Protective Role And Potential Mechanism Of Baicalein Against High Fat Induced-hepatic Iron Disorder

Objective: To investigate the role of lysosome and NCOA4-mediated ferritin degradation and lysosomal V-ATPase assembly in non-alcoholic fatty liver disease(NAFLD)induced iron disorder and potential mechanism of the protection of baicalein.Methods: 1.105 male C57BL/6J mice(6 weeks,18-20 g)were randomly divided into seven groups(15/group)each as follows:(1)control group(C,10% fat of low fat diet);(2)high fat diet group(H,60% fat);(3)baicalein control group(B)received baicalein(baicalein: 400 mg/kg.bw);(4)iron group(Fe)was administrated by carbonyl iron powder(w/w: 0.2%);(5)high fat diet plus baicalein group(baicalein: 400 mg/kg.bw;H + B);(6)high fat diet plus iron group(Fe + H);(7)high fat diet,baicalein plus iron group(baicalein: 400 mg/kg.bw;Fe + H + B).Mice were fed for 12 weeks until sacrificed after an overnight fasting.Serum and liver tissue sample were collected rapidly for further biochemical assays.Serum aspartate aminotransferase(AST),alanine aminotransferase(ALT)and lipid level were detected.Hepatic pathological change and lipid content were observed with H&E and Oil-Red O staining.Hepatic total cholesterol(TC),triglycerides(TG),oxidative stress and iron-related parameters were detected.The V-ATPase B2 expression in lysosome and lysosomal redox-active iron in the liver were detected.Formalin-fixed,paraffin-embedded liver tissues were incubated V-ATPase B2 and lysosome associated membrane protein 2(LAMP2)antibodies to observe the colocalization of V-ATPase complex V1 subunit and lysosome.2.According to the purpose of experiments,the FFA-stimulated Hep G2 cells were treated with various pharmacological reagents,NCOA4 si RNA and Sestrin2 si RNA at their optimal concentration and exposure time.Hep G2 cell were stained with BODIPY493/503 for lipid staining.NCOA4,Sestrin2,protein carbonyls and autophagy-related protein were detected.lysosomal low mass iron were stained with sulphide-silver method.Cellular liable iron content,lysosomal V-ATPase assembly,lysosomal p H and related indicators were detected.Results: 1.Baicalein supplementation evidently alleviated the leakage of hepatic aminotransferases,the hepatic lipid deposition and oxidative stress induced by high fat diet(HFD).Baicalein intake led to 1.56-fold increase in hepatic total iron content(p<0.05)and 1.27-fold increase in liable iron pool(LIP)(p<0.05),compared with the HFD group.HFD fed resulted in 5.23-fold increase in lysosomes redox-active iron content(p<0.05),increasing cathepsin B activity by 20.8%(p<0.05)and decreased mature cathepsin D level,lysosomal V-ATPase complex V1 subunit expression and the colocalization of cytosol V-ATPase B2 and LAMP2;Such harmful results were partially counteracted by baicalein.2.Baicalein supplement eased FFA-induced cellular TG accumulation,LIP deficiency,lysosomes redox-active iron deposition and LMP alteration.Bafilomycin A1 stimulation resulted in cellular ferritin accumulation,decreased cytosol LIP and increased lysosomal iron in FFA-incubated cells,which partially counteracted the protective effect of baicalein.Rapamycin,preserving lysosomal function and restoring lysosomal p H,increased cellular FTH and LIP(p<0.05),lowered lysosomal low mass iron content.3.Baicalein intervention elevated Sestrin2 expression,inhibited the m TOR phosphorylation,reduced lysosomal ROS and increased MMP(p<0.05).Sestrin2 knockdown led to cellular ferritin accumulation,lysosomal low mass iron deposition,lysosomal p H elevation accompanied by decreasing LIP and colocalization of cytosol V-ATPase B2 and LAMP2,partially counteracting the protective effects of baicalein.Knockdown Sestrin2 increased m TOR phosphorylation and inhibited the effect of baicalein on m TOR;Rapamycin intervention,inhibiting m TOR expression,decreased cellular ferritin,increased LIP(p<0.05)and restored lysosomal p H in baicalein-stimulated cells.Knockdown Sestrin2 elevated protein carbonyls level and inhibited the antioxidant function of baicalein;a decrease in the level of cellular FTH and lysosomal p H,and an increase in FTL expression were observed in N-acetylcysteine and baicalein-incubated cells(p<0.05).4.Proteasome inhibitor did not block baicalein-mediated ferritin degradation and lysosomal acidification;3-MA partially inhibited the effect of baicalein on ferritin degradation and decreased cellular LIP,but autophagy inhibitor had no influence on lysosomal redox-active content.Baicalein supplementation evidently decreased the NCOA4 and p62 expression,elevated Beclin1 level(p<0.05);NCOA4 knockdown increased cellular ferritin(p<0.05),and partially counteracted the effect of baicalein on ferritin degradation;whereas NCOA4 knockdown partially restored LMP stability,lowered lysosomal low mass iron and cellular LIP(p<0.05),and increased colocalization of cytosol V-ATPase B2 and LAMP2 in FFA and FAC-incubated cells.Conclusions: Lysosomal V-ATPase complex assembly,lysosome and NCOA4-mediated ferritin degradation played a major role in HFD-induced iron deficiency in NAFLD.Increasing lysosomal V-ATPase complex assembly,and consequent restoring lysosomal acidification and enhancing ferritin degradation via ferritinophagy and lysosomes by baicalein may be the potential mechanism of protection against HFD-induced iron deficiency in NAFLD.Additionally,baicalein could promote Sestrin2 expression,inhibit m TOR expression and relieve protein oxidative damage to increase lysosomal V-ATPase complex assembly and consequent restore lysosomal acidification.Baicalein regulating Sestrin2/m TOR/ROS signaling pathway may be a major molecular mechanism of the improvement lysosomal V-ATPase complex assembly dysfunction induced by HFD.