氧化镧调控断奶仔兔肠黏膜机械屏障功能的机理研究

Weaning stress leads to the impairment of intestinal mucosal mechanical barrier in weaning rabbits resulting in enteritis, which is the mainly reason for their decreased growth performance and a large number of death. Previous studies have found that lanthanum oxide supplementation improved the growth performance, intestinal morphology and the expression of CaM mRNA of intestinal mucosal epithelial cells in weaning rabbits, indicating that lanthanum oxide played an important role in regulating the function of intestinal mucosal mechanical barrier. However, it is unclear whether lanthanum oxide can repair the impairment of intestinal mucosal mechanical barrier in weaning rabbits, and whether it can regulate its function via GPCR/CaM/cAMP signal pathway of intestinal cells. This project intends to take weaning rabbits as experimental materials. Investigate the effects of dietary lanthanum oxide on growth performance, GPCR/CaM/cAMP signal pathway in intestinal cells, physiological and biochemical and other parameters in weaning rabbits by using feeding trial, transcriptomics, proteomics, ELISA and other technologies. On this basis, RT-PCR and Western-blot were used to verify the function of possible signal pathway obtained in vivo at cellular level. The results of this study will clarify the mechanism of lanthanum oxide in regulating intestinal mucosal mechanical barrier function in weaning rabbits, and also provide a theoretical reference for the application of lanthanum oxide in feedstuffs.

断奶应激导致仔兔肠黏膜机械屏障受损而诱发肠炎，造成其生长性能下降并大量死亡。项目组前期研究发现，日粮添加氧化镧可促使断奶仔兔生长性能提高、肠道形态改善以及肠黏膜上皮细胞CaM基因表达上调，预示着氧化镧在调控肠黏膜机械屏障功能中发挥着重要作用。但是氧化镧能否修复断奶仔兔肠黏膜机械屏障的损伤，是否通过肠道细胞GPCR/CaM/cAMP信号通路对断奶仔兔肠黏膜机械屏障功能进行调控尚不清楚。本项目拟以断奶仔兔为试验材料，采用饲养试验、转录和蛋白组学、ELISA等技术，考察日粮氧化镧水平对断奶仔兔生长性能、肠道细胞GPCR/CaM/cAMP信号通路、生理生化等指标的影响；在此基础上，采用RT-PCR和Western-blot技术对活体试验获得的可能信号通路在细胞水平进行功能验证。研究结果将阐明氧化镧对断奶仔兔肠粘膜机械屏障功能的调控机理，也可为氧化镧在畜禽饲料中的应用提供理论参考。

集约化养殖条件下，断奶仔兔非常容易发生肠黏膜机械屏障受损等断奶应激。本项目通过断奶仔兔饲养试验发现，与对照组比较，氧化镧可以显著改善十二指肠肠道形态，对空肠肠道形态有改善趋势，但对回肠肠道形态无改善作用，表明氧化镧对肠道形态的影响部位主要在小肠前段；氧化镧能显著改善胃肠激素水平和肠道微生物菌落组成；氧化镧处理组仔兔十二指肠黏膜上皮细胞中的与离子转运通道和炎症因子有关的基因（如Slc37a2, CBR1, IL2等）mRNA得到了显著表达；通过对断奶仔兔小肠黏膜上皮细胞蛋白组学的研究发现，氧化镧组有144个上调蛋白，71个下调蛋白，这些差异蛋白包含了较多的与离子转运通道和炎症因子有关的蛋白（如Slc23a1_2, CD3G, CD8B, Slc37a1_2等）。通过MODE-K细胞培养试验，我们发现，镧处理后的MODE-K细胞中G蛋白偶联受体、钙调蛋白、肠上皮细胞连接因子、腺苷酸环化酶和炎症因子等基因（如Gprc5b，Cldn9，Camk2b，Nfkbie，Adcy7，Il7等）的mRNA水平与对照组相比差异显著。与此同时，镧处理后的MODE-K细胞中Adcy7, Camkk2, Nfkbie, TLR7, Cldn9, Gpr162等的蛋白表达量与对照组相比差异显著。通过分析体内（动物饲养试验）和体外试验（细胞培养试验）结果，并对比KEGG数据库，我们发现镧可以通过影响GPCR/cAMP/CaM通路或与离子转运相关的通路来改善断奶仔兔肠道形态、胃肠激素水平和肠道微生物菌落组成，达到修复断奶仔兔肠黏膜机械屏障损伤的作用，从而缓解仔兔断奶应激。