多胺对SGLT1通路介导的葡萄糖吸收的影响及益气健脾方药的干预作用

Intestinal uptake of glucose is one of the spleen's important form that it governs the transportation and transformation of cereal essence. Spleen Qi deficiency has intestinal epithelial cell injury and glucose malabsorption. Replenishing qi to invigorate the spleen is the fundamental principle of TCM treatment of spleen qi deficiency syndrome.Yiqi Jianpi recipe has an obviouse effect on gastrointestinal mucosa protection and the improvement of glucose malabsorption of spleen deficiency syndrome. Sodium-dependent glucose transporter1(SGLT1) pathway is very important in glucose absorption of intestine, SGLT1 expression has close relationship with the intestinal mucosa epithelial cells along the intestinal villus axis of differentiation and cell cycle.Polyamine which has the functions of promoting the intestinal mucosal epithelial cell's proliferation, differentiation et al can improve D-glucose exertion of modle rat in our previous research. Based on these findings, this research puts forward the hypothesis"regulation of glucose uptake by polyamine through SGLT1 pathways is one of the important mechanisms about the spleen transport and Yiqi Jianpi recipe treatment effect ". In order to verify this hypothesis, this paper intends to give an animal experiment to observe the spleen qi deficiency syndrome and polyamines and Yiqi Jianpi recipe treatment before and after the intervention of the small intestinal mucosal morphology, glucose uptake, polyamine content, ODC, SGLT1 and GLUT2 gene and protein expression changes; Used antagonists and RNAi in a cellular model, clear the regulation mechanism of polyamines on SGLT1 pathway and the intervention effect of Yiqi Jianpi recipe. Provide the evident of exploring the essence of deficiency of spleen and the mechanism of Yiqi Jianpi recipe.

葡萄糖小肠吸收是脾主运化水谷精微的重要形式之一。脾气虚证存在着肠黏膜上皮细胞损伤及葡萄糖吸收障碍。益气健脾法是中医治疗脾气虚证的根本法则，益气健脾方药具有明显的胃肠黏膜保护及改善葡萄糖吸收障碍作用。钠依赖性葡萄糖转运载体1（SGLT1）通路在小肠葡萄糖吸收中起重要作用，其表达与肠黏膜上皮细胞沿肠绒毛轴的分化及其细胞周期关系密切。前期研究表明具有促进肠黏膜上皮细胞增殖、分化等作用的多胺能促进模型大鼠的D-木糖排泄率。因此，本研究提出"多胺对SGLT1介导的葡萄糖吸收的调节作用是脾主运化及益气健脾方药治疗作用的重要机制之一"这一假说。为验证该假说，本课题拟以动物实验观察脾气虚证及予以多胺和益气健脾方药治疗干预前后小肠黏膜形态、葡萄糖吸收、多胺含量、ODC和SGLT1、GLUT2基因及蛋白表达的改变；在细胞模型中通过拮抗剂和RNAi的使用，明确多胺对SGLT1调控机制以及益气健脾方药的干预作用。

脾虚证存在着肠黏膜上皮细胞损伤及葡萄糖吸收障碍。钠依赖性葡萄糖转运载体1（SGLT1）通路在小肠葡萄糖吸收中起重要作用，其表达与肠黏膜上皮细胞沿肠绒毛轴的分化及其细胞周期关系密切。前期研究表明具有促进肠黏膜上皮细胞增殖、分化等作用的多胺能促进模型大鼠的D-木糖排泄率提示多胺具有促进脾虚证肠黏膜葡萄糖吸收的可能性。为了揭示多胺对SGLT1介导的葡萄糖吸收的调节作用是脾主运化及益气健脾方药治疗作用的重要机制之一，复制了利血平脾虚大鼠模型，观察模型组及给予多胺、表皮生长因子和益气健脾方药干预后小肠黏膜形态、葡萄糖吸收、多胺含量、ODC和SGLT1、GLUT2基因及蛋白表达的改变；研究表明脾虚大鼠葡萄糖的吸收显著降低，空肠粘膜损伤明显，肠黏膜多胺含量，ODC和SGLT1、GLUT2基因及蛋白表达下调，经过多胺、表皮生长因子及四君子汤干预，以上指标得到不同程度的恢复；为进一步探讨多胺及四君子汤促进葡萄糖吸收是否通过促进肠黏膜修复从而激活SGLT1通路而产生，在Caco-2细胞模型中通过多胺合成抑制剂DFMO及SGLT1拮抗剂根皮苷的使用，观察Caco-2细胞增殖、微绒毛结构、葡萄糖摄取、SGLT1及GLUT2 mRNA及蛋白表达、钙离子（[Ca2+]cyt）水平、P-MLC蛋白的表达及多胺含量的变化，实验结果表明，DFMO抑制Caco-2细胞增殖，降低多胺含量，破坏微绒毛结构，使葡萄糖摄取及SGLT1及GLUT2 mRNA及蛋白表达下调，使钙离子（[Ca2+]cyt）水平及P-MLC蛋白表达下调，多胺及四君子汤对DFMO及根皮苷作用下的上述指标具有修复作用。本研究证实了脾虚证肠黏膜损伤可影响SGLT1通路介导的葡萄糖吸收，具有修复肠黏膜作用的多胺可通过促进SGLT1通路相关基因和蛋白表达从而提高葡萄糖的吸收，治疗脾虚证四君子汤可通过提高肠黏膜多胺含量达到肠黏膜修复及改善葡萄糖吸收障碍，以上研究为揭示了“脾主运化”分子机制及健脾方药作用机理提供了实验数据。