FOXO介导的β-AR/NF-кB通路在制动应激致猪妊娠早期子宫免疫应答紊乱中的作用机制

The rate of embryo loss at early stage of swine was about 30% which brought numerous economic losses to husbandry and serious animal welfare problem. The possible reasons may be intensive breeding and the use of pregnancy cage. We have found previously that restraint stress led to local immune disorder in uterus and embryo loss of model animals (mice) but its signal pathway still needs further study. To explore the mechanism of restraint stress on local immune disorder in uterus of swine by signal pathway which stress hormones (catecholamine) combined with β-AR and activated the migration of transcription factor (FOXO) and expression of nuclear transcription factor (NF-кB, AP-1), in the project with restraint stress to pregnant swine, it will be studied the dynamic changes of the immune cells and cytokines in uterus, then screened microRNA by microarray chip and regulation targets of restraint stress on uterus immune disorder by cell culture, adding blocking agent and RNA interfering technology in based of β-AR signal pathway of the key stress hormone receptors (catecholamine) as the breakthrough point. The result is expected to not only provide a development progress for further researching pathogenic mechanism of embryo implantation failure caused by restraint stress but also provide theoretical basis for a better breeding way in production.

我国妊娠母猪早期胚胎丢失率高达30%，与目前普遍采用妊娠笼和集约化养殖方式有密切关系，这不仅对畜牧生产造成巨大的经济损失而且引发严重的动物福利问题。我们前期研究发现制动应激诱发模式动物（孕鼠）发生严重的子宫局部免疫紊乱和胚胎丢失现象，但其胞内外信号通路尚需进一步探讨。本项目以关键应激激素（儿茶酚胺）的β-AR信号通路为切入点，采用不同妊娠笼应激处理孕猪，分析制动应激影响孕猪子宫局部免疫细胞活性、细胞因子表达的动态变化，通过microRNA微阵列芯片技术筛选上游microRNA分子，利用细胞培养、添加抑制剂和RNA干扰技术研究制动应激影响子宫局部免疫紊乱的调控靶点，探究应激激素结合β-AR，激活胞内转录因子FOXO迁移及核内NF-кB、AP-1表达的信号通路及作用机制。研究结果不仅为深入探讨制动应激致猪早期流产的致病机制提供一个新的研究进展，而且为生产中进一步优化饲养方式提供理论指导。

我国妊娠母猪早期胚胎丢失率高达30%，与目前普遍采用妊娠笼和集约化养殖方式有密切关系，这不仅对畜牧生产造成巨大的经济损失而且引发严重的动物福利问题。本项目以关键应激激素（儿茶酚胺）的β-AR信号通路为切入点，采用应激处理孕猪和孕鼠，细胞培养等技术，研究了制动应激对孕猪胚胎着床和应激水平的影响，制动应激对孕猪子宫局部免疫水平的影响，制动应激影响子宫局部免疫应答的β-AR通路，FOXO介导的胞内通路在子宫局部免疫应答中的作用。研究结果表明制动应激导致母猪的应激水平升高，子宫内膜组织的β2-AR激活；子宫内膜组织发生了局部炎症，且子宫内膜组织的抗氧化功能障碍，氧化应激水平升高；子宫内膜发生了明显的凋亡；子宫内膜基质细胞中存在β2-AR蛋白的表达且表达最高，并且刺激β2-AR抑制ESCs细胞增殖并促进ESCs细胞凋亡，体内体外研究发现主要作用信号通路是cAMP-PKA-ERK；制动应激导致c-jun和c-fos mRNA高水平，应激可能启动了子宫内膜组织的一系列信号；进一步研究发现FOXO1在应激中升高尤为显著，低程度过氧化氢导致FOXO1出核乙酰化，促进细胞自噬的发生，而高程度的过氧化氢导致FOXO1入核，促进细胞凋亡的发生。研究结果不仅为深入探讨制动应激致猪早期流产的致病机制提供一个新的研究进展，而且为生产中进一步优化饲养方式提供理论指导。