【摘要】 子宫内膜炎是奶牛养殖业常见的一种繁殖疾病，目前主要利用抗生素进行治疗。本试验利用已成功构建的子宫内膜细胞炎症模型，研究添加1μmol/L虾青素对脂多糖(LPS)诱导奶牛子宫内膜细胞调亡的影响，处理24 h后利用流式细胞仪检测细胞的凋亡比例，利用荧光定量RT-PCR和Western Blot分别检测Bax、Bcl-2、Fas、Caspase 8、Caspase 3基因的mRNA和蛋白表达水平，并利用酶标仪检测培养液中FasL含量。结果表明，脂多糖(LPS)作用子宫内膜细胞6 h后，细胞凋亡比例显著增加(P<0.05);线粒体介导的促凋亡关键基因Bax表达升高(P<0.05),抗凋亡基因Bcl-2表达降低(P<0.05);Fas/FasL信号通路的关键基因Fas、Caspase 8表达升高(P<0.05),FasL蛋白水平也显著升高(P<0.05);凋亡执行效应酶Caspase 3基因表达显著升高(P<0.05)。说明LPS从线粒体和Fas/FasL两条通路诱导子宫内膜细胞发生凋亡。加入1μmol/L虾青素培养细胞24 h后，细胞凋亡比例显著下降(P<0.05);Bax、Fas、Caspase 8和Caspase 3表达水平均显著降低(P<0.05),Bcl-2表达水平显著升高(P<0.05),说明虾青素通过线粒体和Fas/FasL两条信号通路抑制LPS诱导的子宫内膜细胞凋亡。本试验可为奶牛子宫内膜炎的预防和治疗提供理论参考。更多还原

【Abstract】 Endometritis is a common reproductive disease in dairy cattle breeding industry, and antibiotics are mainly used for treatment currently. In this study, the endometrial cells inflammation model successfully constructed in vitro was used to study the effects of treating with 1 μmol/L astaxanthin for 24 h on apoptosis of bovine endometrial cells induced by lipopolysaccharide(LPS). The ratio of cell apoptosis was detected by flow cytometry. The mRNA and protein expression levels of Bax, Bcl-2, Fas, Caspase 8 and Caspase 3 were detected by fluorescent quantitative RT-PCR and Western Blot, and the FasL content in culture medium was detected with a microplate reader. The results showed that the proportion of cell apoptosis in LPS group increased significantly(P<0.05) after treated for 6 hours; the expression of Bax which was a key pro-apoptosis gene mediated by mitochondria increased(P<0.05), however, the anti-apoptotic gene Bcl-2 decreased(P<0.05). The expression of key genes Fas and Caspase 8 of Fas/FasL signaling pathway increased(P<0.05), and the level of FasL protein also significantly increased(P<0.05); the expression of Caspase 3 which was an apoptosis executive effector enzyme significantly increased(P<0.05). It indicated that endometrial cell apoptosis was induced by LPS through mitochondrial-mediated and Fas/FasL pathways. After cultured for 24 h in the medium containing 1 μmol/L astaxanthin, the proportion of endometrial cell apoptosis significantly decreased(P<0.05); the expression levels of Bax, Fas, Caspase 8 and Caspase 3 significantly reduced(P<0.05) on average, and the expression level of Bcl-2 significantly increased(P<0.05). These results showed that astaxanthin inhibited LPS-induced endometrial cell apoptosis through mitochondria-mediated and Fas/FasL signaling pathways. This research could provide theoretical references for prevention and treatment of bovine endometritis.更多还原