黄芪多糖通过AQP5调节ERK/MLCK通路干预肠黏膜通透性改善脾虚水湿运化失常机制研究

Dysfunction of dampness transportation due to spleen deficiency can lead to various diseases or occur at different stages of disease progression and make its recurrent and persistent. Spleen deficiency is the basic pathogenesis of dysfunction of dampness transportation, which related to intestinal mucosa changes. Our previous studies have shown that Astragalus polysaccharides can significantly improve disorders of the intestinal function and water metabolism in rats with dampness stagnancy due to spleen deficiency syndrome, up-regulate low expression of AQP5. AQP5 not only promotes water and fluid excretion, but also induces ERK/MLCK pathway to enhance tight junction and decrease intestinal mucosal permeability. Therefore, we propose the scientific hypothesis: "Astragalus polysaccharide enhances tight junction and reduces intestinal mucosal permeability to attenuate dysfunction of dampness transportation due to spleen deficiency via ERK/MLCK pathway mediated by AQP5". In this study, the rats model with dampness stagnancy due to spleen deficiency syndrome and AQP5 silencing cells are established, gene transfection, qRT-PCR, western blot will be applied to study the regulatory effect of Astragalus polysaccharide on dysfunction of dampness transportation due to spleen deficiency caused by tight junction reduction and intestinal permeability increase because of AQP5-induced activation of ERK/MLCK pathway, and ERK inhibitor will be applied to verified that. This study will provide new research idea for revealing the mechanism action of Astragalus Polysaccharide in attenuating dysfunction of dampness transportation due to spleen deficiency.

脾虚水湿运化失常可引发各种病证或出现在疾病进展的不同阶段，并使其迁延不愈。脾虚失运是脾虚水湿运化失常的基本病机特征，与肠黏膜改变关系密切。我们前期研究表明，黄芪多糖可明显改善脾虚水湿不化大鼠肠道功能及水液代谢障碍，上调AQP5低表达。AQP5既促进水液排泌又可诱导ERK/MLCK通路增强紧密连接降低肠黏膜通透性。而据此提出科学假说：“黄芪多糖通过AQP5诱导的ERK/MLCK通路增强紧密连接，降低肠黏膜通透性，改善脾虚水湿运化失常”。本研究拟使用脾虚水湿不化动物模型和AQP5沉默细胞模型，采用基因转染、qRT-PCR、蛋白印迹等方法，研究黄芪多糖对AQP5低表达诱导ERK/MLCK通路激活破坏紧密连接增加肠黏膜通透性而导致脾虚水湿运化失常的调节作用，并使用ERK抑制剂进行反证，为揭示黄芪多糖改善脾虚水湿运化失常的分子机制提供实验依据和研究思路。

脾虚水湿不化是中医临床实践中常见的一种证型，也称为脾虚湿困，可引发各种病证或出现在疾病进展的不同阶段，并使其迁延不愈。脾虚失运是脾虚水湿运化失常的基本病机特征，与肠黏膜通透性改变密切相关。药食同源中药材黄芪具有健脾益气的功效，而黄芪多糖是从黄芪干根中提取的一种大分子物质，是黄芪健脾作用的主要成分，具有调节免疫、抗炎、保肝、抗肿瘤及利尿等多种药理作用。我们前期研究表明，脾虚水湿不化大鼠肠道AQP5表达水平降低，黄芪多糖可明显改善脾虚水湿不化大鼠水湿运化失常，提高AQP5水平。AQP5既促进腺体的水液排泌又可诱导ERK/MLCK通路增强紧密连接降低肠黏膜通透性。本项目主要研究了黄芪多糖“健脾祛湿”通过AQP5调节ERK/MLCK通路干预肠黏膜通透性改善脾虚水湿运化失常机制。研究中，在体复制了脾虚水湿不化大鼠模型，体外建立了AQP5-shRAN干扰细胞并构建了肠黏膜屏障体外模型，通过体重、一般状况评分、胃肠功能及水液代谢等指标评价模型大鼠脾虚水湿运化的情况，通过荧光素葡聚糖FITC-Dexran、单层细胞跨膜电阻（TEER）及荧光素钠跨膜通量测定肠黏膜通透性，透射电镜检测肠黏膜细胞间紧密连接状况，qRT-PCR、Western Blot技术检测AQP5、Occludin、ZO-1、Claudin-2、ERK1/2、p-ERK1/2、MLCK及p-MLC2表达水平，并采用ERK抑制剂进行反证。研究结果显示，黄芪多糖能够减轻脾虚水湿不化大鼠的症状、增加体重，增加AQP5表达水平，抑制ERK/MLCK通路，增加紧密连接蛋白Occludin和ZO-1表达水平，降低Claudin-2水平，增强紧密连接，降低肠黏膜通透性，改善脾虚水液运化失常，验证了项目"“黄芪多糖通过AQP5诱导的ERK/MLCK通路增强紧密连接，降低肠黏膜通透性，改善脾虚水湿运化失常”科学假说，为脾虚水湿不化证的研究及黄芪多糖治疗脾虚水湿不化的机制研究提供了新的科学依据。