艾灸干预克罗恩病免疫相关GTP酶M介导中性粒细胞自噬的作用机制研究

Recurrent chronic non-specific colitis is the most intractable problem in the treatment of Crohn’s disease (CD). Immunity-related GTPase M (IRGM) is genetically associated with CD. Studies have found that moxibustion was effective in treating CD, regulating the immunologic function and autophagy level of colonic epithelial cells of CD rats. This research will focus on the mediation of IRGM in selective autophagy of neutrophil. The histology, cytology, molecular biology, etc techniques and methods will be applied and combined with colonic tissues and peripheral blood to analyze the mechanisms. ①To confirm that IRGM induces abnormal autophagy of neutrophil and causes persistent inflammation of CD colon. To illuminate that moxibustion relieves colonic inflammation and impairment of CD via regulating IRGM and mediating neutrophil autophagy. ②To elucidate that IRGM abnormally regulates NLRP3 and ASC activation in neutrophil, and suppresses their autophagic degradation, activates the release of neutrophil extracellular traps (NETs). To reveal that moxibustion effectively inhibits colonic inflammation of CD via regulating the expression of IRGM to mediate NLRP3 and ASC autophagic degradation, suppressing the release of NETs and inhibiting activation of inflammasome, and down regulating the expression of inflammatory cytokines. We expect that this research will provide scientific evidences of moxibustion for treating CD.

慢性非特异性肠道炎症反复发作是克罗恩病(CD)治疗最为棘手的难题，免疫相关GTP酶M(IRGM)与CD遗传易感性显著相关。前期发现，艾灸能有效调节CD患者机体免疫功能，调控CD大鼠结肠上皮细胞的自噬水平、控制炎症。本课题拟从IRGM介导的中性粒细胞选择性自噬为切入点，运用组织学、细胞学、分子生物学等技术和方法，结合结肠组织和外周血检测及相关性分析：①证实IRGM诱导中性粒细胞自噬异常，诱发CD结肠持续性炎症反应，拟阐明艾灸通过调控IRGM、介导中性粒细胞自噬，减轻CD结肠炎症损伤的效应；②阐释IRGM异常调控中性粒细胞NLRP3和ASC蛋白活性，阻抑其自噬降解，激活中性粒细胞胞外诱捕网(NETs)释放，揭示艾灸可能通过调节IRGM的表达，介导NLRP3和ASC自噬降解，抑制NETs的释放和炎症小体的激活，下调炎症因子表达水平，从而达到抑制CD结肠炎症反应之目的，为艾灸治疗CD提供实验资料。

背景：克罗恩病(CD) 发病机制仍未完全阐明，具有慢性反复发作的特点。前期发现，艾灸能有效调节CD患者机体免疫功能，调控CD大鼠结肠上皮细胞的自噬水平、控制炎症。免疫相关GTP酶M(IRGM)与CD遗传易感性显著相关。.研究内容：以IGRM介导中性粒细胞自噬诱发CD持续炎症为切入点，采用组织病理学、细胞培养、透射电镜、免疫荧光、免疫组化、ABC-ELISA、RT-qPCR等方法，评价艾灸干预CD大鼠的结肠效应，观察IRGM调控CD大鼠NLRP3和ASC蛋白活性及介导中性粒细胞自噬的情况，观察艾灸干预CD大鼠IRGM调控中性粒细胞自噬及对NETs释放的影响。.主要结果：艾灸能改善CD大鼠体重减轻、便血等一般情况，降低疾病活动指数，改善黏膜病理损伤。与正常组相比，模型组大鼠中性粒细胞自噬体减少，中性粒细胞和结肠组织中IRGM蛋白及mRNA表达增高，p62等自噬相关分子蛋白及mRNA表达增高，LC3II蛋白及mRNA表达降低，NETs复合物浓度升高，蛋白表达增高，NLRP3、ASC、IL-1β、IL-18等炎症因子蛋白和mRNA表达增高，差异有统计学意义；与模型组相比，隔药灸组大鼠中性粒细胞自噬体增多，中性粒细胞和结肠组织中IRGM蛋白及mRNA表达降低，p62等自噬相关分子蛋白及mRNA表达降低，LC3II蛋白及mRNA表达增高，NETs复合物浓度和蛋白表达降低，NLRP3、ASC、IL-1β、IL-18等炎症因子蛋白和mRNA表达均降低，差异有统计学意义。.科学意义：①证实IRGM诱导中性粒细胞自噬异常，CD大鼠外周血和结肠组织中大量炎症因子堆积，诱发CD大鼠结肠持续性炎症反应，艾灸可通过抑制CD大鼠外周血和结肠组织中IRGM的表达，促进中性粒细胞自噬，起到改善CD大鼠结肠炎症损伤的效应。.②证实艾灸可抑制IRGM在CD大鼠外周血和结肠上皮组织中的表达， 负调控 NLRP3和ASC蛋白活性，促进其自噬降解，抑制中性粒细胞胞外诱捕网NETs 释放和炎症小体激活，进而起到缓解CD大鼠结肠炎症反应之目的。.为艾灸治疗CD进一步提供了科学的实验数据，也为艾灸在临床的应用和推广提供更多理论依据。