Protective Role And Potential Mechanism Of Quercetin Against Lysosomal Redox Active Iron Mediated Alcoholic Liver Damage And Autophagy Dysfunction

Objective:To investigate the role of lysosomal redox active iron mediated lysosome damage and autophagy dysfunction in ALD and the potential mechanism of the protection of quercetin.Methods:1. The male C57BL/6J mice (n=90) were divided randomly into six groups of fifteen each:Normal control group (C); Ethanol group (E,30%of total calories as ethanol); Ethanol plus Quercetin group (EQ, quercetin:100mg/kg.bw); Quercetin control group (Q); Ethanol plus DFO group (ED, DFO:100mg/kg.bw) and DFO control group (D). Quercetin and DFO were received by gavage and intraperitoneal injections respectively according to the body weight. The mice were pair-fed with either regular or ethanol-containing Lieber De Carli liquids diets for15weeks and the related parameters were assayed.2. Primary hepatocytes were isolated by two-step collagenase method and incubated with ethanol (100mmol/L). According to the purpose of experiments, the hepatocytes were pretreated with various pharmacological reagents at their optimal concentration and exposure times.Result:1. The weight gain of all ethanol-fed mice was lower than those of non ethanol-challenged groups, and ethanol led to the marked increase in the liver ratio against body weight, whereas quercetin suppressed these adverse effects. And, quercetin supplementation evidently alleviated the leakage of aminotransferases, the hepatic lipid accumulation, the liver oxidative damage and hepatocytes apoptosis induced by ethanol.2. Chronic ethanol feeding gave rise to intracellular accumulation of iron in liver. A1.4-fold,1.5-fold and1.6-fold increase of liver total iron content, LIP and lysosome redox active iron than normal control was observed, respectively. Quercetin intervention significantly reduced the total iron, LIP and lysosomes redox-active iron levels. Additionally, long-term ethanol expose evidently increased lysosome ROS production, caused LMP alteration and cathepsin B leakage; Upon LMP, mitochondria were damage and subsequent cytochrome C released to cytoplasm. Such harmful results were counteracted by quercetin. The hepatotoxicity was aggravated by escalating lysosomal iron with FeCl3while relieved by reducing lysosomal iron with Bafilomycin A1and DFO. As well as DFO, quercetin eased hepatocytes oxidative injury and apoptosis by chelating lysosomeal iron.3. Chronic ethanol feeding suppressed liver autophagic flux. Quercetin ameliorated the accumulation of autophagosome, decreased the abnormal LC3-â…¡ and p62expression, accelerated the autophagosome-lysosome fusion and increased the expression of LAMP1, LAMP2and Rab7. Besides, queretin reversed the inhibition of TFEB nuclear translocation incited by ethanol. At the same time, quercetin decreased them TORC1activity. Compaired with ethanol group, the phosphorylation level of mTOR and p70s6k decreased50%.4. Lysosomal inhibitor bafilomycin A1caused LC3-â…¡ accumulation compared with normal control, however, bafilomycin A1did not further increase ethanol-delivered LC3-â…¡ accumulation. Quercetin exhibited the similar effect to Torin1which is themTORC1activity inhibitor. Moreover, pretreatment with Totin1could promote nuclear translocation TFEB, and a2.1-fold increaseof TFEB nuclear translocation than ethanol group was observed. Quercetin also promoted TFEB nuclear translocation similar to Torin1.Conclusion:Lysosomal redox-active iron played a major role in alcoholic liver oxidative damage and apoptosis. Lowering lysosomal iron and consequent the mitigation of lysosome damage by quercetin may be the potential mechanism of protection against ALD. Additionally, quercetin could promote TFEB nuclear translocation to enhance lysosomal function and autophagic flux, regulating mTORCl-TFEB signaling pathway may be a major molecular mechanisms of the improvement lysosomal autophagy dysfunction induced by ethanol of quercetin.