Bisphenol A promotes autophagy in ovarian granulosa cells by inducing AMPK/mTOR/ULK1 signalling pathway
Bisphenol A promotes autophagy in ovarian granulosa cells by inducing AMPK/mTOR/ULK1 signalling pathway
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双酚A通过诱导AMPK/mTOR/ULK1信号通路促进卵巢颗粒细胞自噬
DOI：
10.1016/j.envint.2020.106298
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发表时间：
2021-02-01
影响因子：
11.8
通讯作者：
Quan, Song
中科院分区：
文献类型：
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作者：
Lin, Miaoling;Hua, Rui;Quan, Song
背景：双酚A （BPA）是一种广泛存在的内分泌干扰化学物质，具有雌激素样作用，它可以通过破坏颗粒细胞（GCs）的正常功能来干扰人类生殖系统，导致卵巢功能异常。然而，其对人体GCs的毒性作用机制迄今尚未明确描述。方法：招募106例进行第一次体外受精-胚胎移植（IVF-ET）周期的正常促性腺激素不育妇女。分析尿双酚a水平及IVF-ET早期结局。以尿中位BPA浓度作为临界值，将患者分为低BPA暴露组和高BPA暴露组。使用小鼠和人颗粒细胞系（KGN细胞）进行体内和体外研究。以6 ~ 8周龄雌性昆明小鼠为研究对象，每天1次灌胃不同剂量（1、10、100 μ g/kg）的BPA，连续2周，同时以浓度为1、10、100 nM的双酚a对KGN细胞进行24 h、48 h、72 h的双酚a处理，通过组织病理学观察双酚a对卵巢的影响。分别采用CCK-8、TUNEL和流式细胞术检测细胞活力和凋亡。采用ELISA检测激素水平，免疫荧光、RT-PCR和western blots检测分子机制。结果：随着尿液BPA浓度的升高，卵母细胞回收率、成熟率和胚胎着床率显著降低。高BPA组E2峰值水平较低，但无统计学意义。在BPA处理的小鼠中，组织病理学观察到囊性扩张，GCs数量减少。激活AMPK/mTOR/ULK1信号通路，体内和体外均可检测到E2、P4和AMH水平降低，以及GCs自噬。在KGN细胞中证实，磷酸化AMPK和ULK1增加，磷酸化mTOR减少，通过敲低AMPK或3-MA抑制自噬，可以逆转体外BPA暴露的不良影响。结论：BPA暴露可能通过AMPK/mTOR/ULK1通路激活GCs自噬，从而异常影响人卵巢功能，导致卵泡发生异常。
Background: Bisphenol A (BPA) is a widespread endocrine-disrupting chemical with estrogen like effects, which could interfere with the human reproductive system by disrupting the normal function of granulosa cells (GCs) leading to abnormal ovarian function. However, the mechanism of its toxicity on human GCs has not been clearly described thus far.Methods: 106 normogonadotropic infertile women undergoing their first in-vitro fertilization-embryo transfer (IVF-ET) cycle were recruited. Urinary BPA level and the early outcomes of IVF-ET were analysed. Patients were divided to low and high BPA exposure groups using the median urinary BPA concentration as the cut-off value. In-vivo and in-vitro studies were conducted using mice and human granulosa cell line (KGN cells). Female Kunming mice approximately 6-8 weeks of age were poisoned with BPA at different dosages (1, 10 or 100 mu g/kg) by oral gavage once daily for 2 weeks, while KGN cells were exposed to BPA at the concentration of 1, 10 or 100 nM for 24 h, 48 h or 72 h. BPA-induced ovarian morphologic changes were analysed by histopathology investigation. Cell viability and apoptosis were evaluated using CCK-8, TUNEL and flowcytometric, respectively. Hormone levels were determined using ELISA and the molecular mechanism studies were conducted using immunofluorescence, RT-PCR and western blots.Results: The oocyte retrieval rate, maturation rate and embryo implantation rate significantly decreased with the higher level of urinary BPA concentration. Peak E2 level was lower in high BPA group, but no statistical significance could be observed. In BPA treated mice, cystic dilation of the follicles with a decreased number of GCs could be observed histopathologically. Decreased E2, P4 and AMH level and GCs autophagy could be detected both in-vivo and in-vitro with the activation of AMPK/mTOR/ULK1 signalling pathway. As being confirmed in KGN cells, phosphorylated AMPK and ULK1 increased while phosphorylated mTOR decreased, and by inhibition autophagy using knockdown of AMPK or 3-MA, adverse effects of BPA exposure in-vitro could be reversed.Conclusion: BPA exposure might abnormally influence human ovarian functions leading to abnormal folliculogenesis by activation of autophagy in GCs through AMPK/mTOR/ULK1 pathway.