从PI3K/Akt/mTOR信号通路调控ICC 自噬探讨温肾健脾法治疗溃疡性结肠炎肠动力紊乱的分子机制

Ulcerative colitis (UC) is a great harm, recurrent inflammatory bowel disease. The latest study found that intestinal motility disorder is an important mechanism of the incidence of UC, but ICC autophagy of the PI3K-Akt-mTOR signal mediated is closely related to intestinal motility disorders. Previous studies have shown that the method of warming kidney and invigorating spleen plays a regulatory role for intestinal mucosal immune disorders in UC, which can relieve some symptoms such as tenesmus and intestinal motility disorders. However, the mechanism of improving intestinal motility disorders is unknown. Based on this, The hypothesis about the method of warming kidney and invigorating spleen in treating intestinal motility disorders mechanism in UC and regulating ICC autophagy of PI3K-Akt-mTOR signal mediated has been established. Spleen kidney Yang deficiency syndrome UC rats model will be set up by adopting TNBS/ethanol solution clyster+subcutaneous injection of hydrocortisone+fenxieye gavage. Applying some technological means such as immunohistochemistry, immunoblotting, RT-PCR and TEM are used to test intestinal mucosa ICC autophagy, intestinal motility and the expression of PI3K-Akt-mTOR signal related factors for the treatment of Sishen Wan. The project is attended to explore the method of warming kidney and invigorating spleen in treating molecule mechanism of intestinal motility disorders in UC and provide experimental evidence for developing Chinese patent medicine that can adjust immunity and improve intestinal motility.

溃疡性结肠炎（UC）是危害巨大、反复发作的炎症性肠病。最新研究发现，肠道动力紊乱是 UC 发病的重要机制，而PI3K-Akt-mTOR信号介导的ICC 自噬与UC肠动力紊乱密切相关。我们前期研究表明，温肾健脾法对UC肠黏膜的免疫失调具有较好的调节作用，且能缓解里急后重、便次增多等肠道动力紊乱症状，但其改善肠动力紊乱的具体机制不明。基于此，项目建立“温肾健脾法”治疗UC肠动力紊乱的机制与其调节PI3K-Akt-mTOR信号介导的ICC自噬相关”的假说，采用TNBS/乙醇溶液灌肠+皮下注射氢化可的松+番泻叶灌胃建立脾肾阳虚型 UC大鼠模型，以四神丸进行治疗，运用免疫组化、免疫印迹、RT-PCR、透射电镜等技术检测肠黏膜ICC自噬、肠道动力及PI3K-Akt-mTOR信号相关因子的表达，探讨温肾健脾法治疗UC肠动力紊乱的分子机制，为开发具有免疫调节和改善肠动力双重作用的优势中成药提供实验依据。

溃疡性结肠炎是严重危害人体身心健康的炎症性肠病，易反复发作、治疗难度大，是结肠癌的癌前病变。目前西医缺乏特异性治疗，临床观察以四神丸为代表方的温肾健脾法可收获良效。前期研究发现四神丸对肠黏膜炎症损伤具有明显改善作用，本研究在前期研究的基础上，探讨“温肾健脾法”治疗UC的作用机制与ICC自噬对肠道动力紊乱影响的密切相关性，番泻叶灌胃给药+氢化可的松皮下注射诱导+DNBS/乙醇溶液灌肠法进行模型建立。以四神丸灌胃治疗，运用免疫组化、免疫印迹、RT-PCR、免疫荧光等技术检测大鼠肠道动力、肠黏膜ICC自噬、及PI3K-Akt-mTOR信号相关因子的表达，探讨温肾健脾法治疗UC肠动力紊乱的分子机制。实验结果：（1）空白组结肠黏膜上皮完整；模型组结肠黏膜及黏膜下层可见大量炎性细胞浸润，杯状细胞大量损坏、丢失；用药后，各组炎性细胞均有不同程度的减少，黏膜结构均有不同程度的改善且趋于完整。(2) 与空白组比较，模型组大鼠结肠组织c-kit蛋白及mRNA均显著减少（p＜0.01），Beclin1、LC3、p-PI3K、Akt、p-Akt、mTOR、p-mTOR蛋白及mRNA均显著增多（p＜0.01）；与模型组相比，用药后大鼠结肠组织Beclin1、LC3、PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR蛋白及mRNA均呈不同程度减少（p＜0.01或p＜0.05），c-kit蛋白及mRNA均呈不同程度增多（p＜0.01或p＜0.05）。结论：温肾健脾法及其代表方剂四神丸可有效改善脾肾阳虚型溃疡性结肠炎模型大鼠肠道动力紊乱、ICC自噬异常并有效下调脾肾阳虚型UC大鼠PI3K/Akt/mTOR信号通路关键基因和蛋白的表达量，调控炎症因子并改善 UC 大鼠的整体症状，提示四神丸可能通过调控PI3K/Akt/mTOR信号通路、调节自噬从而改善肠道动力紊乱有关。本项目对具有脾肾阳虚型UC的研究具有重大意义，温肾健脾法可通过调控PI3K/Akt/mTOR信号途径、调节肠道ICC自噬，使肠道动力得以恢复。从细胞自噬途径揭示中医理论“阳化气”的科学内涵及温肾健脾法“温阳化气、促肠动力”的分子机制，为中医从协调脾肾关系途径干预 UC 提供新的视角和靶标，为临床拓展中医经典效方的广泛运用提供实验依据，亦可为充分发挥中医药多靶点、多效应优势，开发防治溃疡性结肠炎的优势中成药提供理论支撑。