积雪草酸对实验性胆汁淤积型肝损伤的影响  

Asiatic acid alleviates the liver injury induced by experimental cholestasis

作　　者：周炳杰[1] 赵梦蝶[2] 耿骥[3] 郭文洁[4] 高静[1] ZHOU Bing-jie ZHAO Meng-die GENG Ji GUO Wen-jie GAO Jing(School of Pharmacy, Jiangsu University, Zhenjiang Jiangsu 212013 School of Medicine, Jiangsu University, Zhenjiang Jiangsu 212013 School of Pharmacy, Soochow University, Suzhou Jiangsu 215123 School of Life Sciences, Nanjing University, Nanjing Jiangsu 210093, China)

机构地区：[1]江苏大学药学院,江苏镇江212013 [2]江苏大学医学院,江苏镇江212013 [3]苏州大学药学院,江苏苏州215123 [4]南京大学生命科学学院,江苏南京210093

出　　处：《江苏大学学报（医学版）》2017年第2期107-113,共7页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81402938;81373400);江苏省自然科学基金资助项目(BK2014575)

摘　　要：目的:研究积雪草酸改善胆管结扎小鼠肝纤维化的作用及其机制。方法:在体实验,小鼠麻醉后打开腹腔,用缝合线结扎胆总管,建立淤胆型肝损伤小鼠模型。手术后将C57小鼠随机分为假手术组、模型组、积雪草酸15 mg/kg组和积雪草酸30 mg/kg组。假手术组和模型组给予0.5%的羧甲基纤维素钠(CMC-Na),其余两组给予0.5%CMC-Na稀释的积雪草酸,各组均连续口服灌胃5 d。末次给药后空腹12 h,眼眶采血检测天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)。处死小鼠,取肝脏及脾脏称重并计算肝脏指数、脾脏指数;将肝脏组织切片进行HE染色、Masson染色和TUNEL染色;采用实时荧光定量PCR检测肝组织炎症因子基因水平,包括前列腺素内过氧化物合成酶2(PTGS2),IL-6,TNF-α和趋化因子配体3(CCL3);蛋白质印迹法检测细胞凋亡相关蛋白表达水平。细胞实验采用甘氨鹅脱氧胆酸钠诱导人肝HL-7702细胞损伤,分别加入0.1、0.3、1和3μmol/L积雪草酸,MTT法检测细胞存活率,AnnexinⅤ/PI流式细胞术检测细胞凋亡。结果:积雪草酸给药后的小鼠血清AST、ALT水平明显低于模型组;显微镜下观察,给药组肝组织胶原纤维和细胞凋亡明显减少;炎症因子基因水平降低,Bax蛋白表达水平降低,Caspase3活化减少,Bcl2表达水平增加。细胞实验显示,低浓度积雪草酸能够促进HL-7702细胞增殖,抑制凋亡。结论:积雪草酸通过抑制肝细胞凋亡,减轻胆管结扎模型小鼠肝纤维化,同时能缓解炎症,改善肝损伤。Objective：To investigate the effect and mechanism of asiatic acid（AA） on experimental cholestasis-induced liver injury.Methods：In vivo,a total of 32 C57 mice were divided into sham group,bile duct ligation（BDL） model group,AA 15 mg/kg group and AA 30 mg/kg group.Mice were anesthetized by an intraperitoneal（i.p） injection of pentobarbital（60 mg/kg）.After anaesthetized,the common bile duct was double-ligated by suture.The sham group and model group were intragastric administration by 0.5% CMC-Na solution,the other two groups were intragastric administration by AA（dissolved in 0.5% CMC-Na）.All groups were intragastric administration with CMC-Na or AA per day for 5days.The morphology of liver were observed,and also the content of glutamic-oxalacetic transaminase（AST） and glutamic-pyruvic transaminase（ALT） biochemically determined.Liver tissue sections were used for HE staining,Masson staining and Tunel staining.PCR assay tested the expression levels of inflammatory factors including prostaglandin-endoperoxide synthase（PTGS2）,IL-6,TNF-α,Chemokineligand 3（CCL3）.The expression levels of apoptosis-related proteins（Caspase3,cleaved-caspase3,Bax,Bcl2） were determined using Western blotting.In vitro,the cell viability ratio and apoptosis of HL-7702 treated with sodium glycochenodeoxycholate（GCDC） detected by MTT and flow cytometry assay,respectively.Results：In vivo,after treated with AA,the liver index and spleen index of mice were significantly reduced,compared with model group.AA decreased the content of AST and ALT,and reduced the hepatocyte apoptosis showed by Tunel staining.And also,AA decreased the level of pro-inflammation genes and regulated the expression of apoptosis-related proteins.AA can inhibit GCDC-induced HL-7702 cells apoptosis in a concentration-dependent manner.Conclusion：Asiatic acid can alleviate liver injury by inhibiting hepatocyte apoptosis.

关 键 词：积雪草酸 胆管结扎 肝纤维化 HL-7702细胞 凋亡 

分 类 号：R966[医药卫生—药理学]