从Ras/Raf/MEK/ERK通路探讨黄芪多糖调节脾虚水湿不化大鼠肝脏AQP3基因表达的机制研究

Abnormal expression of AQPs in liver is closely related to the occurrence of syndrome of dampness retention due to spleen deficiency. And the expression of AQPs is regulated by Ras/Raf/MEK/ERK pathway, which is one of the major pathways regulating the water transport. Our previous studies had demonstrated that Astragalus polysaccharide could significantly reduce the expression of AQP3 gene, increase serum total protein and albumin levels, improve the abnormal morphology and function of liver, and enhance the capacity of water transport in rats with syndrome of dampness retention due to spleen deficiency. It had been proved that Astragalus Polysaccharide affected the Ras/Raf/MEK/ERK pathway by inhibition of ERK phosphorylation. Therefore, we supposed that Astragalus polysaccharide could decrease the expression of AQP3 gene by regulating Ras/Raf/MEK/ERK pathway, and thereby, strengthen spleen and remove dampness. In this project, the mechanism of the gene expression of AQP3 in liver regulated by Astragalus polysaccharide based on Ras/Raf/MEK/ERK pathway was studied by using immunohistochemistry, RT-PCR and Western Blot in vivo and in vitro, in order to clarify the mechanism of strengthening spleen and removing dampness of Astragalus Polysaccharide.

肝脏AQPs表达异常与脾虚水湿不化证的发生密切相关。Ras/Raf/MEK/ERK通路是调控AQPs基因表达，改善水液运化的主要信号通路。项目组前期研究表明，黄芪多糖能显著降低脾虚水湿不化大鼠肝脏AQP3基因的表达，上调血清总蛋白和白蛋白水平，纠正肝脏形态和功能的异常，提高水液运化能力。研究证实，黄芪多糖可以抑制ERK磷酸化，影响Ras/Raf/MEK/ERK通路的调控作用。因此提出科学假说，黄芪多糖可以通过抑制Ras/Raf/MEK/ERK信号通路，降低AQP3基因的表达，发挥其健脾祛湿的功效。本项目通过体内、体外实验，运用免疫组化，RT-PCR以及Western Blot方法研究黄芪多糖通过干预Ras/Raf/MEK/ERK通路关键靶点Ras、Raf、MEK、ERK调节肝脏AQP3基因表达的机制，使用ERK抑制剂PD98059反证科学假说，为阐明黄芪多糖健脾祛湿的机制研究提供实验依据。

肝脏AQPs表达异常与脾虚水湿不化证的发生密切相关。Ras/Raf/MEK/ERK通路是调控AQPs基因表达，改善水液运化的主要信号通路。本项目提出科学假说，黄芪多糖可以通过抑制Ras/Raf/MEK/ERK信号通路，降低AQP3基因的表达，发挥其健脾祛湿的功效。本项目通过体内、体外实验，运用免疫组化，RT-PCR以及Western Blot方法研究了黄芪多糖通过干预Ras/Raf/MEK/ERK通路关键靶点Ras、Raf、MEK、ERK调节肝脏AQP3基因表达的机制，并使用ERK抑制剂反证科学假说，为阐明黄芪多糖健脾祛湿的机制研究提供实验依据。结果表明，黄芪多糖能明显改善大鼠脾虚水湿不化症状，提高其自主活动；促进胃肠功能，胃排空率、小肠推进率和D-木糖排泄率显著升高；明显改善脾虚水湿不化大鼠水液运化功能，尿量显著升高，水负荷指数显著降低；提高脾虚水湿不化大鼠血清蛋白水平，改善其血脂紊乱；缓解肝脏功能和形态的异常。体内外研究均表明，黄芪多糖各剂量组和ERK抑制剂组能显著降低AQP3基因和蛋白水平，提示Ras/Raf/MEK/ERK通路对肝脏 AQP3 有调节作用，黄芪多糖可能以AQP3为靶点影响脾虚水湿不化大鼠，从而改善其脾虚水湿不化症状。此外，黄芪多糖各剂量组能不同程度抑制Ras/Raf/MEK/ERK通路中Ras、 Raf、 MEK、ERK基因和蛋白表达以及其活性，其中，黄芪多糖高剂量组作用最为显著，表明黄芪多糖可能通过抑制Ras/Raf/MEK/ERK通路关键靶点降低肝脏AQP3水平，从而发挥其健脾祛湿功效。结论：黄芪多糖可以改善脾虚水湿不化症状，促进胃肠功能，提高蛋白水平，调节脂质代谢，促进水液运化，改善肝脏形态和功能的异常，发挥其健脾祛湿的功效。黄芪多糖健脾祛湿的分子机制可能与抑制Ras/Raf/MEK/ERK通路关键靶点从而降低肝脏AQP3水平有关。

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