化瘀通阳方对溃疡性结肠炎大鼠肠屏障通透性的作用及机制

The study is the hot spot that intestinal barrier damage theories are Ulcerative colitis (UC) pathogenesis.Intestinal barrier breakdown, blocking intestinal microcirculation, lead UC hypercoagulable state, and hypercoagulable state is consistent with TCM blood stasis,while, currently, blood stasis injury UC intestinal barrier are rarely reported. Traditional Chinese Medicine indicate" diarrhea for a long time, too much blood stasis;enteropathy is trapped,clear air does not rise" ,that yang deficiency and blood stasis is the key to the pathogenesis of this disease,treatment with Huayutongyang method.Huayutongyang Formulae is the effective prescription which is verifed by clinical. The mechanism may be that it through to improve UC intestinal hypercoagulable state, reduce the permeability of the intestinal mucosa and repair intestinal barrier.Intestinal mucosa permeability changes may reflect the degree of injury of the intestinal barrier,so the study proposed to measure intestinal mucosa permeability by Blood fatty acid binding protein (I-FABP) content、Urinary lactulose and mannitol ratio (LMR)、occludin in Ulcerative colitis rats.From its treatment of DSS induced UC rat model start,investgate whether UC blood hypercoagulation states are related to intestinal mucous membrane permeability increase synchronous,whether improve the hypercoagulation states have a positive influence on repair intestinal mucosa barrier,and illustrate the relations between Intestinal barrier injury and TCM blood stasis.The study aim at providing a new practical basis for the clinical treatment of ulcerative colitis.

肠屏障损伤学说是溃疡性结肠炎（UC）发病机制的研究热点。肠屏障破坏阻塞肠黏膜微循环，导致高凝状态UC形成，而高凝状态与"血瘀"一致，但目前血瘀与UC肠屏障损伤相关性研究鲜有报道。中医认为"泻肚日久,瘀血过多；肠病则陷，清气不升"，阳虚血瘀是本病的病机关键，治以化瘀通阳法。化瘀通阳方是经临床验证的有效方剂，其机制可能是通过改善UC肠道高凝状态，降低肠黏膜通透性，修复肠屏障说。因肠黏膜通透性改变可反映肠屏障损伤程度，本课题拟通过UC大鼠血脂肪酸结合蛋白含量，尿乳果糖与甘露醇比值,闭锁蛋白表达监测肠黏膜通透性。从建立DSS诱导UC大鼠模型切入，通过观察该方对UC大鼠肠黏膜通透性、高凝状态、炎症活动性三方面的作用，阐明修复肠屏障损伤的机制，探讨UC高凝状态与肠黏膜通透性增高是否具有相关性，改善高凝状态是否对肠黏膜屏障修复产生积极影响，阐述肠屏障损伤与"血瘀"的关系，为UC临床治疗提供新的实验依据。

在前期临床实践中证实化瘀通阳方可通过改善溃疡性结肠炎（UC）患者高凝状态，控制炎症进展，文献学习发现UC发病机制与肠黏膜微循环障碍而形成的高凝状态密切相关的基础上，本研究旨在探讨化瘀通阳方对肠黏膜损伤修复的具体机制，观察其对肠黏膜通透性、高凝状态和炎症活动性三方面的作用。实验结果显示研究建立的UC大鼠模型病理评价接近于人UC病变情况，且经过化瘀通阳方治疗后，UC大鼠的一般状态及形态学结构得以改善，肠黏膜损伤趋向愈合；乳果糖/甘露醇排出率间接反映的肠黏膜通透性情况提示化瘀通阳方和阳性对照药物美沙拉嗪一样可以改善UC大鼠肠黏膜通透性；然而由于模型鼠存在小肠粘膜受损，故间接反映肠黏膜通透性的另一指标肠型脂肪酸结合蛋白在各组间差异不大；为了更直观了解肠黏膜情况，实验通过免疫组化和透射电镜直接观察了肠黏膜Occludin蛋白和肠黏膜细胞超微结构，结果提示化瘀通阳方能够增加结肠Occludin蛋白的表达，改善肠上皮细胞间紧密连接状态，改善线粒体及粗面内质网等关键细胞器的结构；在炎症控制方面，实验筛选出与UC密切相关的26种细胞因子，且发现MIP-2、CRP、P选择素和粪钙卫蛋白与炎症程度正相关，化瘀通阳方可以通过降低其表达，增加血清中IL-10、IL-4的含量而发挥抗炎作用；在高凝状态研究方面，化瘀通阳方可以有效降低血小板计数、D-二聚体和凝血酶原片段F1+2浓度，起到抗凝作用。此外，本研究发现肠道菌群代谢产物短链脂肪酸的含量不仅可以反映UC大鼠粪便中的肠道菌群状态，还可以评价肠黏膜生物屏障。由此本研究完善了化瘀通阳方治疗UC的有效机制。