基于强致病菌感染探讨四君子汤保护肠粘膜屏障的作用及对紧密连接蛋白表达的影响

Mucosal barrier is the most important barrier in the intestine. It constitutes with integrity of the intestinal epithelial cells(IEC) and the junction between adjacent intestinal epithelial cells with the function of regulate transepithelial water and solute transport(such as simple sugars, amino acids, nucleotides, vitamins and hormones, etc.).There are several kinds of connection between adjacent epithelial cells, such as tight junction(TJ), and gap junction(GJ), adherence junction(AJ), and desmosome and others. TJ is most important connections in the epithelial cell, its function is to allow ions and small molecules soluble substances through while block toxic marcomolecules and microorganisms. This particular physiological function plays an important role in the maintenance of the intestinal barriers. A significant body of evidence has demonstrated that intestinal barrier defects are involved in several intestinal and metabolic diseases, such as inflammatory bowel disease, food allergies obesity and alcoholic liver disease In these diseases, inflammatory cytokines, reactive oxygen species andpathogenic bacteria have been found to impair intestinal TJ function..We have seen in literature review about Sijunzi-decoction and other benefit formulas that the current study of these kinds of benefit formulas is mainly concentrated in weak, elderly and low immunity and mainly reflected the effects of tonic nursing Qi. The function of get rid of pathogenic factors (in term of "Xie") for benefit formulas was rarely involved. Chinese traditional medicine has a saying "Righteousness exist, evil can not enter". But there is still no clear experimental data to support. What is the role of benefit formulas in the role of get rid of pathogenic factor is still unclear..Goodwin published the "leaky roof" in 1998, he said gastric mucosal barrier likened to the roof. Similarly, we can also be seen as the epithelial barrier is the "roof". Pathogenic bacterial infection firstly destroyed the cell tight junctions, resulting in the leaking of the roof and in the body incidence. Combine the features of the tonic of strengthen Qi, whether we can speculate: the tonic could repair the "roof",restore of damaged tight junctions and finally get rid of the pathogenic factors?.This study attempts to build the research system consisted with tonic, tight junctions and foreign pathogenic factors. Within this system, tight junction on behalf of "righteousness Qi ", foreign bacterial infection on behalf of "evils". We will study the barrier function changes, as well as the role played by the tonic when body was invaded by the pathogenic bacteria. Our study also provide the experimental basis for "Righteousness exist, evil can not enter".

紧密连接蛋白（Tight Junction，TJ）是目前黏膜屏障的研究热点，是细胞与细胞间最为重要的连接方式，它可选择性的区分小分子，以及细菌、病毒等外来侵入性大分子，在肠道屏障的维护中起着举足轻重的作用。而这一作用，与中医"正气"的概念极为接近。以往对补益药的研究多为直接对某种病理模型的改善，很少能区分出机体本身的"正气"，以及补益药所谓"补"和"益"的作用。.本研究试图构建由补益药、机体屏障及外来邪毒组成的研究体系，以屏障功能代表"正气"；以细菌感染代表"外邪"； 采用整体动物模型与体外细胞培养相结合的方式，通过对上皮细胞形态及行为、细菌侵袭力及细胞通透性考察、紧密连接结构破坏、炎症介质释放以及TNF-α/NF-kB/MLCK信号转导的调控等角度研究在细菌侵入之时，机体屏障功能的变化，以及补益药四君子汤所可能发挥的顾护、治疗及补益作用，为"正气存内，邪不可干"提供实验依据。

四君子汤可用于治疗胃肠道溃疡、慢性腹泻等多种疾病，但其作用机制不明确。课题主要采用动物模型和体外细胞，观察四君子汤（SJTP）、其含药血清、四君子汤多糖(TPSJ)以及羧甲基茯苓多糖（CMP）对肠道粘膜屏障的修复与作用机制。.结果表明TNBS损伤大鼠结肠给予SJZD治疗之后，大鼠体重明显增加，DAI、CMDI、TDI等评分均降低，以中剂量（2.8g/kg）及高剂量(5.6g/kg)明显。病理结果表明SJZD明显促进结肠上皮黏膜修复，减少炎症细胞浸润。SJZD治疗后结肠occludin, claudin明显上调，同时可降低结肠TNF-α、IFN-γ表达与血清IL-6含量。.体外研究表明5-20%的SJZD含药血清可促进TNBS损伤的Caco2细胞增殖，抑制凋亡，升高TEER值，改善酚红透过率。15% 含药血清可改善claudin-2基因与蛋白表达，抑制激活的NF-κB/MLCL信号通路。采用过氧化氢叔丁基（t-BHP）制备细胞氧化应激损伤模型，发现10% 含药血清可降低LDH泄漏量，降低MDA含量，提高T-SOD活性，与SOD-1基因水平。.120-150μg/ml 四君子汤多糖（TPSJ）作用可恢复TNBS以及TNF-α导致的Caco2细胞增殖受损，对claudin1,ZO3以及occludin基因与蛋白表达作用明显，TPSJ对升高的NF-κB转录活性以及p65蛋白表达均具有明显的抑制作用，下调MLCK表达，对肠道单层屏障发挥保护作用。.我们也检测了TPSJ对炎症状态下巨噬细胞RAW264.7的影响。结果表明TPSJ可降低NO,LDH，MDA含量，升高SOD水平，抑制细胞上清中TNF-α，IL-1β含量，抑制TNFα mRNA表达，抑制NF-κB转录活性，以及p65与TLR4蛋白表达。采用NF-κB与TLR4特异性抑制剂后，TPSJ作用减弱，表明TPSJ通过下调LPS/TLR4/NF-κB信号通路起到抑制炎症的作用的。.最后我们检测了羧甲基茯苓多糖CMP体外对肠道屏障损伤的修复作用。结果也证实CMP具有肠道屏障保护作用，机制与抑制MLCK信号通路有关。.我们的研究结果证实四君子汤及多糖主要是通过影响TNF-α/NF-κB/MLCK信号通路而影响紧密连接蛋白表达，以及通过抗氧化与抗炎而起到修复肠道粘膜屏障作用的。我们也首次证实CMP具有肠道屏障保护作用。