肝爽颗粒对CCl_4诱导的慢性肝损伤小鼠模型和肝损伤细胞模型的保护作用  

Ganshuang granules protect mouse liver from chronic injury induced by CCl_4 via autophagy

作　　者：孙海青[1] 王小琪[1] 时红波[1,2] 娄金丽[1] 陈煜[1] 段钟平[1,2] 

机构地区：[1]首都医科大学附属北京佑安医院,北京100069 [2]北京市肝病研究所,北京100069

出　　处：《临床肝胆病杂志》2015年第7期1114-1119,共6页Journal of Clinical Hepatology

基　　金：国家自然科学基金(81300349);北京市科技新星计划(Z131107000413016);北京市自然科学基金(7144216)

摘　　要：目的本研究拟初步探讨肝爽颗粒通过细胞自噬在保护四氯化碳(CCl4)诱导小鼠慢性肝损伤模型和肝损伤细胞模型中的作用和机制,为其临床应用提供依据。方法 CCl4腹腔注射构建小鼠慢性肝损伤模型,CCl4体外诱导肝细胞系7702细胞构建肝损伤细胞模型。建模成功后给予肝爽颗粒进行干预,同时设立正常对照组和未给予肝爽颗粒的CCl4组。在此基础上,应用自噬抑制剂3-甲基腺嘌呤(3-MA)抑制细胞自噬并观察肝爽颗粒对细胞凋亡的影响。HE染色观察肝组织损伤情况;生化法测定血清ALT、AST水平;免疫荧光染色法、Western Blot观察细胞自噬情况;流式细胞术及Annexin V/PI双标记免疫荧光染色法检测肝细胞凋亡情况。多组间比较采用方差分析,方差齐采用LSD检验,方差不齐采用Dunnett T3检验。结果 HE染色显示肝爽颗粒干预组小鼠肝组织损伤程度较CCl4组明显减轻,3组间ALT和AST总体表达水平差异有统计学意义(F值分别为15.76、13.35,P值均<0.05);体内细胞实验和体外动物实验免疫荧光结果均显示肝爽颗粒干预组细胞自噬表达较CCl4组明显增加,肝细胞凋亡较CCl4组明显减少。加入3-MA后肝爽颗粒的保护性作用减弱,细胞凋亡增加。流式细胞仪检测结果显示5组7702细胞凋亡比例差异有统计学意义(F=25.637,P<0.01)。结论肝爽颗粒对肝脏损伤具有保护性作用,其抗凋亡机制可能与肝爽颗粒增强细胞自噬有关。Objective To investigate the role and action mechanism of Ganshuang granules in the protection against CCl4- induced chronic liver injury in mice via autophagy,and to provide a basis for its clinical application. Methods We established a chronic liver injury model in mice by intraperitoneal injection of CCl4,and a cell model of liver damage in cell line HL- 7702 induced by CCl4 in vitro. The animals were divided into three groups,Ganshuang granule intervention group,normal control group,and CCl4 group without receiving Ganshuang granules. In addition,we exposed the cells to 3- methyladenine（ 3- MA）,an autophagy inhibitor,and observed the effects of Ganshuang granules on cell apoptosis. Liver tissue injury was evaluated by HE staining; serum alanine aminotransferase（ ALT） and aspartate aminotransferase（ AST） levels were determined by biochemical assays. Autophagy was assessed by immunofluorescence staining and Western blotting. Liver apoptosis was analyzed by flow cytometry and Annexin V / PI double staining. Comparisons between groups were performed using analysis of variance. Results HE staining showed that liver tissue injury was significantly milder in the Ganshuang granule intervention group than in the CCl4 group. Serum ALT and AST levels were significantly differences between Ganshuang granule intervention group,normal control group,and CCl4group（ F = 15. 76、13. 35,P 〈0. 05）. Both in vivo and in vitro tests showed that autophagy increased significantly in cells treated with Ganshuang granules than in cells exposed to CCl4 alone,while apoptosis was significantly reduced in the former.The administration of 3- MA weakened the protective effect of Ganshuang granules and increased apoptosis. Flow cytometry showed that apoptosises were significiantly differences between five groups（ F = 25. 637,P 〈0. 01）. Conclusion Ganshuang granules have protective effects against chronic liver injury by inhibiting apoptosis,possibly through enhanced autophagy.

关 键 词：肝疾病 自噬 细胞凋亡 肝爽颗粒 小鼠 近交BALB C 

分 类 号：R285.5[医药卫生—中药学] R-332[医药卫生—中医学]