The scoring scale used for histology.

Irritable bowel syndrome with predominant diarrhea (IBS-D) is characterized by increased intestinal permeability. Previous studies have shown that the microRNA-29 gene is involved in the regulation of intestinal permeability in patients with IBS-D. NF-κB was proved to play a key role in inflammatory response of intestine and resultant disruption of tight junction integrity, whose activity could be inhibited by TNF Receptor-Associated Factor 3 (TRAF3). However, the exact mechanism that induces increased intestinal permeability in IBS-D patients has not been clarified. In this study, we found that microRNA-29b‑3p (miR-29b-3p) was significantly upregulated, while TRAF3 was decreased and the NF-κB-MLCK pathway was activated within the colonic tissue of IBS-D patients. Subsequently, we confirmed the targeting relationship between miR-29b-3p and TRAF3 through a double-luciferase reporter assay. Lentivirus transfection of NCM460 cells with miR-29b-3p-overexpressing and -silencing vectors demonstrated that the expression of TRAF3 was negatively correlated with the level of miR-29b-3p. The NF-κB/MLCK pathway was activated in the miR-29b-3p-overexpressing group and inhibited to some extent in the miR-29b-3p-silencing group. Results in WT and miR-29 knockout mice showed that miR-29b-3p levels were increased, TRAF3 levels were decreased, and the NF-κB/MLCK signaling was activated in the WT IBS-D group as compared with the WT control group. The protein levels of TRAF3 and TJs in the miR-29b-/- IBS-D group were partially recovered and NF-κB/MLCK pathway indicators were, to a certain extent, decreased as compared with the WT IBS-D group. These results suggested that miR-29b-3p deletion enhances the TRAF3 level in IBS-D mice and alleviates the high intestinal permeability. In brief, through the analysis of intestinal tissue samples from IBS-D patients and miR-29b-/- IBS-D mice, we showed that miR-29b-3p is involved in the pathogenesis of intestinal hyperpermeability in IBS-D via targeting TRAF3 to regulate the NF-κB-MLCK signaling pathway.

以腹泻为主要表现的肠易激综合征（Irritable bowel syndrome with predominant diarrhea, IBS-D）以肠道通透性增高为核心特征。既往研究表明，微小RNA-29（microRNA-29）参与调控IBS-D患者的肠道通透性。已有研究证实，核因子κB（NF-κB）在肠道炎症反应及由此引发的紧密连接完整性破坏中发挥关键作用，其活性可被肿瘤坏死因子受体相关因子3（TNF Receptor-Associated Factor 3, TRAF3）抑制。然而，导致IBS-D患者肠道通透性升高的确切机制尚未阐明。本研究发现，IBS-D患者结肠组织中微小RNA-29b-3p（microRNA-29b-3p, miR-29b-3p）表达显著上调，TRAF3表达水平降低，且核因子κB-肌球蛋白轻链激酶（NF-κB-MLCK）通路被激活。随后，通过双荧光素酶报告基因实验验证了miR-29b-3p与TRAF3之间的靶向结合关系。利用携带miR-29b-3p过表达及沉默序列的慢病毒载体转染NCM460细胞，证实TRAF3的表达水平与miR-29b-3p呈负相关。在miR-29b-3p过表达组中，NF-κB/MLCK通路被激活；而在miR-29b-3p沉默组中，该通路受到一定程度的抑制。野生型（wild type, WT）及miR-29基因敲除小鼠的实验结果显示，与野生型对照组相比，野生型IBS-D模型组小鼠结肠组织中miR-29b-3p水平升高、TRAF3水平降低，且NF-κB/MLCK信号通路被激活。与野生型IBS-D模型组相比，miR-29b-/- IBS-D模型组小鼠的TRAF3及紧密连接（tight junctions, TJs）蛋白水平得到部分恢复，NF-κB/MLCK通路相关指标也有一定程度的下调。上述结果表明，敲除miR-29b-3p可提高IBS-D模型小鼠体内TRAF3的表达水平，并缓解肠道高通透性状态。简言之，通过对IBS-D患者结肠组织样本及miR-29b-/- IBS-D模型小鼠的分析，本研究证实miR-29b-3p通过靶向TRAF3调控NF-κB-MLCK信号通路，参与IBS-D肠道高通透性的发病过程。