猪胆粉抗溃疡性结肠炎效应成分调节CD4+T细胞亚群平衡的作用机制研究

Ulcerative colitis (UC) is a common inflammatory bowel disease and the incidence rate increases every year. The World Health Organization calls it a “refractory disease”. The medicines for UC therapy have severe adverse reactions currently. Therefore, the development of high effective and safe medicines from traditional Chinese medicine has vast importance to UC treatment. The pathogenesis and molecular biological mechanism of UC have not yet been elucidated, but imbalances between CD4+T cell subsets Thl/Th2、Thl7/Treg cells are believed to play an important role in the pathophysiology of this disease. The regulation of Th cell specific transcription factors and cytokines balance, reduction of colonic epithelial cells injury and inhibition occurrence and development of inflammation are the key factors in UC treatment. Pulvis Fellis Suis is a traditional Chinese medicine which has been used for treatment of diarrhea and dysentery. Our previous research showed that conjugated cholic acids in Pulvis Fellis Suis have anti-inflammatory effects in mice with UC. On the basis of previous study, this project prepares to establish TNBS induced UC model in mice, confirm the regulatory effect of conjugated cholic acid on specific transcription factors and cytokines of Th cell according to the differentiation characteristics of CD4+T cell subsets Thl/Th2/Thl7/Treg cells, elucidate molecular mechanism of conjugated cholic acid on UC treatment by using flow cytometry, western blot, RT-PCR, ELISA and other methods. The accomplishment of this project will have theoretical and practical significance in to UC therapy and conjugated cholic acid utilization.

溃疡性结肠炎(Ulcerative colitis，UC)是一种常见的肠道炎症性疾病，近年来发病率逐年上升，被世界卫生组织列为现代难治病。目前治疗UC的药物普遍存在严重的不良反应，因此开发高效安全的UC治疗药物具有重要意义。近年研究发现CD4+T细胞亚群免疫失衡在UC发病中起关键作用。如何用药物调节CD4+T细胞亚群相关炎症因子水平，抑制炎症的发生及发展，是UC治疗的关键。猪胆粉是治疗泄泻、痢疾的传统中药，前期研究发现猪胆粉结合型胆酸具有抗溃疡性结肠炎活性，对UC有较好疗效。本项目拟在此研究基础上建立TNBS诱导的UC动物模型，采用流式细胞术、Western blot、RT-PCR等方法，探明结合型胆酸通过调节CD4+T细胞亚群平衡治疗UC的分子机制。本项目的完成将对UC的治疗和含结合型胆酸中药应用具有重要的理论和实践意义。

溃疡性结肠炎（Ulcerative colitis，UC）是一种非特异性肠道炎症性疾病，发病率呈逐年上升趋势，已成为我国消化系统常见病，是造成慢性腹泻的主要原因。CD4+T细胞亚群平衡失调所致的免疫异常被视为UC的重要发病机制。如何用药物调节CD4+T细胞亚群平衡，抑制炎症的发生与发展，是UC治疗的关键。目前UC尚无理想治疗药物，从中药中寻找高效、安全的UC治疗药物具有重要意义。猪胆粉是治疗泄泻、痢疾的传统中药，前期研究发现猪胆粉结合型总胆酸对UC有较好疗效，其主要抗炎活性成分牛磺猪去氧胆酸（THDCA）能抑制炎症介质释放，减轻氧化损伤，具有抗溃疡性结肠炎作用。然而，结合型胆酸治疗UC的作用机制尚不明确。据此，本课题采用三硝基苯磺酸诱导的UC小鼠模型，系统研究THDCA对UC小鼠结肠及脾脏中CD4+T细胞亚群的调节作用，以期明确结合型胆酸治疗UC的作用机制。实验结果显示：（1）THDCA能减轻UC小鼠结肠组织损伤程度，降低炎性细胞浸润程度；（2）THDCA能降低UC小鼠结肠中Th1细胞因子IFN-γ、IL-12含量和mRNA表达水平，抑制Th1细胞转录因子T-bet的mRNA和蛋白表达；THDCA能增加UC小鼠结肠中Th2细胞因子IL-4含量和mRNA表达水平，促进Th2细胞转录因子GATA3的mRNA和蛋白表达；（3）THDCA能降低UC小鼠结肠中Th17细胞因子IL-6、IL-17A含量和mRNA表达水平，抑制Th17细胞转录因子RoRγt、STAT3的mRNA和蛋白表达；THDCA能增加UC小鼠结肠中Treg细胞因子IL-10、TGF-β含量和mRNA表达水平，促进Treg细胞转录因子Foxp3、Smad3的mRNA和蛋白表达；（4）THDCA能调节UC小鼠脾脏中Th1/Th2、Th17/Treg细胞比例；抑制脾脏淋巴细胞中Th1细胞因子IFN-γ和转录因子T-bet的mRNA表达，增加Th2细胞因子IL-4和转录因子GATA3的mRNA表达；抑制脾脏淋巴细胞中Th17细胞因子IL-17A和转录因子RoRγt的mRNA表达，增加Treg细胞因子IL-10和转录因子Foxp3的mRNA表达。以上实验结果表明THDCA能通过调节CD4+T细胞亚群特异性转录因子和相关细胞因子表达，维持CD4+T细胞亚群Th1/Th2、Th17/Treg细胞平衡，发挥治疗UC的作用。