【作者】 张燕；

【导师】 高中洪；

【作者基本信息】 华中科技大学 ， 无机化学， 2009， 博士

【摘要】 随着生活水平的提高，膳食中肉类比重的增加，体内铁超载（Iron overload）的现象值得引起重视。铁超载是指由于遗传因素或病理条件导致机体出现一系列病理变化。近几年来，铁超载所带来的负面效应，已经引起了人们越来越多的关注。铁除了可以通过催化产生自由基而导致脂质过氧化以及蛋白质氧化外，它还可能促进活性氮的生成甚至直接催化活性氮与蛋白质反应而导致蛋白质硝化，同时它还会对体内其它一些微量元素产生影响。铁超载病理条件下，肝中蛋白质硝化情况及对其它微量元素的影响，尚未有人研究过。本文在整体动物、细胞水平和生化水平上，分别从铁超载对微量元素的影响及促进蛋白质氧化和硝化两个方面来研究铁超载肝损伤病理机制，并在此基础上探讨了黄芩苷对铁超载肝损伤的干预作用。主要内容包括以下几个方面：1、选用成年雄性大鼠，采用两周内5次腹腔注射铁右旋糖苷（每次铁量分别为30，60，100 mg Fe／kgbw）的方法造成大鼠慢性铁超载模型；与此同时，对总铁剂量为500 mg Fe／kg的大鼠分别喂以含0．3％和1％黄芩苷的饲料。通过紫外可见分光光度法测定一些基本生化指标、毛细管电泳检测尿液中硝酸盐含量、免疫沉淀及免疫组化分析肝中蛋白质硝化等，来研究铁超载导致肝损伤的情况及黄芩苷的干预作用。结果显示，铁超载可以导致血清铁、转铁蛋白饱和度、谷丙转氨酶（ALT）、谷草转氨酶（AST）、肝中铁含量、丙二醛（MDA）含量、尿液中硝酸盐含量及肝中3-硝基酪氨酸含量显著增加，而肝中总铁结合力及巯基（SH）含量则随给铁剂量的增加而明显降低；补充黄芩苷后能显著降低由铁超载所导致大鼠血清转铁蛋白饱和度、ALT、AST、肝中铁含量、MDA含量、尿液中硝酸盐含量以及蛋白质硝化水平的增加。这表明铁超载除引起肝脏常规指标改变外，同时还可引起蛋白质发生硝化；黄芩苷的摄入能显著减轻铁超载所导致的肝脏损伤。2、选用成年雄性小鼠，采用两周内5次腹腔注射铁右旋糖苷（每次铁量为100 mgFe／kg bw）的方法造成小鼠慢性铁超载模型；同时喂以含1％黄芩苷的饲料。利用HPLC-ICP-MS对肝线粒体、微粒体及上清液三种亚细胞器中微量元素（铁、铜、锌及镉）的含量及分布进行检测。结果显示，铁在肝中主要以高分子量蛋白形式存在，而铜、锌及镉在各种分子量蛋白中均含有。铁超载可导致肝中微量元素锌及镉的含量显著增加，同时对铜和镉在肝中的分布产生影响；黄芩苷的摄入显著降低由铁超载所导致的铁、锌及镉的含量增加。这表明，过多铁在体内聚积将对其它微量元素的代谢产生影响；黄芩苷的摄入能显著降低由铁超载所导致的肝中微量元素的增加。3、选用肝中重要的酶之一一谷氨酸脱氢酶（GDH）做为研究对象，采用蛋白质硝化的两种主要途径（ONOO^-及铁原卟啉（hemin）-H₂O₂-NO₂^-）来引起蛋白质氧化和硝化，通过紫外可见光谱、圆二色谱、凝胶电泳、免疫印迹以及液质联用等方法来研究蛋白质氧化和硝化各自对GDH活性的影响。研究结果表明：（1）在生理pH条件下，Hemin-H₂O₂-NO₂^-和SIN-1均可催化GDH发生蛋白氧化和硝化而使其灭活，但是蛋白质氧化（非巯基氧化）对活性的作用要显著强于蛋白质硝化；（2）Hemin-H₂O₂-NO₂^-和SIN-1虽然都可以导致GDH发生蛋白质氧化和硝化，但两者的作用方式却是不尽相同的。Hemin-H₂O₂-NO₂^-催化GDH发生硝化的酪氨酸残基是Tyr²⁶²和Tyr⁴⁷¹，而SIN-1催化GDH发生硝化的酪氨酸残基则是Tyr⁴⁰¹和Tyr⁴⁹³。4、采用外源性硝化体系（Hemin-H₂O₂-NO₂^-及柠檬酸铁-H₂O₂-NO₂^-）来造成HepG2细胞中氧化（硝化）应激增加，利用免疫沉淀法从其中提取谷胱甘肽硫转移酶（GSTs），然后检测其表达水平、蛋白氧化水平、硝化水平及酶活力情况，以此来探讨铁卟啉和柠檬酸铁在催化H₂O₂-NO₂^-所导致蛋白质氧化和硝化上的差异，并在此基础上探讨黄芩苷的干预作用。研究结果表明：（1）铁卟啉和柠檬酸铁在催化H₂O₂-NO₂^-体系导致蛋白质氧化和硝化的能力上有所差异。Hemin具有较强的催化蛋白质氧化和硝化的作用；而柠檬酸铁仅具有较强的催化蛋白质氧化的作用。（2）黄芩苷的摄入可显著降低由Fe（Ⅲ）-H₂O₂-NO₂^-体系所导致的GSTs蛋白氧化和硝化损伤。更多还原

【Abstract】 With the improving of living standards,the proportion of the meat in daily meal isincreasing,and the iron overload in our body should be deserved our attention.Ironoverload was defined that a series of pathological changes in the body which induced bygenetic factors or pathological conditions.In recent years,the deleteriousness of ironoverload has been given more and more attention.Ironcan catalyze the generation of freeradicals which are able to cause lipid peroxidation and protein oxidation.Meanwhile,itcan promote the generation of reactive nitrogen or even directly catalyze the reaction ofreactive nitrogen and protein which resulted in protein tyrosine nitration.It also has animpact on the metabolism of other trace elements in the body at the same time.However,little was known about the role of tyrosine nitration and the effect of excessive iron onother trace elements in iron overload induced hepatic injury.In this dissertation,theimpacts of excessive iron on other trace elements and oxidative/nitrosative injury of ratliver which induced by iron overload were studied through aimimal model,cellular modeland biochemical analysis.Moreover,the role ofbaicalin was also studied.1) The male rats were used to induce chronic iron overload by peritoneal injection ofiron-dextran five times in two weeks,and supplemented with baicalin containing diet（0.3% and/or 1% w/w） at the same time.Some basic biological indicators,nitrate contentin urine and protein nitration in liver were assayed and the effect of baicalin was studied.The results showed that iron stimulated liver injury in a dose-dependent manner,the levelsof serum non-heme iron,transferin saturation,aspartate aminotransferase （AST） andalanine aminotransferase （ALT） contents were increased and total iron binding capacitywas decreased;the contents of iron,malondialdehyde （MDA）,protein nitration in liverand nitrite content in urine were elevated,whereas liver glutathione （GSH） content wasreduced.Supplemented with baicalin could significantly reduce the increased transferin saturation,ALT,AST,liver iron content,MDA content,nitrate content and proteinnitration caused by iron overload.It meant that iron overload could induce proteinnitration besides of changes of some conventional indicators;and baicalinsupplementation could attenuate liver injury caused by iron overload.2) The mice were peritoneal injection with iron-dextran （100 mg/kg each） was used toinduce chronic iron overload,and supplemented with baicalin containing diet （1% w/w） atthe same time,to study its intervention effects.The high performance liquidchromatography and inductively coupled plasma mass spectrometry （HPLC-ICP-MS） wasused to analyze the subcellular distribution of metal-containing proteins in mouse liver.The results showed that Fe was present only in high-molecular-weight （HMW） protein;Cu,Zn and Cd were found in different MW proteins.And excessive iron accumulation invivo could increase the contents of Zn and Cd,and also affect the distribution of Cu andCd,which demonstrated that iron overload has an impact on the metabolism of other traceelements （such as Cu,Zn and Cd）.Moreover,the contents of Fe,Zn and Cd decreasedafter supplemented with baicalin.3) Glutamate dehydrogenase （GDH）,which is an important enzyme in liver,waschosen to study the difference between the effects of protein nitration and oxidation onprotein function which caused by ONOO^- and/or heme peroxidases （hemin）-H₂O₂-NO₂^-.The results showed that （1） Both hemin-H₂O₂-NO₂^- and SIN-1 could catalyze proteinoxidation and tyrosine nitration of GDH at physiological conditions,and the effect ofprotein oxidation on enzyme activity was stronger than that of tyrosine nitration.（2）Protein oxidation and tyrosine nitration of GDH induced by hemin-H₂O₂-NO₂^- weredifferent from those induced by SIN-1.Mass spectrometric analysis indicated that nitratedtyrosine residues by hemin-H₂O₂-NO₂^- were Tyr²⁶² and Tyr⁴⁷¹ while by SIN-1 were Tyr⁴⁰¹and Tyr⁴⁹³.4) HepG2 cell was incubated with exogenous nitrating agents （hemin-nitrite-H₂O₂ andferric citrate-H₂O₂-NO₂^-）,and then glutathione S-transferase （GSTs） extracted from it was used to study the differences between ferric citrate and heme in catalyting H₂O₂-NO₂^- tocause protein oxidation and tyrosine nitration.Moreover,the effects of baicalin on bothmodels have also been discussed.It was found that there were some differences betweenhemin-H₂O₂-NO₂^- and ferric citrate-H₂O₂-NO₂^- in inducing protein oxidation and nitrationof GSTs of HepG2 cell.Hemin has a strong catalytic role in oxidation and nitration,andferric citrate only has a strong catalytic role of protein oxidation.GSTs expression wassignificantly decreased in the heme-H₂O₂-NO₂^-treated HepG2 cell,while the proteinoxidation and tyrosine nitration was increased.However,ferric citrate-H₂O₂-NO₂^- had nosignificant effect on GSTs expression and tyrosine nitration,and only significantlyincreased GSTs oxidation.Moreover,after supplemented with baicalin,both oxidative andnitrative injuries of GSTs of HepG2 caused by Fe（Ⅲ）- H₂O₂-NO₂^- were reduced.更多还原