基于 Wnt/β-catenin 通路研究芪地通便方对慢传输型便秘小鼠的影响

Effects of Qidi Tongbian Prescription on mice with slow transit constipation based on Wnt/β-catenin pathway

关键词:  慢传输型便秘  芪地通便方  Wnt/β-catenin 信号通路

Key words:  slow transit constipation  Qidi Tongbian Prescription  Wnt/β-catenin signaling pathway

摘要:

[目的] 研究芪地通便方基于Wnt/β-catenin信号通路对慢传输型便秘小鼠的具体调节作用。[方法] 将30只BALB/c小鼠随机分为6组，每组5只。除空白组外，其余各组以盐酸洛哌丁胺（10 mg/kg）灌胃14 d建立慢传输型便秘小鼠模型，随后予高、中、低剂量（18.906、9.453、4.727 g/kg）芪地通便方及阳性药物乳果糖（3 g/kg）灌胃干预14 d。测量粪便含水率与数量及小肠推进率，苏木精-伊红（HE）染色评价结肠组织学病变，荧光实时定量聚合酶链反应（Real-time PCR）和免疫组化方法检测Wnt-3a和β-catenin的基因和蛋白表达水平，免疫荧光染色检测Cajal间质细胞（ICC）标记物Ano1蛋白的表达水平，酶联免疫吸附实验（ELISA）检测闭合蛋白（Occludin）、5-羟色胺（5-HT）、水通道蛋白3（AQP3）蛋白表达水平。[结果] 与空白组比较，模型组小肠推进率、粪便数量与含水率均显著降低（P<0.01），芪地通便方干预可以有效提高小肠推进率、粪便数量与含水率（P<0.05），改善组织学病变。与空白组比较，模型组Wnt-3a、β-catenin mRNA（P<0.05）和蛋白（P<0.01）表达升高，Ano1蛋白表达降低（P<0.01），Occludin蛋白表达降低（P<0.01），AQP3（P<0.01）蛋白表达升高；芪地通便方干预后，Wnt-3a蛋白、β-catenin mRNA和蛋白表达降低（P<0.05），Ano1蛋白表达提高（P<0.01），Occludin蛋白表达升高（P<0.01），AQP3（P<0.05）蛋白表达降低。[结论] 芪地通便方可能通过抑制Wnt/β-catenin信号通路过度激活发挥治疗慢传输型便秘的作用。

Abstract:

[Objective] To study the specific regulatory effects of Qidi Tongbian Prescription on slow transit constipation （STC） mice based on Wnt/β-catenin signaling pathway. [Methods] The 30 BALB/c mice were randomly divided into 6 groups，five mice in each group. Except the blank group，the STC mouse model was established by intragastric administration of loperamide hydrochloride （10 mg/kg） for 14 days，followed by intragastric intervention of high，medium and low doses（18.906，9.453，4.727 g/kg） of Qidi Tongbian Prescription and positive drug lactulose（3 g/kg） for 14 days. Fecal moisture content，fecal quantity and small intestine advancement rate were measured. Colon histological changes were evaluated by HE staining. Gene and protein expression levels of Wnt-3a and β-catenin were detected by PCR and immunohistochemistry. And the expression level of Ano1 protein in Cajal interstitial cells （ICC） was detected by immunofluorescence staining. The expression levels of Occludin，5-hydroxytryptamine（5-HT） and aquaporin 3（AQP3） were detected by ELISA. [Results] Compared with blank group，the small intestinal propulsion rate，fecal quantity and water content in model group were significantly decreased（P<0.01）. The intervention of Qidi Tongbian Prescription could effectively improve the small intestinal propulsion rate，fecal quantity and water content（P<0.05），and improve the histological lesions. Compared with blank group，the expression of Wnt-3a，β-catenin mRNA（P<0.05） and protein（P<0.01） increased in model group，while the expression of Ano1 protein decreased（P<0.01），Occludin protein decreased（P<0.01）. The protein expressions of AQP3（P<0.01） were increased. The prognosis showed that Wnt-3a protein，β-catenin mRNA and protein expression decreased（P<0.05），Ano1 protein expression increased（P<0.01），Occludin protein expression increased（P<0.01）. The protein expressions of AQP3 were decreased（P<0.05）. [Conclusion] Qidi Tongbian Prescription may play a role in the treatment of STC by inhibiting the overactivation of Wnt/β-catenin signaling pathway.