大蒜素对TGF?β_1诱导人胆管癌细胞上皮间质化的影响及机制

The inhibition effects of allicin on TGF-β_1 induced epithelial ? mesenchymal transition of human cholangiocarcinoma cell and its mechanism

目的胆管癌的转移机制可能与上皮间质化(EMT)有关。文章旨在探讨大蒜素对TGF?β_1诱导的人胆管癌细胞上皮间质化的影响及其作用机制,为大蒜素应用于胆管癌的治疗提供理论依据。方法采用MTT法检测不同浓度大蒜素对人胆管癌RBE细胞的增殖抑制作用,以24h的IC50确定为大蒜素给药浓度,将其分为空白对照组、大蒜素组(130.7μmol/L)、TGF?β_1组(10ng/mL)及大蒜素+TGF?β_1组(130.7μmol/L+10ng/mL);采用划痕实验和Transwell小室试验分别检测24h后RBE细胞的迁移及侵袭能力;Western blot分别检测各组EMT相关蛋白E?钙黏蛋白(E?Cadherin)、N?钙黏蛋白(N?Cadherin)、Vimentin、Snail及核因子?κB(NF?κB)信号通路表达情况。结果与空白对照组及TGF?β_1组迁移率(28.19%±0.66%、49.22%±0.27%)比较,大蒜素组及大蒜素+TGF?β_1组(9.25%±0.36%、13.91%±0.75%)均下降(P<0.05);与空白对照组及TGF?β_1组侵袭率(33.48%±0.04%、40.21%±0.12%)比较,大蒜素组及大蒜素+TGF?β_1组(6.59%±0.06%、9.40%±0.05%)亦均明显下降(P<0.05);与空白对照组相比,大蒜组的E?Cadherin蛋白表达上调(P<0.05),而N?Cadherin、Vimentin、Snail、NF?κB及p?NF?κB蛋白表达下调(P<0.05);与TGF?β_1组相比,大蒜素+TGF?β_1组的E?Cadherin蛋白表达上调(P<0.05),N?Cadherin、Vimentin、Snail、NF?κB及p?NF?κB蛋白表达下调(P<0.05)。结论大蒜素可抑制TGF?β_1诱导的人胆管癌细胞发生上皮间质化,其机制可能与阻断NF?κB信号通路有关。

Objective The metastasis mechanism of cholangiocarcinoma is complex,which may be related to epithelial-mesenchymal transition(EMT).This study focused on investigating the inhibition effects of allicin on TGF-β1 induced epithelium mesen-chymal transition of human cholangiocarcinoma cells and its related mechanism,and providing theoretical basis for the application of allicin in the treatment of cholangiocarcinoma.Methods MTT assay were used to detect the inhibition effects of different concentrations of allicin on the human cholangiocarcinoma RBE cell proliferation,and the drug concentration of allicin was determined by IC50 of 24 h.The RBE cells were cultured and divided into control group,allicin group(130.7μmol/L),TGF-β1 group(10ng/mL)and allicin+TGF-β1 group(130.7μmol/L+10ng/mL).Wound scratch and tran swell invasion assay were performed to detect the migration and invasion ability of RBE cells after 24 hours.Western blots were applied to detect expression of EMT-related proteins(E-Cadherin,N-Cadherin,Vimentin,Snail)and NF-κB signaling pathways.Results The migration rates in allicin group and allicin+TGF-β1 group were both decreased compared with that in the control group(9.25%±0.36%vs 28.19%±0.66%,P<0.05)and TGF-β1 group(13.91%±0.75%vs 49.22%±0.27%,P<0.05).The invasion rates in allicin group and allicin+TGF-β1 group were also decreased compared with that in the control group(6.59%±0.06%vs 33.48%±0.04%,P<0.05)and TGF-β1 group(9.4%±0.05%vs 40.21%±0.12%,P<0.05).Compared with the control group,E-Cadherin expression was significantly increased,and N-Cadherin,Vimentin,Snail,NF-κB and p-NF-κB expression were significantly decreased in the allicin group(P<0.05).Compared with TGF-β1 group,E-Cadherin expression was significantly up-regulated,and N-Cadherin,Vimentin,Snail,NF-κB and p-NF-κB expression were significantly down-regulated in the allicin+TGF-β1 group(P<0.05).Conclusion These results indicate that allicin can inhibit the EMT induced by TGF-β1 on the human cholangiocarcinoma cell by blocking NF-κB signaling pathway,which may have potential value to be the drug candidate for the treatment of human cholangiocarcinoma in future.