鳖甲煎丸通过NF-κB信号通路抑制肝癌细胞上皮间质转化的作用机制  

Mechanism of Biejiajian Wan Against EMT of Hepatocellular Carcinoma Cells Through NF-κB Signaling Pathway

作　　者：钟晓丹 文彬 孙海涛[1] 孙嘉玲 杨雪梅[1] 陈炜聪 招文婷 何春雨 刘洋 李彤 贺松其[1] ZHONG Xiao-dan;WEN Bin;SUN Hai-tao;SUN Jia-ling;YANG Xue-mei;CHEN Wei-cong;ZHAO Wen-ting;HE Chun-yu;LIU Yang;LI Tong;HE Song-qi(School of Chinese Medicine,Southern Medical University,Guangzhou 510515,China;Air Force Hospital of Southern Theater Command of PLA,Guangzhou 510602,China;Traditional Chinese Medicine-Integrated Hospital of Southern Medical University,Guangzhou 510315,China)

机构地区：[1]南方医科大学中医药学院,广州510515 [2]解放军南部战区空军医院,广州510602 [3]南方医科大学中西医结合医院,广州510315

出　　处：《中国实验方剂学杂志》2022年第1期24-32,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81774037);广东省自然科学基金项目(2021A1515012591)。

摘　　要：目的:研究鳖甲煎丸对转化生长因子-β_(1)(TGF-β_(1))诱导Hep G2细胞上皮间质转化(EMT)的影响,探讨其抑制肝癌细胞EMT的作用机制。方法:以Hep G2细胞为研究对象,将细胞随机分为空白组,模型组(10μg·L^(-1)TGF-β_(1)),鳖甲煎丸低(10μg·L^(-1)TGF-β_(1)+0.55 g·kg^(-1)鳖甲煎丸)、中(10μg·L^(-1)TGF-β_(1)+1.1 g·kg^(-1)鳖甲煎丸)、高(10μg·L^(-1)TGF-β_(1)+2.2 g·kg^(-1)鳖甲煎丸)组,索拉非尼组(10μg·L^(-1)TGF-β_(1)+0.03 g·kg^(-1)索拉非尼)。用10μg·L^(-1)TGF-β_(1)诱导Hep G2细胞发生EMT,构建EMT模型。分别给予相应的含药血清处理后,采用细胞增殖与活性检测(CCK-8)法检测细胞增殖情况,分别采用细胞划痕实验,transwell迁移实验检测细胞迁移情况,采用免疫荧光、蛋白免疫印迹法分别检测EMT,核转录因子-κB(NF-κB)信号通路相关蛋白的表达情况。结果:与空白组比较,TGF-β_(1)刺激4 d后,细胞呈梭形改变,细胞之间变得松散,间隙增宽,伸出触角,同时E-钙黏素(E-cadherin)蛋白表达降低(P<0.05),N-钙黏素(N-cadherin),波形蛋白(vimentin)蛋白表达升高(P<0.05),说明TGF-β_(1)刺激4 d成功构建了Hep G2细胞的EMT模型组。与模型组比较,用相应含药血清处理48 h,各用药组均能抑制已发生EMT作用的Hep G2细胞增殖,其中以鳖甲煎丸低、高剂量组最为显著(P<0.01)。与模型组比较,鳖甲煎丸中剂量组E-cadherin蛋白表达升高(P<0.05),磷酸化(p)-p65,N-cadherin,vimentin蛋白表达降低(P<0.05)。与空白组比较,细胞划痕实验和transwell迁移实验表明TGF-β_(1)增强了Hep G2细胞的迁移能力(P<0.05,P<0.01);与模型组比较,鳖甲煎丸低、中、高剂量组和索拉非尼组抑制Hep G2细胞迁移能力(P<0.05,P<0.01)。与空白组比较,模型组促进p65,Snail入核表达;与模型组比较,鳖甲煎丸低、中、高剂量组和索拉非尼组抑制p65和Snail入核表达。结论:鳖甲煎丸可能通过抑制NF-κB信号通路来抑制TGF-β_(1)诱导Objective:To investigate the effect of Biejiajian Wan(BJJW)on transforming growth factor-β_(1)(TGF-β_(1))-induced epithelial-mesenchymal transition (EMT) of HepG2 cells,and explore its mechanism against EMT of hepatocellular carcinoma cells.Method:HepG2 cells were randomly divided into a blank group,a TGF-β_(1)model group(10μg·L^(-1)TGF-β_(1)),a low-dose BJJW group(10μg·L^(-1)TGF-β_(1)+0.55 g·kg^(-1)BJJW),a medium-dose BJJW group(10μg·L^(-1)TGF-β_(1)+1.1 g·kg^(-1)BJJW),a high-dose BJJWgroup(10μg·L^(-1)TGF-β_(1)+2.2 g·kg^(-1)BJJW),and a sorafenib group(10μg·L^(-1)TGF-β_(1)+0.03 g·kg^(-1)sorafenib).The EMT model was induced by 10μg·L^(-1)TGF-β_(1)in HepG2 cells.After treatment with corresponding medicated serum,cell counting kit-8(CCK-8)assay was used to detect cell proliferation.Cell migration ability was detected by the Transwell assay and wound healing assay.The protein expression related to EMT and nuclear factor-kappa B(NF-κB)signaling pathway was detected by cell immunofluorescence assay and Western blot.Result:Compared with the blank group 4 days later,the TGF-β_(1)model group showed fusiform and loose cells with widened gap and antennae reaching out,decreased protein expression of E-cadherin(P<0.05),and increased protein expression of N-cadherin and vimentin(P<0.05),which indicated that the EMT model was properly induced in HepG2 cells by TGF-β_(1)stimulation for 4 days.After 48 hours of treatment with the corresponding medicated serum,each medication group showed inhibited proliferation of HepG2 cells that had undergone EMT,especially the low-and high-dose BJJW groups(P<0.01),and the medium-dose BJJW group showed increased E-cadherin protein expression(P<0.05)and decreased p-p65,N-cadherin,and vimentin protein expression(P<0.05),as compared with the TGF-β_(1)model group.As revealed by the transwell assay and wound healing assay,TGF-β_(1)enhanced the migration ability of HepG2 cells(P<0.05,P<0.01)compared with the results in the blank group,compared with the TGF-β_(1)model gro

关 键 词：肝癌 上皮间质转化 鳖甲煎丸 核转录因子-κB(NF-κB)信号通路 转化生长因子-β_(1)(TGF-β_(1)) 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]