The Expression Of CD36 And Palmitoylation In High-fat Induced Hepatocyte Apoptosis

Background and Objective:With lifestyle and dietary changing,non-alcoholic fatty liver disease(NAFLD)as the part of metabolic syndrome has been the most common liver disease involving a third of the world’s population.NAFLD covers a wide spectrum of liver injury ranging from simple steatosis to nonalcoholic-steatohepatitis(NASH),cirrhosis and fibrosis.Studies showed that Non-alcoholic steatohepatitis(NASH),defined as the progressive liver of NAFLD,is an important cause of fibrosis and hepatoma.Therefore,it is important to early prevent and treat NASH for preventing cirrhosis and hepatoma.However,there is no effective control method for NASH.Previous extensive scientific research and clinical data show that hyperlipidemia is one of the important pathogenic factors of NASH,a large portion of elevated circulating free fatty acids(FFAs)in NASH patients and is highly associated with severity and progressing of disease.Furthermore,more obvious hepatocyte apoptosis was observed in NASH patients and mice NASH models and hepatocyte apoptosis is one of the important morphological and pathological hallmarker of NASH.AS a result,the study of high-fat induced hepatocyte apoptosis may be a potential target for treatment of NASH.Fatty acid translocase(FAT/CD36)is widely located on a variety of cell,such as mononuclear macrophages,adipose cells and hepatocytes.As one member of the scavenger receptor family,CD36 can bind with various ligands,such as long chain fatty acid(LCFA)and oxidized low density lipoprotein(oxLDL)and so on.CD36 expression in normal liver was weak,while CD36 expression was significantly increased in liver tissues of NAFLD patients.Studies showed that CD36 was associated with the LCFA uptake and lipid accumulation of liver.However,it is not clear whether CD36 is involved in the hepatocyte apoptosis in NAFLD.Therefore,this study intends to explore the relationship and mechanism between CD36 and hepatocyte apoptosis in NASH.Methods: In the first part,in vivo experimentation,we established a NASH model by feeding male C57BL/6J mice high fat diet(HFD)for 22 weeks and the mice were randomly divided into two groups: the normal chow diet(NCD)group or high fat diet(HFD)group.In vitro experimentation,HepG2 cells were divided into two treatment groups: control group(0.2% BSA containing DMEM medium)and palmitate treatment group(0.2% BSA+ PA).The mice serum was collected and detected the activity of transaminase.Hepatic steatosis was observed by HE staining;The expression of CD36,bcl-2 and bax were determined by Western blot.The level of apoptosis was detected by TUNEL stain,caspase-3 activity detection and Annexin-V/PI flow cytometry.The second part,we gave the male C57BL/6J mice/HepG2 cells lentivirus via tail injection to establish a overexpression wild-type CD36(CD36OE)mice or cell model,and the control group was injected NC lentivirus(NC).The two mice group were fed to HFD for eight weeks and the cells were treated with PA for 24 before experiments.Furthermore,we also gave siRNA interference on HepG2 cells,the apoptosis grade was measured by flow cytometry.In the third part,we established CD36 palmitoylation site mutant by lentivirus(AA-SS)in HepG2 cells.The level of CD36 palmitoylation were identified by ABE;the expression of apoptosis-related proteins were detected by Western Blot;the activity of caspase-3 were examined by kit;the level of bcl-2 promoter acetylation were detected by ChIP assay.Results: In the first part: in the HE staining,compared with NCD group,there are a large number of inflammatory cell infiltration in HFD group(P<0.05).The level of liver function transaminase enhanced suggested liver function injury.Western blot results show that the expression of CD36 was increased in HFD or PA treated group.TUNEL positive cells and caspase-3 activity were increased in HFD or PA group(P<0.05).The expression of bax were increased in total and mitochondria.The expression of bcl-2 were reduced and the phosphorylation level of bcl-2 were increased.The above results indicated that high fat promoted apoptosis of liver cells and increased CD36 expression.In the second part: compared to the NC group,the employment of lentivirus(CD36OE)were more TUNEL positive cells and higher caspase-3 activity(P<0.05).Furthermore,siCD36 can reduce the apoptosis cells(P<0.05).In the third part: in the cell experiment,PA treatment can promote CD36 palmitoylation of HepG2 cells;CD36 palmitoylation can significantly promote the HepG2 apoptosis,inhibit the expression of bcl-2 protein and phosphorylation level,and promote the expression of bax in mitochondria(P<0.05).The results of chromatin immunoprecipitation showed that inhibition of palmitoylation could significantly promote the acetylation of bcl-2 promoter,thus promoting bcl-2 transcription,which reduced hepatocyte apoptosis.Conclusion:1.High fat diet triggered hepatocyte lipid accumulation and increased CD36 expression and promoted hepatocyte apoptosis.2.CD36 overexpression can promote hepatocyte apoptosis;CD36 interference can reduce liver apoptosis.3.Inhibition of CD36 palmitoylation may reduce apoptosis by promoting bcl-2 phosphorylation and promoter acetylation resulted in the promotion of bcl-2 transcription,thereby relieving hepatocyte apoptosis.