基于CFTR调控AQP3阐释薏苡仁油健脾利湿的机制

Spleen deficiency and wet dampness ZHENG (SDWD) is a one of the common syndrome of traditional Chinese medicine (TCM). It can be expressed in obesity, cancer and other diseases, and it will also make a block on the road of clinical treatment. Previous studies have found that the oils of Coix seed can improve the syndrome score of SDWD in rat model. And its effect is related to the regulation of AQP3 expression, but its mechanism is unknown..Recent studies have confirmed that the expression of AQP3 is regulated by cAMP-PKA signal-pathway. And some studies found CFTR can regulate the expression of AQP3. CFTR is the typical cAMP-dependent protein. Accordingly, we speculated that the Coix seed oils plays a major role in regulating SDWD by inhibiting the expression of CFTR in order to influence the expression of AQP3 through the cAMP-PKA signaling pathway..The hypothesis is confirmed in vivo and in vitro. In vivo the syndrome scores is calculated by PLS-DA method and the effect of Coix seed oil is monitored in SDWD rat model. In vitro the water movement is monitored in human intestinal epithelial cells. The level of CFTR and AQP3 are detected by immunohistochemistry, qRT-PCR and western blot. The main role of Coix seed oils in treating SDWD is explained based on CFTR and AQP3 regulation.

脾虚湿阻证是常见的中医证候之一，常见于肥胖、肿瘤等多种疾病，健脾利湿法对疾病的防治具有重要意义。前期研究发现，薏苡仁油能显著提高脾虚湿阻大鼠的证候评分，改善脾运化功能；其功效与AQP3有关，但上游机制未明。.cAMP-PKA通路是AQP3表达的主要调控机制，CFTR同为cAMP依赖蛋白；有报道称CFTR可以影响AQP3表达、改善水转运。由此假设：薏苡仁油能作用于cAMP-PKA通路，抑制CFTR，进而调节AQP3表达和活性，影响水液转运，发挥健脾利湿作用。.项目拟从两方面验证假说。体内构建动物模型，PLS-DA量化证候程度，观察薏苡仁油健脾利湿功效；体外以cAMP激活剂诱导AQP3高表达，观察薏苡仁油对水液转运的影响，并以CFTR激活剂反证。并运用免疫组化、qRT-PCR、WB等技术检测CFTR、AQP3水平，验证薏苡仁油影响CFTR、调控AQP3的关键作用，阐释其健脾利湿的机制。

脾虚湿阻证是中医常见证候之一，与多种疾病密切相关。健脾利湿能够显著改善脾运化功能，其功效与受CFTR调控的AQP3有关。本项目提出假说，薏苡仁油能够作用于cAMP-PKA信号通路，抑制CFTR表达，进而调节AQP3的表达和活性，影响水液转运，发挥健脾利湿作用。项目通过体内构建动物模型，PLS-DA量化证候程度，观察薏苡仁油干预效果，体外以cAMP激活剂诱导AQP3高表达，观察薏苡仁油对水液转运的影响，并以CFTR激活剂验证，运用免疫组化、qRT-PCR、WB等多种技术手段，检测CFTR、AQP3水平，验证薏苡仁油影响CFTR、调控AQP3的作用，阐释其健脾利湿的机制。结果表明，薏苡仁油能够显著改善模型大鼠脾虚湿盛证候评分，改善血浆及肠道组织MTL、GAS、VIP、ADH、cAMP、PKA、AQP3和CFTR含量，影响Ht-29细胞水通透性；同时体内外实验均显示，CFTR、AQP3及cAMP/PKA变化在蛋白层面具有相似变化趋势，使用cAMP激活剂后，AQP3和CFTR mRNA均出现显著增加，抑制AQP3后，CFTR表达也降低； CFTR激活后AQP3含量明显增加，抑制后AQP3含量降低；薏苡仁油同样能够降低CFTR激活细胞的AQP3含量，证明薏苡仁油可能通过CFTR调控AQP3的表达。结论： CFTR和AQP3均受cAMP-PKA调控，同时AQP3表达受CFTR影响。薏苡仁油健脾利湿功效的发挥，与CFTR调控的AQP3有关，而这一过程受cAMP-PKA通路调节。