Metformin Protects Against Nonalcoholic Fatty Liver Disease By Inhibiting Ferroptosis

Background:With the increasing incidence of non-alcoholic fatty liver disease（NAFLD）year by year globally,NAFLD has become a major priority of global public health.The etiology of NAFLD and its mechanism are complicated.Recent research indicates that ferroptosis is closely related to the occurrence and development of NAFLD.Metformin is the first-line pharmacologic treatment for type 2 diabetes and it has been reported to improve NAFLD greatly.However,the cellular mechanism of metformin protective role on hepatocyte remains unclear.Evidence suggests that adenosine 5’-monophosphate-activated protein kinase（AMPK）is closely related to ferroptosis.Metformin is an AMPK activator and alleviates hepatic steatosis,inflammation,and fibrosis by reducing lipid peroxidation and reactive oxygen species（ROS）accumulation in NAFLD.We speculate that metformin attenuates NAFLD through anti-ferroptotic effects.Thus,our study aims to explore the potential mechanism of metformin in improvement of NAFLD,and the inhibitive effects of metformin on ferroptosis.Objective:1.The molecular mechanism of metformin in improvements of NAFLD based on ferroptosis in vivo and in vitro experiments;2.The molecular mechanism of metformin on ferroptosis in NAFLD based on iron overload.Methods:1.To establish a NAFLD model with metformin intervention in vivo experiment.32 male Sprague-Dawley rats were randomly divided into two groups,rats on a normal diet（ND,n=8）,or on a high fat diet（HFD,n=24）for 8 weeks.At the beginning of 9th week,HFD rats were randomly divided into three groups:HFD group,low-dose metformin treatment group（HFD+Metformin 150）,high-dose metformin treatment group（HFD+Metformin 300）（n=8 each）.At the ending of 16th week,all the rats were sacrificed.Body weight,serum liver function,inflammatory factors,liver histopathology,liver fibrosis,serum iron and ferritin,hepatic iron content,liver malondialdehyde（MDA）,glutathione（GSH）,total superoxide dismutase（T-SOD）,the relative expression of ferroptosis-associated biomarkers,such as nicotinamide adenine dinucleotide phosphate oxidase 1（NOX1）,prostaglandin-endoperoxide synthase 2（PTGS2）,glutathione peroxidase 4（GPX4）mRNA and hepatic GPX4 protein expression were compared among groups.2.In vitro experiment,WRL68 cells were randomly divided into three groups:normal control group,palmitic acid（PA）intervention group,palmitic acid+metformin intervention groups with different concentrations（PA+Metformin 0.5mM,1.0mM,2.5mM）.After intervention for 24h,cell viability,the concentrations of lipid peroxidation and ROS in WRL68 cells were compared among groups.3.Cell viability,the concentrations of lipid peroxidation,ROS,iron content and GPX4 protein expression of WRL68 cells were observed in erastin-induced ferroptosis model with metformin intervention.After metformin intervention for 24h,the concentrations of lipid peroxidation,ROS,and iron content were compared in PA+erastin,and PA+FAC induced-WRL68 cells.4.After metformin intervention for 24h,the relative expression of hepatic ferroportin（FPN）protein in NAFLD was assessed by western blotting.AMPK agonist AICAR,and AMPK antagonist compound C were incubated with PA in WRL68 cells.The relative expression of FPN protein was also determined by western blotting.WRL68 cells were transfected with FPN si RNAs and overexpression GFP/FPN.The concentrations of total iron,Fe²⁺,and Fe³⁺were compared among groups.After adding chloroquine and proteasome inhibitor-132,the relative expression of FPN protein in WRL68 cells was determined by western blotting.The ubiquitin level of FPN protein in each group was determined by co-immunoprecipitation.Result:1.Body weight,the concentrations of serum alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）,hepatic steatosis,fibrosis,and inflammation were significantly increased in HFD group than ND group（P<0.05）.Compared with HFD group,body weight,serum liver function,hepatic histopathology,fibrosis,and inflammatory factors were significantly reduced in HFD+Metformin 150,HFD+Metformin 300groups.2.Compared with ND group,serum ferritin,hepatic iron content and MDA were significantly higher,hepatic GSH,T-SOD content were significantly reduced（P<0.05）.Hepatic GPX4 mRNA expression was decreased in HFD group compared with ND group（P=0.06）.Compared with HFD group,serum ferritin,hepatic iron content,MDA,PTGS2,NOX1 mRNA expression were significantly reduced,and hepatic GSH,GPX4 protein and mRNA expression were significantly increased in HFD+Metformin 300 group.3.Compared with control group,cell viability,intracellular GSH,T-SOD content,GPX4 protein expression were significantly reduced.Intracellular lipid drop,MDA,iron content,and ROS were increased in PA group（P<0.05）.Metformin significantly increased cell viability,reduced the concentrations of intracellular lipid drop,MDA,iron content,ROS,and upregulated GPX4 protein expression in PA-induced WRL68cells.4.Compared with control group,intracellular MDA,ROS levels were significantly higher,intracellular GSH,T-SOD levels were significantly reduced in PA+erastin,erastin,and PA+FAC-induced WRL68 cells.Metformin not only significantly reduced the concentrations of intracellular lipid peroxidation,ROS,in PA+erastin,erastin,and PA+FAC induced WRL68 cells,but also decreased intracellular iron content in erastin,and PA+FAC induced WRL68 cells.5.Si FPN reversed the amelioration of iron overload in NAFLD by metformin.Overexpression GFP/FPN reduced intracellular total iron,Fe²⁺,and Fe³⁺in PA+FAC induced WRL68 cells.6.Metformin and AICAR decreased the lysosomal degradation of FPN protein in PA induced WRL68 cells.Conclusion:Metformin alleviates hepatic iron overload by upregulating FPN protein through AMPK pathway,inhibits ferroptosis and exerts a protective effect on HFD-induced NAFLD.