DEN诱导大鼠肝癌形成中miR-199a的表达机制及健脾解毒法的干预作用

Involvement of miR-199a downregulation in the preventive effects of Jianpi Jiedu Recipe against experimental hepatocarcinoma in rats

目的:探讨中药复方健脾解毒方对二乙基亚硝胺(DEN)诱导的大鼠肝癌的预防作用及其调控miR-199a的表达机制.方法:♂Wistar大鼠,随机分为正常组(n=25)、模型组(n=40)及中药预防组(n=40).除正常组外,其他组在1-12wk饮用含DEN80mg/L的饮水[mg/(kg?d)]以诱癌,中药组同时给予含生药1.75g/mL的健脾解毒方灌胃(10mL/kg),正常组给予10mL/kg生理盐水灌胃,每日1次,共12wk.于4、8、12、16wk时相点,各组随机取5只大鼠处死取肝,20wk将剩余大鼠全部处死取肝,观察肝脏外观,计算死亡率和腹水生成率,比较肝脾指数,肝组织进行HE染色,应用Real-timePCR检测肝组织miR-199a的表达.结果:在20wk实验结束时,正常大鼠无死亡,而模型大鼠死亡率为42.5%(17/40),预防组死亡率为17.5%(7/40),16-20wk时模型组腹水发生率为87.5%(7/8),预防组为44.4%(8/18),与模型组有差异性(P<0.05).正常组没有肿瘤形成,模型组和预防组在16wk后成瘤率均为100%.16wk模型组肝癌Ⅲ级发生率为100%(5/5),而预防组Ⅰ、Ⅱ、Ⅲ级肝癌发生率分别为40%(2/5)、40%(2/5)及20%(1/5),两组比较有差异(P<0.05).与模型组比较,中药预防组有显著降低肝脾指数的作用(均P<0.01).PCR结果显示,模型组肝组织miR-199a较正常组明显上调,中药预防组除16wk外均有显著下调miR-199a表达的作用(均P<0.01).结论:健脾解毒方有预防DEN诱导大鼠肝癌发生的作用,其机制可能部分与下调miR-199a的表达有关.

AIM：To investigate the preventive effects of Jianpi Jiedu Recipe against diethylnitrosamineinduced hepatocarcinoma in rats and explore the involvement of miR-199a downregulation in this process.divided into three groups：normal control group （n=25）,model group （n=40） and prevention group （n=40）.The normal control group was given normal saline [10 mL/（kg·d） ig] for 12 weeks,the model group was given diethylnitrosamine dissolved in drinking water （80 ppm） at a dose of 8 mg/（kg·d） for the same duration,and the prevention group was given both diethylnitrosamine at the same dose and Jianpi Jiedu Recipe [17.5 g/（kg·d）,ig] for the same duration.Five rats in each group were executed at weeks 4,8,12 and 16,respectively,and the remaining rats were killed at week 20.Meanwhile,the mortality,incidence of ascites,and liver and spleen index were measured.Hematoxylin-eosin staining （HE） was used to examine hepatic pathological changes.The expression of miR-199a mRNA was detected by real-time reverse transcriptionpolymerase chain reaction （RT-PCR）.RESULTS：At week 20,the mortality rate and the incidence rate of ascites were 0% and 0% in the normal control group,42.5% and 87.5% in the model group,and 17.5% and 44.4% in the prevention group,respectively.The mortality rate and the incidence of ascites were significantly lower in the prevention group than in the model group （both P 〈0.05）.At week 16,the incidence of HCC was 100% in both the model group and prevention group and 0% in the normal control group,the incidence of grade III liver cancer was 100% （5/5） in the model group,and the incidences of grade I,II and III liver cancer were 40% （2/5）,40% （2/5） and 20% （1/5） in the prevention group,respectively.Compared with the model group,the liver and spleen index in the prevention group were significantly reduced at all time points （all P〈 0.01）.Real-time RT-PCR results showed that the expression of miR-199a was significantly upregulated in the model group.However,miR-199a expression was significantly downregulated in the prevention group at all time points except at week 16 （all P 〈0.01）.CONCLUSION：Jianpi Jiedu Recipe has good preventive effects against hepatocarcinoma in rats perhaps partly by downregulating miR-199a expression.