miR-146a-5p介导TLR4/MAPKs途径在热应激鸡肠道损伤中的作用机制及白藜芦醇的干预研究

microRNA regulation as a novel method to explore the target genes and molecular signaling pathway in the study of livestock and poultry immunological stress. In our previous study, we constructed small RNA libraries of the jejunum mucosa of full-sib chickens (Gallus. gallus domesticus) under normal and thermal stress conditions, and identified candidate miR-146a-5p as down-regulated significantly miRNA in response to heat stress using Solexa miRNA high-throughput sequencing analysis. Based on the sequences of miRNAs, the potential target genes of miR-146a-5p were predicted by using TargetScan and miRanda database, which participate in the Toll-like receptor 4 (TLR4) mediated mitogen activated protein kinases (MAPKs) signaling pathway. The chicken intestinal epithelial cells in vitro will be chosen to establish immune stress model induced by high temperature treatment in this study, simultaneously, many methods including miRNA transfection, real-time quantitative PCR, western blotting, fluorescence in situ hybridization, immunohistochemical detection and enzyme-linked immunosorbent assay, and so forth, will be implemented to explore the regulation of overexpressed or inhibited miR-146a-5p on immune function in chickens intestinal epithelial cells, miR-146a-5p mediated TLR4/MAPKs signaling pathway and theirs key role genes and proteins expression and effects of resveratrol on attenuating intestinal immune stress. According to the distribution of immune factors and the changes of villi ultrastructure in intestinal mucosa of chickens suffered from heat stress, combined to analyze the relation between miR-146a-5p and genes involving in TLR4/MAPKs signaling pathway. Then, the negative feedback-regulate effect of miR-146a-5p targeted genes and protein will be verified and regulated intestinal immune response via TLR4/MAPKs signaling pathways in chicken exposure to heat stress, and revealed the protective effect of resveratrol on intestinal mucosal barrier. Thus, this research will aim at providing theoretical basis for the study of miR-146a-5p role, intestinal immune mechanisms and resveratrol biological function in chicken exposure to heat stress.

在microRNA层面上探索热应激的免疫应答靶点和调控途径是当今畜禽应激研究中的新兴手段。前期研究我们构建了全同胞仔鸡空肠黏膜在正常和热应激下的small RNA文库，经Solexa测序筛选出受热应激诱导显著下调的miR-146a-5p，生物信息学预测其靶标参与TLR4/MAPKs通路。本项目拟建立体外热应激模型，采用miRNA转染、荧光定量PCR、Western-blot、荧光原位杂交、免疫组化等方法，探讨过表达或抑制miR-146a-5p对鸡小肠上皮细胞TLR4/MAPKs信号通路中关键基因和蛋白的表达及白藜芦醇的干预效果，结合热应激仔鸡肠黏膜损伤中免疫因子分布及绒毛超微结构的变化，分析miRNA与信号途径中各基因间的交互关系，揭示miR-146a-5p负反馈靶基因调控热应激仔鸡肠道损伤中免疫应答的作用机理及白藜芦醇对肠黏膜屏障的保护效应，为家禽肠道抗热应激的免疫机制研究提供理论依据。

miR-146a-5p是一种免疫调控因子，在先天免疫应答及炎症因子信号传导途径中起重要的调节。本研究探讨了miR-146a-5p介导TLR4/MAPKs途径在热应激鸡肠道损伤中的作用机制及白藜芦醇的干预效果，结合鸡空肠黏膜形态学超微结构、TLR4/MAPKs途径相关基因和免疫细胞因子指标进行综合分析。通过扫描电镜结果发现，热应激导致鸡空肠绒毛排列紊乱，表面粗糙不平，局部破溃现象，而白藜芦醇明显改善空肠黏膜的形态结构。qRT-PCR结果表明，白藜芦醇能够显著上调热应激鸡空肠黏膜miR-146a-5p的表达，降低空肠黏膜中TLR4、c-fos、c-Jun、p38、Caspase-3 mRNA的表达量，以及增加Bcl-2的表达量（P<0.05）；免疫组化结果发现，白藜芦醇有利于增加鸡空肠黏膜紧密连接蛋白Occludin、ZO-1、Claudin1的平均光密度值，降低热应激鸡空肠黏膜TLR4/MAPKs信号通路中 p38和JNK的表达水平；Westblot检测结果表明，热应激鸡肠黏膜p53、MKK3和MYD88等蛋白表达显著增加（P<0.05），白藜芦醇能有效抑制p53和MYD88蛋白表达水平，并显著降低热应激鸡空肠黏膜组织中TNF-α的含量（P<0.05），推断白藜芦醇可能对热应激鸡肠黏膜的氧化损伤及炎症反应起调控作用。