补中益气汤经葡萄糖转运体SGLT1的调控作用治疗脾虚泄泻证的分子机制研究

“Governing transportation and transformation” and “ascending lucid Yang” are two basic physiological function of spleen. Diarrhea due to spleen deficiency is often associated with nutrient absorption barriers caused by the spleen’s failure in transportation and transformation. Bu-zhong-yi-qi Tang (BZYQT) is common used as a “tonifing Qi and raising Yang” formula in Chinese Medicine and has significant effect of treating diarrhea due to spleen deficiency. SGLT1 is the main carrier of glucose by the small intestine absorption. NHE3 activity status and diarrhea is closely related. SGLT1 is the key factor to start NHE3 transposition to the intestinal brush border, and this process requires p38MAPK/Akt2 signal transduction pathway mediated. Bu-zhong-yi-qi decoction can increase the expression of SGLT1 in the mucous membrane of small intestine . So we propose that BZYQT in the treatment of diarrhea due to spleen deficiency is related to mediating the expression of NHE3 by regulating SGLT1 by p38MAPK/Akt2 signal transduction pathway. This subject intends to observe the changes of glucose absorption capacity and related substances of spleen deficiency syndrome and before and after BZYQT intervention from animal level. The correlation between SGLT1 and NHE3 will be verified at the cellular level. The molecular mechanism of SGLT1 regulating NHE3 and interfering effects of BZYQT will be explored from different levels of genes and protein by using siRNA technology too. In all, this project will illustrate the physiologic behaviors of diarrhea due to spleen deficiency, and provide the basis for the mechanism of “tonifing Qi and raising Yang”.

“主运化”和“升清阳”是脾的两大基本功能，脾虚泄泻常伴随着脾失运化所致的营养物质吸收障碍。补中益气汤治疗脾虚泄泻有显著的疗效。钠依赖葡萄糖转运体1（SGLT1）是葡萄糖经小肠吸收的主要载体，钠-氢交换体3（NHE3）的活性状态与泄泻发生密切相关，而SGLT1是启动NHE3转位至小肠刷状缘上的关键因素，该过程的发生需要p38MAPK/Akt2信号转导途径的介导。补中益气汤可提高小肠粘膜SGLT1的表达。推论该方治疗脾虚泄泻与其调控SGLT1经p38MAPK/Akt2信号转导途径介导NHE3表达有关。项目拟从动物水平观察脾虚证及予补中益气汤干预前后葡萄糖吸收及相关物质的变化，并在细胞水平上对SGLT1与NHE3之间的关联性进行验证，采用siRNA技术，在基因、蛋白水平探讨SGLT1对NHE3调控的分子机制及补中益气汤的干预效果，为探明脾失运化所致泄泻的科学本质及益气升阳法的作用机理提供依据。

项目的背景：项目以通过探讨补中益气汤对脾虚泄泻大鼠SGLT1/NHE3通路的影响，以及补中益气汤含药血清对caco-2细胞SGLT1、P38MAPK通路及NHE3蛋白及基因表达的影响，以进一步阐明该方的作用机制。.主要研究内容：(1)以“大黄+利血平+控制饮食”多因素法建立脾虚泄泻大鼠模型，随机分为补中益气汤低、中、高剂量组和脾虚泄泻模型组，观察大鼠造模及给药后泄泻指数及肠粘膜Na+-K+ATP酶的变化，采用western blot法检测钠依赖性葡萄糖转运体(SGLT1)、葡萄糖转运体2(GLUT2)、钠氢交换体3（NHE3）、P38MAPK、p- P38MAPK、MAPKAPK2、p-MAPKAPK2、AKT2、p-AKT2、EZRIN、p-EZRIN蛋白的表达。(2)根据血清药理学方法制备补中益气汤含药血清，将caco-2细胞分为正常组、根皮苷组、根皮苷+补中益气汤含药血清组和根皮苷+空白大鼠血清组。检测各组SGLT1蛋白和基因、P38MAPK/Ezrin通路蛋白和NHE3蛋白的表达。.重要结果：(1)补中益气汤给药后腹泻指数有不同程度的的下降，以补中益气汤中、高剂量组较为明显（P<0.01或P<0.05），脾虚泄泻大鼠小肠粘膜Na+-K+ATP酶活性与正常对照组比较显著降低（P<0.01），而补中益气汤有提高Na+-K+ATP酶活性的作用。脾虚泄泻大鼠小肠黏膜中SGLT1、GLUT2、NHE3、 P- P38MAPK、P-MAPKAPK2、P-Akt2、P-Ezrin蛋白表达量较正常组减低（P<0.01），经补中益气汤治疗后SGLT1、GLUT2、NHE3及P- P38MAPK、P-MAPKAPK2、P-Akt2、P-Ezrin蛋白表达量均有所提高（P<0.01或P<0.05）。(2)与根皮苷组比较，根皮苷剂+补中益气汤含药血清组caco-2细胞SGLT1蛋白表达有所上调（P <0.05），且呈浓度依赖关系；根皮苷剂+15%补中益气汤含药血清组caco-2细胞SGLT1mRNA，NHE3蛋白及P38MAPK/Ezrin通路磷酸化蛋白表达均有所增加，与根皮苷组比较差别有显著性（P <0.05）；根皮苷剂+补中益气汤含药血清组的SGLT1mRNA及NHE3、P-P38MAPK、P-Ezrin蛋白表达均高于根皮苷剂+15%空白血清组，差异有统计学意义（P <0.05