The Mechanism Of CREBH Regulating Mitochondrial Oxidative Stress Of Hepatocytes In Non-alcoholic Steatohepatitis

Objective:We constructed the cell and mouse models of non-alcoholic steatohepatitis(NASH)to investigate the mitochondrial oxidative stress of hepatocytes and the change of cyclic AMPresponsive element binding protein H(CREBH)level in NASH,and to reveal the effect of regulating CREBH expression on mitochondrial oxidative stress of liver cells in NASH and the enrolled molecular mechanism.Methods:1.We used palmitic acid(PA)to stimulate AML12 hepatocytes to induce the model of hepatocyte lipid overload.The superoxide levels and change of membrane potential of mitochondrial in hepatocytes were measured.The expression of CREBH,sirtuin 3(Sirtuin 3,SIRT3)and NLR family pyrin domain containing 3(Nlrp3)inflammatory pathway,the total protein lysine acetylation level of hepatocytes and the molecular activity of manganese superoxide dismutase(MnSOD)was determined.2.We constructed the AML12 cell lines stably overexpressing CREBH and applied PA to induce the model of hepatocyte lipid overload.The superoxide levels and change of membrane potential of mitochondrial in hepatocytes were evaluated.Levels of SIRT3 and Nlrp3 inflammatory pathway were calculated.The expression,lysine acetylation and activity of MnSOD and the total protein lysine acetylation level of hepatocytes were detected.3.We constructed CREBH-OE-siSIRT3 AML12 cell lines and used PA to induce the model of hepatocyte lipid overload.Content of mitochondria superoxide levels and level of the mitochondrial membrane potential were measured.Levels of SIRT3 and Nlrp3 inflammatory pathway were calculated.The expression,lysine acetylation and activity of MnSOD and the total protein lysine acetylation level of hepatocytes were detected.4.Systemic CREBH knockout mice were constructed,and treated with high-fat(HF)diet and a methionine-choline-deficient(MCD)diet to simulate NASH in animal models.The levels of CREBH,SIRT3,antioxidant enzymes and Nlrp3 inflammatory signaling pathway were detected.Results:1.In the model of hepatocyte lipid overload,the level of mitochondrial superoxides significantly increased,the activity of MnSOD decreased,the mitochondrial membrane potential decreased,and the Nlrp3 inflammasome was activated;mitochondrial oxidative stress and CREBH increased depending on the dose of PA.SIRT3 had a significant increasing after stimulated by 400μM PA for 24h.Total protein lysine acetylation degree in hepatocytes was upregulated depending on the dose of PA.2.In the model of hepatocyte lipid overload,CREBH overexpression alleviated PA-induced mitochondrial oxidative stress of hepatocytes and upregulated the expression of SIRT3.CREBH overexpression antagonized the PA-induced MnSOD decreasing and downregulated the lysine acetylation level of MnSOD and the total protein lysine acetylation level of hepatocytes.After silencing the expression of SIRT3,the effect of CREBH protecting the hepatocyte mitochondria was weakened.3.In NASH mouse models fed with HF and MCD diet,the expression of CREBH,SIRT3,and MnSOD decreased;after CREBH gene was knockout,the expression of SIRT3 and MnSOD decreased,the lysine acetylation level of MnSOD increased,and the Nlrp3 inflammasome was activated.Increased mitochondrial oxidative stress was observed in hepatocytes.Conclusions:During the occurrence and development of NASH,mitochondrial oxidative stress of hepatocytes is induced by lipid overload,resulting in changes in the expression of CREBH and SIRT3.CREBH/SIRT3 signaling pathway regulates the acetylation of MnSOD and affects its activity,thereby reducing mitochondrial oxidative stress of hepatocytes and preventing inflammatory response and even the formation of fibrosis in hepatocytes.