咖啡酸对玉米赤霉烯酮诱导小鼠卵巢颗粒细胞凋亡的保护作用

doi:10.11843/j.issn.0366-6964.2020.12.016

摘要/Abstract

摘要： 卵巢颗粒细胞通过与卵母细胞相互作用及其自身分泌作用为卵泡的形成和发育成熟提供特殊的微环境。多种有害刺激可引起颗粒细胞凋亡和代谢失调从而降低卵母细胞质量，对胚胎的形成产生负面影响。玉米赤霉烯酮（zearalenone，ZEA）是畜禽养殖业中常见的造成卵巢颗粒细胞损伤的霉菌毒素，且缺少有效治疗药物。因此，本试验用ZEA造成小鼠卵巢颗粒细胞损伤，探究咖啡酸对ZEA诱导的小鼠卵巢颗粒细胞凋亡的保护作用。通过机械法分离小鼠卵巢颗粒细胞；运用间接免疫荧光法对分离出的细胞进行鉴定；使用MTT法测定咖啡酸对正常小鼠卵巢颗粒细胞活性的影响；选取200、100和50 μg·mL^-1咖啡酸分别与ZEA共处理颗粒细胞，同时设置细胞对照组和ZEA模型组，24 h后显微镜观察细胞形态及贴壁情况，MTT法检测细胞活力；qRT-PCR技术检测caspase-3 mRNA的表达；Western blot检测cleaved-caspase-3和cleaved-PARP蛋白水平。结果发现，FSHR阳性染色大量出现在试验组细胞胞浆中，提示分离到的细胞是小鼠卵巢颗粒细胞；咖啡酸对卵巢颗粒细胞没有毒性作用且细胞活力均在90%以上；与空白对照组细胞相比，ZEA组细胞体积较小，贴壁较差，细胞间隙增大，细胞活力显著降低（P<0.001），caspase-3 mRNA相对表达量以及cleaved-caspase-3和cleaved-PARP蛋白表达水平显著升高（P<0.001），而给予咖啡酸处理后细胞间隙减小，贴壁紧密，细胞活力显著升高（P<0.001），显著降低ZEA诱导的caspase-3 mRNA含量增加（P<0.001），显著降低凋亡相关蛋白cleaved-caspase-3和cleaved-PARP的表达（P<0.001）。本研究发现，咖啡酸可通过抑制ZEA诱导的细胞凋亡，恢复颗粒细胞活性。

关键词: 咖啡酸, 玉米赤霉烯酮, 卵巢颗粒细胞, 细胞凋亡

Abstract: Ovarian granulosa cells provide a special microenvironment for follicle formation and maturation through interaction with oocytes and their own secretion. A variety of harmful stimuli can cause granulosa cell apoptosis and metabolic disorders, reduce the quality of oocytes and have a negative impact on embryo formation. Zearalenone (ZEA) is a common cause of ovarian granulosa cells injury in the livestock industry, which is produced by mycotoxins, and lack of effective treatment drug. Therefore, in the current study zearalenone was used to induce ovarian granulosa cell injury and to explore the protective effect of caffeic acid on zearalenone-induced ovarian granulosa cell apoptosis in mice. Mouse ovarian granulosa cells were isolated by mechanical method, and indirect immunofluorescence was used to identify the isolated cells. MTT assay was used to determine the effect of caffeic acid on the activity of normal mouse ovarian granulosa cells.After granulosa cells were co-treated with caffeic acid (200, 100 and 50 μg·mL^-1) and ZEA for 24 hours, and control and ZEA group were set up at the same time, cell morphology and adherence were observed under a microscope. MTT was also used to detect cell viability. Caspase-3 mRNA expression level was detected by qRT-PCR. Cleaved-caspase-3 and cleaved-PARP protein expre-ssion levels were determined by Western blot. The results showed that positive FSHR staining appeared in cell cytoplasm of the test group, which confirmed that the isolated cells were mouse ovarian granulosa cells. The cell viability was above 90% which showed that caffeic acid had no toxic effect on granulosa cells. Compared with control group, ZEA group had smaller cell size, poor adherence, increased cell gap, and significant reduction in cell viability (P<0.001). Furthermore, the relative expression of caspase-3 mRNA, and cleaved-caspase-3 and cleaved-PARP protein level were significantly increased (P<0.001) compared with the control group. After caffeic acid treatment, cell gap was reduced, adherence was tight, cell viability was significantly increased (P<0.001). Caffeic acid significantly reduced zearalenone-induced increase in caspase-3 mRNA, and cleaved-caspase-3 and cleaved-PARP protein expression level (P<0.001). This study indicated that caffeic acid can restore granulosa cell viability by inhibiting ZEA-induced apoptosis.

Key words: caffeic acid, zearalenone, ovarian granulosa cells, apoptosis

中图分类号:

Q255

裴亚萍, 赵瑾, 孙娜, 孙盼盼, 孙耀贵, 范阔海, 尹伟, 李宏全. 咖啡酸对玉米赤霉烯酮诱导小鼠卵巢颗粒细胞凋亡的保护作用[J]. 畜牧兽医学报, 2020, 51(12): 3068-3075.

PEI Yaping, ZHAO Jin, SUN Na, SUN Panpan, SUN Yaogui, FAN Kuohai, YIN Wei, LI Hongquan. The Effect of Caffeic Acid on Zearalenone-induced Ovarian Granulosa Cell Apoptosis in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(12): 3068-3075.

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