牛病毒性腹泻病毒感染诱导线粒体自噬功能缺陷调控胃肠道炎症反应机制的研究

Bovine viral diarrhea virus （BVDV）infection caused high fever, diarrhea, neutropenia and other clinical symptoms in cattle and other animals, especially infection of neonate caused severe diarrhea and mucosal disease, the mortality rate is up to 100%, which seriously affect the healthy and sustainable development of animal husbandry. However, the mechanisms of BVDV infection cause diarrhea inflammation of neonate have not been reported, and it needs research and exploration. A large number of studies have confirmed that there is a close relationship between the changes of interstitial cells of Cajal (ICC) and inflammatory diseases of diarrhea. Our previous studies found that numerous ICC cells occur morphological change and decreased in number causing by BVDV Xinjiang epidemic strain TC infection. We also found elevating of ROS, increasing of mitochondrial membrane permeability, and increasing of IL-1 and IL-18 secretion, suggesting that mitochondrial respiratory dysfunction and ICC cell inflammation. But its molecular mechanism is not yet clear. Basing on the previous study, this project takes BVDV infection induced ICC cells changes, mitophagy dysfunction and ICC cell inflammatory reaction as research objects, and explores the relationship between three parties and their molecular mechanisms. The results will provide theoretical basis for elucidating the mechanism of BVDV infection leading to inflammatory lesions of diarrhea, and also provide a new target for the prevention and control of BVD.

牛病毒性腹泻病毒BVDV感染牛羊等动物后引起高热、腹泻、白细胞减少等临床症状，尤其造成幼畜严重腹泻和粘膜病，病死率高达100%，严重影响畜牧业健康和可持续性发展；然而BVDV感染引起幼畜腹泻炎症病变的机制尚未见报道，亟待研究探索。大量研究报道证实腹泻炎症病变与胃肠道运动起博细胞-Cajal间质细胞（ICC）改变之间存在密切关系。前期研究发现，BVDV新疆流行株TC感染造成ICC细胞数量减少和形态发生变化；此外发现ROS水平升高，线粒体膜通透性增加，IL-1和IL-18分泌水平上调，提示线粒体呼吸功能发生异常及ICC细胞发生炎症反应，然而其分子机制尚未明确。所以本项目基于前期研究发现，以BVDV感染诱导ICC细胞改变、线粒体自噬功能变化及ICC细胞炎症反应三者为研究对象，深入探索三者关系及其相互调控分子机制，为阐明BVDV感染导致腹泻炎症病变机理提供理论依据，也为防控BVD提供新靶点。

牛病毒性腹泻病毒属于黄病毒科瘟病毒属，感染主要引起犊牛腹泻、持续性感染等，持续性感染犊牛再次感染BVDV造成严重的腹泻粘膜病，致死率高达100%。前期研究发现BVDV感染造成胃肠道炎性病变，其具体机制尚不清楚。本研究成功分离和鉴定绵羊胃肠道ICC细胞，建立BVDV感染ICC模型，RNA-Seq构建BVDV感染ICC细胞转录组学图谱，筛选获得调控BVDV复制的关键宿主因子早期应答基因3（Immediate Early Response 3，IER3）和紧密连接蛋白（Occludin，OLCN）等，探明IER3和OCLN调控BVDV复制中的重要作用，并利用蛋白邻近标记技术筛选获得IER3和OCLN下游互作蛋白，发现IER3和OCLN通过调控细胞内吞和宿主天然免疫信号通路等发挥重要功能，后续研究获得国家自然科学基金资助；同时研究发现BVDV感染造成胃肠道细胞炎症因子改变、造成ICC细胞出现双层膜的自噬体或线粒体自噬结构，为进一步提供重要依据；研究发现BVDV感染造成ICC细胞出现线粒体膜通透性增加、膜电位降低、Parkin和Pink1等因子转运到线粒体上，并与线粒体表面标志物共定位、激活Pro-caspase-3和Pro-caspase-9加工成熟等数据综合表明BVDV感染激活线粒体自噬；进一步研究线粒体自噬功能缺陷/异常调控ICC细胞炎症反应和BVDV复制，结果发现使用线粒体自噬诱导剂CCCP和抑制剂Brefeldin A能显著性改变线粒体自噬水平，其中CCCP处理造成线粒体自噬活性增加，显著性上调IL-1β、IL-6、IL-8、IL-10和TNFα mRNA水平，增加Parkin和Pink1在线粒体上的分布，抑制BVDV复制。综上结果表明，BVDV感染ICC细胞增加线粒体自噬水平，导致线粒体结构改变，造成ICC细胞结构功能改变，最终造成细胞炎症反应。本项目实施共发表科研论文11篇，其中第一标注8篇，第二标注1篇，第三标注2篇，培养中青年科技人才1人、硕士研究生4人，完成预期目标和成果。