HMGB1-TLR4-MyD88途径对溃疡性结肠炎中黏液屏障功能的调控及机制研究

Recent studies have shown that UC is not triggered by the immune response against pathogens, but by the priming of the immune response to the normal intestinal flora. Intestinal mucus barrier is essential to prevent the interaction between bacteria and the intestinal epithelial cells, therefore has an important role in the pathogenesis of UC. Once the bacteria penetrate the inner mucus layer, intestinal inflammation is triggered. Mucins such as MUC2 and intestinal ion transporters such as DRA, NHE3 are important factors affecting inner mucus layer function. However, the initial regulatory factor for impaired inner mucus barrier is not yet understood. Our preliminary study shows that early stage danger signal HMGB1 protein can suppress NHE3 by direct combination of it, or inhibit NHE3 and MUC2 expression by activation of TLR4-MyD88 pathway. Therefore, this project aims at: firstly, to study the effects of HMGB1 on intestinal mucus barrier function and intestinal inflammation in UC patient as well as control colonic biopsies using in vitro tissue culture and inner mucus layer detection techniques. Secondly, to study the regulatory effect of HMGB1-TLR4-MyD88 pathway and NHE3 on the function of the inner mucus layer by application of knockout mice. Finally, to apply HMGB1-siRNA to block HMGB1 from the source to see the regulatory effect on mucus barrier and intestinal inflammation, so as to provide new therapeutic strategies for UC.

新近研究显示，肠道黏液屏障阻止细菌接触肠道上皮细胞，因此在UC的发病中有重要作用，一旦细菌穿透肠道受损的黏液层内层，即可促发炎症反应。黏蛋白及肠道离子转运体DRA、NHE3是影响黏液层内层功能的重要因素，然而，何为其使动调控因素尚无定论。本小组前期研究显示，早期危险信号分子HMGB1蛋白可通过直接结合的方式抑制NHE3表达，或通过激活TLR4-MyD88途径抑制NHE3、MUC2表达功能。因此，本项目主要采用结肠活检组织离体培养+干预及荧光颗粒法、细菌探针法黏液层内层检测技术，在UC患者及对照肠道活检标本中研究HMGB1对肠道黏液层内层功能及肠道炎症的影响，并应用基因敲除小鼠研究HMGB1-TLR4-MyD88途径及其所调控的因素如NHE3对黏液层内层功能的影响，最后研究应用HMGB1-siRNA从源头阻断HMGB1对黏液屏障及肠道炎症的影响，以期为UC提供新的干预策略。

背景：本研究旨在小鼠DSS-诱导的结肠炎模型中阐明采用HMGB1中和抗体（HnAb）治疗是否减轻肠道炎症及其可能的作用机制，及其下游MyD88 MyD88对结肠炎症和肠道菌群的影响。.方法：在HnAb治疗结肠炎症的实验中，小鼠随机分为结肠炎模型HnAb治疗组（HnAb）；结肠炎模型IgY对照组（DSS-C）以及正常对照组 (Ctrl)三组。评估炎症活动指数（DAI）、结肠长度、结肠炎组织学评分(HS)、MPO活性及细胞因子水平。采用流式细胞学分析巨噬细胞活化，免疫荧光Mucin2染色观察黏液层结构，紧密连接（T-J）蛋白结合电镜评估T-J结构。在探讨MyD88作用的实验中，MyD88敲除小鼠和野生型小鼠被随机分为正常饮用水(WT和MyD88 KO组)或DSS(WT-DSS和MyD88-DSS组)四组。对HS、DAI、TGF-β1、EGF、cox - 2和T-J蛋白进行检测评估。采用WB法评估NF-κB激活情况。高通量测序16srDNA分析肠道黏膜菌群。采用one-way ANOVA结合Post Hoc LSD test 及Tamhane’s T2 test进行统计学分析，p<0.05为有统计学差异。.结果：在HnAb治疗实验中，与DSS-C组相比，HnAb治疗从结肠长度、DAI、HS及MPO活性方面显著改善小鼠DSS结肠炎，其TNF-α, IFN-γ及 IL-1β mRNA表达明显低于DSS-C组，黏液层结构、T-J蛋白ZO-1，claudin-5、 occludin表达及结构亦明显改善。经HnAb治疗后，胞核及胞浆HMGB1蛋白水平均明显下降， TLR4及MyD88表达亦明显下降，巨噬细胞活化明显减少。.在MyD88敲除小鼠的实验研究中，相比WT-DSS组，MyD88-DSS组的结肠NF-κB活性被显著抑制。然而，与WT-DSS组相比，敲除MyD88并不能改善DSS小鼠的DAI和HS。结肠IL-1β,TNF α and IFN γ mRNA、TGF-β1、EGF, cox - 2和T-J蛋白mRNA表达也无显著性差异。4组间结肠黏膜微生物alpha多样性无显著性差异。MyD88 KO小鼠的变形菌门比例显著升高到41.9%，其中假单胞菌目、伯克氏菌目和肠杆菌目等病原菌在目的水平上明显增加；乳酸菌等益生菌比例下降(31.5%)。.结论： 早期阻断HMGB1不仅阻断HM