线粒体-细胞色素C途径在兔非酒精性脂肪肝肝细胞凋亡中的作用  

Effect of mitochondrial cytochrome c on hepatocyte apoptosis in non-alcoholic fatty liver disease in rabbits

作　　者：吴方[1] 余陈欢[2] 张欢欢[2] 马月[2] 俞冰[1] 应华忠[2] 

机构地区：[1]浙江中医药大学药学院,杭州浙江310053 [2]浙江省医学科学院浙江省实验动物与安全性研究重点实验室,杭州浙江310013

出　　处：《中国比较医学杂志》2016年第4期7-13,23,共8页Chinese Journal of Comparative Medicine

基　　金：国家自然基金资助项目(81573591);浙江省科技计划项目(2011C37095)

摘　　要：目的探讨线粒体-细胞色素C途径在兔非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)肝细胞凋亡中的作用。方法雄性日本大耳兔40只,随机分为正常对照组(8周)和NAFLD模型组(4、6、8周组),每组各10只。模型组按1.2 m L/kg皮下注射花生油,2次/周,建立日本大耳兔NAFLD模型。按各组时间点处死兔子,测定兔血清中生化指标水平;HE染色观察肝组织病理形态改变,流式细胞仪检测肝细胞凋亡率;提取肝脏线粒体,分光光度法检测线粒体通透转换孔(mitochondrial permeability transition pore,MPTP)的状态变化;免疫组织化学法检查Bcl-2、Bax、细胞色素C和caspase-3在肝组织中的表达情况;Western blot检测细胞色素C以及caspase-3含量变化。结果与正常对照组相比,模型组肝脏脂质代谢指标和相关炎症因子表达水平升高,模型组肝细胞凋亡率与对照组比较显著增加(P<0.01)。造模4周时出现肝功能损伤,脂质代谢紊乱,随着NAFLD病程的发展,Bcl-2、Bax、细胞色素C和caspase-3表达量逐渐增加,线粒体通透转换孔开放率随高脂饮食饲养时间延长明显增加(P<0.01)。结论线粒体-细胞色素C途径在NAFLD引起的肝细胞凋亡中起到了一定的调控作用。Objective To investigate the role of mitochondrial cytochrome c on hepatocyte apoptosis in nonalcoholic fatty liver disease in rabbits and its pathogenesis. Methods Forty Japanese white rabbits were randomly assigned to control group and model group. The model group was divided into three subgroups： 4-week,6-week,and 8-week groups,with 10 rabbits in each group. The model groups were subcutaneously injected with peanut oil（ 1. 2 m L / kg）,twice a week for 4 weeks,6 weeks or 8 weeks. The rabbits of all groups were killed at the right time. Serum samples were collected to detect the serum biochemical index levels. Liver tissue samples were taken for pathological observation using HE staining. The hepatocyte apoptosis index（ AI） was measured by flow cytometry,and mitochondrial permeabilitytransition pore（ MPTP） was evaluated by ultraviolet spectrophotometry. Immunohistochemistry was employed to detect the hepatic expressions of Bcl-2,Bax,CYT C and caspase-3. Western blot was performed to detect the changes of CYT C and caspase-3 protein expressions. Results The model groups showed hepatic injury and high level of TC,TG,CRP,IL-6and TNF-α beginning from 4 weeks. With the NAFLD process,the hepatocyte apoptosis index was significantly increased at4-8 weeks and the MPTP was gradually increased. In the model group,hepatic Bcl-2,Bax,CYT C and caspase-3expressions were increased steadily with the time passing. Conclusions NAFLD-induced liver damage is associated with apoptosis,and the mitochondrial cytochrome c-mediated apoptotic pathway plays a role in the occurrence of NAFLD.

关 键 词：非酒精性脂肪肝 凋亡 细胞色素C 胱冬肽酶-3 线粒体通透转换孔 

分 类 号：R33[医药卫生—人体生理学]