SARA产生的DAP诱导反刍动物乳腺炎症反应的分子机制研究

Subacute ruminal acidosis (SARA) is the most frequent nutritional and metabolic clinical disease in high-yielding dairy cows. Much abnormal metabolic product diaminopilemic acid (DAP) is produced in the rumen. Our initial study showed that DAP produced by SARA could be trans-located and induced inflammatory response in mammary gland of dairy cows. However, the exact mechanism involved in it is still unclear. Based on the former study, in this project lactating goats will be used as model animals. Rumen fistula and catheters of mammary gland artery and vein will be installed by surgical operation. SARA will be induced by feeding high grain diet. CHART-PCR, DNA methylation analysis, RNAi and signal pathway analysis techniques will be taken. The contents of the project are as follows: the regulation of DAP produced in the rumen and trans-located into the mammary glands; transcription control of DAP on gene expression of NOD1 in mammary glands; effects of DAP on NOD1/RIP2 signal transduction pathway of mammary glands; molecular mechanism of DAP produced by SARA on inflammatory response in mammary glands will be further revealed by DAP infusion experiment from mammary gland artery, DAP treatment on primary epithelial cell culture in vitro and NOD1-siRNA trans-infection; by manipulating rumen metabolism to interfere inflammatory response of mammary glands. The results of this project will provide new insight and theoretical basis for veterinary clinical comprehensive prevention and treatment on mammary gland inflammation of dairy cows.

亚急性瘤胃酸中毒(SARA)是高产奶牛临床上最为常见的营养代谢病，瘤胃中产生大量的异常代谢产物二氨基庚二酸（DAP）。前期研究发现，SARA产生的DAP发生移位，导致奶牛乳腺发生炎症反应，但其确切机制尚不清楚。本课题拟在此基础上，以奶山羊为模型，手术安装瘤胃瘘管和乳腺动静脉血管瘘，以高精料日粮诱发SARA，应用CHART-PCR、DNA甲基化分析、RNAi、信号通路分析等方法研究：SARA产生的DAP在瘤胃中形成与转运到乳腺的规律；DAP对乳腺组织中NOD1基因表达的转录调控；DAP对乳腺组织中NOD1/RIP2信号转导通路的影响；通过乳腺动脉DAP灌注试验、体外乳腺上皮细胞DAP刺激和NOD1-siRNA转染试验进一步阐明SARA产生的DAP诱导乳腺炎症反应的分子机制；通过调控瘤胃代谢，干预DAP引起的乳腺炎症反应。研究结果可为兽医临床上奶牛乳腺炎的综合防治提供新的思路和理论依据。

在高产奶牛泌乳早期和中期，为了满足高产的营养需求，经常饲喂高精粗比的日粮，使奶牛采食量和能量吸收最大化，以增加产奶量。但是采食高精粗比的日粮，会导致瘤胃微生物迅速发酵，产生过多的挥发性脂肪酸和乳酸，使奶牛处于亚急性瘤胃酸中毒状态。在集约化饲养的奶牛场中，高产奶牛在泌乳早期和泌乳中期亚急性瘤胃酸中毒的发病率高达20%以上，导致瘤胃内微生态紊乱，革兰氏阴性菌大量死亡、崩解，释放有害代谢物二氨基庚二酸（DAP）。大量蓄积的DAP经受损的瘤胃屏障进入血液循环到达乳腺，诱发反刍动物乳腺炎症反应，但其确切机制尚不清楚。本项目通过饲喂高精料日粮成功建立了亚急性瘤胃酸中毒病理模型，手术安装瘤胃瘘管和乳腺动静脉血管插管，系统研究了瘤胃中有害代谢物DAP诱发反刍动物乳腺炎症反应的机制。建立了柱前手性衍生化高效液相色谱法检测瘤胃液和乳静脉中DAP含量的方法。结果表明，亚急性瘤胃酸中毒状态瘤胃液与乳静脉中iE-DAP 含量显著升高。研究结果表明，亚急性瘤胃酸中毒状态下DAP在瘤胃中释放，并可转运到达乳腺，诱导乳腺组织中DAP特异性配体NOD1的基因和蛋白表达升高，NOD1启动子区域染色质发生重塑，由压缩转为疏松状态，NOD1启动子区域CpG岛的甲基化水平显著降低，明确了NOD1基因转录调控机制。同时，NOD1/NF-κB信号转导通路被激活，NF-κB进入乳腺细胞核内增强炎性因子基因的表达，诱发乳腺组织的炎性反应。乳腺动脉DAP灌注试验进一步证明了以上结果。体外培养乳腺上皮细胞，进行DAP刺激试验，通过NOD1基因功能分析，进一步揭示了DAP可以通过NOD1/NF-κB、MAPK、ERK/Nrf2信号通路诱导乳腺细胞炎性反应与氧化应激。通过日粮添加丁酸钠，调控瘤胃消化代谢，可以显著提高瘤胃的缓冲容量，降低瘤胃中DAP的产生，缓解DAP诱发的乳腺炎症反应。同时发现，谷氨酰胺、丙戊酸钠也可降低DAP对奶牛乳腺上皮细胞的炎性反应。本项目通过在体综合性研究和体外分析性研究，揭示了SARA状态下瘤胃产生的DAP诱导乳腺炎症反应的分子机理，明确了DAP对乳腺组织中NOD1基因表达的转录调控机制，研究结果可为兽医临床上奶牛乳腺炎的综合防治提供重要依据和新的思路。