The Role And Mechanism Of Targeting FoxO1 To Regulate Endoplasmic Reticulum Stress And Necroptosis In Relieving Nonalcoholic Fatty Liver Disease

Background: Nonalcoholic fatty liver disease(NAFLD)is a high risk factor for liver cirrhosis and liver cancer,but its pathogenesis is unknown,so treatment options are limited.In recent years,it has been found that intrahepatic inflammatory response and cellular metabolism play an important role in NAFLD.Forkhead Box O1(FoxO1)is thought to play an important role in regulating glucose metabolism and lipid metabolism.The progression of NAFLD is accompanied by endoplasmic reticulum stress and necroptosis.However,the relationship between FoxO1 in regulating endoplasmic reticulum stress and necroptosis in nonalcoholic fatty liver disease has not been fully elucidated.This study was to investigate the role of FoxO1 in a mouse model of nonalcoholic fatty liver disease and to observe the effects of FoxO1 inhibitor AS1842856,and to investigate the role of FoxO1 in regulating endoplasmic reticulum stress and necroptosis in vitro,providing new ideas for the treatment of nonalcoholic fatty liver disease.Objective: To investigate the role and mechanism of FoxO1 in nonalcoholic fatty liver disease Methods: FoxO1 participates in the development of nonalcoholic fatty liver disease 1.In vivo experiment: the NAFLD mouse model was constructed,and the success of the NAFLD model was identified by the detection of serum triglycerides,serum cholesterol,oil red staining and HE staining.Western Blot was used to detect the expression levels of FoxO1 and p-foxo1 in the liver of normal and NAFLD mice.2.In vitro experiment: the NAFLD cell model was constructed: human hepatocellular carcinoma cell line HepG2 was selected as the research object,and palmitic acid treatment medium with different concentrations was used to cultivate the cells at the optimal concentration for 24 h.The expression levels of FoxO1 and p-foxo1 in cells were detected by Western Blot.Role of er stress and necroptosis in the development of NAFLD 1.In vivo experiment: a NAFLD mouse model was constructed and Western Blot was used to detect the expression levels of endoplasmic reticulum stress-related proteins(PERK,BIP,CHOP)and necroptosis-related proteins(RIP1,RIP3,p-mlkl)in the liver.2.In vitro experiments: the NAFLD cell model was constructed and the expression levels of endoplasmic reticulum stress-related proteins(PERK,BIP,CHOP)and necroptosis-related proteins(RIP1,RIP3,p-MLKL)were detected by Western Blot.Study on the mechanism of FoxO1 regulating endoplasmic reticulum stress and necroptosis affecting the development of NAFLD 1.In vivo experiment: C57BL/6 mice were divided into three groups: control group,high-fat diet group,high-fat diet +FoxO1 inhibitor group.After 16 weeks,the expression levels of endoplasmic reticulum stress related proteins(PERK,BIP,CHOP)and necroptosis-related proteins(RIP1,RIP3,p-MLKL)in mouse liver were detected by Western Blot.The shape of endoplasmic reticulum in mouse liver was observed by electron microscopy.2.In vitro experiments: NAFLD cell model was constructed: HepG2 cells were pretreated with FoxO1 inhibitor AS1842856 at a concentration of 1.0umol/L for 2h,and then cultured with 0.75mmol/L of palm acid for 24 h.Western Blot was used to detect total FoxO1 and p-foxo1 proteins,endoplasmic reticulum stress related proteins(PERK,BIP,CHOP),necroptosis-related proteins(RIP1,RIP3,p-mlkl)in HepG2 cells.The expressions of CDKN1B/P27KIP1 mRNA,PMAIP1/NOXA mRNA,BCL2L11/BIM mRNA and TNFSF10/TRAIL mRNA were detected by qrt-pcr in HepG2 cells.Results: 1.High-fat diet could make mice gain weight,increase ALT,AST,TC,TG,liver steatosis,and activate FoxO1 and decrease FoxO1 phosphorylation.With the increase of palmitate concentration,FoxO1 expression increased in HepG2 cells and pfoxo1 expression decreased,suggesting that FoxO1 is involved in the development of NAFLD.2.High-fat diet can activate endoplasmic reticulum stress and necroptosis in mouse liver,and the expression of related proteins is increased.With the increase of palmitate concentration,the expression of endoplasmic reticulum stress and necroptosis-related proteins in HepG2 cells also increased,demonstrating that endoplasmic reticulum stress and necroptosis are involved in the development process of NAFLD.3.Inhibition of FoxO1 can reduce the expression of proteins related to endoplasmic reticulum stress and necroptosis in mouse hepatocytes,and the results of electron microscopy showed that the injury of endoplasmic reticulum in the inhibitor group was reduced.FoxO1 inhibitor can reduce the expression of endoplasmic reticulum stress and necroptosis related proteins in HepG2 cells,and reduce the expression of FoxO1 downstream TRAIL mRNA,which proves that FoxO1 can regulate endoplasmic reticulum stress and necroptosis to affect the development of NAFLD,and this process may be mediated by FoxO1 downstream target gene TRAIL.Conclusion: in the NAFLD mouse model,inhibition of FoxO1 can alleviate er stress and necroptosis,thereby alleviating the progression of NAFLD,which may be a target for the treatment of NAFLD.