The Protective Effects Of Quercetin On Ferritinophagy In Alcohol-induced Liver Iron Overload

Objective: To investigate the effects of quercetin and iron treatment on hepatic iron overload and the level of ferritinophagy induced by chronic-plus-binge ethanol feeding,and further elucidate the intervention mechanisms of quercetin against ethanol-induced liver iron overload by focusing on ferritinophagy.Methods:1.Ninety male C57BL/6J mice,weighting 18-20 g,were randomly divided into nine groups as the following:1)Normal control group(C)received regular Lieber De Carli liquid diets;2)Chronic-plus-single-binge ethanol group(E)was fed ethanol-containing Lieber De Carli liquid diets(28% of total calories as ethanol);3)Quercetin control group(Q)received quercetin(100mg/kg body weight,ig)and regular Lieber De Carli liquid diets;4)Ethanol plus quercetin group(E+Q)received quercetin and ethanol-containing Lieber De Carli liquid diets(28% of total calories as ethanol);5)High iron control group(HI)was administrated by carbonyl iron powder(w/v:0.2%)and regular Lieber De Carli liquid diets;6)High iron group plus ethanol group(HI+E);7)Quercetin,high iron group plus ethanol group(HI+E+Q);8)Low iron control group(LI)was fed regular Lieber De Carli liquid diets for removing iron from the diet;9)Low iron plus ethanol group(LI+E).The mice were pair-fed with Liber De Carli liquid diets for 12 weeks.Alcoholic liver injury was established by the method of chronic-plus-single-binge ethanol feeding.Body weight was measured weekly and food consumption was calculated daily.At the end of the experiment,plasma samples were collected to measure the level of triglyceride(TG)and transaminase.HE and Oil-Red O staining,and enzyme dynamic methods were used to observe liver pathological changes and hepatic steatosis.Prussian blue staining was used to observe hepatic iron accumulation and flame atomic absorption spectrophotometry was used to detect liver total iron and liable iron pool(LIP)level.Western blot was used to measure the expression of autophagy and ferritinophagy-related proteins.Double immunofluorescence was used to observe the co-localization of liver ferritin heavy chain(FTH)and nuclear receptor coactivator 4(NCOA4).The software predicted that CEBPβ and FOXO1 might be the transcription factors of NCOA4.Western blot was used to measure the expression of nuclear CCAAT enhancer bingding protein β(CEBPβ)and Forkhead box protein O1(FOXO1)for verification.Immunohistochemistry was used to observe FOXO1 nuclear translocation.2.Vitro experiments:Adenovirus transfection technique was used to obtain the overexpressed NCOA4 adenovirus(Ad-NCOA4)or adenovirus empty vector(Ad-Null)-transfected HepG2 cells,and the cells were treated with ethanol(100 mmol/L)and/or quercetin(100 μmol/L)for 72 h.Western blot was used to detect the expression of autophagy and ferritinophagyrelated proteins,and ferritin light chain(FTL)protein.Prussian blue staining was used to observe cell iron deposition and colorimetry was used to measure LDH activity.On this basis,the FOXO1 transcriptional activity inhibitor(AS,1 μmol/L)was added to HepG2 cells which were treated with ethanol(100 mmol/L)and/or quercetin(100 μmol/L).When the cells were treated for 72 h,the lysosome pH was detected by the probe of lysoTrackor Red DND-99 and cell viability was measured by CCK-8 assay kit.Western blot was used to measure the expression of sirtuin1(SIRT1),p-FOXO1,transcription factor EB(TFEB),FTL and ferritinophagy-related proteins.Results:1.After 12 weeks of pair-feeding,the body weight of all ethanol-fed mice was lower than that of non-ethanol-challenged mice,which was slightly increased by quercetin intervention but there was no obvious differences.Compared with normal control group,the liver index of chronic-plus-binge ethanol-fed mice or high-ironexposed mice,especially ethanol-high-iron co-exposed mice was significantly elevated,which obviously alleviated by quercetin intervention.2.Compared with normal control mice,liver lipid accumulation of chronic-pussingle-binge ethanol-fed mice increased,the TG level of serum and hepatic,and serum ALT and AST level elevated by 73.5%(P < 0.01),24.5%(P < 0.01),116%(P < 0.01),and 162%(P < 0.01),respectively.Compared with ethanol exposure group,the above changes were further aggravated in mice ingesting ethanol in combination with 0.2% carbonyl iron powder,but ethanol-exposed mice treated with low iron alleviated these changes at some extent.When quercetin was supplied to the ethanol-fed mice and ethanol-high-iron co-exposed mice,hepatic lipid accumulation and contents of serum and liver TG were evidently reduced,and serum ALT and AST levels were declined compared to the corresponding ethanol-fed group and the co-exposed group.3.Compared to normal control mice,liver total iron content,LIP level,FTL,p62 and FTH expression of chronic-plus-binge ethanol exposure mice increased 46.7%(P < 0.01),45.7%(P < 0.01),198%(P < 0.01),969%(P < 0.01)and 309%(P < 0.01),respectively;and liver LC3 II and NCOA4 protein level reduced by 72.8%(P < 0.05)and 71.2%(P < 0.05),respectively,accompanying with the reduced of liver FTH and NCOA4.In comparison with ethanol-fed group,ethanol and high iron co-fed mice further exacerbated the above manifestations,but ethanol-exposed mice treated with low iron could normalize these changes to a certain extent.When quercetin was given to the ethanol-fed mice and ethanol-high-iron co-exposed mice,the elevation of liver total iron,LIP content,FTL,p62 and FTH expression were evidently attenuated,liver NCOA4 and LC3 II protein level was obviously increased,and the FTH and NCOA4 co-localization areas was increased in comparison with the corresponding ethanol model group and cotreated group.Quercetin or low iron per se could obviously increase NCOA4 protein expression and the co-localization of liver FTH and NCOA4.Compared with vector control group,ethanol decreased the expression of NCOA4 and LAMP1 proteins,increased the level of p62,FTH and FTL proteins,raised the iron accumulation and increased LDH release level.Compared to vector ethanol group,NCOA4 overexpressed HepG2 cells treated with ethanol evidently raised LAMP1 protein level,lowered the expression of p62,FTH and FTL proteins,attenuated the cell iron deposition and eased the increased of LDH level.The effects of quercetin on cells treated with vector-ethanol was similar to that of NCOA4 overexpressed group,which significantly alleviated these changes induced by ethanol.4.Compared with normal control mice,chronic-plus-binge ethanol obviously elevated nuclear CEBPβ protein,reduced the level of nuclear FOXO1 protein and weaken the co-localization of nuclear and FOXO1.Quercetin supplement significantly declined nuclear CEBPβ level,elevated the expression of nuclear FOXO1 and increased the positive areas of nuclear and FOXO1 co-localization.5.When HepG2 cells were treated with ethanol or AS alone,especially ethanol in combination with AS,p-FOXO1 expression was significantly increased,along with elevation of p62,FTH and FTL level,TFEB protein expression and cell activity were decreased,and lysosome pH was increased.However,AS did not further reduce the NCOA4 protein level in ethanol-incubated HepG2 cells,but when quercetin was given to the ethanol-AS co-incubated cells,TFEB expression was increased,lysosome specific pH fluorescence labeling and cell activity were elevated.Conclusions:1.Ethanol intake blocked the ferritinophagy initiation and impaired lysosme function by reducing the expression of liver NCOA4 and TFEB,and thereby induced secondary liver iron overload and injury.Quercetin intervention promoted ferritinophagy initiation and improved lysosome function by raising NCOA4 and TFEB expression level,and further antagonized ethanol-induced liver overload and damage.2.Ethanol increased the expression of CEBPβ and FOXO1 phosphorylation,decreased FOXO1 nuclear translocation,but the inhibition of FOXO1 transcription activity did not further decrease the NCOA4 expression,suggesting that CEBPβ and FOXO1 might not be a transcriptional regulator of NCOA4,while FOXO1 might be involved in TFEB transcriptional regulation.And it’s necessary to be further explored the transcriptional regulation mechanisms of NCOA4.