基于RIP1-RIP3/DRP1/Mit信号通路调控NLRP3炎性小体在溃疡性结肠炎中的作用探讨祛瘀生新方的调控机制

The proposal is based on the clinical effects of the Traditional Chinese Medicin methods of “Qu yu sheng xin,QYSX” (removing stasis tissues to stimulate the growth of new mucosa) on ulcerative colitis，and our published data including QYSX herbs can regulation of macrophage to reduced IL-1β、TNF-αand promoted IL-10 expression and colonic mucosal epithelial cell. Combining the result of present research that the action of the RIP1-RIP3/DRP1/Mit signal pathway on NLRP3 inflammasome is the key pathway in colonic mucosal ulcer healing, we design to verify our hypothesis that TCM QYSX method could regulate RIP1-RIP3/DRP1/Mit pathway to control NLRP3 inflammasome to expression of IL-1βand IL-18 which are the key inflammatory cytokines of ulcerative colitis to promote colonic mucosal regeneration. For explore this hypothesis, we will use intestinal epithelial cell culture and UC mice model, RT-PCR, Western blot, ELISA, FCM, enzyme immunoassay and Immunofluorescence co-localization,JC-1 method to demonstrate the RIP1-RIP3/DRP1/Mit pathway and NLRP3 inflammasome regulates intestinal epithelial cell multiplication, cell cycles and cell apoptosis and other biological behavior by Nec-1, CP-456773 and DRP1-DNA vector liposome transfection method multipoint-switch function of RIP1-RIP3/DRP1/Mit pathway and NLRP3 inflammasome in the refractory colonic ulcer healing, and explore the new mechanism of TCM QYSX method affecting RIP1-RIP3/DRP1/Mit pathway to promotec IECs regenration in ulcerative colitis.

基于UC的难治性及发病机制的不明确，和祛瘀生新法确切的疗效，以及课题组前期研究提出“祛瘀生新法治疗UC的作用机制的关键环节与调节RIP1-RIP3/DRP1/Mit信号通路调控NLRP3炎性小体的作用密切相关”的假说。项目离体实验拟采用多点阻断和重新激活通路的方法，验证该通路调控NLRP3炎性小体对UC细胞坏死、凋亡等的影响；探讨该通路调控NLRP3炎性小体及其下游通路在UC中的作用机制。在体实验拟通过RT-PCR、免疫酶标法、Western blot、ELISA、免疫荧光共定位及JC-1法等技术对干预后的UC小鼠结肠粘膜溃疡中该通路RIP1-RIP3、DRP1、MLKL、NLRP3炎性小体、ASC 、caspase-1、IL-1β及IL-18等关键蛋白及其mRNA和线粒体自噬、ROS水平的检测分析，明确该信号通路调节NLRP3炎性小体参与UC过程，探讨祛瘀生新法对细胞分子生物学物学机制。

研究背景：溃疡性结肠炎(Ulcerative colitis UC)是一种难治性的自身免疫性非特异性炎症性肠病，发病机制不明确，祛瘀生新方以“祛瘀补虚、敛疮生新”为主要治则，疗效确切，提出“该方治疗UC的作用机制可能与RIP1-RIP3/DRP1/ NLRP3信号通路调控线粒体自噬密切相关”的假说。.方法：细胞实验以小鼠肠上皮细胞(IECs)为平台，动物实验以UC小鼠模型为平台，采用NEC-1、CP-456773抑制通路及RIP1-DNA vetor/脂质体转染方式重新激活信号通路的方法，采用RT-PCR、Western Blot、ELISA检测细胞中该信号通路中相关蛋白及mRNA水平的表达；流式法、免疫荧光共定位、酶标仪分别检测IECs膜电位、线粒体自噬及ROS的水平；HE染色观察小鼠结肠黏膜组织学变化情况。.结果：细胞实验显示：NEC-1、CP-456773可抑制RIP1、RIP3、DRP1、NLRP3、IL-1β、IL-18的表达； RIP1-DNA vetor/脂质体可促使RIP1、RIP3明显升高，而NLRP3、IL-1β、IL-18升高不明显；祛瘀生新方血清能明显下调炎性IECs后RIP1、RIP3、NLRP3、Drp1、IL-1β、IL-18、caspase-1、ASC的表达，并能抑制ROS的升高和降低线粒体膜电位及LC3、FIS1的水平。动物实验结果显示：药物干预7d后祛瘀生新方对小鼠中RIP1、RIP3有明显的抑制作用；而第14、21天的作用不明显；对DRP1、NLRP3、IL-1β、IL-18、caspase-1、ASC、LC3a/b、FIS1也有明显的下调作用；能降低结肠黏膜细胞中ROS荧光值；该方对该信号通路中关键mRNA的表达也有一定的抑制作用。.结论：祛瘀生新方可能是通过RIP1-RIP3/DRP1/NLRP3信号通路调控线粒体自噬来达到降低炎症反应；祛瘀方、生新方对该通路的相关蛋白具有一定的降低作用，但作用明显弱于祛瘀生新方和美莎拉嗪；祛瘀方、生新方两者的效果并无明显差异，两者需要配伍合用才能达到更好疗效。

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