SARA产生的内源性LPS对结肠黏膜上皮细胞凋亡的影响及GCs的参与

Due to lack of quality forage, dairy cows are usually fed with a high proportion of grain concentrate to meet the needs of high milk production. Subacute ruminal acidosis (SARA) is often induced because of the rapid fermentation of grain concentrate in the rumen. When SARA occurs, a large amount bacterial endotoxin (lipopolysaccharide, LPS) is released in the rumen and the large intestine, which is far beyond the intestinal detoxification ability, then resulting in LPS translocation to the bloodstream and causing endotoxemia as well as. Nutrients repartipation in the body occurs during systemic inflammation caused by SARA, which will reduce the precursors of milk composition from the blood into the mammary gland, and ultimately lead to lower milk quality and milk production. Therefore, LPS translocation from the digestive tract into blood is the key process to affect milk quality during SARA. To date, the site of LPS translocation has not been determined, however, lower gut is much more "leaky" because of the monolayer structure compared to the rumen. How does LPS damage the intestinal mucosal barrier during SARA? How to defense this damage by the body? In this project, Saanen dairy goat during lactation period was employed as an animal model, it is the first time to investigate the effects of LPS produced in colonic mucosa on mucosal barrier and epithelial cell apoptosis, as well as the interaction between LPS and glucocorticoids (GCs) during SARA induced by the high proportion of grain concentrate. In vitro colonic mucosal epithelial cells culture system will be used for further evaluating the effects of LPS on epithelial cells apoptosis and the signaling pathway and the interaction with GCs (cross-talk). Through this project we will provide new targets and new ideas of preventing SARA in ruminants.

由于缺乏优质牧草，常给奶牛饲喂高比例谷物精料以满足高产奶的需求，而导致亚急性瘤胃酸中毒（SARA）的发生。SARA时瘤胃和后段肠道内G-菌大量裂解并释放菌壁成分-脂多糖（LPS），超出肠道脱毒能力而致LPS移位至血液，引起内毒素血症和全身性炎症反应。体内营养物质发生"重分配"，由血液入乳腺的乳成分前体物减少，导致乳质和量降低。因此，消化腔内LPS移位是SARA造成乳质量下降的关键环节。迄今，有关LPS移位的解剖位置尚无定论。与瘤胃相比，后段肠道更为易"漏"。SARA产生的LPS如何损伤肠黏膜屏障？机体如何对抗这一损伤？本课题以萨能奶山羊为动物模型，首次研究高比例谷物精料诱发SARA时结肠腔内LPS对黏膜上皮细胞凋亡的影响，以及糖皮质激素（GCs）对此过程的抵抗作用与信号通路。本项目以整合生理学基本原理为指导，深入研究反刍动物慢性酸中毒时尚待解决的科学问题,为该病的防治提供靶点和理论依据。

由于优质牧草的缺乏，常给反刍动物饲喂高比例谷物精料而导致亚急性瘤胃酸中毒（SARA）的发生，而患SARA动物更易患其他代谢性疾病，如蹄叶炎、子宫内膜炎、乳房炎等，但其生理机制仍不清楚。本项目以泌乳奶山羊为研究对象，研究短期或长期饲喂高精料日粮对其瘤胃微生物发酵、盲结肠发酵、发酵异常产物以及对肠道黏膜屏障的影响，并且进一步研究发酵产物经胃肠道吸收后对肝脏物质代谢和乳腺泌乳性能的影响及机制。研究发现，1. 短期饲喂高精料日粮导致泌乳奶山羊瘤胃和后段肠道微生物区系不稳定；而长期饲喂高精料日粮则导致瘤胃和后段肠道内微生物多样性和丰富度降低、有害菌和产甲烷菌数量增加；瘤胃和后段肠道微生物发酵异常，表现在发酵类型的改变、异常产物如内毒素LPS和组胺含量的升高。2. 短期饲喂高精料日粮导致应激轴(下丘脑-垂体-肾上腺HPA)轴活性增强，应激激素—皮质醇分泌增加；而长期饲喂高精料日粮则使得HPA轴活性降低，皮质醇分泌减少，因机体负反馈调节而使得各组织中皮质醇受体GR表达上调。3. 因异常代谢产物和应激内分泌激素分泌的异常，瘤胃和后段肠道（尤其是结肠）黏膜上皮损伤、亚细胞结构（尤其是线粒体）损伤、细胞氧化应激增强、凋亡升高、抗炎因子下降、炎症反应增强，从而导致营养物转运能力下降。4. 长期饲喂高精料日粮引起肠道炎症、氧化应激和炎症的同时，导致肝脏组织抗氧化能力下降、应激反应增强、分解代谢增强以产生更多能量应对不良刺激，而合成代谢减弱，从而导致乳合成前体物（如氨基酸、脂肪酸和糖）降低。5. 长期饲喂高精料日粮导致乳脂下降，乳腺中乳脂合基因表达下调、基因启动子区域甲基化水平升高是乳脂下降的主要原因。6. C12-HSL通过细胞损伤（细胞凋亡与氧化应激）抑制肠道杯状细胞分泌黏蛋白mucin的功能，导致黏蛋白合成及硫酸盐化作用受阻，从而破坏肠道黏膜的屏障功能。TQ416（对氧磷酶抑制剂）显著缓解C12-HSL引起的细胞活力及分泌功能的损伤。以上研究结果提示：瘤胃和后段肠道微生物区系的紊乱（丰富度降低、有害菌增加）、异常代谢产物的增加、应激轴HPA的紊乱（糖皮质激素GCs）增加了患病动物的易感性，从而导致临床上高比例普通群发性代谢病的发生。因此，维护瘤胃正常pH、微生物菌群则是防控代谢病发生的第一道防线。