基于泛素化修饰酶A20调控探讨艾灸对TNF-α介导克罗恩病肠上皮屏障损伤的关键作用机制

On the basis of previous National Natural Science Foundation, with abnormalities of TNF-alpha-mediated TNFR1-associated death domain(TRADD)-Fas-associated death domain(FADD) apoptotic pathway and nuclear factor κB(NF-κB)-myosin light chain kinase(MLCK) tight junction pathway, which are dually negatively regulated by the expression of ubiquitination enzyme A20, being the main link of causing intestinal epithelial barrier damage in Crohn's disease(CD) as the starting point, and using the techniques of gene and molecular biology, the project has studied through intestinal epithelial gene A20-knockout CD animal model and gene A20-silenced cell model, in order to ① prove that, A20 is the main regulatory proteins of CD intestinal epithelial barrier damage by deubiquitination of important adapter molecules TRADD, receptor-interacting protein(RIP) and tumour-necrosis factor receptor-associated factor 6(TRAF6), and negative regulation of TNF-alpha / TRADD-FADD apoptotic pathway and TNF-alpha / NF-κB-MLCK tight junction pathway; ② preliminarily clarify that moxibustion may promote or regulate expression of CD intestinal epithelial A20, thereafter regulate abnormalities of the two TNF-alpha-mediated pathways, thus reduce excessive apoptosis of intestinal epithelial cells and improve abnormal closely connection of intestinal epithelial cells, which is the key mechanism of moxibustion in protecting or repairing TNF-alpha-mediated CD intestinal epithelial barrier damage.

本项目在上一自然科学基金基础之上，以TNF-α介导的TRADD-FADD细胞凋亡途径和NF-κB-MLCK紧密连接途径异常是导致克罗恩病(CD)肠上皮屏障损伤的主要环节为切入点，运用基因技术和分子生物学等技术，旨从对这二条途径具有双重负调控作用的泛素化修饰酶A20表达角度，通过肠上皮A20基因敲除CD动物模型和A20基因沉默细胞模型整体与细胞两个层面进行研究：①拟证实A20可通过对重要接头分子TRADD、RIP、TRAF6去泛素化，负调控TNF-α/TRADD-FADD 细胞凋亡途径和TNF-α/NF-κB-MLCK紧密连接途径，是CD肠上皮屏障损伤的重要调控蛋白；②初步阐明艾灸可能通过促进或调节CD肠上皮A20表达，从而调控TNF-α介导的这二条途径的异常，由此减少肠上皮细胞过度凋亡和改善肠上皮细胞紧密连接异常，是艾灸保护或修复TNF-α介导CD肠上皮屏障损伤的关键作用机制。

本项目以TNF-α介导的TRADD-FADD细胞凋亡途径和NF-κB -MLCK紧密连接途径异常是引起克罗恩病(CD)肠上皮屏障损伤的主要环节为切入点，探讨由TNF-α诱导产生的泛素化修饰酶A20是否能对CD肠上皮屏障TNF-α所介导的这二条途径具有调控作用，同时研究艾灸是否可通过影响CD肠上皮A20，来达到保护或修复CD肠上皮屏障损伤之效应。分别从A20基因沉默细胞模型和肠上皮A20基因敲除CD动物模型两个层面开展研究。结果显示：TNF-α能明显降低CD大鼠肠上皮A20表达，显著增加其介导的TRADD-FADD细胞凋亡途径中TNFR1、TRADD、RIP、FADD表达、肠上皮细胞凋亡以及NF-κB-MLCK紧密连接途径中NF-κBp65、MLCK、MLC、TRAF6、RIP1表达、降低肠上皮occludin、claudin、ZO-1、F-actin表达。观察到隔药灸能明显改善CD模型大鼠肠黏膜组织损伤状况和肠上皮屏障通透性；明显降低CD大鼠肠上皮TNF-α/TRADD-FADD细胞凋亡途径中TNF-α、TNFR1、TRADD、RIP、FADD表达和抑制肠上皮细胞的凋亡率；明显降低CD大鼠肠上皮TNF-α/NF-κB-MLCK紧密连接途径中NF-κBp65、MLCK、MLC、TRAF6、RIP1表达和促进肠上皮occludin、claudin、ZO-1、 F-actin表达；同时隔药灸在能显著促进或增加CD大鼠肠上皮A20表达的同时降低TRADD、RIP1、TRAF6表达。提示A20可能是通过去泛素化TRADD、RIP1、TRAF6，进而负调控TNF-α/TRADD-FADD 细胞凋亡途径和TNF-α/NF-κB-MLCK紧密连接途径；艾灸可能是通过促进或增加A20表达，从而有效调控TNF-α所介导的这二条途径异常，是艾灸保护或修复TNF-α介导CD肠上皮屏障损伤的关键作用机制。