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

[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。

[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 andβ-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.