基于蛋白激酶A（PKA）及丝裂原活化蛋白激酶（MAPK）传导通路探讨通便汤治疗STC的机制研究

Slow transit constipation (STC) is an intractable digestive tract disease which is caused by abnormality of colon motility. Because of unknown exact etiology and lack of effective therapy at present, it has been considered to be one of the world’s refractory diseases. Zhu Bingyi, a famous TCM doctor from our hospital, created an empirical formulanamed “Tongbian Decoction ”according to the theory that “deficiency of yin fluid causesfailure of distributing lung-fluid”. The decoction has a remarkable clinical efficacy on the treatment of STC and shows superiority of TCM treatment on chronic diseases. However, its specific mechanism of action still exists unknown. In the stage of preliminary study, we created Tongbian Granule which is made from Tongbian Decoction and tested the expression, distribution and relative amount of aquaporin 3 (AQP3) and aquaporin 8 (AQP8) in colons of STC rats by immunohistochemistry. It is initially thought that Tongbian Granule may treats constipation by inhibiting the expression of AQPs and thus reducing the water absorption of intestinal tract. On this basis, the study used Western blot and fluorescent quantitative PCR (FQ-PCR) to test AQPs and its genes more accurately and adopted the clinical empirical Tongbian Decoction to observe its effect on the expression of AQPs of experimental animals with polyethylene glycol powder in contrast and then discussed further the effect of protein kinase A (PKA) and mitogen-activated protein-kinases( MAPA) signal pathway on the AQPs regulatory process.It aims at discussing the partial mechanism of action and pathway of Tongbian Decoction on the treatment of STC in molecular level and then provides new thinking and methods of the prevention and cure of constipation.

慢性传输型便秘是一类以结肠动力障碍所致的顽固性消化道疾病，因其病因不明，目前缺乏有效的治疗方法，已被确定为世界难治性疾病之一。我院名老中医朱秉宜根据“津不足，津不布”创立经验方“通便汤”在临床上治疗STC疗效显著，发挥了中医药治疗慢性疾病的优势，但其具体作用机制尚不清楚。我们前期研究将通便汤制成通便颗粒并运用免疫组化检测STC大鼠结肠水通道蛋白3、8表达分布及相对含量，初步认为通便颗粒可能是通过抑制AQPs的表达从而减少肠道对水分的吸收来治疗便秘。在此基础上，本研究运用Western blot和荧光定量PCR更为准确地对AQPs及其基因进行检测，采用临床经验方通便汤，以聚乙二醇散剂作对照，观察本方对实验动物AQPs表达的影响，并进一步探讨蛋白激酶A（PKA）及丝裂原活化蛋白激酶（MAPK）信号传导通路在通便汤对AQPs调节过程中的作用，旨在从分子水平探讨通便汤治疗STC的部分作用机制与途径

水通道蛋白(aquaporins，AQPs)是维持结肠内水代谢平衡的分子学基础，尤其是AQP3和AQP4，AQP3在mRNA水平上被环磷酸腺苷(cyclic adenosine monophosphate，cAMP)所上调，而这一调节又是通过蛋白激酶A(protein kinase A，PKA)信号通路来实现的，AQP4的磷酸化是由PKA所介导，并且随后发生胞化内化，磷酸化可能是AQP4囊泡循环的机制；丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)信号传导通路中亚型细胞外信号调节的蛋白激酶(extracellular signal rehulating kinase，ERK)，c-JunN端激酶(c-Jun Nterminal kinase，JNK)，p38丝裂原活化蛋白激酶(p38 MAPK)的激活与水通道蛋白的表达有一定的相关性，在STC的发生发展中发挥着重要作用。本课题分别通过一期和二期实验STC模型验证通便汤治疗STC的有效性，通过阻断PKA/MAPK信号通路从而下调AQP3、AQP4调节肠内水分吸收、分泌，初步诠释本方治疗STC的作用机制；课题组继而通过STC大鼠的肺肠组织，运用ELISA法观察本方对于肺肠组中神经递质的SP、VIP的影响，初步诠释通便汤方治疗STC的“肠病及肺”的生物学机制。通过酶解大鼠肠道组织的方法，获得ICC细胞，经通便汤方干预后，观察ICC细胞增殖或者凋亡情况，探讨了本方对ICC自噬可能的影响，取得阳性结果。.结果：①PKA/MAPK信号通路实验：通便汤可明显改善便秘症状，通过阻断PKA/MAPK信号通路从而下调AQP3、AQP4调节肠内水分吸收、分泌，治疗慢传输便秘效果良好。②初探“肠病及肺”机制实验：肺肠神经递质SP、VIP及肺AQP1、肠AQP3可能是便秘“肠病及肺”的生物学基础，通便汤治疗便秘可能是通过上调肺肠组织SP含量，下调肺肠VIP含量及肺AQP1、肠AQP3蛋白含量能明显改善大鼠便秘症状，促进肠道蠕动，并能减少结肠留存大便量，能够有效治疗STC。③ICC自噬机制：通便汤治疗STC与用药剂量有一定联系，高剂量通便汤治疗STC效果最佳，主要机制可能与下调Beclin1、LC3表达而抑制ICC过度