Regulation Mechanism Of PPAR? Signaling Pathway In Steatosis Hepatocytes Of Hens By Resveratrol And Establishment Of PPAR? Lentiviral Over-Expression Vector

To investigate the regulation mechanism of resveratrol(Res)on PPAR signaling pathway in steatosis hepatocytes of laying hens.Primary hepatocytes of laying hens with high activity were obtained by modified via situ two-step perfusion method.According to the in vitro model of human non-alcoholic fatty liver,the hepatocytes were treated with mixed fatty acids(oleic acid: palmitic acid 2:1)to establish the steatosis model.The experiment was divided into four groups: Control group(normal cultured primary hepatocytes),FA group(Control group + 1.0mm fatty acid),FA+Res group(FA group +80?M Res),Con+Res group(Control group +80 ?M Res).Then,the cell activity,antioxidant indexes(SOD,MDA),lipid-related indexes(TG,TC,HDL-ch,LDL-ch),apoptotic genes(Bcl-2,BAX,Caspase-3)and PPAR signaling pathway-related genes(ADFP,FABP1,FABP3,PPAR,Srebp-1c,Apo-A?,Apo-B100,SCD-1)mRNA expression levels were measured.In addition,lentiviral over-expression vectors were constructed to overexpress the PPAR gene in liver cells.The results were as follows:(1)The results of cellular activity and lipid-related indicators indicated that 1.0 mM fatty acid was the best modeling condition at 48 h.Compared with the control group,TG content increased significantly when 0.5-1.2 mM fatty acid was added.(2)Compared with the Control group,SOD activity was significantly decreased and MDA concentrations were significantly increased in the FA group(P<0.05)whereas the FA+Res group was significantly improved.Compared with the Control group,TG and TC concentrations were highly significantly increased in the FA group(P<0.01).However,Compared with the FA group,TG and TC contents were highly significantly decreased in the FA+Res group(P<0.01).(3)Compared with the Control group,the expression levels of BAX and caspase-3mRNA were significantly increased while the Bcl-2 was significantly decreased in the FA group(P<0.05).However,compared with the FA group,the expression levels of caspase-3mRNA were significantly decreased in the FA+Res group(P<0.05).(4)Compared with the Control group,the expression levels of ADFP ? FABP1 ?FABP3?Srebp-1c?Apo-A1 mRNA were highly significantly increased(P<0.01)while the PPAR,Apo-B100 and SCD-1 were significantly decreased in the FA group(P<0.05 or P<0.01).However,compared with the FA group,the expression levels of Srebp-1c,FABP1,FABP3 and ADFP mRNA were significantly decreased in FA+Res group(P<0.01).(5)Lentivirus overexpression of PPAR ? transfection results: no fluorescence was observed when the MOI value was 125 or below,while green fluorescence was observed when the MOI value reached 150 or above.However,the transfection efficiency ofprimary hepatocytes was very low and did not reach the expected value.When the MOI reached 5,the transfection efficiency reached more than 80%.Conclusion: In this study,a model of primary hepatocyte steatosis in laying hens was successfully established,which was induced via 1 mM fatty acid(palmitic acid: oleic acid=2:1).Res can improve lipid deposition level by regulating the expression of PPAR ?signaling pathway related genes in laying hens steatosis hepatocytes.