针灸阻抑克罗恩病肠纤维化上皮间质转化的分子调控机制

Intestinal fibrosis is the common while the most intractable problem in Crohn's disease (CD). Based on a large number of previous effective acupuncture treatments for Crohn's disease and intestinal fibrosis, this research group takes intestinal epithelial-mesenchymal transition can initiate or result in the testinal fibrosis in Crohn's disease as the cut point. Histological, cellular, molecular biological and other techniques are applied to research from cellular level in vitro, whole animal experiments and clinical observation for the following purposes: ① To confirm that Obviously abnormal expressions of the major media tumor necrosis factor-alpha(TNF-alpha)and transforming growth factor-β1(TGF-β1) of Crohn's disease-mediated chronic inflammation and damaging repair cause intestinal epithelial-mesenchymal transition through TNF-alpha-nuclear factor κB(NF-κB)-SNAIL and TGF-β1-Smads-SNAIL pathways and initiate or result in the testinal fibrosis in Crohn's disease; to prove that transcription factor SNAIL can inhibit the expression of Crohn's disease intestinal epithelial markers and is an important regulatory protein in intestinal epithelial-mesenchymal transition of these two pathways; ② To clarify that acupuncture may deter the intestinal fibrosis of Crohn's disease through inhibition of intestinal epithelial-mesenchymal transition of these two pathways by inhibiting the abnormal TNF-alpha and TGF-β1 expression, and expect to provide the scientific evidence for the effect mechanism of acupuncture and moxibustion therapy for Crohn's disease and intestinal fibrosis.

肠纤维化是目前克罗恩病(CD)中常见且治疗最为棘手的难题。本项目在课题组以往大量针灸治疗CD及肠纤维化有效的基础上，以"肠上皮间质转化可引发或导致CD肠纤维化"为切入点，运用组织学、细胞学及分子生物学等技术，试图从体外细胞水平、在体动物实验与临床观察多层面相结合研究：①拟证实介导CD肠道慢性炎症与损伤修复的主要介质TNF-α和TGF-β1表达异常，两者分别通过TNF-α-NF-κB-SNAIL和TGF-β1-Smads-SNAIL途径所致的肠上皮间质转化是引发或导致CD肠纤维化的主要环节之一；同时证明转录因子SNAIL能够抑制CD肠上皮主要标志物的表达是这二条途径肠上皮间质转化中的重要调控蛋白；②初步阐明针灸可能是通过抑制CD肠组织TNF-α、TGF-β1的异常表达进而阻遏这二条途径所致的肠上皮间质转化，由此达到阻抑CD肠纤维化效应，以期为针灸治疗CD及肠纤维化的效应机制研究提供科学依据。

本项目以“肠上皮间质转化(epithelial mesenchymal transition, EMT)可引发或导致CD肠纤维化”为切入点，探讨介导CD肠道慢性炎症与损伤修复的主要介质TNF-α和TGF-β1表达异常，两者是否可通过NF-κB-SNAIL和Smads-SNAIL途径促使肠EMT，是引发或导致CD肠纤维化的主要环节之一；观察转录因子SNAIL是否能够抑制CD肠上皮主要标志物的表达，是这二条途径肠EMT中的重要调控蛋白；研究针灸是否可通过调控CD肠上皮TNF-α和TGF-β1介导的这二条途径所致的肠EMT，进而达到阻抑CD肠纤维化效应。结果显示：CD大鼠和CD患者可见明显的肠上皮再生和组织纤维化表现，TNF-α所介导的NF-κB-SNAIL途径中TNF-α、TNFR1、NF-κB p65、SNAIL1表达和TGF-β1所介导的Smads-SNAIL途径中TGF-β1、TβR2、Smad3、SNAIL1表达均有显著性增高，转录因子SNAIL1 mRNA和间质细胞标志物fibronectin mRNA和蛋白表达均有显著性增高而上皮细胞标志物E-cadherin mRNA和蛋白表达均有显著性降低；针灸治疗则可明显改善CD肠EMT病理学改变，显著降低TNF-α-NF-κB-SNAIL途径中TNF-α、TNFR1、NF-κB p65、SNAIL1表达和TGF-β1-Smads-SNAIL途径中TGF-β1、TβR2、Smad3、SNAIL1表达，抑制fibronectin和促进E-cadherin的mRNA和蛋白表达。提示CD肠上皮TNF-α和TGF-β1的异常/过度表达，可分别通过NF-κB-SNAIL和Smads-SNAIL途径介导肠EMT；针灸可能是通过抑制CD肠组织TNF-α、TGF-β1的异常表达进而阻遏这二条途径所致的肠EMT，由此达到阻抑CD肠纤维化效应。