Effect Of TNF-α On The SCAP-SREBP-1c Pathway Of Lipid Synthesis In A Model Of Hepatocyte Steatosis

Objective:To study the effect of TNF-α(Tumor Necrosis Factor-α) on the expression of Sterol Regulatory Element Binding Protein(SREBP) Cleavage-Activating Protein (SCAP) and SREBP-1c in a model of hepatocyte steatosis,so as to explore the role and possible mechanism by which TNF-αacts on the SCAP-SREBP-1c pathway of lipid synthesis in nonalcoholic fatty liver disease(NAFLD).Methods1.A model of oleic acid-induced steatosis of human hepatocytes was established.2.L-02 strain hepatocytes(normal adult human hepatocytes) underwent subcultures with 1640 medium containing 10%fetal bovine serum and the cultures were divided into the control group(Group C,cultured with the normal medium) and the model group(Group F,cultured with the normal medium + oleic acid).In addition,the two groups were added with TNF-α,Namely,C1(group C added with TNF-α),F1(group F added with TNF-α).The final concentration of TNF-αwas 200ng/mL.3.The accumulation of lipid droplets in hepatocytes was observed by light microscopy after oil red-0 staining.4.The change of the expression of SCAP,SREBP-1c,FAS(Fatty Acid synthetase) mRNAs was detected by RT-PCR.5.The change of the expression of SCAP and SREBP-1c proteins was detected by Western blotting.6.The change of the FAS activity in hepatocytes was detected by substrate-binding reaction.7.The content of triglyceride(TG) in hepatocytes was also determined.Results:1.The model of oleic acid-induced steatosis of L-02 strain hepatocytes was established successfully.No red lipid droplet was observed in hepatocytes in the control group by optical microscopy after oil red-0 staining.A small number of steatotic hepatocytes were found in the model group only 24 h after culture and many orange-red lipid droplets were found in the cytoplasm 72h after culture.Lipid droplets may be occasionally observed by electron microscopy in the cytoplasm in the control group.In the model group,the size of hepatocytes was relatively large,with many lipid droplets in the cytoplasm,and the nuclear membrane was tortuous,with an obscure contour;the number of mitochondria was reduced. with swollen matrix and sparse crista.2.In group C,no steatosis of hepatocytes was observed by optical microscopy after oil red-0 staining;hepatocytes distributed densely,with clear contour and nuclei.A small number of steatotic hepatocytes were observed in group C1.However,a great number of steatotic hepatocytes were observed in group F,particularly,in group F1.In group F1,the number of lipid droplets in hepatocytes was increased significantly,with some lipid droplets fused into larger ones that pushed nuclei to one side of the cell.3.Comparison of the expression of SCAP,SREBP-1c,and FAS mRNAs in hepatocytes between groups:The expression was upregulated in groups C1,F,and F1 as compared with that in the control group(P＜0.01),with the most significant upregulation seen in group F1.4.The expression of SCAP and SREBP-1c proteins and that of their mRNAs changed in the same way.5.Comparison of the activity of FAS:The activity of FAS was enhanced in groups C1, F,and F1 as compared with that in group C(P＜0.01),with the most significant enhancement seen in group F1.There were significant differences in the activity of FAS between group C1 and group C as well as between group F1 and group F(P＜0.01).6.Comparison of the content of TG in hepatocytes:The content of TG was increased in groups C1,F,and F1 as compared with that in group C(P＜0.01),with the highest TG content seen in group F1.There were significant differences between group C1 and group C as well as between group F1 and group F(P＜0.01),indicating that TNF-αacts on group C, particularly,on group F.Conclusions:1.A model of steatosis of human hepatocytes can be established successfully with L-02 strain of hepatocyte. 2.TNF-αmay upregulate the expression of SCAR SREBP-1c,and FAS in normal and steatotic L-02 strain hepatocytes to promote the synthesis and accumulation of TG in hepatocytes.3.When acting on normal hepatocytes,TNF-αcauses steatosis only in a small number of hepatocytes;however,when acting on hepatocytes of the model group,it causes steatosis in more hepatocytes and higher TG contents.TNF-αmay promote the expression of SCAP and SREBP-1c in steatotic hepatocytes;therefore,TNF-αis an important factor worsening the disorder of lipid metabolism in steatotic hepatocytes.