still poor. Peroxisome proliferators-activated receptorγ(PPARγ), a member of the nuclear hormone receptor superfamily of ligand-actived transcription factors, is ubiquitously expressed within the lung. Recent studies have indicated PPARγcan protect lung for its anti-inflammatory properties. But the reason that PPARγdecreases in the inflammation and whether PPARγcan protect typeⅡalveolar epithelial (AT-Ⅱ) cells in LPS-stimulation are still not clear. In this study, we detected the effects of NF-κB on PPARγexpression in macrophages and the protective effect and mechanism of Troglitazone, one of agonists of PPARγ', in rat AT-Ⅱcells. The study provided the strong evidence for the usage of PPARγagonist in the prevention and treatment of ALI in clinic.ObjectiveTo determine the effects of NF-κB on PPARγexpression in the murine macrophage cell line Ana-1 and the protective effect and mechanism of PPARγin rat AT-Ⅱcells.Methods1. Ana-1 cells were divided randomly into seven groups: control group, LPS groups (cells were activated by 0.1 mg/L LPS in 1,2,4,8h respectively), SN50 group (cells were stimulated by 50 mg/L SN50 in 4h) and NF-κB transfected group. PPARγand tumor necrosis factorα(TNF-α) mRNA level were assayed by reverse transcription polymerase chain reaction(RT-PCR)and the TNF-αprotein was measured by enzyme linked immunosorbent assay (ELISA) after being activated by LPS. The eukaryotic expression vector of murine NF-κB p65 gene was constructed and stably transfected into Ana-1 cells with DOTAP liposome. The PPARγand NF-κB protein were extracted from Ana-1 cells'nuclear, which were stable transfected of p65 gene or stimulated by LPS and SN50, were assayed by Western blot.2. The lung tissues of the rat were digested by trypsase and collagenase. AT-Ⅱcells were isolated and purified by immuno-adherence, and than primarily cultured AT-Ⅱcells were divided randomly into four groups: Control group, LPS group, Troglitazone group and Troglitazone+GW9662 group. Pulmonary surfactant-associated protein A (SP-A) and TNF-αmRNA level were assayed by RT-PCR. SP-A protein was measured by immunofluorescence and Western blot. And TNF-αprotein was assayed by ELISA. Apoptosis and necrosis rates of cells were detected by flow cytometry after being stained by AnnexinⅤ/PI.Results1. PPARγwere decreased when cells were stimulated by LPS(P <0.05),which were related to the activity of NF-κB and TNF-αin Ana-1 cells(P <0.05).2. The eukaryotic expression vectors containing murine p65 gene were successfully constructed and stably transfected into Ana-1 cells.3. NF-κB overexpressed or stimulated by LPS which upregulated NF-κB could downregulate PPARγexpression in Ana-1 cells(P<0.05). But downregulated NF-κB by SN50 could upregulate PPARγexpression in Ana-1 cells(P<0.05).4. Primary culture of rat AT-Ⅱcells were successfully established as confirmed by the expression of AKP, SP-A and unltrastructural features.5. SP-A was decreased when AT-Ⅱcells were stimulated by LPS (P<0.05), which was related to the activity of TNF-αand the increase of apoptosis and necrosis of cells.6. When Troglitazone were used, the apoptosis and necrosis rates and TNF-αwere decreased (P<0.05). SP-A was increased at the same time (P<0.05).The protective effect could be blocked by GW9662, one of antagons of PPARγ'. Conclusion1. LPS can markedly decrease the expression of PPARγin Ana-1 cells, which may be related to its enhancing NF-κB and TNF-αactivity, suggesting PPARγis involved in the inflammatory response.2. NF-κB were overexpressed by stably transfected in Ana-1 cells as same as cells were in the LPS-stimulated could downregulate PPARγexpression. But downregulated NF-κB by SN50 could upregulate PPARγexpression in them.All of above proves NF-κB can control PPARγexpression in the reverse direction in Ana-1 cells.3. LPS can markedly increase the expression of TNF-αand the apoptosis and necrosis of AT-Ⅱcells. SP-A was decreased at the same time. Troglitazone,one of agonists of PPARγ', can protect AT-Ⅱcells after being stimulated by LPS, which may be related to the decrease of the TNF-α, the apoptosis and necrosis of cells and the increase of SP-A.