补肾解毒散结方对脱氧胆酸诱导的TGR5活性及人结肠癌细胞侵袭转移的影响

Effects of Bushen Jiedu Sanjie Recipe on deoxycholic acid-induced TGR5 activity and invasion and metastasis of human colon cancer cells

目的:探讨补肾解毒散结方对脱氧胆酸(DCA)诱导的TGR5活性和人结肠癌细胞侵袭转移的影响及其可能机制。方法:分别以TGR5稳转HEK293细胞和人结肠癌HCT-116细胞为对象进行实验。将细胞分为对照组和DCA组、DCA+中药低剂量组、DCA+中药高剂量组。除对照组外,其他各组均先给予DCA(30μmol/L)干预;DCA+中药低剂量组和DCA+中药高剂量组再分别给予20μg/ml和60μg/ml补肾解毒散结方醇提液;对照组仅给予等体积的培养基。细胞培养24 h后,双荧光素酶报告基因检测HEK293细胞中TGR5转录活性;划痕实验观察0、12、24 h各组HCT-116细胞的迁移情况,计算细胞迁移率;Western blot检测各组HCT-116细胞表皮生长因子受体(EGFR)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)的蛋白表达;ELISA检测各组HCT-116细胞血管内皮生长因子(VEGF)表达。结果:①双荧光素酶报告基因检测显示,DCA组细胞TGR5转录活性较对照组显著上调(P<0.01);高、低剂量补肾解毒散结方作用后均可显著抑制DCA诱导的TGR5过表达(P<0.01),且DCA+中药高剂量组细胞的TGR5活性明显低于DCA+中药低剂量组(P<0.01)。②细胞划痕实验显示,DCA组细胞12、24 h时迁移率较对照组明显升高(P<0.01);高、低剂量补肾解毒散结方作用后均可显著抑制DCA诱导的HCT-116细胞的迁移,细胞迁移率较DCA组显著降低(P<0.01),且DCA+中药高剂量组的细胞迁移率明显低于DCA+中药低剂量组(P<0.05)。③蛋白表达检测显示,DCA组细胞MMP-2、MMP-9、EGFR蛋白表达和VEGF含量较对照组显著上调(P<0.05,P<0.01);与DCA组相比,高、低剂量补肾解毒散结方作用后均可显著下调MMP-2、MMP-9、EGFR蛋白表达,降低VEGF含量(P<0.01),且DCA+中药高剂量组细胞的MMP-9、EGFR蛋白表达水平和VEGF含量明显低于DCA+中药低剂量组(P<0.05,P<0.01)。结论:补肾解毒散结方可下调DCA诱导的TGR5过表达,并抑制DCA诱导的人结肠癌细胞侵袭转移。

Objective:To investigate the effects and possible mechanisms of Bushen Jiedu Sanjie Recipe on deoxycholic acid(DCA)-induced TGR5 activity and invasion and metastasis of human colon cancer cells. Methods:The HEK293 cells stably transfected with TGR5 and the human colon cancer HCT-116 cells were taken as the subjects for the experiment, respectively. The cells were divided into the control group, DCA group, DCA+Chinese medicine low-dose group and DCA+Chinese medicine high-dose group. Except the control group, the other groups were treated with DCA at 30 μmol/L;then the DCA+Chinese medicine low-dose group and the DCA+Chinese medicine high-dose group were treated with the alcohol extract of Bushen Jiedu Sanjie Recipe at 20 μg/ml and 60 μg/ml, respectively;and the control group was only treated with the medium at equal volume. After cell culture for 24 hours, the transcription activity of TGR5 in HEK293 cells was detected by double luciferase reporter gene assay;the migration of HCT-116 cells at 0, 12 and 24 h was observed in the scratch test, and the cell migration rate was calculated;the protein expressions of epidermal growth factor receptor(EGFR), matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) in HCT-116 cells of each group were detected by Western blot;and the expression of VEGF in HCT-116 cells was detected by ELISA. Results:① The results of double luciferase reporter gene assay showed that the transcription activity of TGR5 in the DCA group was significantly higher than that in the control group(P<0.01);Bushen Jiedu Sanjie Recipe at high-and low-dose could significantly inhibit the overexpression of TGR5 induced by DCA(P<0.01);and the activity of TGR5 in the DCA+Chinese medicine high-dose group was significantly lower than that in the DCA+Chinese medicine low-dose group(P<0.01). ② The cell scratch test showed that the cell migration rate of the DCA group was significantly higher than that of the control group at 12 and 24 h(P<0.01);Bushen Jiedu Sanjie Recipe at high-and low-dose could significantly inhibit the migration of HCT-116 cells induced by DCA, and the cell migration rate was significantly lower than that of the DCA group(P<0.01);the cell migration rate of the DCA+Chinese medicine high-dose group was significantly lower than that of the DCA+Chinese medicine low-dose group(P<0.05). ③ The protein expressions of MMP-2, MMP-9, EGFR and the VEGF content in the DCA group were significantly higher than those in the control group(P<0.05, P<0.01);compared with the DCA group, the protein expressions of MMP-2, MMP-9, EGFR and the VEGF content were significantly down-regulated in the DCA+Chinese medicine low-dose group and DCA+Chinese medicine high-dose group(P<0.01);and the protein expressions of MMP-9 and EGFR and the VEGF content in the DCA+Chinese medicine high-dose group were obviously lower than those in the DCA+Chinese medicine low-dose group(P<0.05, P<0.01). Conclusion: Bushen Jiedu Sanjie Recipe can down-regulate DCA-induced overexpression of TGR5, and inhibit the invasion and metastasis of human colon cancer cells.