Haem Oxygenase 1 Mediated Ferroptosis Promotes Non-alcoholic Steatohepatitis

Objective:With the increasing incidence of obesity and diabetes,non-alcoholic fatty liver disease(NAFLD)has become the most common chronic liver disease worldwide,and about 25%of NAFLD patients are prone to progress to nonalcoholic steatohepatitis(NASH).The pathological features of NASH include steatosis,hepatocellular damage,and varying degrees of fibrosis.Among them,hepatocyte death and inflammation are important factors that cause NASH to develop into endpoint liver diseases such as cirrhosis and liver cancer.As a regulatory mode of cell death closely related to hepatic iron deposition and oxidative stress,ferroptosis is one of the main ways of hepatocyte death in NASH,which can aggravate liver inflammation and promote liver fibrosis.However,which molecules affect the occurrence and development of NASH by regulating ferroptosis have not been fully elucidated.Heme oxygenase-1(HO-1)is an important molecule that regulates oxidative damage and is also a rate-limiting enzyme for the liver to break down heme.The regulation of HO-1 in ferroptosis is controversial by its antiferroptotic or pro-ferroptotic role in different tissues and diseases.Therefore,the association between HO-1 and ferroptosis in nonalcoholic steatohepatitis was determined in this research.Methods:1.Establishment of NASH model in vivo and analysis of the correlation between ferroptosis and HO-1.Mouse model of NASH was established by feeding C57BL/6 mice on a methionine-choline deficiency(MCD)diet for 8 weeks.Pathological sections of mouse liver were prepared,and HE staining and oil red O staining were performed.The degree of liver fat deposition and inflammation was observed under the microscope.Biochemical indices were determined,including serum alanine aminotransferase(ALT),serum aspartate aminotransferase(AST)and liver triglyceride(AG).The mRNA relative expression levels of TNFα,IL-1β and IL-6 in liver were detected by RT-qPCR.Malondialdehyde(MDA),a biochemical marker of ferroptosis,was detected by a test kit.mRNA and protein relative expression levels of HO-1 and molecular markers of ferroptosis were detected by RT-qPCR and Western blot.Molecular markers of ferroptosis include solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),transferrin receptor 1(TFR1),ferritin light chain(FTL),ferritin heavy chain 1(FTH1),ferroportin(FPN),acyl-CoA synthetase long chain family 4(ACSL4).2.Establishment of NASH model in vitro and analysis of the correlation between ferroptosis and HO-1.Human hepatoma cell line HepG2 cells were treated with palmitic acid(PA)and arachidonic acid(AA)for 24h in vitro to construct cell model.Ferroptosis inhibitors ferrostatin-1(Fer-1)and deferiprone(DFP)were added to the model group for 24h,respectively.Cell viability was detected by CCK8 method.AG and MDA were detected using kits.The relative protein expression levels of HO-1 and the molecular marker of ferroptosis were detected by Western blot.3.Investigating the effect of HO-1 on ferroptosis in HepG2 cells.Model group was treated with HO-1 inhibitor zinc protoporphyrin(ZnPP)for 24h.The ZnPP concentration gradient was set,and the survival rate of ZnPP treated cells after 24h was detected by CCK8 method.FerroOrange fluorescence probe was used to detect the change of intracellular free iron bivalent.Western blot was used to detect iron metabolism-related proteins.Results:1.Association between ferroptosis and HO-1 in NASH mouse models(1)Establishment of MCD diet-induced NASH mouse model.Compared with the control group,ALT and AST levels in serum,AG content in liver tissue,and mRNA levels of liver inflammatory cytokines TNFα,IL-1β and IL-6 were increased in model group.HE staining and oil red O staining showed hepatic lipid deposition and inflammatory cell infiltration.(2)Changes of ferroptosis markers in liver tissue of NASH mice.MDA content in liver homogenate of mice in model group was significantly higher than that in control group.The results of RT-qPCR showed that the mRNA relative expressions of SLC7A11,GPX4,TFR1,FTL,FTH1,FPN and ACSL4 in the liver tissues of mice in the model group were up-regulated.Western blot results showed that,compared with the control group,the relative expression levels of SLC7A11 protein in liver tissue of model group were decreased,while the relative expression levels of TFR1,FTL,FTH1,FPN and ACSL4 protein were increased.(3)Changes of HO-1 expression in liver tissue of NASH mice.Compared with the control group,mRNA and protein relative expression levels of HO-1 in the model group were increased.2.Association between ferroptosis and HO-1 in NASH cell model.(1)Establishment of fatty acid induced NASH model in HepG2 cells.Compared with the control group,the AG content in the model group was significantly increased,while the cell survival rate was significantly decreased.(2)Changes of ferroptosis markers in HepG2 cells.Compared with the control group,the cell survival rate of the model group was decreased,while the levels of AG and MDA were increased;Compared with model group,cell survival rate in Fer-1 and DFP treatment groups increased,while AG and MDA levels decreased.Western blot results showed that the protein expressions of GPX4,FTL,FTH1 and FPN in the model group were up-regulated,while the protein expressions of SLC7A11,TFR1 and ACSL4 were down-regulated.(3)Changes of HO-1 expression in HepG2 cells.The protein expression level of HO-1 in the model group was up-regulated.3.Effect of HO-1 on ferroptosis in HepG2 cells and relevant mechanism.(1)Effect of HO-1 on ferroptosis in HepG2 cells.Compared with the model group,the cell survival rate in ZnPP treated group was increased,while the levels of MDA and AG were decreased.(2)Effect of HO-1 on free iron bivalent in HepG2 cells.Compared with the control group,the fluorescence intensity of free ferric bivalent in the cytoplasm of the model group was stronger;The fluorescence intensity of ZnPP treatment group was weaker than that of model group.Western blot showed that the protein expressions of TFR1 and FPN in ZnPP treatment group were increased,while the protein expressions of FTL and FTH1 were decreased.Conclusions:1.Ferroptosis is correlated with HO-1 and HO-1 expression is up-regulated in liver tissues and cells of NASH with ferroptosis.2.HO-1 promotes ferroptosis by increasing the content of free iron bivalent in cells.