健脾解毒方通过Wnt/β-catenin信号通路下调幽门螺杆菌诱导的胃癌血管生成因子的研究

Jianpi Jiedu Recipe down-regulates angiogenic factors in Helicobacter pylori-induced gastric cancer cells by inhibiting Wnt/β-catenin signaling pathway

目的研究健脾解毒方对幽门螺杆菌(HP)感染胃癌细胞诱导的血管生成因子(VEGF)、血管生成素-2(Ang-2)、成纤维细胞生长因子(b FGF)的影响,并探讨其可能的机制。方法采用HP标准株NCTC11637与人胃癌MKN45细胞共培养的方法感染MKN45细胞,分为对照组、HP感染组、健脾解毒方醇提物低中高剂量组(低中高剂量分别为0.5 g/L、1.0 g/L和2.5 g/L),采用ELISA法检测健脾解毒方对HP感染诱导的人胃癌MKN45细胞VEGF、b FGF、Ang-2表达的影响;使用Wnt/β-catenin特异性抑制剂FH-535(20μmol/L)抑制Wnt/β-catenin活性后,再进行HP感染,ELISA法检测人胃癌MKN45细胞VEGF、b FGF、Ang-2蛋白表达的变化。Western blotting检测健脾解毒方对HP感染激活的人胃癌MKN45细胞Wnt/β-catenin信号通路活性的调控作用。结果 HP感染MKN45细胞12 h后,VEGF、b FGF、Ang-2蛋白的表达明显上调(P<0.01)。健脾解毒方醇提物低中高剂量组均可下调HP诱导的人胃癌MKN45细胞VEGF、b FGF、Ang-2蛋白的表达,并呈一定的剂量依赖效应。采用Wnt/β-catenin特异性抑制剂FH-535抑制Wnt/β-catenin活性后可明显抑制HP诱导的VEGF、b FGF、Ang-2蛋白的表达(P<0.01)。HP感染胃癌MKN45细胞12 h后,胞浆和胞核β-catenin蛋白表达明显上调(P<0.01),健脾解毒方可下调胞浆和胞核β-catenin蛋白表达,并呈一定的剂量依赖效应。结论健脾解毒方可下调HP诱导的人胃癌细胞VEGF、b FGF、Ang-2表达,抑制Wnt/β-catenin信号通路是其机制之一,这可能是该方抗胃癌作用的重要机制。

Objective To study the influence of Jianpi Jiedu Recipe on the expression of vascular endothelial growth factor （VEGF）, angiopoietin-2 （Ang-2） and basic fibroblast growth factor（bFGF） in Helicobacter pylori （HP） -infected gastric cancer ceils, and discuss its possible mechanism. Methods Human gastric cancer MKN45 cells were infected by HP standard strain NCTC11637 by co-culture. The cells were divided into the control group, HP infection group, alcohol extract of Jianpi Jiedu Recipe（JPJD） groups with low-, medium- and high-dose（ as 0.5 g/L, 1.0 g/L and 2.5 g/L respectively）. The effects of Jianpi Jiedu Recipe on the expression of VEGF,bFGF and Ang-2 in HP infected MKN45 cells were detected by ELISA. After the inhibition on Wnt/13-catenin activity by the specific inhibitor FH-535 at concentration of 20 μmol/L, the cells were infected by HP once again, and the expression changes of VEGF, bFGF and Aug-2 in MKN45 ceils were detected by ELISA. The regulation of Jianpi Jiedu Recipe on the activity of Wnt/β-catenin signaling pathway in HP infected MKN45 ceils were determined by Western blotting. Results After 12 hours of HP infection, the expression of VEGF, bFGF and Aug-2 in MKN45 cells were obviously up-regulated （ P 〈 0.01 ）. The JPJD alcohol extract with low-, medium- and high-dose could all down-regulate the expressions of VEGF, bFGF and Ang-2 in HP infected MKN45 cells, with a certain dose-dependent manner. After the intervention of FH-535, the activity of Wnt/β-catenin was suppressed, the expressions of VEGF, bFGF and Ang-2 were obviously inhibited in HP infected MKN45 cells （P 〈0. 01 ）. After 12 hours of HP infection, the protein expression of β-catenin in cytoplasm and nucleus of MKN45 ceils was obviously up-regulated（ P 〈0.01 ）. Jianpi Jiedu Recipe could down-regulate the protein expression of β-catenin in cytoplasm and nucleus,with a certain dose-dependent manner. Conclusion Jianpi Jiedu Recipe could down-regulate the expressions of VEGF,bFGF and Ang-2 in HP-infected human gastric cancer cells, and the inhibition on Wnt/β-catenin signaling pathway is one of its mechanisms, which may be its important mechanism in the treatment of gastric cancer.