Study On The Regulatory Mechanism Of Sodium Butyrate On Steatosis Of Primary Chicken Hepatocytes Through AMPK/PPARα Pathway

The study aims to illustrate the protective effect and mechanism of sodium butyrate(NaB)on free fatty acid-induced steatosis in primary chicken hepatocytes.In this experiment,12-day-old chicken embryos were selected,and chicken embryo hepatocytes were isolated by collagenase digestion and cultured in serum-free medium.The chick hepatocyte steatosis model was established by adding sodium oleate/sodium palmitate(2:1)at a total concentration of 1 mM,and the model was treated with NaB at concentrations of 0.25mM,0.5mM and ImM.The cells were divided into 6 groups:Control group(0mM sodium oleate/sodium palmitate),FFA group(1mM sodium oleate/sodium palmitate),FFA+LNaB group(1mM sodium oleate/sodium palmitate+0.25mM NaB),FFA+MNaB group(1mM sodium oleate/sodium palmitate+0.5mM NaB),FFA+HNaB group(1mM sodium oleate/sodium palmitate+1mM NaB)and Control+HNaB group(0mM sodium oleate/sodium palmitate+1mM NaB).After 24 hours of cell treatment,hepatocytes were identified by cytokeratin CK8 and CK18 immunofluorescence detection and glycogen staining,the effect of different concentrations of NaB on cell viability was detected by CCK8 method,the effects of NaB on hepatic lipid metabolism-related indexes(TG,T-CHO,HDLC,LDL-C),oil red O staining,the mRNA transcriptional level and protein expression of lipid metabolism-related genes in chicken hepatocyte steatosis model were detected,and the AMPK inhibitor Compound C(15μM)and the PPARα inhibitor GW6471(4μM)were used to analyze the role of AMPK and PPARα in alleviating chicken hepatocyte steatosis by NaB,respectively.The specific results are as follows:1.The primary chicken hepatocytes were treated with different concentrations of NaB,It was found that the cell viability extremely significantly decreased(P<0.01)at the concentration of NaB in 1.5mM,so 0.25mM,0.5mM and 1mM NaB were selected as the follow-up experimental concentrations.2.Compared with Control group,hepatocytes in FFA group showed a large amount of red lipid droplets,and the contents of TG,T-CHO and LDL-C were extremely significantly increased(P<0.01).Compared with FFA group,the lipid droplets,TG and LDL-C of hepatocytes in FFA+LNaB group,FFA+MNaB group and FFA+HNaB group were extremely significantly decreased(P<0.01),but there was no significant difference(P>0.05)in T-CHO and HDL-C content.3.Compared with Control group,the mRNA and protein of AMPKα1,PPARα,CPT1 and the protein of p-AMPKα1 in FFA group were extremely significantly decreased(P<0.01),while the mRNA of SREBF1,ACC,LXRα,HMGR and the protein of SREBF1 were extremely significantly increased(P<0.01),the mRNA and protein of FASN were significantly(P<0.05)and extremely significantly increased(P<0.01).Compared with FFA group,the mRNA and protein of AMPKα1,PPARα,CPT1 and the protein of p-AMPKα1 in hepatocytes of the FFA+LNaB group,FFA+MNaB group and FFA+HNaB group were all increased,especially the mRNA of AMPKα1,and the above proteins in FFA+MNaB group and FFA+HNaB group were increased most significantly(P<0.01),while the mRNA of SREBF1,ACC,FASN,SCD1,LXRα and the proteins of SREBF1,FASN were decreased,especially the mRNA of ACC,FASN,LXRα and the proteins of FASN were decreased most signifi cantly(P<0.01).4.After using Compound C and GW6471 inhibitors,the lipid droplets and TG content of hepatocytes in FFA+MNaB+Compound C group and FFA+MNaB+GW6471 group were extremely significantly increased(P<0.01)compared with FFA+MNaB group,At the same time,the effects of NaB on promoting fatty acid oxidation and inhibiting lipid synthesis were weakened.Conclusion:NaB can regulate AMPK/PPARα pathway to promote fatty acid oxidation and inhibit lipid synthesis,thus alleviating steatosis of chicken primary hepatocytes induced by free fatty acids.