丁酸通过NF-κB信号通路调控瘤胃上皮细胞单羧酸转运蛋白表达的影响机制研究

Subacute ruminal acidosis (SARA) is a common nutritional metabolic disease in ruminant production. SARA activate cellular inflammation-related signaling pathways such as NF-κB signaling pathway in rumen epithelial cells. Increasing the ratio of concentrate and forage affects the expression of MCT1 in rumen epithelial cells, Butyric acid has anti-inflammatory effects as the main short-chain fatty acid produced in the rumen. Adding butyrate can inhibit the inflammation of rumen epithelial cells, and butyrate also can inhibit the degradation of IκB-α and NF-κB activity by reducing protease activity.,and promote the expression of MCTs, but it is unclear that the mechanism of the effect of butyrate on the expression of MCTs. Therefore, this project hypothesize that butyrate could regulate the expression of MCTs in rumen epithelial cell through NF-κB signaling pathway under subacute rumen acidosis. In this project, the primary culture of rumen epithelial cells and induction of inflammatory responses in rumen epithelial cells in vitro, the project reveale butyric acid as a signaling molecule regulates the expression of MCT in rumen epithelial cells through subacute rumen acidosis NF-κB signaling pathway by culture cell with butyrate in vitro, enrich nutrient absorption and translocation regulation in rumen under subacute ruminal acidosis, and improve ruminant feeding level.

亚急性瘤胃酸中毒是反刍动物生产中常见营养代谢病。亚急性瘤胃酸中毒会激活瘤胃上皮细胞中细胞炎症相关信号通路如NF-κB信号通路，提高日粮中精粗比例会影响瘤胃上皮细胞中MCT1表达，丁酸作为瘤胃中产生的主要短链脂肪酸具有抗炎症作用。添加丁酸能抑制瘤胃酸中毒导致的炎症作用，丁酸能通过降低蛋白酶活性抑制IκB-α降解影响NF-κB活性，并促进MCTs表达，但目前丁酸对MCTs表达的影响机制仍不清楚。因此，本项目假设在反刍动物处于亚急性瘤胃酸中毒状态下，丁酸能通过NF-κB信号通过调控瘤胃上皮细胞MCTs的表达。项目通过原代瘤胃上皮细胞的分离培养和体外诱导瘤胃上皮发生细胞炎症反应，通过添加丁酸、以及对NF-κB信号通路的阻断，揭示丁酸作为信号分子通过亚急性瘤胃酸中毒细胞NF-κB信号通路调控瘤胃上皮细胞MCT的表达，丰富亚急性瘤胃酸中毒瘤胃内营养物质吸收转运调控理论，提高反刍动物饲养水平。

短链脂肪酸在瘤胃内大量积累和瘤胃pH值急剧降低会导致亚急性瘤胃酸中毒（SARA）。单羧酸转运载体MCTs作为瘤胃短链脂肪酸的主要转运载体，丁酸作为信号分子在对瘤胃上皮细胞中MCTs的表达是否有调控作用尚不清楚。本项目中我们首先摸索建立了体外培养原代瘤胃上皮细胞方法，并构建了体外瘤胃上皮细胞SARA模型，然后利用体外模型研究了不同水平丁酸对SARA状态下瘤胃上皮细胞中MCTs表达的影响，在细胞水平探索了丁酸影响瘤胃上皮细胞中MCTs表达的细胞炎症信号通路。结果表明：（1）瘤胃上皮细胞在体外呈S型生长曲线，同时对CK147免疫荧光鉴定等确认成功培养出原代瘤胃上皮细胞；（2）组胺添加能显著增加细胞中IL-6基因mRNA和蛋白表达，但会降低TNF-α基因mRNA表达但其蛋白表达反而升高(p<0.05)，在含组胺培养基中添加SCFA会降低IL-1β和IL-6蛋白表达，组胺和SCFA会显著增加瘤胃上皮细胞Claudin-1和Occludin基因mRNA表达，仅添加高浓度SCFA会显著减低MCT1和CD147基因mRNA表达(p<0.05)和增加MCT1蛋白表达，添加组胺和正常浓度SCFA会增加MCT1和CD147蛋白表达，添加组胺和SCFA会显著降低MCT4 mRNA表达(p<0.05)，而转录因子NF-κβ和p-NF-κβ的mRNA和蛋白表达均随SCFA浓度增加而显著增加(p<0.05)；（3）瘤胃上皮细胞MCT1、MCT4和CD147基因mRNA表达量随丁酸添加显著增加(p<0.05)，组胺添加组随丁酸增加MCT4和CD147基因表达增加幅度较无组胺组高，但均在丁酸在2mM出现最大值。随丁酸添加会显著降低组胺添加组中瘤胃上皮细胞IL-6、NF-κβ和p-NF-κβ基因表达量(p<0.05)；（4）添加NF-κβ抑制剂会显著降低细胞炎症因子IL-1β和IL-6蛋白表达量(p<0.05)，增加瘤胃上皮细胞MCT1和CD147蛋白表达(p<0.05)，丁酸会减低细胞炎症因子IL-1β和IL-6以及NF-κβ和p-NF-κβ蛋白表达量(p<0.05)，增加上皮细胞中MCT1和CD147蛋白表达(p<0.05)。以上结果说明瘤胃上皮亚急性瘤胃酸中毒会引发细胞炎症影响上皮细胞结构进而参与SCFA转运的MCTs表达变化，而丁酸可影响NF-κβ信号通路抑制炎症因子表达和促进MCTs表达。