黄芪-莪术-重楼配伍降低血管内皮通透性抑制结肠癌转移作用的研究

Study on the Inhibitory Effect of Huangqi-Ezhu-Chonglou Combination on Metastasis of Colon Cancer by Reducing Vascular Endothelial Permeability

目的观察黄芪-莪术-重楼配伍对人脐静脉内皮细胞株(HUVEC)通透性与细胞紧密连接的影响及其抑制人结肠癌细胞系(HCT116)血行转移的作用机制。方法制备空白对照血清和黄芪-莪术-重楼配伍含药血清。构建HUVEC-HCT116共培养体系,实验分为共培养模型组,黄芪-莪术-重楼配伍低、中、高剂量(2.1、4.2、8.4 g·kg^(-1))组。另设HUVEC单培养对照组。CCK-8法检测细胞增殖;FITC-dextran法、Transwell法检测HUVEC通透性与HCT116跨内皮迁移数量;免疫荧光法检测ZO-1蛋白的分布;Western blot法检测RhoA、ROCK及ZO-1蛋白表达量。结果黄芪-莪术-重楼配伍不同剂量含药血清处理48 h后,HUVEC增殖均显著减少(P<0.01)。与单培养对照组相比,共培养模型组FITC-dextran渗透量、跨越内皮迁移的HCT116细胞数量明显增加(P<0.01),ZO-1蛋白表达下调(P<0.01),RhoA、ROCK蛋白表达上调(P<0.01);与共培养模型组相比,黄芪-莪术-重楼配伍各剂量组FITC-Dextran渗透量、跨越内皮迁移的HCT116细胞数量均减少(P<0.05),ZO-1蛋白表达上调(P<0.05),RhoA、ROCK蛋白表达下调(P<0.05),且呈现剂量依赖性。结论黄芪-莪术-重楼配伍可能通过抑制RhoA/ROCK通路调控HUVEC紧密连接相关蛋白ZO-1的表达,降低血管内皮通透性,进而抑制结肠癌血行转移。

OBJECTIVE To observe the effects of Huangqi-Ezhu-Chonglou combination(HEC)on the permeability and tight junction of human umbilical vein endothelial cells(HUVEC)and to explore the mechanism of HEC on inhibiting the hematogenous metastasis of colon cancer cells(HCT116).METHODS Control serum and HEC-medicated serum were prepared.The co-culture system of HUVEC-HCT116 was constructed and divided into single culture control group,co-culture model group and HEC low,medium and high dose(2.1,4.2,8.4 g·kg^(-1))groups.In addition,HUVEC single culture control group was set up.Cell proliferation was detected by CCK-8 method;FITC-dextran method and transwell method were used to detect the permeability of HUVEC and the number of cross endothelial migration of HCT116 cells;The distribution of ZO-1 protein in HUVEC was detected by immunofluorescence;The protein expressions of RhoA,ROCK and ZO-1 were detected by Western blot.RESULTS The proliferation of HUVEC was inhibited after 48 h of treatment with different doses of HEC medicated serum(P<0.01).Compared with the single culture control group,FITC-dextran penetration and the number of transendothelial migration of HCT116 in the co-culture model group significantly increased(P<0.01),the expression of ZO-1 was down-regulated(P<0.01),and the expressions of RhoA and ROCK were up-regulated(P<0.01);Compared with the co-culture model group,the FITC-dextran penetration and the number of transendothelial migration of HCT116 in the HEC group significantly decreased(P<0.05),the expression of ZO-1 protein was up-regulated(P<0.05),and the expressions of RhoA and ROCK were down-regulated in a dose-dependent manner(P<0.05).CONCLUSION HEC may regulate the expression of tight junction associated proteins ZO-1 in HUVEC by inhibiting RhoA/ROCK pathway,reduce vascular endothelial permeability,and then inhibit colon cancer hematogenous metastasis.