Gpnmb/MAPK信号通路与羔羊断奶应激的关系及其营养调控

Early weaning can shorten the breeding cycle of ewes, benifit to breed multiple lambs, and ensure the survival rate of newborn lambs. It is of great theoretical and practical value to alleviate weaning stress. In the early study of the applicant's team, early feeding of alfalfa can alleviate weaning stress, Gpnmb (glycoprotein nonmetastatic melanoma protein b) may be a potential target. Therefore, this project will evaluate the gene expression of MAPK signaling pathway and production of proinflammatory cytokine of the rumen epithelium of lambs supplemented with concentrate/concentrate-roughage mix before weaning respectivily, then use LPS-induced Inflammatory rumen epithelial cells as study model, through RNA interference to silence Gpnmb gene and MAPK signaling pathway respectivily, to explore whether Gpnmb can negative regulate the phosphorylation of MAPKs and production of proinflammatory cytokine, and the possible biological mechanism of Gpnmb to regulate inflammatory response. Based on analysis of the mechanism, the effects of exogenous addition of conjugated linoleic acid before weaning on the proliferation, absorption and barrier function of gastrointestinal tract epithelium of lambs after weaning were further studied, to confirm the possitive effect of Gpnmb by nutritional upregulate its expression. The aim of this project is to provide new ideas and targets for alleviating weaning stress of young ruminants and optimizing the early weaning technology.

早期断奶能够缩短母羊的繁殖周期，发挥多羔母羊的繁殖优势，保证多羔群体的仔羊成活率。缓解断奶应激具有重要的理论和应用价值。申请者所在团队前期研究发现，早期补饲苜蓿减缓断奶应激，Gpnmb可能是潜在的作用靶点。为此本项目在评定饲喂精料型/精粗混合型开食料对羔羊断奶前和断奶一周后瘤胃上皮MAPK信号通路相关基因表达的影响的基础上，通过LPS诱导瘤胃上皮细胞炎症反应，借助RNA干扰分别沉默Gpnmb基因和MAPK信号通路，探究Gpnmb是否通过负反馈调节MAPKs的磷酸化和促炎细胞因子的生成而缓解断奶应激，以及Gpnmb调控炎症反应可能的生物学作用机制。在此作用机制分析的基础上，通过饲养试验进一步验证外源添加共轭亚油酸诱导Gpnmb表达，对羔羊断奶后胃肠道组织上皮增殖、吸收和屏障功能的影响。为缓解幼龄反刍动物断奶应激和优化早期断奶技术提供新的思路和靶点。

早期断奶能够缩短母羊的繁殖周期，发挥多羔母羊的繁殖优势，保证多羔群体的仔羊成活率。缓解断奶应激具有重要的理论和应用价值。申请者所在团队前期研究发现，早期补饲苜蓿减缓断奶应激，Gpnmb可能是潜在的作用靶点，为此，本项目以前期发现为基础，结合体外瘤胃上皮细胞分离培养、转录组学、微生物高通量测序、RNA干扰、Western blot等技术，研究了Gpnmb作用于MAPK或其他信号通路缓解瘤胃上皮细胞炎症反应的可能生物学机制，并通过动物饲养试验进一步验证了外源添加CLA诱导Gpnmb表达，对羔羊断奶后瘤胃上皮组织吸收代谢和屏障功能的影响，以及瘤胃微生物菌群多样性和功能的影响。本项目首先利用胰酶消化及差速贴壁法成功分离了原代瘤胃上皮细胞，利用SV40T转染法构建了永生化瘤胃上皮细胞系模型，并借助此模型研究发现，Gpnmb的高表达可以通过抑制MAPKs活化降低瘤胃上皮细胞炎症因子生成；沉默MAPKs后，Gpnmb亦可通过抑制NF-κB和TNF等其他信号通路来缓解LPS诱导的瘤胃上皮细胞炎症反应；100 μM的顺-9，反-11-CLA和反-10，顺-12-CLA均可显著上调Gpnmb表达，而CLA对瘤胃上皮细胞炎症发生的调控作用具有同分异构体特异性，与顺-9，反-11-CLA相比，反-10，顺-12-CLA通过上调PPARγ表达和下调TLR4-NF-κB信号通路等分子机制表现出更好的抗炎作用。借助动物饲养试验发现，补饲1%的CLA可以降低羔羊早期断奶后5天瘤胃中Acinetobacter、Neisseria、Streptococcus等病原菌属的相对丰度，下调瘤胃上皮组织中的炎症反应和原发性免疫缺陷的发生；增加瘤胃中Bacteroides、Treponema、Prevotellaceae_UCG_001和Rikenellaceae_RC9_gut_group等菌属的相对丰度，提高瘤胃发酵功能，上调瘤胃上皮组织中花生四烯酸代谢、半胱氨酸和蛋氨酸代谢、谷胱甘肽代谢、碳水化合物的消化吸收和叶酸的生物合成等生理过程，一定程度上缓解羔羊的早期断奶应激。且CLA对瘤胃微生物和生理功能的调控作用可以持续到断奶后21天。该项目为缓解幼龄反刍动物断奶应激和优化早期断奶技术提供了新的思路和靶点。