基于肠道菌群紊乱研究奶牛乳腺炎易感性增强的分子机制

The increased incidence of dairy cattle mastitis can be caused by feeding concentrate. However, the associated mechanism remains unclear. According to the pathomechanism that alterations in intestinal microbiota will lead to subclinical endotoxemia which causes chronic systemic low-grade inflammation, and combined with our previous studies which confirmed that LPS could disrupt the Milk-Blood Barrier and decrease the threshold of neutrophil cross Milk-Blood Barrier, we suggest a scientific hypothesis that stress factors such as feed change could lead to the increases of ‘harmful bacteria’ in the intestines and LPS in blood which disrupts the Milk-Blood Barrier and enhance the susceptibility of mastitis. Therefore, we carried out the following studies: firstly, we validate the relationship between alterations of intestinal microbiota and the increases of blood LPS and analysis the effects of LPS on Milk-Blood Barrier through the model of subclinical endotoxemia. Secondly, we detect the susceptibility of mastitis through the stimulation of pathogenic bacteria. Finally, we detect the relationship between intestinal microbiota and the occurrence of dairy cattle mastitis through metagenomics and metabonomics method to reveal the mechanism of the relativity on increased incidence of dairy cattle mastitis with alterations in intestinal microbiota. This project will provide a valuable theoretical base to develop functional additives, probiotics, and traditional Chinese medicine products and develop new ideas for prevention and treatment of mastitis.

针对奶牛增喂精饲料可引发乳腺炎发病率升高但关联机制不明这一临床问题，依据肠道菌群紊乱能导致亚临床内毒素血症并引发机体系统性慢性低度炎症的病理机制，结合课题组已证实乳腺灌注LPS可破坏血乳屏障，降低中性粒细胞跨血乳屏障阈值的研究基础，提出饲料变更等应激因素，导致肠道菌群紊乱，大量“有害菌”滋生，产生过量LPS入血，破坏血乳屏障，增加乳腺炎易感性这一科学假设，展开如下研究：首先构建肠源性亚临床内毒素血症模型，验证肠道菌群紊乱与血中LPS升高的相关性，分析LPS对血乳屏障机能的影响；然后通过病原刺激，检测肠道菌群紊乱时乳腺炎的易感性变化；最后采用宏基因组和代谢组学方法，观测奶牛胃肠道菌群构成、血中LPS浓度与乳腺炎发生的相关性，筛出“有害菌”，揭示肠道菌群紊乱与奶牛乳腺炎易感性增加的关联性机制，从而为探寻相关功能性饲料、微生态制剂和中医药产品研发，开辟奶牛乳腺炎新的防治途径提供研究思路和理论指导

乳腺炎是奶牛尤其是高产奶牛最为重要的疾病。传统观念认为，乳腺炎是细菌等病原微生物感染乳腺引起的局部炎症反应。但在临床实践中，有些乳腺的乳汁中只是表现为体细胞数升高，却分离不到病原菌；此外，奶牛乳腺炎常继发或伴发于前胃迟缓和亚急性瘤胃酸中毒（SARA）等消化紊乱性疾病；并且，针对乳腺的局部治疗效果有限。因此，我们推测乳腺炎的发生除与外源性病原微生物感染有关外，尚存在内源性致病途径。据此我们开展了以下研究：.首先，通过混合抗生素扰乱小鼠肠道菌群，检测肠道菌群紊乱与乳腺炎的关系，结果表明，肠道菌群紊乱可以导致血乳屏障渗透性增加，诱发乳腺炎症（内源性乳腺炎）。当存在金葡菌感染时，肠道菌群紊乱的小鼠乳腺炎症更加明显，而给与肠道菌群紊乱的小鼠移植健康小鼠粪便显著缓解了乳腺炎症反应，表明肠道菌群紊乱加剧乳腺炎的严重程度。.进一步研究发现，抗生素处理导致肠道内短链脂肪酸（SCFAs）产生菌以及SCFAs含量显著降低，对小鼠补充SCFAs和丁酸产生菌显著缓解了金葡菌诱导的血乳屏障渗透性的增加以及乳腺炎症反应。.随后，通过对奶牛饲喂8周高精料诱导SARA这一典型的瘤胃菌群紊乱动物模型发现，瘤胃菌群紊乱诱发奶牛乳腺炎，进一步研究表明，瘤胃源的LPS可通过血液循环到达泌乳的乳腺，诱发乳腺炎，此外SARA奶牛瘤胃内丰度大量升高的寡养单胞菌同样是诱发乳腺炎的重要诱因。.据此推测，肠道菌群与乳腺炎之间存在密切关联，提示机体可能存在“肠-乳轴”，并且肠道细菌和/或其代谢产物迁移可能是“肠-乳轴”相互关联的重要途径之一。该研究同时还提示我们，机体各组织器官是一个相互关联的整体，局部器官疾病（如乳腺）的防治不能只关注局部（如乳腺），还要考虑整体，特别是维护肠道（瘤胃）菌群的平衡，可能是防治乳腺炎以及其他其器官疾病重要因素。这不仅为临床防治奶牛乳腺炎提供新的理论指导和实验依据，而且对动物其他系统性、代谢性和感染性疾病的防治具有重要的借鉴意义。