微生物群感信号分子acy-HSL在SARA引起的结肠黏膜上皮细胞凋亡中的作用及机制研究

Due to lack of quality forage, dairy cows are usually fed with high level of grain concentrate diet to meet the demands of high milk production. Subacute ruminal acidosis (SARA) is a common metabolic disorders induced by the rapid fermentation of carbohydrates in rumen. It’s well known that the level of endogenous endotoxin (LPS) will be largely increased in the colonic lumen due to the rapid multiply of gram negative (G-) bacteria. Our previous results showed that SARA goats exhibited profound damages of mucosa barrier with structural disruption in villi and cells shedding, and paralleled a marked activation of epithelial cells apoptosis. However, the increase of LPS in the colonic lumen was not involved in the process of epithelial apoptosis, indicating the chronically adaptive “tolerance” to endogenous endotoxin. Recent research showed that Quorum sensing (QS) is a widespread mechanism of bacterial communication in which individual cells produce and respond to small chemical signals (also called autoinducer). In most G- bacteria, N-acylhomoserine lactones (acy-HSL) plays roles as signal molecules in QS system. Acy-HSL is lipid soluble, which can easily diffuse from the lumen into the epithelium and underlying tissues. It’s reported that acy-HSL with low concentrate rapidly triggered the intrinsic apoptotic pathway in a variety cells, particularly in the epithelial cells. To date, it’s reasonable to speculate that the rapid increase of G- in lumen will produce a large amount of QS signals, which is largely contributed to cell apoptosis in the mucosa epithelial cells. In this project, Saanen dairy goat was employed as an animal model, it is the first time to investigate QS signal on mucosal barrier and epithelial cell apoptosis during SARA. Our results will provide new targets and new ideas of preventing SARA and intestinal inflammation in ruminants.

因缺乏优质牧草，常给奶牛饲喂高谷物精料以满足其高产奶的能量需求，而导致亚急性瘤胃酸中毒（SARA）的发生。SARA时常伴随肠道内（G-）菌大量繁殖及内毒素LPS累积。我们的研究结果表明，SARA山羊结肠黏膜屏障受损、上皮细胞凋亡严重；但LPS不是诱发细胞凋亡的主要因素，提示机体对内源性LPS的适应性“耐受”。细菌间主要依赖“群体感应（QS）”产生的小分子物质进行交流、协调菌群行为。（G–）和（G+）菌都存在QS，酰化高丝氨酸内酯（acy-HSL）是绝大多数（G–）菌的信息分子。新近发现，acy-HSL具有快速而强烈的促进哺乳动物细胞凋亡的作用。那么，SARA时后端肠道内（G–）菌的大量繁殖是否伴随acy-HSL升高，从而激发上皮细胞的凋亡？迄今尚未见相关报道。本课题以萨能奶山羊为动物模型，首次研究SARA时结肠腔内QS信号分子对上皮细胞凋亡的作用及机制，为慢性炎症的防治提供靶点和理论依据。

本项目主要研究长期饲喂高比例精饲料日粮对泌乳反刍动物肠道黏膜屏障结构的影响,旨在揭示后段肠道内菌群感应分子N-酰化高丝氨酸内酯（C12-HSL）对宿主肠上皮细胞的作用及机制。研究结果表明，与饲喂低精料（35%精料）组泌乳山羊相比，长期（19周）或短期（4周）饲喂高精料（65%精料）日粮引起：（1）亚急性瘤胃酸中毒SARA，后段肠道内容物中游离内毒素（Lipopolysaccharide, LPS）和C12-HSL浓度均显著升高。（2）肠道内微生物多样性下降，且在细菌的属水平上，结肠内容物中的梭菌、颤螺菌、普氏菌和拟杆菌等的丰度显著升高，而瘤胃球菌显著降低。（3）长期饲喂高精料日粮严重破坏泌乳期奶山羊后段肠道上皮形态结构，引起肠道上皮细胞凋亡增加、细胞增殖能力降低、局部炎症反应的发生和紧密连接受损，引起肠道上皮通透性增大。（4）QS信号分子C12-HSL通过引起线粒体肿胀、线粒体膜电位去极化、线粒体功能紊乱和细胞凋亡诱导肠道杯状细胞稳态失衡。此外，C12-HSL能够抑制杯状细胞黏蛋白的合成和硫酸盐化，破坏肠道黏液屏障。（5）C12-HSL通过磷氧酶PON2诱导肠道杯状细胞氧化应激，进而激活caspase-1&3级联信号，最终导致细胞凋亡及黏蛋白分泌和成熟紊乱。先天免疫同样参与C12-HSL对肠道杯状细胞的不良反应，细胞因子特别是IL-8可能参与其中。综上所述，泌乳反刍动物长期采食高精料日粮引起SARA，同时后段肠道发酵异常、微生态紊乱，微生物群体感应分子C12-HSL浓度升高，通过磷氧酶PON2信号途径诱导肠黏膜上皮的损伤，而肠黏膜结构受损是引起有害代谢物转移入血液的关键环节，从而导致机体代谢性疾病的发生与发展。