鳖甲煎丸通过TGF-β/Smad信号通路抑制DEN诱导肝癌大鼠上皮间质转化的机制  

Mechanism of Biejia Jianwan in Suppressing Epithelial-mesenchymal Transition in DEN-induced Rat Hepatocellular Carcinoma via TGF-β/Smad Pathway

作　　者：陈炜聪 文彬 孙海涛[1] 孙嘉玲 杨雪梅[1] 招文婷 陈冠新 贺松其[1] CHEN Wei-cong;WEN Bin;SUN Hai-tao;SUN Jia-ling;YANG Xue-mei;ZHAO Wen-ting;CHEN Guan-xin;HE Song-qi(School of Traditional Chinese Medicine(TCM),Southern Medical University,Guangdong 510515,China;Air Force Hospital of Southern Theater Command of Chinese People's Liberation Army,Guangdong 510602,China;Dongguan Hospital of TCM,Dongguan 523000,China)

机构地区：[1]南方医科大学中医药学院,广州510515 [2]中国人民解放军南部战区空军医院,广州510602 [3]东莞市中医院,广东东莞523000

出　　处：《中国实验方剂学杂志》2020年第20期9-15,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(8177140072)。

摘　　要：目的:通过研究鳖甲煎丸对二乙基亚硝胺(DEN)诱导肝癌模型大鼠中转化生长因子-β(TGF-β)/Smad通路信号分子及下游靶基因的影响,探讨鳖甲煎丸抗肝癌侵袭转移的分子机制。方法:大鼠分为正常组、模型组、鳖甲煎丸组(2.2 g·kg^-1·d^-1),对模型组及鳖甲煎丸组大鼠使用DEN进行腹腔注射,诱导大鼠肝脏发生肝炎-肝硬化-癌变病理改变,鳖甲煎丸组在肝硬化阶段给予鳖甲煎丸连续灌胃6周,收集血清及肝脏标本。采用试剂盒检测大鼠血清丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),总胆红素(TBIL),白蛋白(Alb),γ-谷氨酰转肽酶(GGT),碱性磷酸酶(ALP);苏木素-伊红(HE)染色观察大鼠肝脏病变;实时荧光定量聚合酶链式反应(Real-time PCR)检测转化生长因子(TGF)-β1的mRNA转录水平;蛋白免疫印迹法(Western blot)检测TGF-β/Smad通路中TGF-β1,Smad2/3,磷酸化Smad2/3及上皮间质转化标志物N-钙黏蛋白(N-cadherin),E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的蛋白表达水平。结果:鳖甲煎丸组与模型组之间肝功能指标差异均无统计学意义。与模型组比较,鳖甲煎丸能改善大鼠肝癌组织中细胞呈气球样变性、水肿、凝固性坏死的病理变化,降低肝癌组织中TGF-β1mRNA转录及蛋白表达水平(P<0.01),下调磷酸化Smad2蛋白表达水平(P<0.01),同时下调下游靶基因蛋白N-cadherin,Vimentin蛋白表达水平(P<0.01)。结论:鳖甲煎丸可能通过减少肝癌组织中TGF-β1的表达水平,抑制由TGF-β/Smad通路激活介导的肝癌细胞上皮间质转化(EMT),从而达到抗肝细胞癌转移侵袭的作用。Objective: To study the effect of Biejia Jianwan on expressions of signal molecules and target genes of transforming growth factor-β(TGF-β)/Smad pathway in diethylnitrosamine(DEN)-induced rat hepatocellular carcinoma, and explore the mechanisms of Biejia Jianwan suppressing the invasion of hepatocellular carcinoma. Method:The rats were divided into three group,namely normal group,model group and Biejia Jianwan group(2.2 g·kg^-1·d^-1). Rats in Biejia Jianwan group and model group received intraperitoneal injections of DEN to induce sequential chronic inflammation,cirrhosis and hepatocellular carcinoma. At the sign of cirrhosis,rats in Biejia Jianwan group began taking Biejia Jianwan by gavage for 6 weeks. Rat blood was collected to measure serum levels of biochemical markers of liver function tests, including alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),albumin(Alb),γ-glutamyl transpeptadase(GGT), alkaline phosphatase(ALP). Rat livers were fixed in formalin and stained with hematoxylin-eosin(HE)staining,quantitative real-time PCR was used to test the mRNA expressions of TGF-β1,and Western blot was used to test protein expressions of TGF-β1,Smad2/3,p-Smad2/3,N-cadherin,E-cadherin and Vimentin. Result: All of the levels of biochemical markers showed no difference in Biejia Jianwan group and model group. Biejia Jianwan could improve the pathological changes of balloon-like degeneration,edema,and necrosis in liver cancer tissues. Importantly,the treatment dramatically decreased the mRNA expression of TGF-β1(P<0.01),and the protein expressions of TGF-β1,p-Smad2(P<0.01). Besides,the protein expression of N-cadherin and Vimentin were decreased significantly(P<0.01). Conclusion: Biejia Jianwan can inhibit epithelial-mesenchymal transition(EMT)in hepatocellular carcinoma cells activated via TGF-β/Smad pathway by reducing TGF-β1 expression,so as to suppress the metastasis and invasion of hepatocellular carcinoma.

关 键 词：鳖甲煎丸 肝癌模型大鼠 转化生长因子-β(TGF-β)/Smad信号通路 上皮间质转化 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]