大鼠肝癌发生蛋白激酶/磷酸肌醇-3-激酶信号通路的表达及健脾解毒法的调节作用  

Study on AKT/PI3K signaling pathway and prevenion effect of Jianpi Jiedu Recipe in rats with experimental hepatocarcinogenesis

作　　者：张斌[1] 李琦[1] 殷佩浩[1] 赵成根[1] 高虹[1] 孙珏[1] 范忠泽[1] 

机构地区：[1]上海中医药大学附属普陀医院中医肿瘤科,上海200062

出　　处：《中国中西医结合消化杂志》2010年第1期4-9,共6页Chinese Journal of Integrated Traditional and Western Medicine on Digestion

基　　金：中国博士后基金资助项目(20080440639);教育部肝肾疾病病证重点实验室(上海中医药大学)开放课题基金资助项目(GS090203);上海市重点学科资助项目(S30302)

摘　　要：[目的]探讨中药复方健脾解毒方对二乙基亚硝胺（diethylnitrosamine,DEN）诱导的大鼠肝癌的预防作用及其调控蛋白激酶/磷酸肌醇-3-激酶（AKT/PI3K）信号通路的机制。[方法]雄性Wistar大鼠,随机分为正常组（n=25）、模型组（n=40）及中药组（n=40）。除正常组外,其他组1-12周饮用含DEN 80 mg/L水[8 mg/（kg.d）]诱癌;中药组同时给予含生药1.75 g/ml的健脾解毒方（10 ml/kg）灌胃,正常组给予10 ml/kg 0.85%氯化钠灌胃,1次/d,共12周。于4、8、12、16周时相点,各组随机取5只大鼠处死取肝,20周时剩余大鼠全部处死取肝,观察肝脏外观,计算病死率和腹水生成率,肝组织进行苏木精-伊红染色,应用RT-PCR半定量检测肝组织AKT、PI3K及p70s6k mRNA的表达,Western blot法检测肝组织p-AKT的表达。[结果]在20周实验结束时,正常组无死亡,模型组死亡率为42.5%（17/40）,中药组死亡率为17.5%（7/40）;16-20周时模型组腹水发生率为87.5%（7/8）,中药组为44.4%（8/18）,2组比较差异有统计学意义（P〈0.05）。正常组没有肿瘤形成,模型组和中药组在16周后成瘤率均为100%;同时模型组肝癌Ⅲ级发生率为100%（5/5）,而中药组Ⅰ、Ⅱ、Ⅲ级肝癌发生率分别为40%（2/5）、40%（2/5）及20%（1/5）,2组比较差异有统计学意义（P〈0.05）。RT-PCR结果显示,与正常组比较,模型组4-20周的AKT mRNA均显著上调,p70s6k mRNA均显著下调,PI3K mRNA除第8周外均显著上调。与模型组比较,中药组可阶段性下调AKT/PI3K及上调p70s6k的表达。Western blot显示,模型组与中药组的p-AKT显著上调,尤其在8-12周最为明显,其中药组的上调幅度显著低于模型组。[结论]AKT/PI3K信号通路与肝癌的发生密切相关,表现为AKT、PI3K表达上调,p70s6k表达下调,健脾解毒方有预防DEN诱导大鼠肝癌的作用,其机制可能部分与中药下调AKT/PI3K及上调p70s6k的表达有关。[Objective]To investigate the prevention effects and regulating AKT/PI3K mechanism of Jinpi Jiedu Recipe on experimental hepatocarcinoma rats induced by diethylnitrosamine.[Methods]Male Wistar rats were randomly divided into normal control group（n=25）,model group（n=40） and prevention group（n=40）.In addition to the normal group,other groups were given DEN drinking water（80ppm） at the dosage of 8mg kg^-1 d^-1 for continuous 12 weeks to induce hepatoma,while the prevention group were given Jianpi Jiedu Recipe（17.5g kg^-1 d^-1,ig）,the normal group were given saline 10ml/kg,1/d for 12 weeks.Each groups were randomly executed 5 rats at the 4th,8th,12th and 16th week respectively,and other rats were all sacrificed in the 20th week.Hematoxylin-eosin staining（HE）were used to examine the changes of liver pathology,and the mRNA expression of AKT,PI3K and p70s6k were tested by RT-PCR methods.At the same time,the protein levels of p-AKT in liver tissue were analyzed by Western blot.[Results]During 16th week ～ 20th week,the mortality rate and ascite occur rate of normal control group was 0%、0% respectively,while the model group was 42.5%、87.5%,the prevention group was 17.5%,44.4%,（P〈 0.05）.By the end of the 16th week,the incidence of HCC was 100% in model group and prevention group,while the normal control group was 0%.In the 16th week time,the grade Ⅲ liver cancer occurrence rate of the model group was 100%（5/5）,while the grade Ⅰ、Ⅱ、Ⅲ liver cancer occurrence rate of prevention group were 40%（2 / 5）,40%（2 / 5） and 20%（1 / 5）,respectively.To be compared with normal group,the AKT mRNA expressions of model group were increased significantly during 4 ～ 20w（P〈0.01）.The PI3K mRNA expression of model group had a upward trend except 8th week.On the other hand,the p70s6k mRNA decreased every week in model group.At the same time,AKT and PI3K mRNA of prevention group could periodically decreased,while the p70s6k mRNA can raised partly.Western blot showed that the p-AK

关 键 词：原发性肝癌 健脾解毒方 二乙基亚硝胺 蛋白激酶/磷酸肌醇-3-激酶 

分 类 号：R273[医药卫生—中西医结合] R735[医药卫生—中医肿瘤科]