Slc26A3转运体在炎症性肠病中加强肠上皮细胞屏障的作用研究

Inflammatory bowel disease(IBD) is a chronic non-specific intestinal inflammation, and the incidence is worldwide increasing in recent years. Though the pathogenesis is not totally clear, the damage of epithelial barrier is known to be the central role in the process of onset of intestinal inflammation.Slc26A3 ion transporter is a Cl-/HCO3- exchanger, and in our previous studies, we demonstrated that Slc26A3 deficiency is associated with loss of colonic HCO3- secretion, absence of a firm mucus layer, and HCO3-/mucus barrier impairment in mice. However, whether Slc26A3 had an effect on intestinal epithelial barrier is not clarified yet. Our researches demonstrated that Slc26A3 mRNA and protein expression dramatically decreased in DSS-induced colitis in mice and the tight junction and adherent junction protein expressions were also modified in Slc26A3-/- mice, thus it suggested that Slc26A3 play an important role in epithelial barrier. According to our further studies, we found that the absence of Slc26A3 can upregulate the level of TNF-α, therefore, we pose a hypothesis that the absence of Slc26A3 can exacerbate colon inflammation by increasing TNF-α expression. Based on the present theories and our experiments, we intend to over-express or silence the Slc26A3 gene expression in colon epithelial cells, to observe the corresponding changes of the integrity of epithelial barrier ,then detect the molecular mechanisms of silencing Slc26A3 upregulate TNF-α through MAPK/NF-κB pathway in cell and animal level. In this way we want to illustrate the role and mechanisms of Slc26A3 enhancing the epithelial barrier function to explore potential intervention targets of IBD.

肠上皮细胞屏障在炎症性肠病（IBD）发生中有着重要地位。Slc26A3是Cl-/HCO3-离子交换体，我们前期研究证实Slc26A3的缺乏会削弱HCO3-/黏液屏障，但其对上皮细胞屏障的作用并不清楚。本课题组前期研究结果显示在小鼠结肠炎模型中Slc26A3表达下调，Slc26A3-/-小鼠细胞间紧密连接蛋白表达异常，提示其在上皮屏障中有着重要作用。我们进一步研究发现Slc26A3-/-小鼠结肠TNF-α水平增高，据此推测Slc26A3缺失可通过上调TNF-α影响上皮细胞屏障。因此，本研究拟构建Slc26A3siRNA和Slc26A3高表达慢病毒荧光表达载体，观察其对上皮屏障的影响，从细胞和动物水平研究沉默Slc26A3通过MAPK/NF-κB上调TNF-α表达的分子机制，阐明Slc26A3、MAPK/NF-κB、TNF-α与上皮屏障之间的关系及其分子机制，以期从中寻找IBD的潜在干预靶点。

肠上皮细胞屏障在溃疡性结肠炎发生中有着重要地位。我们研究发现Slc26A3（DRA）在溃疡性结肠炎患者中表达明显下降，同时血清中TNF-α升高；在人肠上皮细胞株Caco2BBE细胞中，DRA敲除后细胞TNF-α表达升高，而过表达DRA后TNF-α表达下降；DRA可和紧密连接蛋白ZO-1共定位并且两者之间可相互作用，激光共聚焦显示沉默DRA后ZO-1染色减弱且形态不规则，而稳定过表达DRA后细胞形态规则且ZO-1染色增强。进一步研究发现敲除DRA的Caco2BBE细胞中Occludin、F-actin染色减弱且形态不规则，Claudin2染色增强，ZO-1、Claudin1、5表达降低，而稳定过表达DRA后可逆转紧密连接蛋白（TJ）变化，提示DRA可影响肠上皮细胞TJ蛋白的表达。我们用TNF-α与Caco2BBE细胞共孵育后，DRA表达下降并从细胞连接处转移至胞质，ZO-1、Occludin、Claudin1、5表达降低而Claudin2表达增加，而过表达DRA后可部分逆转TNF-α的效应。TEER值提示沉默DRA或TNF-α孵育后上皮屏障功能下降，而过表达DRA可部分逆转TNF-α引起的TEER值降低。为进一步探讨其分子机制，我们在Caco2BBE细胞中加入NF-κB的特异性抑制剂发现其可抑制TNF-α诱导的DRA表达下降，维持细胞形态的完整性，启动子突变实验验证了NF-κB可介导TNF-α调控DRA的表达。在DSS小鼠结肠炎模型中我们发现，以DRA重组腺病毒灌肠后小鼠结肠炎活动指数、组织学评分、肠道通透性降低，结肠长度增加，小鼠大便含水量明显减少，HE染色提示小鼠肠上皮状态明显好转，电镜下观察到细胞间连接较完整，DRA表达升高，同时紧密连接蛋白包括Occludin、Claudins、ZO-1等均接近正常对照组，细胞因子TNF-α、IL-6下降，IL-10升高；用TNF-α单抗干预DSS结肠炎小鼠后，DRA表达上调，紧密连接蛋白相应改变，电镜下细胞间连接较完整，小鼠结肠炎症显著减轻。综上，我们的研究表明溃疡性结肠炎中DRA可作为上皮屏障的组成部分与ZO-1相互作用影响紧密连接蛋白的表达从而破坏上皮屏障，而TNF-α可通过NF-κB途径下调DRA的表达，进而破坏正常肠上皮结构，促进肠道炎症的发展。