Mechanism Of PPARγ In CML/RAGE Mediating Liver-vascular Crosstalk

ObjectiveTo explore the role of CML/RAGE signal in the progression of atherosclerosis and non-alcoholic fatty liver.MethodSix-week-old 75 male ApoE^-/-mice were randomly divided into Control group（common diet+tail vein injection of normal saline,n=15）,Model group（high-fat diet+tail vein injection of normal saline,n=15）,CML group（high-fat diet+tail vein injection CML 10mg/kg/day,n=15）,AAV-shscramble group（high-fat diet+tail vein injection CML10mg/kg/day+AAV-shscramble,n=15）,AAV-shRAGE group（high-fat diet+tail vein injection CML 10 mg/kg/day+AAV-shRAGE,n=15）were fed continuously for 16weeks.Morphological observation of the liver and aorta（HE,Oil red O,Masson staining）;related biochemical indicators include total cholesterol（TC）,triglyceride（TG）,low density lipoprotein LDL-C),high-density lipoprotein（HDL-C）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,superoxide dismutase（SOD）,malondialdehyde（MDA）,glutathione peroxidase（GSH-Px）and molecular biology testing.Results（1）At the time of 4 weeks,HE staining suggested that model group has balloon-like changes in liver cells,and hepatic steatosis was obvious after CML intervention;aortic HE staining showed no obvious plaque formation.（2）At the time of 8 weeks,liver HE staining showed significant liver steatosis in the CML group;aortic HE staining suggested intravascular plaque formation.（3）At the time of 16 weeks,the levels of liver index,blood lipids,liver enzymes in the model group were significantly higher,HE and Oil Red O staining observed hepatocellular bullous degeneration,and significant lipid accumulation in liver tissue,indicated non-alcoholic fatty Liver model was successful;hepatic steatosis significantly increased after CML intervention.（4）At the time of 16 weeks,Compared with the model group,the content of SOD and GSH-Px decreased,the content of MDA increased in the CML group,suggested that CML aggravated the oxidative damage of high-fat fed ApoE^-/-mice.（5）At the time of 16 weeks,HE staining of the aorta showed that compared with the model group,the area of aortic plaque was significantly increased in the CML group a large amount of cholesterol crystals could be seen.（6）CML promotes the expression of IL-1β,IL-6,TNF-α,CRP pro-inflammatory cytokine genes in the liver and aorta,the liver and aortic lesions are significantly relieved after silencing RAGE,and the expression of IL-1β,IL-6,TNF-αand CRP pro-inflammatory cytokine genes were significantly down-regulated.Conclusion（1）CML promotes the occurrence of non-alcoholic fatty liver and atherosclerosis.（2）CML/RAGE up-regulates the expression of pro-inflammatory cytokines and accelerates the progression of non-alcoholic fatty liver and atherosclerosis.Objective To investigate the mechanism of PPARγ in CML/RAGE mediating non-alcoholic fatty liver and atherosclerosis in Apo E-/-mice.Method（1）In vivo study 6-week-old male Apo E-/-mice,which were randomly divided into 4groups after a week of basic feed adaptive feeding: control group（common diet + tail vein injection of normal saline,n = 5）,model group（high-fat diet + tail vein injection of normal saline,n = 5）,CML group（high-fat diet + CML10 mg / kg / day,n = 5）,pioglitazone group（high-fat diet + CML10 mg / kg / day + pioglitazone 10 ml / kg / day,n= 5）After 16 weeks of continuous feeding,morphological tests（HE staining,Oil red O staining）and related biochemical indicators were performed.（2）In vitro study First observe the expression of PPARγ in Hep G2 cells stimulated by CML;Second silenced RAGE expression by si RNA,and explores whether CML affects PPARγ expression through RAGE;Third observe the effect of stimulating PPARγ on RAGE and proinflammatory cytokines IL-1β,IL-6,TNF-α,CRP;Finally,Hep G2 was cocultured with endothelial cells,observe the effect of agonistic PPARγ expression on endothelial cells.Methods include: Oil red O staining,ELISA,Western blot and PCR detection.Results（1）Compared with CML group,pioglitazone group significantly improved liver lipid deposition,reduced TC,TG,AST,ALT levels,and relieved the formation of nonalcoholic fatty liver.（2）Compared with the CML group,the pioglitazone group inhibited lipid accumulation in the plaque and delayed the formation of atherosclerosis.（3）CML inhibits the expression of PPARγ in Hep G2 cells by up-regulating the receptor RAGE.（4）Compared with CML group,stimulated PPARγ expression down-regulate the expression levels of pro-inflammatory cytokines IL-1β,IL-6,TNF-α and CRP protein of Hep G2 cells by inhibiting CML / RAGE signaling.（5）Compared with the CML group,the expression of inflammatory cytokines IL-1β,IL-6,TNF-α,CRP protein of endothelial cells was significantly reduced after stimulating PPARγ expression.（6）Compared with CML group,the expression of NOX2 in endothelial cells was significantly reduced after stimulating PPARγ expression.Conclusion Excited PPARγ expression inhibits the progression of non-alcoholic fatty liver and atherosclerosis mediated by CML / RAGE signaling.