Study On The Mechanism Of PPARγ Phosphorylation In Macrophages In Atherosclerosis

ObjectivesAtherosclerosis is a multifactorial disease caused by dyslipidemia,cell inflammation,cell proliferation,hypertension,oxidative stress and insulin resistance.Previous studies have shown that PPAR γ phosphorylation plays a significant role in regulating glucose and lipid metabolism,so it has an indirect effect on atherosclerosis.The purpose of this study was to investigate the role of PPAR phosphorylation in the formation of foam cells and inflammatory response in Raw264.7 macrophage,and to explore the mechanism of its participation in the regulation of the occurrence and development of atherosclerosis.MethodsIn this study,Raw264.7 macrophages were induced by oxidized low density lipoprotein to construct foam cell model,and Roscovitine(PPAR γ phosphorylation indirect inhibitor)was used to intervene.The experiment was divided into three groups: control group,ox-LDL group and inhibitor group.The expression of pPPAR γ,PPAR γ,CDK5 and its activator p35 protein was detected by Western blot,the intracellular lipid accumulation was analyzed by oil red O staining and isopropanol extraction experiment,the intracellular cholesterol content was determined by enzymatic method,the protein expression and mRNA levels of ox-LDL uptake related genes CD36,SRA1 and cholesterol efflux related genes ABCA1 and ABCG1 were detected by Westernblot and PCR,the secretion and expression of pro-inflammatory cytokines TNF α and IL-1 β were detected by ELISA and PCR.Observed the relationship between PPAR γSer273 phosphorylation and foam cell formation or inflammation.In order to verify the above changes,PPAR γ 273 site mutation was carried out,and then constructed and screened stable expression of PPAR γ WT and Ser273 site mutant Raw264.7 macrophages.The macrophages were induced by ox-LDL and were divided into four groups: WT group,WT+ox-LDL group,S273 A group and 273A+ox-LDL group.The expression levels of pPPAR γ and PPAR γ in macrophages were detected by Western blot,Westernblot and PCR methods were used to detect the protein and mRNA expression levels of CD36,SR-A1,ABCA1 and ABCG1 in macrophages.ELISA and PCR methods were used to detect the secretion and mRNA expression of TNF α and IL-1 β in macrophages.Results1.The MTT results of Raw264.7 macrophages treated with different concentrations of ox-LDL or Roscovitine showed that ox-LDL larger than 50mg/L and Roscovitine more than 15 umol / L had obvious inhibitory effect on cells,so 50mg/Lox-LDL and 15 umol/LRoscovitine were selected for follow-up experiments.2.The formation of foam cells in different groups was observed.The results showed that the intracellular lipid accumulation,total cholesterol content,free cholesterol content and cholesterol ester/total cholesterol ratio increased after treated by ox-LDL.After the addition of inhibitor,the above indexes decreased,indicating that the inhibitor successfully interfered with foam cell formation.3.Ox-LDL induced the increase of pPPAR γ / PPAR γ and p35/CDK5 ratio,and the addition of Roscovitine could antagonize the above changes,indicating that the phosphorylation of PPAR γ Ser273 was related to the activity of CDK5.Combined with the results of foam cell formation,it can be seen that CDK5-mediated PPAR γ Ser273 phosphorylation may be involved in foam cell formation..4.The results of protein and mRNA showed that the expression levels of CD36 and SR-A1 protein and mRNA in ox-LDL group were consistent with PPAR γ Ser273 phosphorylation,while the expression levels of cholesterol efflux related genes ABCA1 and ABCG1 protein and mRNA were opposite to those of PPAR γ Ser273 phosphorylation,indicating that PPAR γ Ser273 phosphorylation was involved in the mechanism of foam cell formation.5.ELISA and mRNA showed that the secretion and mRNA expression of pro-inflammatory cytokines TNF α and IL-1 β were consistent with PPAR γ Ser273 phosphorylation,indicating that PPAR γ Ser273 phosphorylation was involved in inflammatory response.6.Raw264.7 macrophages stably expressing PPAR γ WT and Ser273 site mutants were successfully constructed.Firstly,PPAR γ was silenced,and the silencing efficiency was detected by Westernblot.The results showed that compared with untreated group and untreated control group,PPAR γ was not detected in the experimental group,indicating that PPAR γ silencing was successful.The WT type and Ser273 point mutant plasmids were transferred into the silenced cells.The Westernblot results showed that compared with the WT group,the PPAR γ phosphorylation level was no longer detected in the S273 A group,indicating that the cell line was constructed successfully.7.PPAR γ WT and Ser273 mutant cells were treated by ox-LDL,and observed the relationship between the changes of CD36,SR-A1,ABCA1 and ABCG1,inflammatory cytokines TNF α and IL-1 β and PPAR γ Ser273 phosphorylation.We founded that whether stimulated by ox-LDL or not,the expression of CD36 and SR-A1 decreased,the expression of ABCA1 and ABCG1 increased,and the levels of TNF α and IL-1 β also decreased after mutation.Results were further verified the role of PPAR γ Ser273 phosphorylation in foam cell formation and inflammation.Conclusions1.PPAR γ Ser273 phosphorylation promoted foam cell formation.2.PPAR γ Ser273 phosphorylation regulated the expression of ox-LDL uptake related genes CD36,SR-A1 and cholesterol efflux related genes ABCA1 and ABCG1 in Raw264.7 macrophages,induced cholesterol lipid accumulation in macrophages to promote foam cell formation,and then accelerated the occurrence of atherosclerosis.3.PPAR γ Ser273 phosphorylation induced the inflammatory response of Raw264.7 macrophages by promoting the secretion and expression of inflammatory cytokines TNF α and IL-1 β,further accelerate the formation of foam cells,thus promoted the progression of atherosclerosis.4.The purpose of this study was to explore the role of PPAR γ Ser273 phosphorylation in atherosclerosis and to provide a powerful basis for the application of anti-atherosclerotic drugs based on this mechanism.