健脾通里中药对胃切除大鼠肠黏膜上皮紧密连接的影响及其机制

Serious stress, including surgical stress etc, can cause dysfunction of intestinal mucosa barrier, thus lead to bacterial translocation, endotoxemia, systemic inflammatory response syndrome(SIRS) and multiple organdysfunction syndrome(MODS),which seriously influence the outcome and prognosis of disease. Tight junctions in intestinal mucosa epithelial cells, the core part of gut mucosal mechanical barrier, plays a vital role in gut mucosal barrier. The material basis and molecular structure are almost clear, which mainly consist of cytomembrane protein including occludin、claudin and cytoplasm protein ZO-1..Our previous clinical and animal experiments all confirmed that herbs of tonifying spleen and catharsishas have the effect of protecting the gut mucosal barrier. However,it’s unknow whether the mechanism is to realize by influencing tight junctions and regulating the expression of.molecules.This project is planned on the basis of our preliminary studies through the estabilishment of gastrectomy surgical trauma model in rats,dripping herbs of tonifying spleen and catharsis into small intestine to intervention study, aims: (1)To observe the morphological structure of tight junctions via electron microscopy. (2)To detect the co-location of occludin, claudin and ZO-1 proteins by immunofluorescence. (3)To detect the combination that between occludin, claudin and ZO-1 proteins by immunoprecipitation.(4)To detect the gene transcription circumstance of occluding, claudin and ZO-1 proteins by fluorogenetic quantitative RT-PCR and the expression of proteins after transcription by Western blot.This project aims at investigating the influence of herbs on tight junctions and the mechanism of molecules after surgical stress in different ways and levels, laying a foundation for protection of gut mucosal barrier, prevention and treatment of related critical diseases with traditional Chinese medicine.

手术等应激可引起肠屏障功能障碍，导致细菌易位、全身炎症反应综合征和多器官功能障碍综合征，严重影响疾病转归和预后。肠上皮细胞间紧密连接是构成肠黏膜机械屏障的核心，在肠屏障中发挥至关重要的作用，其物质基础主要由细胞膜蛋白occludin、claudin和细胞质蛋白ZO-1构成。我们以往研究证实，健脾通里中药具有保护胃切除后肠屏障功能，但是否通过影响紧密连接及其调节其分子表达而实现，尚不清楚。本项目拟在我们前期研究基础上，通过建立胃切除手术大鼠模型，健脾通里中药小肠内滴注，进而：（1）电镜下观察紧密连接的形态结构；（2）免疫荧光法检测紧密连接分子occludin、claudin、ZO-1共定位；（3）免疫共沉淀法检测结合；（4）荧光定量PCR 检测其转录；Western blot检测其蛋白表达。旨在多途径观察中药对术后紧密连接的影响及其分子机制，为中药保护肠屏障功能及其危重病的防治奠定基础。

手术等应激可引起肠屏障功能障碍，导致细菌易位、全身炎症反应综合征和多器官功能障碍综合征，严重影响疾病转归和预后。肠上皮细胞间紧密连接是构成肠黏膜机械屏障的核心，在肠屏障中发挥至关重要的作用，其物质基础主要由细胞膜蛋白occludin、claudin和细胞质蛋白ZO-1构成。本项目拟在我们前期研究基础上，通过建立胃切除手术大鼠模型，健脾通里中药小肠内滴注，进而：（1）电镜下观察紧密连接的形态结构；（2）免疫荧光法检测紧密连接分子occludin、claudin、ZO-1共定位；（3）免疫共沉淀法检测结合；（4）荧光定量PCR检测其转录；Western blot检测其蛋白表达。结果表明，健脾通里中药芪黄煎剂对胃切除手术创伤引起的肠黏膜上皮细胞间紧密连接破坏具有保护作用。其作用机制通过①促进Occludin、Claudin-1和ZO-1分子mRNA和蛋白表达；②促进受损的紧密连接连续性和完整性得到较好的恢复；③加强Occludin、Claudin-1和ZO-1分子在近细胞处分布强度，加强彼此之间互相结合来实现的。.本研究揭示①芪黄煎剂能够促进紧密连接蛋白Claudin-1、Occludin、ZO-1的表达，进而增强紧密连接的作用，维持肠黏膜的完整性和通透性，对胃切除术后肠屏障功能障碍患者意义重大；②中药芪黄煎剂对肠黏膜紧密连接蛋白影响的研究通过文献检索未见报道。我们的研究填补了这一领域的学术空白，并为临床开展肠黏膜屏障功能的保护和危重病的治疗奠定了理论基础；③本研究首次通过HPLC分析芪黄煎剂主要有效成分，为芪黄煎剂的深入研究奠定了基础。旨在多途径观察中药对术后紧密连接的影响及其分子机制，为中药保护肠屏障功能及其危重病的防治奠定基础。