虾青素通过Fas/FasL信号通路调控奶牛子宫内膜细胞凋亡的研究

A high incidence of early embryonic death has been observed due to failure of attachment to the maternal endometrium during the pre- and peri-implantation period, which results in serious economic losses in dairy industry. Many stimuli can induce apoptosis in endometrial cells, which may damage implantation of the early embryo. Studies have found that Fas/FasL-mediated pathway plays important roles in regulating apoptosis. Astaxanthin is a safe, non-toxic and rich source of oxygen-containing derivatives of red carotenoids, which displays strong anti-apoptotic activity. However, its role in cow endometrial cells has not been reported. Therefore, we will establish a model of apoptotic injury of dairy cow endometrial cells, and investigate endometrial apoptosis, mRNA and protein expressions of apoptosis-related gene in the Fas/FasL signaling pathway, and implantation-associated factors after treated with astaxanthin, using laser scanning confocal microscopy, flow cytometry, RNA interference and overexpression, qRT-PCR, and Western blot. The study was to explore its anti-apoptotic action of astaxanthin in cow endometrial cells, aiming to solve early pregnancy loss in dairy cows using nutrients, increase the rate of calving, and ensure the sustainable and healthy development of the dairy industry.

奶牛早期胚胎向子宫内膜附植前或附植过程中有较高的死亡率，给奶牛生产造成严重的经济损失。许多刺激因素可诱导子宫内膜细胞凋亡，研究者推测这可能影响胚胎的正常附植。研究发现，Fas/FasL信号通路在调控细胞凋亡方面起关键性作用。虾青素是一种安全无毒且来源丰富的红色类胡萝卜素的含氧衍生物，具有很强的抗凋亡能力，然而，其对奶牛子宫内膜细胞凋亡损伤的作用尚未见报道。因此，本课题建立奶牛子宫内膜细胞凋亡损伤模型，加入虾青素处理后，利用激光扫描共聚焦荧光显微镜、流式细胞仪、RNA干扰与过表达、qRT-PCR法、Western-blot等技术手段，检测子宫内膜细胞凋亡情况、Fas/FasL信号通路凋亡相关基因的mRNA及其蛋白表达，以及胚胎附植相关因子的分泌情况，探讨虾青素对奶牛子宫内膜细胞抗凋亡损伤的作用机制，旨在通过营养手段解决生产中奶牛早期流产难题，提高产犊率，确保奶牛产业的持续稳定健康发展。

胚胎早期死亡及流产给奶牛繁殖率和产奶量带来严重影响，造成极大的经济损失。许多刺激因素可诱导子宫内膜细胞凋亡，研究者推测这可能影响胚胎的正常附植。本研究以虾青素为研究对象，利用双氧水（H2O2）及细菌脂多糖（LPS）构建奶牛子宫内膜细胞凋亡损伤模型，利用流式细胞仪、慢病毒RNA干扰、免疫荧光、qRT-PCR、Western-blot等方法，研究了虾青素通过Fas/FasL信号通路对子宫内膜细胞的保护作用及可能的机制。H2O2破坏了子宫内膜细胞抗氧化系统，造成细胞凋亡比例显著升高，Fas/FasL信号通路的关键基因Fas、FasL、Caspase 8、Caspase3在mRNA和蛋白表达水平升高，Fas基因RNA干扰和Fas蛋白直接添加进一步验证了Fas/FasL信号通路在调控子宫内膜细胞凋亡中发挥重要作用。添加虾青素保护细胞免受H2O2导致的氧化应激损伤，降低细胞凋亡，恢复分泌胚胎营养因子的能力，Fas、FasL、Caspase 8、Caspase3在mRNA和蛋白表达水平降低，而且虾青素能修复子宫内膜细胞紧密连接结构损伤，对子宫内膜屏障起保护作用。上述研究为通过营养手段解决生产中奶牛早期流产难题、提高产犊率提供了一定的理论依据。