苦参甘草组方调节紧密连接蛋白对溃疡性结肠炎肠粘膜屏障损伤的修复作用及分子机制研究

The etiology of ulcerative colitis is very complicated, but its drug therapy is only focus on anti-inflammatory approach with poor efficacy so far. A major pathological feature of ulcerative colitis is intestinal mucosal barrier injury. It is significant to repair intestinal mucosal barrier injury with ulcerative colitis. Large studies indicated that the abnormal activating signaling pathway of IL-13/STAT6 and TNF-α/MLCK resulted in alternative expression and cellular localization of tight junctional protein is the molecular pathologic basement of intestinal mucosal barrier injury. Our previous studies suggested that the the flavescen and liquorice prescription (FLP) not noly had the ability to suppressing inflammatory response, but also exhibited synergistic effect on repairing the intestinal mucosal barrier Injury with ulcerative colitis, and the latter effect was closely related to regulating the expression and cellular localization of tight junctional protein. In this study，FLP colon targeted pellets has been prepared，and C57BL/6 mice、HT29/B6 cell are selected to establish the model for intestinal mucosal barrier injury with ulcerative colitis. The RT-PCR、western blot、immunofluorescence and transfection techniques will be applied to illuminate the effect and molecular mechanism of FLP regulating the expression and cellular localization of tight junctional protein for repairing the intestinal mucosal barrier injury with ulcerative colitis. Meanwhile, our studies theoretically offer an idea of multi-targetting and synergetic therapy for the treatment of ulcerative colitis in the investigations of traditional Chinese medicine.

溃疡性结肠炎病因复杂，临床药物集中于简单的抗炎治疗，疗效不佳。肠粘膜屏障损伤是溃疡性结肠炎显著的病理特点，修复肠粘膜屏障损伤是当前重要的研究方向。IL-13/STAT6和TNF-α/MLCK信号通路激活，导致紧密连接蛋白异常表达与错误定位，是溃疡性结肠炎肠粘膜屏障损伤的分子病理基础。我们发现，苦参甘草组方不仅具有抗炎作用，还能修复肠粘膜屏障损伤，该作用与调节紧密连接蛋白密切相关，并具有协同增效的特点。本项目将苦参甘草组方制成结肠靶向微丸，拟采用RT-PCR、免疫印迹和免疫荧光技术，研究其对溃疡性结肠炎模型小鼠肠粘膜紧密连接蛋白表达和细胞定位的调节作用，采用HT29/B6细胞和转基因技术，研究组方对IL-13/STAT6和TNF-α/MLCK信号通路激活的抑制作用。初步揭示苦参甘草组方调节紧密连接蛋白对肠粘膜屏障损伤的修复作用及分子机制；为中药“多靶点协同”治疗溃疡性结肠炎提供新的研究思路。

溃疡性结肠炎病因复杂，肠粘膜屏障损伤是其显著的病理特点，临床症状为持久反复的腹泻。当前临床药物集中于简单的抗炎治疗，疗效不佳。最近的研究表明，修复肠粘膜屏障损伤可能是一条重要的治疗途径。肠上皮细胞间的紧密连接蛋白是构成结肠肠粘膜屏障的重要物质基础。项目主要研究了苦参甘草组方及其活性成分对溃疡性结肠炎模型小鼠肠粘膜关键的紧密连接蛋白claudin2、occludin、claudin7异常表达的调节作用，揭示其通过增强肠粘膜屏障，治疗溃疡性结肠炎的药理机制，并进一步通过细胞实验研究药理作用可能的分子机制。结果表明，苦参甘草组方对溃疡性结肠炎不论是造模同时给药还是造模后给药，都能显著降低模型小鼠的疾病活动度水平，抑制肠粘膜损伤，促进肠粘膜修复，表明苦参甘草组方不仅具有预防作用还具有治疗作用，并且合用组优于单用组；苦参碱、甘草酸能剂量依赖性地抑制模型小鼠体重下降，以及通透性标记物FITC通过肠粘膜入血，表明二者具有增强肠粘膜屏障的作用，合用后效果进一步增强。分子机制研究表明，甘草酸能抑制紧密连接蛋白claudin2的上调和occludin的下调，其分子机制与其能够直接抑制结肠上皮细胞胞内信号ERK1/2和Akt信号通路的激活有关。苦参碱不仅能抑制紧密连接蛋白claudin2的上调和occludin的下调，还能上调claudin7，但未见其对ERK1/2和Akt信号通路的调节作用，因此我们推测苦参碱是通过上调了claudin7的表达，增强肠粘膜对细菌性抗原的特异性屏障作用，降低了胞外免疫因子IL-6、TNF-α的分泌，继而发挥了对claudin2、occludin的间接调节作用。项目研究阐明了苦参甘草组方通过调节紧密连接蛋白对溃疡性结肠炎肠粘膜屏障损伤的修复作用及其分子机制，且初步揭示了苦参碱、甘草酸“协同增效”的作用机制，为中药组方“多靶点”治疗系统性疾病提供了一种研究思路，更进一步开发了药物的结肠靶向制剂，申请了发明专利，为项目的临床转化提供了坚实的前期基础。