Mitochondrial Homeostasis And Mitophagy In Hepatic Steatosis:Regulation Of Quercetin Through Frataxin

Objectives: The aim of this study was to investigate the protective effect of quercetin on hepatic mitochondrial damage and disturbance of mitochondrial homeostasis induced by high-fat diet and to explore the regulatory effect of Frataxin on PINK1-Parkin mitochondrial autophagic signaling pathway.Methods: 48 male C57BL/6J mice were randomly divided into 4 groups: low fat diet(LFD,10% fat-derived calories),high fat diet group(HFD,60% fat-derived calories),quercetin group(HFD + Q,100 mg/kg·bw)and quercetin control group(LFD + Q,100 mg/kg·bw).After 10 weeks of feeding,hepatic lipid deposition and serum lipid concentrate were observed.The genetic expression of lipolism,mitochondrial biosynthesis,mitochondrial fusion and fission,as well as mitophagy,were measured by real-time quantitative qRT-PCR.The ultrastructure of liver mitochondria and mitochondrial autophagy were observed by transmission electron microscope.Immunofluorescence staining was used to observe the translocation of Parkin to mitochondria.Western blot was used to detect the expression of mitophagy pathway related proteins.After design and selection of oligonucleotide interference sequences for FXN gene,lentiviral vectors were transfected into HepG2 cells to obtain shRNA-FXN-interfering HepG2 cells(Lv-FXN-mcherry)and empty vector(Lv-mcherry).HepG2 cells,Lv-FXN-mcherry and LV-mcherry were treated with free fatty acids(FFAs)(oleic acid: palmitic acid = 2: 1,2 mmol/L)and / or 100 μmol/L quercetin for 24 h.The levels of lipid deposition and expression of Frataxin,PINK1,and Parkin were compared in each group of cells.Confocal laser scanning was used to observe the binding of autophagosomes and mitochondria,as well as Parkin’s translocation of mitochondria.HepG2 cells were treated with p53 inhibitor PFT-α and HIF-1α inhibitor PX-478 to observe the change of index above.Results: Compared with the LFD group,serum TG and TC in HFD group increased evidently(P < 0.05),accompanied by lipid accumulation in liver.In addition,the expression of genes involved in lipid anabolism(FAS and SREBP-1c)in liver of HFD group was significantly higher(P < 0.05).Changes in hepatic mitochondrial structure and decreased membrane potential and respiratory control rate were observed in HFD group.The expression of genes involved in mitochondrial biosynthesis,fusion and fission decreased(P < 0.05).The protein levels of LC3II/LC3 I,Beclin1,PINK1 and Frataxin was decreased significantly(P < 0.05),accompanied by increased p62(P < 0.05).Importantly,the expression of Parkin and LC3 II in liver mitochondria of HFD group mice was significantly lower than that of LFD group(P < 0.05).Parkin labeling overlapped less with VDAC1 in the HFD group compared to the LFD group.After treatment with quercetin,the mRNA level of FAS in liver tissue was significantly decreased(P < 0.05).However,the mRNA level of SREBP-1c was further increased by quercetin(P < 0.05).Furthermore,quercetin treatment evidently ameliorated the morphological and functional abnormalities of mitochondria induced by HFD.RCR and MMP were increased(P < 0.05).In addition,the expression of mitochondrial biosynthesis-related genes in the liver of HFD mice was significantly lower than that in the LFD group(P < 0.05),as well as Fis1 and Mfn1(P < 0.05).After quercetin intervention,the changes in protein above can be significantly reversed,with increasing Parkin expression(P < 0.05).Quercetin significantly increased the expression of Parkin and LC3 II in liver mitochondria(P < 0.05).Quercetin also produced larger co-localization of Parkin and VDAC1.In HepG2 cells,compared with Ct group,lipid accumulation and repressive PINK1-Parkin-mediated mitophagy were observed in FFAs-induced HepG2 cells(FFAs group),as well as decreased Frataxin expression(P < 0.05).In addition,the confocal results were showed that TOM20 overlapped less with LC3,and parkin labeling overlapped less with VDAC1 in the FFAs group compared to the Ct group.However,quercetin intervention reversed changes above and up-regulated Frataxin protein levels(P < 0.05).In Lv-FXN-mcherry cells,the expression of LC3II/LC3 I,Beclin1,PINK1 and Parkin was decreased,compared with Lv-mcherry cells(P < 0.05).Moreover,intracellular TG content in Lv-FXN-mcherry cells was significantly higher than of Lv-mcherry cells(P < 0.05),accompanied by futher decreases in mitophagy related proteins including LC3Ⅱ/ LC3Ⅰ,Beclin1,PINK1 and Parkin(P < 0.05).Furthermore,quercetin could stimulate PINK1 expression,which was inhibited by FFAs treatment(P < 0.05).However,quercetin did not improve Parkin,LC3Ⅱ/ LC3Ⅰ and Beclin1 expression in FFAs-induced Lv-FXN-mcherry cells(P > 0.05).On the other hand,PFT-α,a pharmacological p53 inhibitor,inhibited the expression of Frataxin in HepG2 cells(P < 0.05).Under the treatment of FFAs,the Frataxin were further decreased by PFT-α(P < 0.05).Quercetin did not improve the expression of Frataxin when p53 was inhibited(P > 0.05).Conclusions: Frataxin is involved in the regulation of PINK1-Parkin-dependent mitophagy in hepatic steatosis.Quercetin could upregulate Frataxin,which activates PINK1-Parkin-dependent mitophagy to maintain mitochondrial homeostasis and alleviate hepatic steatosis and lipid metabolism disorder.