Protective Role And Potential Mechanism Of Quercetin Against Alcoholic Iron Overload-induced Liver Damage

Objective:To investigate the protection of quercetin against iron overload in C57BL/6J mice fed by ethanol-containned Lieber De Carli diets.Methods:1. One hundred and twelve male C57BL/6J mice were divided randomly into eight groups of fifteen animals each as follows:(1) Normal control group (Ct) received regular-containing Lieber De Carli liquids diets;(2) Ethanol group (Et) was fed ethanol-containing Lieber De Carli liquids diets (30%of total calories as ethanol);(3) Quercetin control group (Qu) received quercetin (100mg/kg.bw, i.g);(4) Quercetin plus ethanol group (Qu+Et) received quercetin and ethanol;(5) Iron group (Fe) was administrated by carbonyl iron powder (w/v:0.2%);(6) Ethanol plus iron group (Et+Fe);(7) Quercetin plus iron group (Qu+Fe);(8) Quercetin, ethanol plus iron group (Qu+Et+Fe).2. Mice were pair-fed for15weeks until sacrificed after an overnight fasting. Serum was collected from blood by centrifuge at3500×g for10min. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and ferritin (Ft) were measured with appropriate kits based on enzymatic kinetic method and ELISA, respectively. Liver tissue samples were fixed and tissue sections were stained with hematoxylin and eosin staining (H&E) and Perls’Prussian blue stain, respectively. Frozen hepatic sections were immediately incubated with DHE to detect the level of ROS. The supernatants of liver homogenates with isopropanol were measured for the levels of hepatic total cholesterol (TC), triglycerides (TG) based on enzymatic colorimetric methods. Measurement of malonaldehyde (MDA), glutathione (GSH) and Superoxide dismutase (SOD) by enzymatic colorimetric methods were based on thiobarbituric acid, improved dithiodimorpholine nitrobenzoic acid and xanthine/xanthine oxidase, respectively. Real time-PCR and Western Blot were used to detect the expression of transferrin receptor (TfR) at mRNA and protein levels.Results:1. During the pair-feeding, there is no difference of the food intake in all groups. However, weights of the mice challenged by ethanol were lower than those of mice fed by non ethanol-containned diets. What’s more, querectin or iron had no effect on weight gain. Compared to normal control group, ethanol or iron caused significant increase in the liver weight ratio which was further enhanced by combined treatment. Importantly, quercetin evidently decreased the liver weight ratio induced by ethanol and/or iron.2. Compared to normal control mice, ethanol or iron incresed fatty infiltration in the liver, accompanying with increased hepatic TC and TG content and serum ALT and AST level, and the extent of fatty infiltration manifested as macrovesicular steatosis was much higher in mice ingesting ethanol in combination with iron with highest content of hepatic TC and TG and highest level of serum ALT and AST. Quercetin supplementation daily to ethanol and/or iron-fed mice evidently alleviated the hepatic lipid accumulation, the levels of hepatic lipids indexes and serum aminotransferases. Quercetin per se has no any effect on pathological changes in the liver, hepatic lipids parameters and serum aminotransferases in normal mice.3. In comparison with normal control, ethanol increased stainable ferric iron in liver, accompanying hepatic total iron content and serum ferritin and transferrin saturation (TS) level resulted in evident oxidative damage, and the extent was much higher in mice challenged by iron. In addition, ethanol further significantly aggravated the indexes of iron-load mice. Importantly, quercetin intervention to ethanol and/or iron-challenged mice significantly lessened the hepatic ferric iron, hepatic total iron content, serum ferritin and TS and hepatic oxidative stress.4. Expression of TfRl at mRNA level in mice challenged by ethanol or iron were reduced compared to normal control, the decreased extent was exaggavated by combinationed treatment. However, compared to normal control, TfRl protein expression in mice challenged by ethanol or iron were increased, and the increased extent was exaggavated by combinationed treatment. Levels of TfRl were partially normalized by quercetin intervention. In addition, ethanol had no contribution to TfR2expression which was significantly enhanced by iron. Furthermore, ethanol had no evident effect on the increase of TfR2expression induced by iron. Quercetin exhibited insignificant changes of TfR2level induced by iron.Conclusion:Quercetin exhibited protective role on alcoholic iron overload-induced liver damage, and regulation of TfR1expression may be the potential mechanism. Objective:The purpose of this part was to explore the mechanisms by which quercetin arrests alcoholic liver "free" iron disorder and the role of critical molecules responsible for "free" iron uptake and release.Methods:1. The groups of animals were the same to part1.2. Detection of hepatic labile iron pool-Fe (LIP-Fe) was based on ultrafiltrated on Micron-30with supernatants homogenated by1mM EDTA to dissociate LIP-Fe. Then, the concentration of hepatic LIP-Fe was measuremed by atomic absorption spectrophotometric assay.3. Plasma non-transferrin bingding iron (NTBI) was measured using fluorescein-labeled apotransferrin which quenched fluorescence upon binding iron.4. Real time-PCR and Western Blot were used to detect the expression of divalent metal transporter1(DMT1), zinc transporter member14(ZIP14), transient receptor potential mucolipin1(TRPML1) and Ft at both mRNA and protein levels.Results:1. In contrast with normal control, serum NTBI concentration as well as expression of DMT1and ZIP14was significantly elevated by chronic ethanol treatment. Iron-fed or co-treated mice also showed the similar change of serum NTBI content and hepatic DMT1and ZIP14levels to ethanol-challenged mice. Importantly, quercetin evidently inhibited the elevation of serum NTBI concentration and hepatic expression of DMT1and ZIP14.2. Compared with normal control, ethanol or iron elevated the increase of hepatic LIP-Fe levels and expression of TRPML1, the increase was also observed in co-dosed mice by ethanol and iron. In comparison with ethanol and/or iron-exposed mice, hepatic LIP-Fe and expression of TRPML1was decreased as a result of quercetin supplementation. Quercetin itself had no overt influence on basal LIP-Fe and TRPML1level of mice fed by normal diet. 3. Based on normal control, alcohol-fed mice showed an evident increase of FL expression. Compared to ethanol-challeged mice, the levels were further increased by iron ingesting. What’s more co-fed with ethanol and iron notably enhanced the expression of FL in contrast with normal control and any single-treated groups. Quercetin intervention effectively reduced the levels of FL in mice ingested with alcohol and iron alone or combination. In contrast, alcohol had no significant effect on the expression of FH.Conclusion:Quercetin prevents ethanol-induced iron disequilibrium, which may be partially attributed to the suppression of ethanol-stimulated overexpression of crucial molecules in response of "free" iron uptake and release. These findings also suggest endosome and lysosome are the potential source of LIP-Fe. Objective:The aim of this section was to explore the effect of naturally-occurring hepatoprotective quercetin on ethanol-induced hepatic iron overload and damage, focusing on the signaling pathway of iron regulatory hormone hepcidin and the porential mechanism of BMP6/Smad4.Methods:1. The groups of animals were the same to part12. Primary hepatocytes were isolated from C57BL/6J mice using a two-step collagenase perfusion procedure. Hepatocytes were incubated with100mmol/L ethanol, with or without quercetin (100μmol/L), human recombination BMP6protein (100ng/ml) and anti-BMP antibody (25μg/ml) for24hours.3. Fixed liver sample incubated with antibody and immunostaining was visualized with3,3’-diaminobenzidine to detect the protein expression of hepcidin. Real time-PCR and Western Blot were used to detect the expression of hepcidin, BMPs and Smad4at both mRNA and protein levels.4. Chromatin Immunoprecipitation (CHIP) was used to detect the binding activity of Smad4to the promoter of HAMP DNA with chromatin isolated from mouse hepatocytes treated with various pharmacological reagents after shearing by sonication and immunoprecipitation by anti-Smad4antibody.Results:1. Mice fed with ethanol-containing diet displayed evident decreased hepcidin expression compared to normal control. Moreover, iron-increased hepcidin expression was decreased by ethanol, remained a higher level compared to normal control. The expression of hepcidin were partially normalized when quercetin supplementation to mice challenged by ethanol, iron, or ethanol in combination with iron. Quercetin had no effect on normal mice.2. Compared with normal control, ethanol resulted in insignificant changes on BMPs expression except BMP6, accompanying a significant down-regulated value of Smad4. Iron remarkably induced BMP6and Smad4expression which was significantly abolished by ethanol and iron combined treatment. Importantly, quercetin partially normalized BMP6and Smad4level disturbed by ethanol, iron, or ethanol in combination with iron. Quercetin had no effect on BMP6/Smad4expression compared to normal mice.3. In contrast with normal control, ethanol decreased mRNA level of Smad4and hepcidin and DNA binding activity of Smad4to HAMP promoter. Furthermore, values were further exacerbated by anti-BMP6but reversed by human recombination BMP6protein or quercetin pre-treatment. Compared to normal control, quercetin had no effect on hepcidin and Smad4mRNA expression and DNA binding activity of Smad4to HAMP promoter.Conclusion:Quercetin effectively prevents ethanol hepatocixity, which may be attributed by the regulation of hepcidin expression via BMP6/Smad4signaling pathway.