microRNA-146b improves intestinal injury in mouse colitis by activating NF-kB and improving epithelial barrier function. Mus musculus

Aim: The precise role of microRNAs in inflammatory disease has been less clear. The present study investigated the effect of microRNA (miR-146b) on improving intestinal inflammation. Methods: The microRNA profile in IL-10-deficient mice was examined using microRNA arrays and miR-146b was selected for the following experiments. The expression vectors containing either the whole sequence of miR-146b or its siRNA were intraperitoneally administered to the dextran sulfate sodium (DSS)-induced colitis mouse. Results: The overexpression of miR-146b activated the NF-kB pathway, improved the epithelial barrier function, relieved intestinal inflammation in the DSS-induced colitis mice, and improved the survival rate of mice with lethal colitis. Furthermore, this amelioration of the intestinal inflammation by miR-146b was negated by the inhibitor for NF-kB pathway. Conclusion: The modulation of miR-146b expression is a potentially useful therapy for the treatment of intestinal inflammation through the activation of the NF-kB pathway. Overall design: Mice: The present studies were approved by the Institutional Animal Care and Use Committee of the Asahikawa Medical College. C57BL/6 and IL-10-/- mice were purchased from Sankyo Labo Service Co., Inc. (Tokyo, Japan) and Jackson Laboratories (Bar Harbor, ME), respectively. Large intestines with or without treatments were removed, rinsed with saline, and the epithelium was gently sheared off with glass slides for protein determination. microRNA arrays: RNA was extracted from the large intestines of mice with Trizol and then was immediately frozen in liquid nitrogen. Next, the microRNA expression profiles of large intestine in wild-type and IL-10-deficient mouse were investigated using the mirVanaTM miRNA bioarray (Filgene, Inc., Japan). Any more than 2-fold differences were considered to indicate a significant change.

研究目的：微小RNA（microRNA）在炎症性疾病中的确切作用迄今尚未完全阐明。本研究探讨了微小RNA miR-146b对肠道炎症的改善作用。
研究方法：采用微小RNA芯片分析IL-10缺陷型小鼠的微小RNA表达谱，筛选出miR-146b用于后续实验。将携带miR-146b全长序列或其小干扰RNA（small interfering RNA，siRNA）的表达载体，通过腹腔注射给药至葡聚糖硫酸钠（DSS）诱导的结肠炎小鼠模型中。
研究结果：在葡聚糖硫酸钠（DSS）诱导的结肠炎小鼠中，过表达miR-146b可激活核因子κB（NF-κB）通路、改善上皮屏障功能、缓解肠道炎症，并提升致死性结肠炎小鼠的存活率。进一步研究表明，miR-146b对肠道炎症的改善作用可被NF-κB通路抑制剂所阻断。
研究结论：通过调控miR-146b的表达以激活NF-κB通路，有望成为治疗肠道炎症的潜在有效策略。
整体实验设计：
实验动物：本研究经旭川医科大学实验动物伦理委员会批准。C57BL/6小鼠与IL-10⁻/⁻小鼠分别购自日本三共实验室服务株式会社（东京，日本）与杰克逊实验室（缅因州巴港，美国）。对经处理或未处理的小鼠大肠组织进行取材，以生理盐水冲洗后，用玻片轻柔刮取上皮组织用于蛋白定量检测。
微小RNA芯片分析：采用Trizol试剂提取小鼠大肠组织总RNA，随后立即置于液氮中冻存。使用mirVana™ miRNA生物芯片（日本Filgene公司）检测野生型与IL-10缺陷型小鼠大肠组织的微小RNA表达谱。以表达差异≥2倍作为显著差异的判定标准。