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1、黄芪甲苷对“铁超载”所致小鼠肝损伤的保护作用 叶枝红 谢海纳 钱知知 叶晶 姜文静 刘霖 谢东浩 张良 江苏大学药学院 南京中医药大学基础医学院 南京中医药大学药学院 上海市徐汇区大华医院 排版定稿印刷版 摘 要： 目的:研究黄芪甲苷对铁超载造成肝损伤保护的作用机制。方法:小鼠按体重随机分为空白组, 模型组、黄芪甲苷 (As) 低、中、高剂量组。连续给药 45 d, 取血清, 检测血清 Fe, 总铁结合力 (total iron binding capacity, TIBC) , 丙氨酸氨基转移酶 (glutamic-pyruvic transaminase, ALT) , 天门冬氨酸氨基转移
2、酶 (glutamic oxalacetic transaminase, AST) , 总胆红素 (total bilirubin, T-BIL) , 丙二醛 (malondialdehyde, MDA) , 谷胱甘肽 (glutathione, GSH) 水平;用苏木素-伊红 (HE) 染色观察肝脏病理组织学变化;用免疫组化学方法分析肝脏组织蛋白硝化的表达变化;用原位末端转移酶标记技术 (TUNEL) 染色法观察肝细胞凋亡的变化。结果:与空白组比较, 模型组小鼠血清中血清 Fe, ALT, AST, MDA, 肝中铁和 T-BIL 含量显著增加 (P0.01) , 而TIBC 和 GSH 含
3、量显著降低 (P0.05, P0.01) ;与模型组比较, 黄芪甲苷组小鼠血清中血清 Fe, ALT, AST, MDA 水平, 及肝中 Fe 和 T-BIL 含量显著降低 (P0.05, P0.01) , 肝中 TIBC 和 GSH 含量显著增高 (P0.05, P0.01) ;HE结果显示, 与空白组比较, 铁超载组可见肝细胞水样变性、肝细胞脂肪变性和炎症细胞浸润等;不同浓度黄芪甲苷组的上述病理改变得以改善;免疫组化结果发现, 与空白组比较, 铁超载组血管周围细胞浆中 3-硝基酪氨酸 (3-NT) 显著增加, 而黄芪甲苷组血管膜周围的 3-硝基酪氨酸则显著减少;TUNEL 染色发现与空白组
4、比较, 模型组肝细胞凋亡显著增多;与模型组比较, 黄芪甲苷组肝细胞凋亡显著减少。结论:黄芪甲苷对铁超载造成的肝损伤具有保护作用。关键词： 黄芪甲苷; 铁超载; 肝损伤; 凋亡; 作者简介：叶枝红, 硕士, 从事中药治疗标准及中药复方抗肝纤维化作用机制研究, E-mail:作者简介：谢东浩, 博士, 主任中药师, 从事中药治疗标准及中药复方抗肝纤维化作用机制研究;E-mail:;张良, 硕士生导师, 副教授, 从事中药毒理学研究, E-mail:zhangl_收稿日期：2017-10-17基金：上海市徐汇区医学科研项目 (SHXH201310) Protective Effect of Astr
5、agaloside on Liver Injury Induced by Iron Overload in MiceYE Zhi-hong XIE Hai-na QIAN Zhi-zhi YE Jing JIANG Wen-jing LIU Lin XIE Dong-hao ZHANG Liang School of Pharmacy, Jiangsu University; School of Basic Medical Sciences, Nanjing University of Chinese Medicine; School of Pharmacy, Nanjing Universi
6、ty of Chinese Medicine; Dahua Hospital of Xuhui District; Abstract： Objective: To investigate the protective role of astragaloside ( As) in the liver injury caused by iron overload. Method: The mice were randomly divided into blank control group, model group, As low-dose group, As middle-dose group
7、and As high-dose group according to their weight. Mice were intraperitoneally injected with As once daily for 45 d. Then their serum was taken to determine the content of serum iron, total iron binding capacity ( TIBC) , alanine aminotransferase ( ALT) , aspartate aminotransferase ( AST) , total bil
8、irubin ( T-BIL) , malondialdehyde ( MDA) , and glutathione ( GSH) ; pathological changes in liver tissues were examined by hematoxylin-eosin ( HE) staining; the expression changes of protein nitration in liver tissues were detected by immunohistochemical staining; and the changes of the liver apopto
9、sis were observed by Td T-mediated d UTP nick end labeling ( TUNEL) staining. Result: As compared with the blank group, the levels of serum Fe, MDA, ALT, and AST in serum as well as Fe and T-BIL in liver were increased in the model group ( P 0. 01) , while the levels of GSH and TIBC were decreased s
10、ignificantly ( P 0. 05, P 0. 01) . However, as compared with the model group, the levels of serum Fe, MDA, ALT and AST in serum as well as Fe and T-BIL levels in liver were significantly decreased in the As groups ( P 0. 05, P 0. 05) , while the levels of GSH and TIBC in liver were significantly dec
11、reased ( P 0. 05, P 0. 01) . HE results showed that as compared with blank group, hepatocyte hydropic degeneration, hepatocyte fatty degeneration and inflammatory cell infiltration were present in iron overload group; but these pathological damages were improved in different dose As groups. Immunohi
12、stochemical results showed that as compared with the blank group, the 3-nitrotyrosine ( 3-NT) level in the perivascular plasma was increased significantly in the iron overload group, but was significantly reduced in the As groups. TUNEL staining results showed that as compared with the blank group,
13、hepatocyte apoptosis was significantly increased in model group; as compared with the model group, the hepatocyte apoptosis was significantly reduced in As groups.Conclusion: As could attenuate the liver injury induced by iron overload.Keyword： astragaloside; iron overload; liver injury; apoptosis;
14、Received： 2017-10-17铁元素是机体必需微量元素之一, 是正常生理过程中不可缺少的物质1, 在细胞中的基本代谢过程具有至关重要的作用2。红细胞的血红蛋白是人体内大部分铁的储存库, 而其余铁则分布在肝、腺、骨髓等组织中3。铁元素在机体正常状态下基本处于平衡状态, 当铁供给超过机体对铁的需要时就会引起体内总铁量过多, 即“铁超载”, 通常会成为脏器疾病病理生化过程的重要机制。在遗传性血色病4、慢性贫血5等疾病中常见铁超载的病理现象。由于肝脏是贮存和代谢铁的主要器官, 铁超载与肝脏疾病之间的关联也尤其密切6。目前, 有关“铁超载”对肝细胞损伤的具体细胞病理学机制还未完全建立, 但铁超
15、载的毒性作用与活性氮氧化物相关这一学说, 得到国内外学者们的广泛认可。研究表明, 铁可促进氧自由基的产生并形成毒性更强的羟自由基, 还能导致 NOs 上调及 NO 生成, 从而导致细胞损伤, 在多种类型肝脏疾病发生发展中占重要地位7。黄芪甲苷为中药黄芪的主要有效成分之一, 具有免疫调节、肝脏保护等广泛的药理作用8-10。本文通过检测小鼠铁超载模型动物中氧化应激相关的生化指标, 在小鼠铁超载造模成功的基础上, 检验铁超载后小鼠肝脏的氧化及硝化损伤情况, 并同时给予不同剂量黄芪甲苷, 以此来研究黄芪甲苷与铁超载导致肝损伤的关系。1 材料1.1 动物 SPF 级 ICR 小鼠 50 只, 体重 20
16、22 g, 雄性合格证号 SCXK (苏) 2012-0004, 购自扬州大学医学动物中心, 分笼饲养, 周围环境通风, 温度 2225、湿度 50%60%, 动物实验操作严格按照江苏省实验动物管理办法规定执行。1.2 药品与试剂黄芪甲苷 (纯度98%, 上海 Aladdin 公司, 批号 A111275) ;铁右旋糖苷 (美国 Sigma 公司, 批号 20151207) ;鼠单克隆抗 3-硝基酪氨酸抗体、生物素化山羊抗小鼠 Ig G 二抗 (英国 Abcam 公司, 批号分别为 ab5609, ab3711) ;原位末端转移酶标记技术 (TUNEL) 染色试剂盒 (Roche 公司, 批号 11435917910) ;血清 Fe, 总铁结合力 (TIBC) , 丙氨酸氨基转移酶 (glutamic-pyruvic transaminase, ALT) , 天门冬氨酸氨
