基于脾肾相关理论研究溃疡性结肠炎粘膜微环境TLR/IL-1信号通路负性调控因子的响应及温肾健脾法的干预机制

Ulcerative colitis (UC) is a kind of inflammatory bowel disease with longer duration and high relapse rate. It is difficult to treat and also is the precancerous lesions of colon cancer. At present, there is no specific treatment for it. Sishen Pill, the classic treatment of diarrhea, using it for treatment of UC has a significant effect, but its role in the mechanism is unclear. Our previous study found, the Sishen Pill had a protective to the damage of rat intestinal mucosa caused by TNBS / ethanol -induced, create the "working hypothesis" has closely related between the Wenshen spleen treatment the UC effect mechanism and regulation TLR/IL-1 of signaling pathways negative regulatory factors ", using TNBS / ethanol + subcutaneous injection hydride cortisone + senna decoction orally to establish UC Syndrome binding model, through Sishen Pill intervention, SP immunohistochemical method, immunoblotting, and real-time quantitative PCR for detecting the expression of negative regulatory factors of UC intestinal mucosa the microenvironment TLR/IL-1 signal pathways, to analyse TLR/IL-1 signaling pathway dynamic expression pattern and Warming spleen treatment intervention mechanism in the UC, clarify the new target and efficacy mechanism of treatment of UC by Wenshen spleen, provide the theoretical support for development and screening of Compound preparation of traditional Chinese medicine to the treatment of UC.

溃疡性结肠炎（UC）是严重危害患者健康，且反复发作的炎症性肠病，容易恶化和癌变，临床缺乏特异性治疗药物。四神丸是治疗泄泻的经典方，临床以四神丸为基本方加减治疗UC具有显著疗效，但其作用的机制不清。前期研究发现, 四神丸对TNBS/乙醇诱导的大鼠肠黏膜损伤具有保护作用，研究基于"脾肾相关"是UC的临床基础和重要论治理论，建立"温肾健脾法治疗UC的疗效机制与调控TLR/IL-1信号通路的负性调控因子密切相关"的工作假说，采用TNBS/乙醇+皮下注射氢化可的松+番泻叶水煎液灌胃建立UC病证结合模型，用四神丸干预，采用SP免疫组化法、免疫印迹、实时定量PCR法检测UC肠粘膜微环境TLR/IL-1信号通路相关负性调控因子的表达，分析TLR/IL-1信号通路在UC 中的动态表达规律及温肾健脾法的干预机理，阐明温肾健脾法治疗 UC的作用新靶点及疗效机制，为开发和筛选治疗UC的特效中医复方制剂提理论支撑。

溃疡性结肠炎（UC）是一种严重危害患者健康的炎性肠病，临床缺乏特异性疗法。我们采用TNBS/乙醇溶液灌肠+皮下注射氢化可的松+番泻叶灌胃制备脾肾阳虚型UC大鼠模型，选用温肾健脾法的代表方四神丸进行干预，运用ELISA法检测各组大鼠血清中促炎细胞因子IL-1、IL-6、IL-8、IL-12、INF-γ、IL-1β和抑炎细胞因子IL-4、IL-5、IL-10、IL-13 、TGF水平，免疫组化SP法、RT-qPCR及Western blot法检测结肠组织中TLR、IL-1、NOD2、IRAK-M、SOCS2、SOCS3、Tollip、SIGIRR mRNA及蛋白表达。实验一通过研究TLR/IL-1信号通路中负性调控因子NOD2、IRAK-M、SOCS、Tollip 、SIGIRR的动态表达，预期证明TLR/IL-1信号通路与UC发生的相关性。研究结果表明，TNBS/乙醇溶液灌肠+皮下注射氢化可的松+番泻叶灌胃可成功建立脾肾阳虚型UC大鼠模型，且结肠损伤随着造模时间不同呈现动态变化趋势，其中以3d 和7d病变最为严重，14d和21d有所修复，阐明了TLR/IL-1信号通路中负性调控因子NOD2、IRAK-M、SOCS、Tollip 、SIGIRR的动态表达与溃疡性结肠炎发病的相关性。实验二采用四神丸干预，阐明温肾健脾法对UC粘膜微环境中TLR/IL-1信号通路负性调控因子NOD2、IRAK-M、SOCS、Tollip、SIGIRR表达的影响，预期证明四神丸治疗UC的机制。实验结果证明了温肾健脾法（四神丸）治疗溃疡性结肠炎的整体调节机制可能是通过调控TLR/IL-1信号通路中负性调控因子的表达，促进了肠粘膜微环境中的免疫恢复平衡，从而达到治疗UC的目的。我们已按计划如期完成该项目，并通过该项目培养了硕士研究生3名，发表了论文6篇，其中CSCD 5篇，一般期刊1篇。