脑源性神经营养因子调控腹泻型IBS肠粘膜屏障通透性的机制研究

Recent evidence indicated that brain-derived neurotrophic factor (BDNF) was significantly increased in the colonic mucosa of patients with diarrhea-predominant irritable bowel syndrome (IBS-D). This study was designed to explore the mechanism of the abnormal intestinal mucosal permeability in IBS-D at the level of BDNF-mediated epithelial tight junction protein expression alterations. ① Detect the expression alterations of BDNF pathway proteins and tight junction proteins in the colonic mucosa of IBS-D patients, and combined with intestinal permeability evaluation, to reveal the underlying mechanisms of BDNF in the regulation of intestinal mucosal barrier function; ② Establish the mice model of bowel dysfunction, using BDNF-gene knockout mice as control, to confirm the role of BDNF pathway in the expression alterations of tight junction proteins and its effects on the intestinal permeability of mice; ③ Culture the BDNF-transfected epithelial cells in vitro, investigate the expressions of the tight junction proteins with intervention of TrkB, Rho kinase and p-MLC antagonists, and combine with transepithelial electrical resistance evaluation, to further clarify the mechanism of BDNF in the regulation of intestinal permeability. In short, this study is meaningful to elucidate the role of BDNF in the modulation of intestinal mucosal permeability, and find a new target for IBS-D treatment.

新近发现腹泻型肠易激综合征（IBS）肠粘膜上皮脑源性神经营养因子（BDNF）表达明显升高。本研究旨在从BDNF调控肠上皮细胞紧密连接蛋白表达的层面探讨腹泻型IBS肠粘膜屏障通透性异常的分子机制：①检测腹泻型IBS患者肠粘膜BDNF通路蛋白及紧密连接蛋白表达的改变，结合肠道通透性检测，揭示BDNF调控肠粘膜屏障功能的潜在机制；②建立肠功能紊乱小鼠模型，结合BDNF基因敲除小鼠，印证BDNF通路在调节肠上皮细胞紧密连接蛋白表达的作用及对小鼠肠道通透性的影响。③BDNF基因转染肠上皮细胞并体外培养，应用TrkB、Rho激酶、p-MLC通路蛋白拮抗剂进行干预，检测肠上皮细胞紧密连接蛋白的表达水平，结合跨上皮细胞电阻检测，进一步阐明BDNF调控肠粘膜屏障通透性的机制。本研究首次系统阐明神经因子BDNF调节腹泻型IBS肠粘膜上皮细胞紧密连接蛋白表达的潜在机制，对寻找腹泻型IBS治疗新靶点具有深远意义。

脑源性神经营养因子(BDNF)可能在结肠粘膜屏障通透性的调节中发挥着重要作用，然而关于BDNF在结肠上皮屏障中作用的系统研究尚未见到报道。在临床试验中，我们收集符合罗马Ш标准腹泻型肠易激综合征患者（IBS-D）与正常健康者直乙交界上皮粘膜，检测发现BDNF在IBS-D患者结肠上皮高表达，同时伴随着occludin与ZO-1表达下调。在动物试验中，取BDNF+/+小鼠与BDNF+/-小鼠结肠粘膜标本，与BDNF+/+小鼠相比，BDNF+/-小鼠电镜下结肠上皮屏障破坏，表现为微绒毛缺失、细胞间隙增宽以及细菌入侵；同时BDNF+/-小鼠结肠上皮通透性增加，同时伴随着紧密连接蛋白occludin、ZO-1以及claudin-1表达下调，而claudin-2表达上调。另外我们发现，与正常对照组小鼠相比，TNBS灌肠28天后，炎症后IBS（PI-IBS）小鼠出现结肠上皮屏障破坏，表现为结肠电阻值降低以及硝酸镧通透性增加。同时，BDNF在PI-IBS小鼠结肠粘膜表达增加，并伴随着紧密连接蛋白的改变。在细胞试验中，在结肠上皮细胞ht-29中导入关于BDNF小干扰RNA（siRNA）可以显著降低BDNF基因与蛋白表达，同时也下调紧密连接蛋白的水平。利用不同浓度的BDNF因子刺激ht-29细胞，我们发现紧密连接蛋白（occludin, ZO-1 and claudin-1）表达下调，而紧密连接蛋白claudin-2表达上调。以上结果表明BDNF通过调节紧密连接蛋白的表达来调控结肠上皮屏障功能。