基于Wnt3a/β-catenin通路研究金银花提取物对脱氧胆酸诱导胃黏膜肠上皮化生的影响

The effect of flos lonicerae extract on deoxycholic acid-induced intestinal metaplasia of gastric mucosa based on Wnt3a/β-catenin pathway

目的探讨金银花提取物(FL)是否通过Wnt3a/β-连环蛋白(β-catenin)通路影响脱氧胆酸(DCA)诱导的胃黏膜肠上皮化生。方法采用四甲基偶氮唑蓝(MTT)法检测1、10和100 g/L金银花提取物对DCA诱导的胃黏膜上皮细胞GES-1增殖的影响。将GES-1细胞分为对照组(未处理),DCA组(500μmol/L DCA),DCA+FL组(500μmol/L DCA+100 g/L FL),DCA+FL+Wnt3a/β-catenin通路激活剂LiCl组(500μmol/L DCA+100 g/L FL+LiCl)。采用流式细胞术和Western blot分别测定GES-1细胞的凋亡和Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、三叶因子2(TFF2)、绒毛蛋白(VIL1)、CDX2、Wnt3a和β-catenin的表达水平。结果1、10和100 g/L金银花提取物降低DCA诱导的GES-1增殖,差异有统计学意义(P<0.05),选择金银花提取物浓度最明显的100 g/L进行后续研究。与对照组比较,DCA组GES-1细胞的凋亡率、Bax蛋白表达水平降低,Bcl-2、TFF2、VIL1、CDX2、Wnt3a和β-catenin蛋白表达水平升高,差异有统计学意义(P<0.05);与DCA组比较,DCA+FL组GES-1细胞的凋亡率、Bax蛋白表达水平提高,并且Bcl-2、TFF2、VIL1、CDX2、Wnt3a和β-catenin蛋白水平降低,差异有统计学意义(P<0.05)。与DCA+FL组比较,DCA+FL+LiCl组GES-1细胞的凋亡率、Bax蛋白水平减少,Bcl-2、TFF2、VIL1和CDX2的蛋白水平增加,差异有统计学意义(P<0.05)。结论金银花提取物通过下调Wnt3a/β-catenin通路,促进DCA诱导的胃黏膜上皮细胞的凋亡和抑制肠上皮化生。

Objective To investigate whether flos lonicerae extract(FL)affects deoxycholic acid(DCA)-induced gastric mucosal intestinal metaplasia through Wnt3 a/β-catenin pathway.Methods The tetramethylazazole blue(MTT)method was applied to detect the effects of 1,10,100 g/L flos lonicerae extract on the proliferation of gastric epithelial cells GES-1 induced by DCA.GES-1 cells were devided into control group(untreated),DCA group(500μmol/L DCA),DCA+FL group(500μmol/L DCA+100 g/L FL),DCA+FL+Wnt3 a/β-catenin pathway activator LiCl group(500μmol/L DCA+100 g/L FL+LiCl).Flow cytometry and Western blot were used to determine the apoptosis of GES-1 cells and protein Bcl-2 related X protein(Bax),B cell lymphoma/leukemia-2(Bcl-2),TFF2,VIL1,CDX2,Wnt3 a,β-catenin expression level.Results DCA-induced GES-1 proliferation were reduced in flos lonicerae extract dose groups(1,10,100 g/L),the difference was statistically significant(P<0.05),the most effective concentration of flos lonicerae extract(100 g/L)was selected for follow-up research.Compared with the control group,the apoptosis rate and Bax protein expression level of GES-1 cells in the DCA group decreased,and the expression levels of Bcl-2,TFF2,VIL1,CDX2,Wnt3 a andβ-catenin were increased,and the difference was statistically significant(P<0.05);Compared with the DCA group,the apoptosis rate of GES-1 cells,the expression level of Bax protein in the DCA+FL group was increased,and the protein levels of Bcl-2,TFF2,VIL1,CDX2,Wnt3 a andβ-catenin were decreased,and the difference was statistically significant(P<0.05).Compared with the DCA+FL group,the apoptosis rate and Bax protein level of GES-1 cells in the DCA+FL+LiCl group were decreased,and the protein levels of Bcl-2,TFF2,VIL1 and CDX2 were increased,the difference was statistically significant(P<0.05).Conclusion Flos lonicerae extract promotes DCA-induced apoptosis of gastric mucosal epithelial cells and inhibits intestinal metaplasia by down-regulating the Wnt3 a/β-catenin pathway.