青黛、参三七介导Zonulin通路调控肠道通透性的机制研究

Ulcerative Colitis (UC) is a disease with the features of yearly climbing prevalence, long disease course, healing difficulty, easily relapse, and huge disease burden for patients and society. Traditional Chinese Medicine (T.C.M.) has incomparable advantage in UC treatment. .Qingchang Suppository is developed by pfofessor Ma Guitong, a renowned T.C.M. doctor in Shanghai. Its efficacy has been demonstrated in our 18 years’ clinical practice and studies, among which, a pre-clinical study has shown that it improved enteric permeability. In this subject, we intend to investigate the mechanisms of Qingdai and Sanqi on enteric permeability from the perspective of Zonulin pathway, based on Dextran Sulphate Sodium induced colitis models and cytology experiment, using methods in cytobiology and molecular biology; to explore the hypothesis of bacterial translocation (through mesenteric lymph node) leads to Zonulin excretion and then causes UC, via 16S rDNA V3-DGGE analysis; to further study the inner link between bacterial translocation and Heat and Toxin Retention, and the biological characteristics of Heat-Resolving and Detoxication in TCM. In order to further improve clinical theory of TCM in UC treatment and demonstrate TCM advantage in UC treatment.

溃疡性结肠炎（Ulcerative Colitis, UC）患病率逐年上升，病程长，反复发作，治愈难度大，给患者和社会造成沉重经济负担，中医药治疗本病具有优势。.清肠栓由上海市名中医马贵同教授研发，临床运用十八年，疗效肯定。前期研究证明清肠栓可改善肠道通透性，本课题以葡聚糖硫酸钠诱导结肠炎大鼠和大鼠小肠上皮细胞和肠道菌群为研究平台，采用细胞生物学、分子生物学等现代生物学研究方法，从调控Zonulin通路的角度研究清肠栓的主药青黛、参三七改善肠道通透性的作用机制；采用16S rDNA V3-DGGE技术探讨肠道菌群经肠系膜淋巴结移位后促进Zonulin分泌引发UC假说的可能性，进一步阐述“菌群移位”与“热毒瘀滞”内在联系以及中医学清热解毒化瘀治疗的生物学特性，完善中医药治疗UC的临床理论依据，从而牢固确立其在UC治疗中的作用和优势。

溃疡性结肠炎（Ulcerative Colitis, UC）患病率逐年上升，病程长，反复发作，治愈难度大 ，给患者和社会造成沉重经济负担，中医药治疗本病具有优势。 清肠栓由上海市名中医马贵同教授研发，临床运用十八年，疗效肯定。前期研究证明清肠栓可维持肠道通透性，基于此，提出了清肠栓、清肠栓主药（青黛、参三七）介导Zonulin通路调控肠道通透性，导致肠道菌群移位的假说。通过研究，我们发现：1、在体内实验中，清肠栓、清肠栓主药（青黛、参三七）能激活Zonulin相关通路PAR2/EGFR/PLC/PPI-3/DAG/ PKC-α从而改变DSS诱导的UC大鼠模型的小肠粘膜通透性，诱导外周免疫应答，抗原负载树突状细胞自小肠迁移到结肠，改善结肠炎症反应。2、用MLN细菌培养发现UC大鼠模型的MLN确实出现了细菌，进一步以各组大鼠肠道粪便为样本进行高通量技术分析得出正常大鼠与UC大鼠小肠粘膜生物群落的确存在差异。应用致病菌E. faecium进行干预caco2细胞，比较各药物主要成分对zonulin通路的影响，论证菌群移位以及肠道菌群-Zonulin 依赖性小肠通透性-结肠炎症间的联系。3、在体外实验中，青黛、参三七的有效提取物靛玉红、人参皂苷Rg1能有效减轻LPS诱导的大鼠结肠上皮细胞及Caco-2细胞UC模型的损伤程度，并上调紧密连接蛋白ZO1、Occludin的表达、改善微丝结构、维持单层膜的完整性及通透性。4、本课题阐述了中医学清热解毒中药青黛和活血化瘀中药参三七治疗UC的生物学特性，并从Zonulin 通路调控肠道通透性的作用机制上以及论证中药治疗“热毒瘀滞” 与“菌群移位 ”的内在联系。本实验进一步完善了中医药治疗UC的临床理论依据，从而牢固确立其在UC治疗中的作用和优势。