理冲汤加减联合5-氟尿嘧啶对人肝癌细胞HepG2上皮间质转化的影响

Effect of Modified Lichongtang Combined with 5-Fluorouracil on Epithelial Mesenchymal Transition of Human Hepatoma Cell Line HepG2

目的:探讨理冲汤加减联合5-氟尿嘧啶(5-FU)对人肝癌细胞HepG2上皮间质转化(EMT)的影响。方法:采用噻唑蓝(MTT)比色法检测理冲汤加减及5-FU单独和联合使用对HepG2细胞生长影响;应用中效原理进行统计分析,确定联合用药时的药物效应-合用指数的关系,判定药物间的相互作用,确定后续实验给药浓度及时间;划痕实验检测理冲汤加减联合5-FU对HepG2细胞迁移能力的影响;细胞侵袭实验(transwell小室)检测理冲汤加减联合5-FU对HepG2细胞侵袭能力的影响;采用实时荧光定量聚合酶链式反应(Real-time PCR)检测理冲汤加减联合5-FU作用HepG2细胞24 h后对EMT相关基因上皮型钙黏蛋白(E-cadherin),神经型钙黏蛋白(N-cadherin),锌指转录因子(Snail,Twist) mRNA表达;采用蛋白免疫印迹法(Western blot)检测理冲汤加减联合5-FU作用HepG2细胞24 h后EMT相关蛋白E-cadherin,N-cadherin,Snail,波形蛋白(Vimentin)的表达。结果:MTT比色法结果显示,随着药物浓度增加,理冲汤加减,5-FU单药或联合用药对HepG2细胞生长的抑制效应也增加;应用中效原理进行统计分析显示,两药在低剂量合用24 h后对HepG2细胞可以产生较好的协同效应,故选用理冲汤加减,5-FU作用HepG2细胞24 h的25%抑制浓度(IC25)即800 mg·L-1理冲汤加减,3. 125 mg·L-15-FU;设空白组,5-FU组,理冲汤加减+5-FU组,理冲汤加减组进行后续实验;划痕和侵袭实验显示,理冲汤加减,5-FU单独及联合均能抑制HepG2细胞迁移侵袭能力(P <0. 05,P <0. 01),与5-FU组比较,理冲汤加减+5-FU组抑制能力更强(P <0. 05,P <0. 01)。与空白组比较,5-FU组,5-FU+理冲汤加减组E-cadherin mRNA表达上调,N-cadherin,Snail,Twist mRNA表达下调(P <0. 05,P <0. 01);与5-FU组比较,5-FU+理冲汤加减组E-cadherin mRNA表达上调,N-cadherin,Snail,Twist mRNA表达下调(P <0. 05,P <0. 01)。与空白组比较,5-FU组,5-FU+理冲汤加减组E-cadherin的表达上调,N-cadherin,Snail,Vimentin蛋白表达下调(P <0. 05,P <0. 01);与5-FU组比较,5-FU+理冲汤加减组E-cadherin的表达上调,N-cadherin,Snail,Vimentin蛋白表达下调(P <0. 05,P <0. 01)。结论:理冲汤加减方与5-FU在低剂量合用24 h后对HepG2细胞可以产生较好的协同效应,并且能明显抑制肝癌细胞的迁移和侵袭能力,上调肝癌细胞内EMT相关标志物E-cadherin的表达,下调N-cadherin,Snail,Vimentin,Twist的表达,据此推测,抑制肿瘤细胞增殖、迁移、侵袭能力及EMT相关mRNA的表达,从而增强化疗药物的疗效,可能是理冲汤加减方联合5-FU协同作用的机制之一。

Objective:To investigate the effect of modified Lichongtang combined with 5-fluorouracil(5-FU)on epithelial-mesenchymal transition(EMT)of human hepatoma HepG2 cells.Method:The growth of HepG2 cells was detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay,and the effect of Chinese medicine and 5-FU alone or combined use on the growth of HepG2 cells was analyzed by the principle of efficacy.The growth curves of HepG2 cells were plotted to determine the relationship between drug effect and combination index as well as the interaction between drugs.Scratch test was used to detect the effect of modified Lichongtang combined with 5-FU on the migration of HepG2 cells.Cell invasion assay(transwell chamber)was used to detect the effect of modified Lichongtang combined with 5-FU on the invasion ability of HepG2 cells.Real-time quantitative polymerase chain reaction(PCR)was used to detect the effect of modified Lichongtang combined with5-FU on EMT-related genes E-cadherin,N-cadherin and Zinc finger transcription factors(snail,twist)mRNA expression after 24 hours of treatment on HepG2 cells.The expression levels of E-cadherin,N-cadherin,Snail and Vimentin in HepG2 cells were detected by Western blot after treatment by modified Lichongtang combined with5-FU for 24 hours.Result:MTT assay showed that with the increase of drug concentration,the inhibitory effect of modified Lichongtang,5-FU alone or combined use on HepG2 cell growth was also increased.Statistical analysis showed that the combined use of these two drugs at a low dosage could produce better synergistic effect on HepG2 cells after 24 hours of treatment.Therefore,modified Lichongtang and 5-FU were selected to treat HepG2 cells for24 hours.25% inhibitory concentration(IC25)was 800 mg·L^-1 modified Lichongtang,3.125 mg·L^-1 5-FU.Blank group,5-FU group,Lichongtang + 5-FU group,and modified Lichongtang group were set for follow-up experiments.Scratch and invasion experiments showed that modified Lichongtang,5-FU alone and combined use can inhibit HepG2 cell migration and invasion ability(P<0.05,P<0.01).As compared with 5-FU group,the inhibitory effect was more obvious in modified Lichongtang + 5-FU group.As compared with the blank group,the mRNA expression of E-cadherin were up-regulated,while the mRNA expression levels of N-cadherin,Snail and Twist were and down-regulated in the 5-FU group and the 5-FU + modified Lichongtang group(P<0.05,P<0.01).As compared with 5-FU group,the mRNA expression of E-cadherin was up-regulated while the mRNA expression levels N-cadherin,Snail and Twist were down-regulated in the 5-FU + modified Lichongtang group(P<0.05,P<0.01).Western blot showed that as compared with the blank group,E-cadherin protein expression was up-regulated while N-cadherin,Snail and Vimentin protein expression levels were down-regulated in 5-FU group and 5-FU + modified Lichongtang group(P<0.05,P<0.01).As compared with the 5-FU group,E-cadherin protein expression was up-regulated while N-cadherin,Snail,and Vimentin protein expression levels were down-regulated in 5-FU + modified Lichongtang group(P<0.05,P<0.01).Conclusion:Modified Lichongtang combined with 5-FU can produce a better synergistic effect on HepG2 cells at a low dosage for 24 hours,and can significantly inhibit the migration and invasion of hepatocellular carcinoma cells,up-regulate the expression of E-cadherin,down-regulate the expression of N-cadherin,Snail,Vimentin and Twist in hepatocellular carcinoma cells.Inhibition of tumor cell proliferation,migration,invasion and EMT-related gene expression may be associated with enhancing the efficacy of chemotherapy drugs,and may act as one of the mechanisms for synergistic effect of modified Lichongtang combined with 5-FU.