炎症性肠病中c-kit/JAK-STAT信号调控炎症机制及香连丸的干预机制研究

The parthenogenesis of inflammatory bowel disease is unclear. The increased prevalence of IBD is beginning to put a serious financial burden on the patient, the patient's family, and the national economy as a whole. Finding a definite cause for IBD is an important problem for pathogeneses around the world. It is widely believed the environment, infection, and immunity are pertinent factors leading to IBD.It is almost universally accepted that immunity is one of the most important factors at the outset of IBD, especially the JAK-STAT pathway. Our research team has previously found the ICC by marked C-Kit is changed and is functionally impaired during the process of IBD which causes an abnormality in the intestinal motility. In our current project we will use gene knockout technology, cell transfection, RANi and other modern technology, and investigate the effects of inflammatory cytokines and ICC in IBD base on C-kit/JAK-STAT pathway, and explore the pathogenesis of inflammatory bowel disease from regulating inflammatory immunity and intestinal motility; We will further explore the role and molecular mechanism of Xianglian Wan on ICC and inflammatory cytokines in IBD and explain that Xianglian Wan is scientific and effective for the treatment of IBD, in order to enrich the scientific connotation of Xianglian Wan effects on IBD.

炎症性肠病病因不明，患病率的日渐增加为患者家庭及国家带来沉重经济负担，明确IBD的病因病机，是全球亟待解决的问题。本病多认为与环境、感染、免疫等相关，免疫异常在发病中的重要性已达成共识，其中JAK-STAT信号途径具有重要作用；课题组前一项青年基金表明，特异性表达C-Kit的ICC在IBD发病过程中有形态改变和功能的损伤，出现肠道动力异常表现。本项目利用基因敲除技术、细胞转染和RANi等现代科学技术，从C-Kit/JAK-STAT信号途径研究炎症细胞因子与ICC在炎症性肠病中的作用机制，从调节炎症免疫配合调控肠动力的新视角探索炎症性肠病的病因病机；并深入研究香连丸在IBD中对ICC和各炎症细胞因子的作用和分子机制，阐明香连丸介导C-Kit/JAK-STAT信号通路调控ICC及各类炎症细胞因子治疗IBD的有效性和科学性，丰富经典方剂香连丸功效主治的科学内涵。

随着社会工业化发展，炎症性肠病（IBD）患病率的日渐增加为患者家庭及国家带来沉重经济负担，明确IBD的病因病机，是全球亟待解决的问题。课题组前期实验表明，特异性表达C-Kit的ICC在IBD发病过程中有形态改变和功能的损伤，出现肠道动力异常表现。本项目利用基因敲除技术、细胞转染和RANi等现代科学技术，从C-Kit/JAK-STAT信号途径研究炎症细胞因子与ICC在炎症性肠病中的作用机制，从调节炎症免疫配合调控肠动力的新视角探索炎症性肠病的病因病机；并深入研究香连丸在IBD中对ICC和各炎症细胞因子的作用和分子机制。实验结果：1）成功构建c-kit基因条件性敲除模型小鼠，基本明确ICC对免疫炎症机制及肠道动力两方面在炎症性肠病发生发展的机制。KO小鼠肠肌电频率减缓，对应激刺激反应减弱，全消化道传输延长，肠道动力发生改变，DSS造模可致肠电生理功能紊乱，消化道传输时间延长；条件性C-Kit基因敲除小鼠的IBD模型全肠道传输时间增加，造模后炎症反应较对照WT组轻，促炎因子，JAK、STAT相对低表达。2) 香连丸可介导C-Kit/JAK-STAT信号通路作用于ICC发挥治疗IBD的作用。KO小鼠中，香连丸可抑制IBD中增高的JAK蛋白，并降低增高的IL-6接近正常组，且在WT小鼠中，香连丸干预后结肠c-kit表达也比模型组高，说明香连丸对ICC具有保护或者恢复其功能的作用，其机制可能是介导C-Kit/JAK-STAT信号通路调节Th1/Th17-Treg细胞的动态平衡来实现。香连丸可通过C-Kit/JAK-STAT信号通路调控ICC的功能活动，抑制炎症反应，调节肠道动力而达到治疗作用。3) 成功分离并体外培养肠ICC，且可进行传代培养。香连丸中萸黄连素生物碱可明显抑制细胞炎症，并促进细胞的生长维持细胞的活性，而木香挥发油对细胞炎症抑制作用一般。运用荧光探针标记Ca2+的变化，LPS诱导的ICC炎症模型基础Ca2+较正常ICC高，细胞内损伤明显，而香连丸药物血清能降低增高的Ca2+。香连丸对损伤细胞有抗炎改善作用，以香连丸组及萸黄连生物碱组明显。香连丸在治疗IBD过程中对ICC有相应的保护或者炎症后的恢复作用。本项目明确了香连丸介导C-Kit/JAK-STAT信号通路调控ICC及各类炎症细胞因子治疗IBD的有效性和科学性，丰富了经典方剂香连丸功效主治的科学内涵。