槲皮素对酒精性肝铁过载的保护效应及其机制研究
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摘要
第一部分槲皮素对酒精性肝铁过载的保护效应
目的：以长期暴露酒精和/或铁的C57BL/6J小鼠为研究对象，探讨槲皮素对酒精性肝铁过载时氧化损伤的保护效应。
方法：
1.分组：120只体重为18-20g的雄性C57BL/6J小鼠随机分为八组：正常对照组（喂饲正常Lieber De Carli饲料）、酒精组（喂饲含酒精（30%能量）的Lieber De Carli饲料）、槲皮素对照组（100mg/kg.bw. ig.）、槲皮素＋酒精组、铁组（羰基铁w/v:0.2%）、酒精＋铁联合组、槲皮素＋铁组、槲皮素＋酒精＋铁组。
2.测定指标：小鼠等能量配对喂养15w至模型成功后，隔夜禁食，眼球取血，分离血清，根据酶动力学法测定血清谷草转氨酶（aspartate aminotransferase，AST）和谷丙转氨酶（alanine aminotransferase，ALT）水平，ELISA方法测定血清铁蛋白（ferritin, Ft）含量。肝脏固定后用于HE染色和铁普鲁士蓝染色；肝脏组织冰冻切片孵育DHE荧光探针检测肝脏ROS水平；酶学比色法测定肝脏总胆固醇（Total Cholesterol, TC）和总甘油三酯（Total triglyceride, TG）水平；硫代巴比妥酸比色法、改良二硫代二硝基苯甲酸比色法和黄嘌呤氧化酶比色法分别测定肝脏丙二醛（Malonaldehyde, MDA）、谷胱甘肽（Glutathione, GSH）和超氧化物歧化酶（Superoxide dismutase, SOD）的水平；实时定量聚合酶链反应（real-timePCR, qRT-PCR）和Western Blot方法分别测定肝脏中转铁蛋白受体（transferrinreceptor, TfR）mRNA和蛋白表达水平。
结果：
1.能量配对喂养期间，各组小鼠日均进食能量无差异，但所有含酒精组小鼠体重轻于不含酒精组小鼠，槲皮素或铁对小鼠体重无影响。与正常对照组比较，酒精或铁喂养小鼠的肝脏体重比增加，酒精和铁的联合组进一步增加，槲皮素可以明显降低酒精和铁单独或者联合作用增加的肝脏体重比系数。
2.与正常对照组相比，酒精或铁均明显引起肝脏脂肪变性，导致肝脏TC、TG含量及血清AST、ALT水平升高；两者联合染毒后脂肪泡进一步增大，肝脏TC、TG与血清AST、ALT水平进一步升高；槲皮素干预有效改善酒精和/或铁单独或联合染毒时引起的肝脏脂肪变性和转氨酶释放。
3.长期酒精暴露尤其是铁染毒后小鼠肝脏总铁含量、血清Ft和转铁蛋白饱和度（transferrin, TS）水平升高，肝脏铁沉积明显，GSH水平与SOD活性下降而ROS、MDA明显升高；两者联合染毒后铁过载与氧化应激程度进一步升高；槲皮素干预有效改善酒精和/或铁单独或联合染毒时引起的肝脏铁过载与氧化应激。
4.与正常对照组相比，长期酒精或铁单独暴露后小鼠肝脏TfR1mRNA水平下降，但其蛋白表达水平升高，联合染毒时TfR1的mRNA水平进一步下降而蛋白水平进一步升高；酒精和铁单独或者联合喂养小鼠经槲皮素干预后，TfR1表达基本恢复正常水平。与正常对照组相比，酒精对TfR2表达无明显影响，但铁单独或联合酒精染毒时均明显增加TfR2的表达（单独与联合染毒时TfR2的表达水平无差异）；槲皮素干预对酒精和/或铁单独或联合染毒小鼠肝脏TfR2表达无明显影响。
5.槲皮素对正常小鼠肝脏损伤、肝脏铁含量和血清Ft和TS水平、氧化损伤和TfR1或TfR2表达均无明显影响。
结论：槲皮素对酒精性肝铁过载以及氧化损伤具有良好的拮抗作用，其机制可能与下调TfR1的蛋白表达有关。
第二部分槲皮素对酒精性肝过载时“游离”铁水平的影响及相关机制
目的：本部分旨在分析慢性酒精暴露诱导小鼠铁过载时“游离”铁水平及其摄取与释放分子的变化，以及槲皮素的拮抗效应及相关机制。
方法：
1.动物分组同前；
2.肝脏LIP测定：含EDTA匀浆介质螯合肝脏中不稳定铁池（labile iron pool,LIP），经超滤管离心后采用火焰原子吸收测定仪测定；
3.血清NTBI测定：采用带有荧光标记比色法的原理测定血清非转铁蛋白结合铁（non-transferrin binding iron, NTBI）；
4. qRT-PCR和Western blot方法分别测定二价金属离子转运蛋白1（Twodivalent metal transporter1, DMT1）、锌转运成员的14（Zinc transporter member14,ZIP14）、瞬时受体潜在粘脂蛋白（transient receptor potential mucolipin1, TRPML1）及Ft的mRNA和蛋白表达水平。
结果：
1.与正常对照组比较，酒精和铁单独或者联合组均升高血清NTBI水平，同时肝脏DMT1和ZIP14的表达均增加，槲皮素干预不同程度的降低酒精和铁增加的血清NTBI水平以及肝脏DMT1和ZIP14表达；
2.与正常对照相比，酒精与铁染毒后肝脏LIP-Fe水平分别增加1.2倍和4.1倍，两者联合染毒后LIP-Fe水平更是增加6.5倍，肝脏TRPML1mRNA与蛋白表达水平也相应增加，槲皮素干预降低酒精、铁以及联合组的LIP-Fe水平及TRPML1的表达，而槲皮素对正常小鼠以上指标无明显影响；
3.与正常对照组相比，长期酒精摄入尤其是铁暴露明显增加肝脏铁蛋白轻链（ferritin light chain, FL）的表达，但对重链（ferritin heavy chain, FH）无明显影响；联合染毒后FL与FH却相较于正常对照组均有明显升高；槲皮素对酒精或铁染毒所致的FL升高及两者联合作用下FL与FH的升高均有明显的拮抗作用；
4.与正常对照组相比，槲皮素组血清NTBI与肝脏LIP-Fe水平及FH、FL表达均没有明显变化，肝脏DMT1、ZIP14、TRPML1和Ft表达也没有明显变化。
结论：槲皮素可拮抗酒精继发性肝铁过载时“游离”铁的紊乱，可能与其调节肝细胞负责摄取血清NTBI和胞浆释放LIP-Fe的关键分子有关,并提示内涵体和溶酶体均是酒精性肝铁过载LIP-Fe的来源。
第三部分槲皮素对hepcidin依赖的酒精性肝铁过载的拮抗效应：BMP6/Smad4的作用
目的：以长期喂饲含酒精和/或铁的液体饲料的C57BL/6J小鼠和酒精孵育的小鼠原代肝细胞为研究对象，探讨hepcidin在槲皮素改善小鼠酒精性肝铁过载中的作用以及骨形态蛋白6（Bone morphogenetic protein6, BMP6）/Smad家族成员4（Smad family member4, Smad4）信号通路潜在效应。
方法：
1.动物分组同前；
2.二步胶原酶法分离培养小鼠原代肝细胞，无水乙醇（100mmol/L）单独或联合槲皮素（Et+Qu,100μmol/L）、人重组BMP6蛋白（Et+BMP6,100ng/ml）、抗BMP6抗体（Et+anti-BMP6,25μg/ml）孵育小鼠原代肝细胞24h；
3.免疫组化方法测定肝脏Hepcidin的表达，qRT-PCR测定肝脏Hepcidin、BMP2、BMP4、BMP6、BMP9和Smad4以及小鼠原代细胞Hepcidin、BMP6和Smad4的mRNA表达水平，Western Blot测定肝脏BMP6和Smad4以及小鼠原代肝细胞胞浆和胞核Smad4的表达；
4.染色质免疫共沉淀方法测定酒精、槲皮素和BMP6抑制剂或激活剂孵育的小鼠原代肝细胞Smad4与HAMP启动区DNA结合活性。
结果：
1.与正常对照组相比，铁明显增加肝脏hepcidin表达，而酒精明显减少hepcidin的表达，且降低铁对hepcidin的诱导（但仍高于正常对照组）；酒精和铁单独或者联合喂养小鼠，经槲皮素干预后肝脏hepcidin表达接近正常对照组水平；
2.酒精对BMP2、BMP4和BMP9的mRNA表达无明显影响，但明显降低BMP6及其下游蛋白Smad4的mRNA和蛋白水平，同时抑制铁对BMP6及SMAD4的诱导，酒精和铁单独或者联合作用引起的BMP6和Smad4的变化，经槲皮素干预后均趋于正常对照组水平；槲皮素对正常小鼠肝脏BMP6及Smad4的表达无明显影响；
3.酒精孵育小鼠原代肝细胞降低Smad4与HAMP的DNA结合力，anti-BMP6进一步降低酒精对Smad4与HAMP的DNA结合力及hepcidin mRNA表达；人重组BMP6蛋白及槲皮素处理酒精孵育的肝细胞，均降低酒精对Smad4与HAMP的DNA结合力的抑制，增加hepcidin的mRNA表达。
结论：槲皮素通过BMP6/Smad4信号通路调节hepcidin的表达，保护酒精性肝铁过载损伤。
Part1Protection of quercetin against ethanol hepatocixity mediatedby iron overload
Objective: To investigate the protection of quercetin against iron overload inC57BL/6J mice fed by ethanol-containned Lieber De Carli diets.
Methods:
1. One hundred and twelve male C57BL/6J mice were divided randomly intoeight groups of fifteen animals each as follows:(1) Normal control group (Ct)received regular-containing Lieber De Carli liquids diets;(2) Ethanol group (Et) wasfed 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 irongroup (Et+Fe);(7) Quercetin plus iron group (Qu+Fe);(8) Quercetin, ethanol plusiron 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 aspartateaminotransferase (AST), alanine aminotransferase (ALT) and ferritin (Ft) weremeasured with appropriate kits based on enzymatic kinetic method and ELISA,respectively. Liver tissue samples were fixed and tissue sections were stained withhematoxylin 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 thelevels of hepatic total cholesterol (TC), triglycerides (TG) based on enzymaticcolorimetric methods. Measurement of malonaldehyde (MDA), glutathione (GSH) andSuperoxide dismutase (SOD) by enzymatic colorimetric methods were based onthiobarbituric acid, improved dithiodimorpholine nitrobenzoic acid andxanthine/xanthine oxidase, respectively. Real time-PCR and Western Blot were usedto 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 micefed by non ethanol-containned diets. What’s more, querectin or iron had no effect onweight gain. Compared to normal control group, ethanol or iron caused significantincrease in the liver weight ratio which was further enhanced by combined treatment.Importantly，quercetin evidently decreased the liver weight ratio induced by ethanoland/or iron.
2. Compared to normal control mice, ethanol or iron incresed fatty infiltration inthe liver, accompanying with increased hepatic TC and TG content and serum ALTand AST level, and the extent of fatty infiltration manifested as macrovesicularsteatosis was much higher in mice ingesting ethanol in combination with iron withhighest 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 alleviatedthe hepatic lipid accumulation, the levels of hepatic lipids indexes and serumaminotransferases. Quercetin per se has no any effect on pathological changes in theliver, hepatic lipids parameters and serum aminotransferases in normal mice.
3. In comparison with normal control, ethanol increased stainable ferric iron inliver, accompanying hepatic total iron content and serum ferritin and transferrinsaturation (TS) level resulted in evident oxidative damage, and the extent was muchhigher in mice challenged by iron. In addition, ethanol further significantly aggravatedthe indexes of iron-load mice. Importantly, quercetin intervention to ethanol and/oriron-challenged mice significantly lessened the hepatic ferric iron, hepatic total iron content, serum ferritin and TS and hepatic oxidative stress.
4. Expression of TfR1at mRNA level in mice challenged by ethanol or iron werereduced compared to normal control, the decreased extent was exaggavated bycombinationed treatment. However, compared to normal control, TfR1proteinexpression in mice challenged by ethanol or iron were increased, and the increasedextent was exaggavated by combinationed treatment. Levels of TfR1were partiallynormalized by quercetin intervention. In addition, ethanol had no contribution to TfR2expression which was significantly enhanced by iron. Furthermore, ethanol had noevident effect on the increase of TfR2expression induced by iron. Quercetin exhibitedinsignificant changes of TfR2level induced by iron.
Conclusion: Quercetin exhibited protective role on alcoholic ironoverload-induced liver damage, and regulation of TfR1expression may be thepotential mechanism.
Part2Quercetin against “free” iron in chronic ethanol induced liveriron overload
Objective: The purpose of this part was to explore the mechanisms by whichquercetin arrests alcoholic liver “free” iron disorder and the role of critical moleculesresponsible 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 ultrafiltratedon Micron-30with supernatants homogenated by1mM EDTA to dissociate LIP-Fe.Then, the concentration of hepatic LIP-Fe was measuremed by atomic absorptionspectrophotometric assay.
3. Plasma non-transferrin bingding iron (NTBI) was measured usingfluorescein-labeled apotransferrin which quenched fluorescence upon binding iron.
4. Real time-PCR and Western Blot were used to detect the expression ofdivalent metal transporter1(DMT1), zinc transporter member14(ZIP14), transientreceptor potential mucolipin1(TRPML1) and Ft at both mRNA and protein levels.
Results:
1. In contrast with normal control, serum NTBI concentration as well asexpression of DMT1and ZIP14was significantly elevated by chronic ethanoltreatment. Iron-fed or co-treated mice also showed the similar change of serum NTBIcontent and hepatic DMT1and ZIP14levels to ethanol-challenged mice. Importantly,quercetin evidently inhibited the elevation of serum NTBI concentration and hepaticexpression of DMT1and ZIP14.
2. Compared with normal control, ethanol or iron elevated the increase of hepaticLIP-Fe levels and expression of TRPML1, the increase was also observed in co-dosedmice 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 quercetinsupplementation. Quercetin itself had no overt influence on basal LIP-Fe andTRPML1level of mice fed by normal diet.
3. Based on normal control, alcohol-fed mice showed an evident increase of FLexpression. Compared to ethanol-challeged mice, the levels were further increased byiron ingesting. What’s more co-fed with ethanol and iron notably enhanced theexpression of FL in contrast with normal control and any single-treated groups.Quercetin intervention effectively reduced the levels of FL in mice ingested withalcohol and iron alone or combination. In contrast, alcohol had no significant effect onthe expression of FH.
Conclusion: Quercetin prevents ethanol-induced iron disequilibrium, which maybe partially attributed to the suppression of ethanol-stimulated overexpression ofcrucial molecules in response of “free” iron uptake and release. These findings alsosuggest endosome and lysosome are the potential source of LIP-Fe.
Part3Quercetin prevents ethanol-induced iron overload byregulating hepcidin through BMP6/Smad4signaling pathway
Objective: The aim of this section was to explore the effect ofnaturally-occurring hepatoprotective quercetin on ethanol-induced hepatic ironoverload and damage, focusing on the signaling pathway of iron regulatory hormonehepcidin and the porential mechanism of BMP6/Smad4.
Methods:
1. The groups of animals were the same to part1
2. Primary hepatocytes were isolated from C57BL/6J mice using a two-stepcollagenase perfusion procedure. Hepatocytes were incubated with100mmol/Lethanol, 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 wasvisualized 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 bindingactivity of Smad4to the promoter of HAMP DNA with chromatin isolated frommouse hepatocytes treated with various pharmacological reagents after shearing bysonication and immunoprecipitation by anti-Smad4antibody.
Results:
1. Mice fed with ethanol-containing diet displayed evident decreased hepcidinexpression compared to normal control. Moreover, iron-increased hepcidin expressionwas decreased by ethanol, remained a higher level compared to normal control. Theexpression of hepcidin were partially normalized when quercetin supplementation tomice challenged by ethanol, iron, or ethanol in combination with iron. Quercetin hadno effect on normal mice.
2. Compared with normal control, ethanol resulted in insignificant changes onBMPs expression except BMP6, accompanying a significant down-regulated value of Smad4. Iron remarkably induced BMP6and Smad4expression which wassignificantly abolished by ethanol and iron combined treatment. Importantly,quercetin partially normalized BMP6and Smad4level disturbed by ethanol, iron, orethanol 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 Smad4andhepcidin and DNA binding activity of Smad4to HAMP promoter. Furthermore,values were further exacerbated by anti-BMP6but reversed by human recombinationBMP6protein or quercetin pre-treatment. Compared to normal control, quercetin hadno effect on hepcidin and Smad4mRNA expression and DNA binding activity ofSmad4to HAMP promoter.
Conclusion: Quercetin effectively prevents ethanol hepatocixity, which may beattributed by the regulation of hepcidin expression via BMP6/Smad4signalingpathway.
目的：以长期暴露酒精和/或铁的C57BL/6J小鼠为研究对象，探讨槲皮素对酒精性肝铁过载时氧化损伤的保护效应。
方法：
1.分组：120只体重为18-20g的雄性C57BL/6J小鼠随机分为八组：正常对照组（喂饲正常Lieber De Carli饲料）、酒精组（喂饲含酒精（30%能量）的Lieber De Carli饲料）、槲皮素对照组（100mg/kg.bw. ig.）、槲皮素＋酒精组、铁组（羰基铁w/v:0.2%）、酒精＋铁联合组、槲皮素＋铁组、槲皮素＋酒精＋铁组。
2.测定指标：小鼠等能量配对喂养15w至模型成功后，隔夜禁食，眼球取血，分离血清，根据酶动力学法测定血清谷草转氨酶（aspartate aminotransferase，AST）和谷丙转氨酶（alanine aminotransferase，ALT）水平，ELISA方法测定血清铁蛋白（ferritin, Ft）含量。肝脏固定后用于HE染色和铁普鲁士蓝染色；肝脏组织冰冻切片孵育DHE荧光探针检测肝脏ROS水平；酶学比色法测定肝脏总胆固醇（Total Cholesterol, TC）和总甘油三酯（Total triglyceride, TG）水平；硫代巴比妥酸比色法、改良二硫代二硝基苯甲酸比色法和黄嘌呤氧化酶比色法分别测定肝脏丙二醛（Malonaldehyde, MDA）、谷胱甘肽（Glutathione, GSH）和超氧化物歧化酶（Superoxide dismutase, SOD）的水平；实时定量聚合酶链反应（real-timePCR, qRT-PCR）和Western Blot方法分别测定肝脏中转铁蛋白受体（transferrinreceptor, TfR）mRNA和蛋白表达水平。
结果：
1.能量配对喂养期间，各组小鼠日均进食能量无差异，但所有含酒精组小鼠体重轻于不含酒精组小鼠，槲皮素或铁对小鼠体重无影响。与正常对照组比较，酒精或铁喂养小鼠的肝脏体重比增加，酒精和铁的联合组进一步增加，槲皮素可以明显降低酒精和铁单独或者联合作用增加的肝脏体重比系数。
2.与正常对照组相比，酒精或铁均明显引起肝脏脂肪变性，导致肝脏TC、TG含量及血清AST、ALT水平升高；两者联合染毒后脂肪泡进一步增大，肝脏TC、TG与血清AST、ALT水平进一步升高；槲皮素干预有效改善酒精和/或铁单独或联合染毒时引起的肝脏脂肪变性和转氨酶释放。
3.长期酒精暴露尤其是铁染毒后小鼠肝脏总铁含量、血清Ft和转铁蛋白饱和度（transferrin, TS）水平升高，肝脏铁沉积明显，GSH水平与SOD活性下降而ROS、MDA明显升高；两者联合染毒后铁过载与氧化应激程度进一步升高；槲皮素干预有效改善酒精和/或铁单独或联合染毒时引起的肝脏铁过载与氧化应激。
4.与正常对照组相比，长期酒精或铁单独暴露后小鼠肝脏TfR1mRNA水平下降，但其蛋白表达水平升高，联合染毒时TfR1的mRNA水平进一步下降而蛋白水平进一步升高；酒精和铁单独或者联合喂养小鼠经槲皮素干预后，TfR1表达基本恢复正常水平。与正常对照组相比，酒精对TfR2表达无明显影响，但铁单独或联合酒精染毒时均明显增加TfR2的表达（单独与联合染毒时TfR2的表达水平无差异）；槲皮素干预对酒精和/或铁单独或联合染毒小鼠肝脏TfR2表达无明显影响。
5.槲皮素对正常小鼠肝脏损伤、肝脏铁含量和血清Ft和TS水平、氧化损伤和TfR1或TfR2表达均无明显影响。
结论：槲皮素对酒精性肝铁过载以及氧化损伤具有良好的拮抗作用，其机制可能与下调TfR1的蛋白表达有关。
第二部分槲皮素对酒精性肝过载时“游离”铁水平的影响及相关机制
目的：本部分旨在分析慢性酒精暴露诱导小鼠铁过载时“游离”铁水平及其摄取与释放分子的变化，以及槲皮素的拮抗效应及相关机制。
方法：
1.动物分组同前；
2.肝脏LIP测定：含EDTA匀浆介质螯合肝脏中不稳定铁池（labile iron pool,LIP），经超滤管离心后采用火焰原子吸收测定仪测定；
3.血清NTBI测定：采用带有荧光标记比色法的原理测定血清非转铁蛋白结合铁（non-transferrin binding iron, NTBI）；
4. qRT-PCR和Western blot方法分别测定二价金属离子转运蛋白1（Twodivalent metal transporter1, DMT1）、锌转运成员的14（Zinc transporter member14,ZIP14）、瞬时受体潜在粘脂蛋白（transient receptor potential mucolipin1, TRPML1）及Ft的mRNA和蛋白表达水平。
结果：
1.与正常对照组比较，酒精和铁单独或者联合组均升高血清NTBI水平，同时肝脏DMT1和ZIP14的表达均增加，槲皮素干预不同程度的降低酒精和铁增加的血清NTBI水平以及肝脏DMT1和ZIP14表达；
2.与正常对照相比，酒精与铁染毒后肝脏LIP-Fe水平分别增加1.2倍和4.1倍，两者联合染毒后LIP-Fe水平更是增加6.5倍，肝脏TRPML1mRNA与蛋白表达水平也相应增加，槲皮素干预降低酒精、铁以及联合组的LIP-Fe水平及TRPML1的表达，而槲皮素对正常小鼠以上指标无明显影响；
3.与正常对照组相比，长期酒精摄入尤其是铁暴露明显增加肝脏铁蛋白轻链（ferritin light chain, FL）的表达，但对重链（ferritin heavy chain, FH）无明显影响；联合染毒后FL与FH却相较于正常对照组均有明显升高；槲皮素对酒精或铁染毒所致的FL升高及两者联合作用下FL与FH的升高均有明显的拮抗作用；
4.与正常对照组相比，槲皮素组血清NTBI与肝脏LIP-Fe水平及FH、FL表达均没有明显变化，肝脏DMT1、ZIP14、TRPML1和Ft表达也没有明显变化。
结论：槲皮素可拮抗酒精继发性肝铁过载时“游离”铁的紊乱，可能与其调节肝细胞负责摄取血清NTBI和胞浆释放LIP-Fe的关键分子有关,并提示内涵体和溶酶体均是酒精性肝铁过载LIP-Fe的来源。
第三部分槲皮素对hepcidin依赖的酒精性肝铁过载的拮抗效应：BMP6/Smad4的作用
目的：以长期喂饲含酒精和/或铁的液体饲料的C57BL/6J小鼠和酒精孵育的小鼠原代肝细胞为研究对象，探讨hepcidin在槲皮素改善小鼠酒精性肝铁过载中的作用以及骨形态蛋白6（Bone morphogenetic protein6, BMP6）/Smad家族成员4（Smad family member4, Smad4）信号通路潜在效应。
方法：
1.动物分组同前；
2.二步胶原酶法分离培养小鼠原代肝细胞，无水乙醇（100mmol/L）单独或联合槲皮素（Et+Qu,100μmol/L）、人重组BMP6蛋白（Et+BMP6,100ng/ml）、抗BMP6抗体（Et+anti-BMP6,25μg/ml）孵育小鼠原代肝细胞24h；
3.免疫组化方法测定肝脏Hepcidin的表达，qRT-PCR测定肝脏Hepcidin、BMP2、BMP4、BMP6、BMP9和Smad4以及小鼠原代细胞Hepcidin、BMP6和Smad4的mRNA表达水平，Western Blot测定肝脏BMP6和Smad4以及小鼠原代肝细胞胞浆和胞核Smad4的表达；
4.染色质免疫共沉淀方法测定酒精、槲皮素和BMP6抑制剂或激活剂孵育的小鼠原代肝细胞Smad4与HAMP启动区DNA结合活性。
结果：
1.与正常对照组相比，铁明显增加肝脏hepcidin表达，而酒精明显减少hepcidin的表达，且降低铁对hepcidin的诱导（但仍高于正常对照组）；酒精和铁单独或者联合喂养小鼠，经槲皮素干预后肝脏hepcidin表达接近正常对照组水平；
2.酒精对BMP2、BMP4和BMP9的mRNA表达无明显影响，但明显降低BMP6及其下游蛋白Smad4的mRNA和蛋白水平，同时抑制铁对BMP6及SMAD4的诱导，酒精和铁单独或者联合作用引起的BMP6和Smad4的变化，经槲皮素干预后均趋于正常对照组水平；槲皮素对正常小鼠肝脏BMP6及Smad4的表达无明显影响；
3.酒精孵育小鼠原代肝细胞降低Smad4与HAMP的DNA结合力，anti-BMP6进一步降低酒精对Smad4与HAMP的DNA结合力及hepcidin mRNA表达；人重组BMP6蛋白及槲皮素处理酒精孵育的肝细胞，均降低酒精对Smad4与HAMP的DNA结合力的抑制，增加hepcidin的mRNA表达。
结论：槲皮素通过BMP6/Smad4信号通路调节hepcidin的表达，保护酒精性肝铁过载损伤。
Part1Protection of quercetin against ethanol hepatocixity mediatedby iron overload
Objective: To investigate the protection of quercetin against iron overload inC57BL/6J mice fed by ethanol-containned Lieber De Carli diets.
Methods:
1. One hundred and twelve male C57BL/6J mice were divided randomly intoeight groups of fifteen animals each as follows:(1) Normal control group (Ct)received regular-containing Lieber De Carli liquids diets;(2) Ethanol group (Et) wasfed 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 irongroup (Et+Fe);(7) Quercetin plus iron group (Qu+Fe);(8) Quercetin, ethanol plusiron 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 aspartateaminotransferase (AST), alanine aminotransferase (ALT) and ferritin (Ft) weremeasured with appropriate kits based on enzymatic kinetic method and ELISA,respectively. Liver tissue samples were fixed and tissue sections were stained withhematoxylin 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 thelevels of hepatic total cholesterol (TC), triglycerides (TG) based on enzymaticcolorimetric methods. Measurement of malonaldehyde (MDA), glutathione (GSH) andSuperoxide dismutase (SOD) by enzymatic colorimetric methods were based onthiobarbituric acid, improved dithiodimorpholine nitrobenzoic acid andxanthine/xanthine oxidase, respectively. Real time-PCR and Western Blot were usedto 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 micefed by non ethanol-containned diets. What’s more, querectin or iron had no effect onweight gain. Compared to normal control group, ethanol or iron caused significantincrease in the liver weight ratio which was further enhanced by combined treatment.Importantly，quercetin evidently decreased the liver weight ratio induced by ethanoland/or iron.
2. Compared to normal control mice, ethanol or iron incresed fatty infiltration inthe liver, accompanying with increased hepatic TC and TG content and serum ALTand AST level, and the extent of fatty infiltration manifested as macrovesicularsteatosis was much higher in mice ingesting ethanol in combination with iron withhighest 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 alleviatedthe hepatic lipid accumulation, the levels of hepatic lipids indexes and serumaminotransferases. Quercetin per se has no any effect on pathological changes in theliver, hepatic lipids parameters and serum aminotransferases in normal mice.
3. In comparison with normal control, ethanol increased stainable ferric iron inliver, accompanying hepatic total iron content and serum ferritin and transferrinsaturation (TS) level resulted in evident oxidative damage, and the extent was muchhigher in mice challenged by iron. In addition, ethanol further significantly aggravatedthe indexes of iron-load mice. Importantly, quercetin intervention to ethanol and/oriron-challenged mice significantly lessened the hepatic ferric iron, hepatic total iron content, serum ferritin and TS and hepatic oxidative stress.
4. Expression of TfR1at mRNA level in mice challenged by ethanol or iron werereduced compared to normal control, the decreased extent was exaggavated bycombinationed treatment. However, compared to normal control, TfR1proteinexpression in mice challenged by ethanol or iron were increased, and the increasedextent was exaggavated by combinationed treatment. Levels of TfR1were partiallynormalized by quercetin intervention. In addition, ethanol had no contribution to TfR2expression which was significantly enhanced by iron. Furthermore, ethanol had noevident effect on the increase of TfR2expression induced by iron. Quercetin exhibitedinsignificant changes of TfR2level induced by iron.
Conclusion: Quercetin exhibited protective role on alcoholic ironoverload-induced liver damage, and regulation of TfR1expression may be thepotential mechanism.
Part2Quercetin against “free” iron in chronic ethanol induced liveriron overload
Objective: The purpose of this part was to explore the mechanisms by whichquercetin arrests alcoholic liver “free” iron disorder and the role of critical moleculesresponsible 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 ultrafiltratedon Micron-30with supernatants homogenated by1mM EDTA to dissociate LIP-Fe.Then, the concentration of hepatic LIP-Fe was measuremed by atomic absorptionspectrophotometric assay.
3. Plasma non-transferrin bingding iron (NTBI) was measured usingfluorescein-labeled apotransferrin which quenched fluorescence upon binding iron.
4. Real time-PCR and Western Blot were used to detect the expression ofdivalent metal transporter1(DMT1), zinc transporter member14(ZIP14), transientreceptor potential mucolipin1(TRPML1) and Ft at both mRNA and protein levels.
Results:
1. In contrast with normal control, serum NTBI concentration as well asexpression of DMT1and ZIP14was significantly elevated by chronic ethanoltreatment. Iron-fed or co-treated mice also showed the similar change of serum NTBIcontent and hepatic DMT1and ZIP14levels to ethanol-challenged mice. Importantly,quercetin evidently inhibited the elevation of serum NTBI concentration and hepaticexpression of DMT1and ZIP14.
2. Compared with normal control, ethanol or iron elevated the increase of hepaticLIP-Fe levels and expression of TRPML1, the increase was also observed in co-dosedmice 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 quercetinsupplementation. Quercetin itself had no overt influence on basal LIP-Fe andTRPML1level of mice fed by normal diet.
3. Based on normal control, alcohol-fed mice showed an evident increase of FLexpression. Compared to ethanol-challeged mice, the levels were further increased byiron ingesting. What’s more co-fed with ethanol and iron notably enhanced theexpression of FL in contrast with normal control and any single-treated groups.Quercetin intervention effectively reduced the levels of FL in mice ingested withalcohol and iron alone or combination. In contrast, alcohol had no significant effect onthe expression of FH.
Conclusion: Quercetin prevents ethanol-induced iron disequilibrium, which maybe partially attributed to the suppression of ethanol-stimulated overexpression ofcrucial molecules in response of “free” iron uptake and release. These findings alsosuggest endosome and lysosome are the potential source of LIP-Fe.
Part3Quercetin prevents ethanol-induced iron overload byregulating hepcidin through BMP6/Smad4signaling pathway
Objective: The aim of this section was to explore the effect ofnaturally-occurring hepatoprotective quercetin on ethanol-induced hepatic ironoverload and damage, focusing on the signaling pathway of iron regulatory hormonehepcidin and the porential mechanism of BMP6/Smad4.
Methods:
1. The groups of animals were the same to part1
2. Primary hepatocytes were isolated from C57BL/6J mice using a two-stepcollagenase perfusion procedure. Hepatocytes were incubated with100mmol/Lethanol, 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 wasvisualized 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 bindingactivity of Smad4to the promoter of HAMP DNA with chromatin isolated frommouse hepatocytes treated with various pharmacological reagents after shearing bysonication and immunoprecipitation by anti-Smad4antibody.
Results:
1. Mice fed with ethanol-containing diet displayed evident decreased hepcidinexpression compared to normal control. Moreover, iron-increased hepcidin expressionwas decreased by ethanol, remained a higher level compared to normal control. Theexpression of hepcidin were partially normalized when quercetin supplementation tomice challenged by ethanol, iron, or ethanol in combination with iron. Quercetin hadno effect on normal mice.
2. Compared with normal control, ethanol resulted in insignificant changes onBMPs expression except BMP6, accompanying a significant down-regulated value of Smad4. Iron remarkably induced BMP6and Smad4expression which wassignificantly abolished by ethanol and iron combined treatment. Importantly,quercetin partially normalized BMP6and Smad4level disturbed by ethanol, iron, orethanol 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 Smad4andhepcidin and DNA binding activity of Smad4to HAMP promoter. Furthermore,values were further exacerbated by anti-BMP6but reversed by human recombinationBMP6protein or quercetin pre-treatment. Compared to normal control, quercetin hadno effect on hepcidin and Smad4mRNA expression and DNA binding activity ofSmad4to HAMP promoter.
Conclusion: Quercetin effectively prevents ethanol hepatocixity, which may beattributed by the regulation of hepcidin expression via BMP6/Smad4signalingpathway.
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