The Protective Effect Of Quercetin On Alcohol-induced Liver Mitochondrial Dysfunction: Regulatory Mechanism Of Iron-frataxin

Data reported by the World Health Organization(WHO)in 2018 showed that 5.1% of the global burden of disease and 5.3% of deaths were attributed to alcohol abuse.Alcoholic liver disease(Alcoholic liver disease,ALD)caused by excessive alcohol consumption is a major kind of alcohol-related chronic disease,alcoholics present early with reversible hepatic steatosis that further develops into steatohepatitis,liver fibrosis,cirrhosis and liver cancer.However,once it progresses to the terminal stage,there is currently no effective treatment strategy other than liver transplantation.The pathogenesis of ALD is intricate and multiple mechanisms coexist.Mitochondria are the main target organelles of alcohol metabolism,and their dysfunction is crucial in the development of ALD.However,the mediating mechanism of mitochondrial damage in ALD remains unclear,so further study is expected to provide the potential theoretical and experimental basis for the early prevention and treatment of ALD.Frataxin is encoded by the nuclear gene FXN and localizes to mitochondria,where it plays an important role as an iron donor in iron-sulfur cluster(iron-sulfur cluster,ISC)assembly and heme synthesis.Notably,the manifestations of mitochondrial damage in disease models with frataxin deficiency are similar to the typical pathological features of ALD,such as defects in a variety of ISC-containing enzymes(e.g.,respiratory chain complex subunits),disturbances in iron metabolism and mitophagy and other mitochondrial dysfunctions.Previous research by our group found that the expression of frataxin in the liver of high-fat or alcohol-fed mice was reduced,which may be related to lipid deposition induced by high-fat diet and ferroptosis caused by alcohol.The above studies reveal the important role of frataxin in liver disease.However,the contradictory results of high-fat-induced hepatic iron deficiency and alcohol-driven hepatic iron overload make the effect of iron on frataxin confusing.In yeast lacking the mitochondrial ABC transporter Atm1(Atm1 deficiency shows features of mitochondrial iron overload and cytoplasmic iron deficiency),frataxin m RNA level was decreased,suggesting that iron may regulate frataxin.In fibroblasts and lymphoblasts from Friedreich's ataxia patients and normal controls,deferoxamine down-regulated frataxin m RNA and protein expression;conversely,ammonium ferric citrate increased frataxin expression.The above results indicated that frataxin expression is closely related to iron level.So,how does frataxin mediate mitochondrial damage in the ALD model and is it dependent on iron regulation? These questions still need further exploration.Quercetin is a plant flavonoid compound widely found in daily fruits and vegetables.In addition to its antioxidant and oxidative effects,numerous studies have also shown the roles of quercetin in iron homeostasis regulation and mitochondrial function protection.Our group has previously shown that quercetin ameliorated alcohol-induced hepatic iron overload and mitochondrial dysfunction(such as reduced membrane potential,opening of mitochondrial permeability transition pores,mitochondrial oxidative stress,and inhibition of mitophagy)in the ALD models.However,the mechanism by which quercetin protects against mitochondrial damage in ALD remains unelucidated.Since frataxin plays an important role in maintaining mitochondrial function and is susceptible to iron level,can quercetin protect mitochondria through iron-frataxin? This issue still needs further study and disclosure.The main contents of this study include the following three parts.Part one Protective effects of quercetin on mice liver mitochondrial dysfunction induced by alcoholObjective: To investigate the protective effects of quercetin on alcohol-induced liver injury and mitochondrial dysfunction.Methods: 70 healthy 8-9 weeks female C57BL/6N mice weighing 18-20 g were randomly divided into control group,ethanol group,ethanol plus quercetin group and quercetin group.The chronic-plus-binge ethanol feeding model was established by gavage on the last day(5% alcohol gavage in the alcohol group)using a Lieber De Carli liquid diet with 28% ethanol-derived calories for 12 weeks,and the control and quercetin groups were iso-energetically paired to feed a control Lieber De Carli liquid diet without ethanol,or supplemented with quercetin intervention(the dose equivalent to 100 mg/kg.bw).Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST) were detected;histopathological changes of the liver(hematoxylin eosin staining and OilRed O staining)were observed;liver differential genes expression were analyzed by transcriptomics;mitochondrial membrane potential level in mice liver was assessed by JC-1;mitochondrial ultrastructure was observed by transmission electron microscopy;the expression of mitophagy-related proteins,mitochondrial respiratory chain complex proteins and mitochondrial iron metabolism-and iron utilization-related proteins were measured by western blot.Results:1.1 Protective effects of quercetin on alcoholic-induced liver injuryCompared with the control group,ethanol increased serum ALT and AST levels(P < 0.05).Hematoxylin eosin staining indicated that the liver tissue of mice in the alcohol group was disorganized,the boundary between hepatocytes was blurred.Oil-Red O staining showed that alcohol induced hepatic lipid droplets accumulation in mice.Quercetin intervention significantly decreased serum ALT and AST levels(P < 0.05)and improved liver tissue histopathological damage.1.2 Effects of quercetin on hepatic gene expression in chronic-plus-binge ethanolfed mice: based on transcriptomic resultsThe results showed that the total number of differential genes was 5750 in the alcohol group compared with the control group;the total number of differential genes was 950 in the alcohol plus quercetin group compared with the alcohol group;844 common genes were changed in the two comparisons.Cell composition analysis showed that the most obvious organelle with differential genes was mitochondria,and the gene enrichment results were mainly reflected in the detoxification of reactive oxygen species,mitophagy,oxidative phosphorylation,tricarboxylic acid cycle and respiratory electron transport,and the assembly of mitochondrial respiratory chain complexes.1.3 Protective effects of quercetin on alcohol-induced mitochondrial dysfunction in mice liverCompared with the control group,alcohol induced abnormal mitochondrial ultrastructure(swelling,fragmentation,disappearance of mitochondrial cristae,and vacuolization of mitochondria),decreased mitochondrial membrane potential(P < 0.05),inhibited the levels of mitochondrial complex I and II proteins,decreased autophagyrelated protein LC3 II and mitophagy-related proteins PINK1,Parkin and Bnip3 levels(P < 0.05),and increased expression of autophagy receptor protein p62,mitochondrial protein TOM20 and VDAC1(P < 0.05).After quercetin intervention,the morphological structure of liver mitochondria was improved,the membrane potential was increased and the inhibition of mitochondrial respiratory chain complex and mitophagy-related proteins were mitigated(P < 0.05).1.4 Effects of quercetin on iron metabolism and iron utilization-related proteins in chronic-plus-binge ethanol-fed mice liver mitochondriaCompared with the control group,alcohol induced the increase in the levels of MFRN2 and FECH,and inhibited ABCB10 expression;quercetin intervention significantly alleviated the disturbance of the above protein expression levels.Conclusion: Quercetin alleviated alcohol-induced mitochondrial dysfunction in mice liver,which may be related to impaired mitochondrial iron metabolism and iron utilization.Part two Quercetin ameliorated ethanol-induced mitochondrial dysfunction in hepatocytes by increasing frataxinObjective: To determine the effect of ethanol on frataxin expression,and to further explore the role of frataxin in ethanol-induced mitochondrial dysfunction in hepatocytes and the protective effect of quercetin.Methods:2.1 The animal model was consistent with the first part,frataxin protein expression in mice liver was detected by western blot.2.2 TMTTM-labeled quantitative proteomics was used to detect the differential proteins of Lv-empty and Lv-FXN group: The interfering sequence of silencing frataxin protein was designed according to the FXN gene,and the lentiviral vector was prepared and infected into Hep G2,then Lv-empty and Lv-FXN silencing group was constructed,respectively.2.3 To investigate the inhibitory effect of ethanol on frataxin:(1)HepG2 was treated with ethanol(100 m M),the pro-oxidant 4-hydroxynonenal(4-HNE)and the antioxidant N-acetyl-L-cysteine(NAC)alone or in combination.(2)Further,HepG2^CYP2E1+/+ overexpressing the ethanol metabolizing enzyme CYP2E1 were divided into control and ethanol groups.Frataxin protein expression was detected.2.4 To investigate the role of frataxin in alcohol-induced mitochondrial dysfunction in hepatocytes and the protective effect of quercetin: Lv-empty and Lv-FXN HepG2^CYP2E1+/+ cells overexpressing CYP2E1 were treated with ethanol and quercetin(50 ?M)alone or in combination.JC-1 was used to evaluate mitochondrial membrane potential level;fluorescence co-localization of mitochondrial(Mitotracker)and lysosomal(Lysotracker)probes were used to measure mitophagy level;mitochondrial iron level was observed by mitochondrial divalent iron fluorescent probe(RPA);and the expression of mitophagy-related proteins was detected.Results:2.1 Effects of alcohol on hepatic frataxin expression in mice: in the animal model of ALD,alcohol inhibited frataxin protein expression in the liver of mice(P < 0.05).2.2 Proteomics results: there were 285 differential proteins in the Lv-FXN and Lvempty groups.A total of 20 differential proteins specifically localized in mitochondria were mainly involved in mitochondrial biogenesis,oxidative phosphorylation and mitochondrial iron utilization.In addition,the proteomic results suggested that frataxin deficiency induced the disturbance of autophagy-related proteins such as VPS33 A,GABARAPL1 and GABARAPL2.2.3 Effects of ethanol on mitochondrial function and the role of frataxin2.3.1 Compared with the control group,ethanol increased frataxin protein at 12 h and 24 h,and decreased the expression of frataxin at 48 h and 72 h in Hep G2(P < 0.05).What's more,in Hep G2 incubated with ethanol for 72 h,NAC alleviated the inhibition of frataxin(P < 0.05).In contrast,4-HNE decreased frataxin expression.2.3.2 In ethanol-challenged HepG2 for 12-48 h,the reduction of frataxin expression was noted(P < 0.05).2.3.3 In Lv-empty HepG2^CYP2E1+/+ cells,ethanol decreased mitochondrial membrane potential;frataxin deficiency exacerbated the inhibition effect of ethanol(P < 0.05).2.3.4 In Lv-empty HepG2^CYP2E1+/+ cells,ethanol inhibited mitophagy(P < 0.05)and induced the decrease of LC3 II,Beclin1 and Bnip3 protein levels and the increase of mitochondrial proteins TOM20 and VDAC1 expression(P < 0.05);frataxin deficiency exacerbated the inhibition effect of ethanol on mitophagy(P < 0.05).2.4 Protective effects of quercetin on mitochondria and the mediating effect of frataxin2.4.1 In the animal model of ALD,alcohol resulted in the inhibition of frataxin,whereas quercetin up-regulated frataxin expression(P < 0.05).Similarly,quercetin increased the ethanol-induced decrease of frataxin in HepG2^CYP2E1+/+(P < 0.05).2.4.2 Compared with ethanol-treated Lv-empty HepG2CYP2E1+/+cells,quercetin upregulated mitochondrial membrane potential and mitophagy and decreased mitochondrial iron level(P < 0.05);frataxin deficiency reversed the protective effects of quercetin(P < 0.05).Conclusion: Alcohol inhibited frataxin protein expression in vitro and in vivo.Further,it was found that quercetin alleviated alcohol-induced hepatic mitochondrial dysfunction by up-regulating frataxin.Part three Quercetin ameliorated ethanol-induced mitochondrial damage by restoring frataxin expression through iron regulationObjective: To investigate whether iron level is involved in quercetin-induced frataxin expression.Methods: 3.1 The animal model is the same as the first part.LIP level in the liver of mice was measured by flame atomic absorption spectrometry,trivalent iron level in the liver tissue was evaluated by Prussian blue staining.The expression of iron metabolismrelated proteins and ferritinophagy-related proteins in the liver were detected by western blot;ferritinophagy level was observed by immunofluorescence staining(co-localization of NCOA4 and FTH).3.2 To investigate whether the effect of quercetin on frataxin is related to iron level:(1)HepG2^CYP2E1+/+,Lv-empty,and Lv-FXN HepG2^CYP2E1+/+ cells were treated with deferiprone(DFP),ferric ammonium citrate(FAC),ethanol and quercetin alone or in combination.(2)HepG2^CYP2E1+/+ were stimulated with ethanol and 3-O methylated quercetin.(3)NCOA4 si RNA was designed to treat HepG2^CYP2E1+/+ incubated with ethanol and quercetin.JC-1 was used to assess mitochondrial membrane potential level;Divalent ferric ion probe(Ferro Orange)was used to measure cellular LIP level;frataxin protein expression was detected.Results: 3.1 Effects of quercetin on hepatic iron level in chronic-plus-binge ethanol-fed miceCompared with the control group,alcohol increased LIP,FTL expression and trivalent iron level(P < 0.05),accompanied by the increase of iron metabolism-related proteins Tfr1,DMT1,FPN and the decrease of IRP1,hepcidin(P < 0.05).Quercetin intervention reduced hepatic LIP,FTL protein and ferric iron level in the alcohol group and improved disturbances of iron metabolism-related proteins(P < 0.05).3.2 Effects of iron on frataxin protein expression and the role of quercetin3.2.1 Compared with the control group,FAC and DFP decreased or increased frataxin expression in HepG2^CYP2E1+/+ in a dose-dependent manner,respectively(P < 0.05).Similarly,an increase of frataxin expression by DFP was also observed in Lv-FXN HepG2^CYP2E1+/+ cells(P < 0.05).3.2.2 In ethanol-stimulated Lv-empty and Lv-FXN HepG2^CYP2E1+/+ cells,quercetin increased frataxin expression;FAC inhibited the induction of frataxin by quercetin(P < 0.05).3.3 The mechanisms of quercetin regulating frataxin protein expression3.3.1 In HepG2^CYP2E1+/+,ethanol increased LIP level and inhibited frataxin protein;3-O methylated quercetin did not affect the increase of LIP level as well as the inhibition of frataxin expression by ethanol(P < 0.05).3.3.2 Compared with the control group,alcohol inhibited NCOA4 expression and increased FTH expression,decreased the co-localization of NCOA4 and FTH of mice liver;quercetin intervention ameliorated disturbances of the above proteins and increased the co-localization of NCOA4 and FTH.Further,in ethanol-treated HepG2^CYP2E1+/+,quercetin enhanced frataxin expression and mitochondrial membrane potential level;silencing NCOA4 decreased the increasing effect of quercetin(P < 0.05).3.3.3 Compared with HepG2CYP2E1+/+ treated with the combination of ethanol and quercetin,FAC decreased the protective effect of quercetin on mitochondrial membrane potential(P < 0.05).Conclusion: Frataxin expression was regulated by iron level.Further analysis revealed that quercetin increased frataxin expression through regulating iron level(chelating iron or NCOA4-mediated),which alleviated mitochondrial dysfunction.