Autophagy Regulates FFA-induced Ferroptosis In Hepatocytes

Objective:The L02 hepatocyte steatosis model was constructed with mixed free fatty acids(Free fatty acids,FFA)to investigate whether autophagy could regulate ferroptosis in steatotic hepatocytes.Methods:1.Normal human hepatocyte line L02 cells were treated with a time gradient of 1mmol/L FFA(Oleic acid:Palmitic acid=2:1)for 24,48 and 72 h.The cells were divided into four groups: control(Control)group,model(FFA)group for 24 h,model(FFA)group for 48 h and model(FFA)group for 72 h.The Control group was treated with normal culture medium culture;model 24,48 and 72 h groups were intervened with 1mmol/L mixed FFA solution(oleic acid:palmitic acid=2:1)for 24 h,48h and 72 h respectively.2.Oil red staining to detect lipid deposition in L02 cells.3.GPO-PAP method for the determination of triglyceride(TG)and cholesterol(TC)in hepatocytes.4.Microplate method for the determination of glutamic oxaloacetic transaminase(AST)and glutamic alanine transaminase(ALT)in hepatocytes.5.Real-Time PCR was performed to detect the levels of ferroptosis related genes SLC7A11,GPX4,FTH1,TFR1 and autophagy related genes LC3-II and ATG7 genes.6.L02 cells were treated with 50umol/L chloroquine(CQ)and divided into three groups: control(Control)group,model(FFA)group and chloroquine(CQ)group.Control group was incubated with normal culture medium;FFA group was intervened with 1mmol/L mixed FFA solution(oleic acid:palmitic acid=2:1)for 24 h;CQ group was treated with FFA group based on The CQ group was post-treated with 50umol/L chloroquine for4 h.7.Real-Time PCR was used to detect the levels of ferroptosis-related genes SLC7A11,GPX4,FTH1,TFR1 and autophagy-related genes LC3-II,ATG7 and NCOA4 genes.8.Western blot was used to detect LC3B、NCOA4 and、FTH1 和 SLC7A11 protein levels.Results:1.Oil red staining showed that FFA-treated cells had red lipid droplet aggregates and that the degree of cellular lipid deposition increased in a time-dependent manner.2.The TG and TC contents could reflect the degree of lipid accumulation in hepatocytes.The TG(P<0.01)and TC(P<0.0001)contents in the model 24 h group,model48 h group and model 72 h group increased significantly with time dependence compared to the Control group.3.The release of two enzymes,AST and ALT,represented the extent of hepatocyte injury.The AST(P<0.0001)and ALT(P<0.0001)contents increased significantly with time dependence in the model 24 h group,model 48 h group and model 72 h group compared to the Control group.4.Real-Time PCR results after FFA time gradient treatment showed that the m RNA levels of ferroptosis-related genes SLC7A11,GPX4 and FTH1 gradually decreased with increasing time of FFA treatment(P<0.05),while the m RNA level of TFR1 gradually increased with increasing time of FFA treatment(P<0.05);The m RNA levels of autophagy-related genes LC3-II and ATG7 gradually increased with the increase of FFA treatment time(P<0.05).5.Real-Time PCR results after adding CQ treatment showed that the expression levels of m RNA of autophagy-related genes LC3-II(P<0.05)and ATG7(P<0.01)were increased in the FFA group,and the expression levels of LC3-II(P<0.01)and ATG7(P<0.0001)were higher after CQ intervention;the expression levels of the autophagy receptor NCOA4 m RNA was increased in the FFA group(P<0.05),and NCOA4 expression level was also higher after CQ intervention(P<0.05);m RNA expression levels of ferroptosis-related genes SLC7A11(P<0.05),GPX4(P<0.0001)and FTH1(P<0.001)were reduced in the FFA group,while CQ intervention resulted in lower expression levels of SLC7A11(P<0.01),GPX4(P<0.0001)and FTH1(P<0.0001);expression levels of m RNA for the ferroptosis-related gene TFR1 were increased(P<0.05),and CQ intervention resulted in higher levels of TFR1 expression(P<0.0001).6.Western blot results showed that the protein expression levels of LC3 B and NCOA4 were upregulated in the FFA group(P<0.01),and those of FTH1 and SLC7A11 were downregulated(P<0.01);the protein expression levels of LC3 B and NCOA4 were higher after CQ intervention(P<0.0001),while the protein expression levels of FTH1 and SLC7A11 were lower(P<0.01).Conclusion:FFA induced steatosis and triggered autophagy and ferroptosis in hepatocytes in vitro.Moreover,inhibition of autophagy increased NCOA4 expression and promoted FTH1 degradation,thereby exacerbating ferroptosis,suggesting that autophagy promotes FFA-induced iron death in steatotic hepatocytes.Objective:To systematically evaluate the association of the presence and pathological changes of non-alcoholic fatty liver disease(NAFLD)with the changes in serum ferritin and serum iron levels.Methods:Pub Med,CNKI,VIP,Wanfang Data,Cochrane Library were searched for Chinese and English articles on the association of the presence and pathological changes of non-alcoholic fatty liver disease(NAFLD)with the changes in serum ferritin and serum iron levels published up to December 2021.Secondary screening,quality assessment and data extraction were performed,and then Stata12.0 were used for meta-analysis.Results:A total of 32 articles with 4177 patients were included.The results showed: compared to controls,Serum ferritin(SMD=0.93,95% Cl:0.85-1.01,P=0.000)and serum iron(SMD=0.57,95% Cl:0.37-0.76,P=0.000)levels were higher in patients with NAFLD;compared to NAFLD patients,Serum ferritin(SMD=0.59,95% Cl:0.41-0.78,P=0.000)levels were higher in NASH patients,while the difference in serum iron(SMD=0.14,95%Cl:0.00-0.29,P=0.057)levels was not statistically significant.Conclusion:The presence of NAFLD correlates with increased serum ferritin and serum iron levels,and the more severe the pathological changes in NAFLD,the higher the serum ferritin levels.Therefore,the causal role of serum ferritin and serum iron in the progression of NAFLD and the pathogenesis of NAFLD merits further research and clinical validation.