The Effect And Mechanism Of PPARδ Agonist GW0742 On Homocysteine-induced Expression Of PP47phox In EA.hy926 Cells

Objective: Atherosclerosis is a common a disease in clinical, it alway causes cerebrovascular disease and coronary heart disease which is a threat to the health and lives of the elderly. HCY activated NADPH oxidase via various signaling pathways, result in the increase of ROS production, cause a series of physiological and pathological changes, is an important part of the formation of atherosclerosis.Recent studies have suggested that PPARδ activation may reduce ROS level, prevents endothelial dysfunction, suppress atherosclerosis development. In this study, EA.hy926 cells were treated with HCY and GW0742, and p47 phox m RNA was measured by RT-PCR, P-p38MAPK、P-p47 phox �'� PPARδ was measured by Western blot analysis, in order to make clear how PPARδinhibited ROS production.Materials and methods: 1. EA.hy926 cells subculture. 2. Cells were grown in serum-free DMEM medium for 24 h, cells were treated with HCY(50μM) for 30 min. m RNA levels of p47 phox were measured by RT-PCR. 3. Cells were grown in serum-free DMEM medium for 24 h, Cells were treated with HCY(50μM) for the indicated times. Protein levels of p47 phox phosphorylation were measured by Western blot analysis. 4. Cells were grown in serum-free DMEM medium for 24 h, Cells were treated(A) withHCY(50μM) for the indicated times;(B) pretreated with or without GW0742(1μM) for 30 min, and then incubated with HCY(50μM) for 30 min. Oxygen species level was measured using the redox-sensitive fluorescent dye DCFH-DA. 5. Cells were grown in serum-free DMEM medium for 24 h, Cells were treated with HCY(50μM) for the indicated times. Protein levels of PPARδ were measured by Western blot analysis. 6. Cells were grown in serum-free DMEM medium for 24 h, Cells were pretreated with or without GW0742(1μM) or GSK0660(1μM) for 30 min, and then incubated with HCY(50μM) for 30 min. Protein levels of PPARδ were measured by Western blot analysis. 7. Cells were grown in serum-free DMEM medium for 24 h, Cells were pretreated with GW0742(1μM) for 30 min, prior to treated with HCY(50μM) for 30 min or one of them; Protein levels of p38 MAPK phosphorylation were measured by Western blot analysis. 8. Cells were grown in serum-free DMEM medium for 24 h, Cells were pretreated with GW0742(1μM) or SB203580(20μM) for 30 min, prior to treated with HCY(50μM) for 30min; Protein levels of p47 phox phosphorylation were measured by Western blot analysis.Results: 1. EA.hy926 Cells the same morphological distribution as found in primary endothelial cells and Biology. 2. HCY increased expression of p47 phox m RNA. 3. HCY induced a time-dependent increase of p47 phox phosphorylation, and the peak of phosphorylation was achieved at 30 min. 4. HCY induced a time-dependent increase ROS production, and ROS production was noticeably increased at 30 min; the increase of reactive oxygen species which induced by HCY was inhibited in the presence of GW0742. 5. HCY induced a time-dependent increase of PPARδ, and the peak of PPARδ was achieved at 30 min.6. PPARδ expression was increased by GW0742 or HCY; GW0742 would significantly increase the expression of PPARδ in inflammation induced by HCY, this change higher than HCY or GW0742 alone. 7. GW0742 would inhibit p38 MAPK phosphorylation. 8. HCY activate p47 phox through p38 MAPK pathway, and GW0742 would inhibit p47 phox phosphorylation.Conclusion: HCY increased p47 phox phosphorylation through P38 MAPK pathway, at the end, enhanced ROS production. However, GW0742 could highly increased PPARδ in oxidative stress induced by HCY, and reduced phosphorylation of p38 MAPK and p47 phox, decreased ROS production.