The Mediating Effect Of Frataxin On Ferroptosis In HepG2 Induced By Alcohol

Objective:To reveal the function of frataxin in HepG2 cell,to investigate the ferroptosis in HepG2 cells induced by alcohol and the role of frataxin in thie process.Methods:1.The results of Proteomic:using lentiviral transfection technology to construct shRNA-FXN interference and its empty control-transfected HepG2 cells?shFXN and NC cells,respectively?,quantitatively labeling shFXN/NC cells with TMT^TM,then conduct LC-MS/MS analysis and the data was compared with the Uniprot database,the differential proteins were screened by the differential folds FC>1.25 or FC<0.8,and P<0.05.Gene Ontology?GO?functional annotation and KEGG pathway enrichment analysis were subsequently performed.2.Cell experiment:?1?Effect of alcohol on frataxin:HepG2 cells were divided into control group and alcohol group?100 mmol/l,72 hours?.Western blotting was used to detect the expression of frataxin protein.?2?The effect of Frataxin deletion on ferroptosis in HepG2 induced by alcohol:shFXN/NC cells were divided into control group,alcohol group to detect LDH and CCK8 release levels,and Western blotting to detect the protein expression levels of GPX4 and xCT.NC cells were divided into control group and alcohol group.The corresponding shFXN cells were divided into control group,alcohol group,alcohol plus Fer-1 group,alcohol plus Erastin group,Fer-1 control group?1?mol/l?and Erastin control group?2.5?mol/l?.The levels of LDH and CCK8 were measured,the mitochondrial LIP-Fe level was observed by RPA fluorescent probe labeling,C11-BODIPY were used to detected the level of lipid ROS,and the MDA was determined by thiobarbituric acid colorimetry.?3?Frataxin reduce the sensibility of ferroptosis in HepG2 induced by alcohol:shFXN cells were divided into alcohol group,alcohol plus Fer-1 group,alcohol plus Erastin group,alcohol plus Ad-control group,alcohol plus Ad-FXN group,alcohol plus Ad-FXN group and Fer-1,alcohol plus Ad-FXN and Erastin group.LDH,LIP-Fe,MDA,and lipid ROS were measured.Results:1.The biological process changes caused by the deletion of frataxin overlap with the mechanism of iron death.?1?Proteomic analysis shFXN/NC cells obtained a total of 285 significant proteins,of which 110 proteins were up-regulated and 175 proteins were down-regulated.?2?Comprehensive GO,KEGG biochemical analysis results and related literature analysis:Frataxin deficiency causes changes in biological processes such as cholesterol synthesis,glutathione metabolism,NADPH production,fatty acid degradation,and cystine and methionine metabolism,and these processes showns great overlap with the cause of ferroptpsis which is iron overload,decreased antioxidant capacity and overlapping lipid peroxide accumulation.2.Alcohol inhibits the expression of frataxin.The expression of frataxin protein in HepG2 cells was reduced by 60%after alcohol treatment?P<0.05?.3.Frataxin deficiency exacerbates alcohol-induced ferroptosis.?1?Frataxin deletion aggravates the decrease in alcohol-induced HepG2 cell viability.?1?After CYP2E1 transfection,the LDH release level was higher than that of the untransfected CYP2E1 control group,and the cell survival rate was decreased.The CYH2E1 transfected shFXN alcohol group had a 35%increase in LDH release compared with the untransfected CYP2E1 control group(?P<0.05?,and the cell viability was reduced by 18%?P<0.05?.The following experimental results are based on CYP2E1 transfected cells.?2?After alcohol treatment,the expression levels of GPX4?P<0.05?and xCT were significantly lower in the shFXN alcohol group than in the NC alcohol group,compared with the shFXN control group,the protein levels of GPX4?P<0.01?and xCT?P<0.01?in shFXN alcohol group were also significantly reduced.?2?Frataxin deficiency exacerbates ferroptosis in HepG2 induced by alcohol.?1?Compared with NC cells,the level of LDH in shFXN cells is increased,and cell viability is decreased after alcohol treatment.Compared with and each group of alcohol,the level of LDH in NC and shFXN were reduced by 14%and 31%,and the cell viability is increased after the combined treatment Fer-1.In contrast,after combined treatment Erastin,NC shFXN with the highest LDH release and cell viability lowest.?2?shFXN cells have higher LIP-Fe than NC cells.After alcohol treatment,the LIP-Fe and MDA levels of NC and shFXN cells increased,and the latter was higher than the former.After treatment with alcohol and Fer-1,Fer-1 treatment reduced LIP-Fe and MDA levels compared with shFXN alcohol group,but after Erastin treatment,LIP-Fe and MDA levels were higher after Erastin treatment compared with shFXN alcohol group.4.Frataxin reduced the sensitivity of alcohol-induced iron death in HepG2.Under the alcohol treatment model,compared with the shFXN control group and the ad-control group,LDH release and LIP-Fe were significantly reduced in the ad-fxn group,and the MDA production and lipid ROS levels were also reduced.Cell LDH release,MDA production,LIP-Fe and lipid ROS levels were all decreased after alcohol combined with Fer-1 and Erastin.Conclusion:Frataxin silencing of HepG2 cells affects downstream cholesterol synthesis,glutathione metabolism,NADPH production,fatty acid degradation and cysteine and methionine metabolism,suggesting that frataxin affects ferroptosis.Alcohol inhibits frataxin expression and induces ferroptosis,while frataxin deficiency further exacerbates alcohol-induced ferroptosis.Restore the protein level of frataxin can reduce the sensitivity of ferroptosis in HepG2.