Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
Reproductive Medicine Center, Guangdong Provincial Family Planning Special Hospital, Guangzhou, 510699, China.
Reprod Biol Endocrinol. 2018 Sep 3;16(1):85. doi: 10.1186/s12958-018-0404-4.
Cisplatin (CDDP), a widely used chemotherapeutic agent, can induce excessive granulosa cell apoptosis, follicle loss and even premature ovarian insufficiency (POI). However, the mechanism remains elusive, although some studies have indicated the involvement of endoplasmic reticulum stress (ERS). The aim of our study was to investigate the possible mechanism ERS in CDDP-induced granulosa cell apoptosis and follicle loss.
A POI mouse model was generated by CDDP. The ovaries samples were collected and processed for isobaric tags for relative and absolute quantification analysis (iTRAQ) to screen out our interested proteins of HSPA5 and HSP90AB1, and the decline in their expression were verified by a real-time quantitative PCR and a western blotting assay. In vitro, human granulosa cells, KGN and COV434 cells were transfected with siRNA targeting HSPA5 and HSP90AB1 and then treated with CDDP, or treated with CDDP with/without CDDP+ 4-phenylbutyric acid (4-PBA) and 3-methyladenine (3-MA). The levels of ERS, autophagy and apoptosis were evaluated by western blotting, DALGreen staining and flow cytometry. In vivo, ovaries from mice that received intraperitoneal injections of saline, CDDP, CDDP+ 4-PBA and CDDP+ 3-MA were assayed by immunofluorescence, hematoxylin and eosin (H&E) staining for follicle counting, and terminal-deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining for cell apoptosis assay. The plasma hormone levels were measured by an enzyme-linked immunosorbent assay (ELISA) kit.
We have clarified the relationships between ERS, autophagy, and apoptosis in CDDP-induced granulosa cell apoptosis, both in vitro and in vivo. Alleviating ERS by inhibiting HSPA5 and HSP90AB1 attenuated CDDP-induced autophagy and apoptosis. 4-PBA treatment significantly attenuated CDDP-induced cell autophagy and apoptosis in cultured KGN and COV434 cells. However, inhibiting cell autophagy with 3-MA negligibly restored the CDDP-induced changes in ERS and apoptosis. In vivo experiments also demonstrated that treatment with 4-PBA, but not 3-MA, prevented CDDP-induced ovarian damage and hormone dysregulation.
CDDP-induced ERS could promote autophagy and apoptosis in granulosa cells, causing excessive follicle loss and endocrine disorders. Alleviation of ERS with 4-PBA, but not of autophagy with 3-MA, protect against CDDP-induced granulosa cell apoptosis and ovarian damage. Thus, 4-PBA can be used to protect the ovary during chemotherapy in women.
顺铂（CDDP）是一种广泛应用的化疗药物，可诱导过多的颗粒细胞凋亡、卵泡丢失，甚至导致卵巢早衰（POI）。然而，其具体机制仍不清楚，尽管一些研究表明内质网应激（ERS）可能参与其中。本研究旨在探讨 ERS 在 CDDP 诱导的颗粒细胞凋亡和卵泡丢失中的可能机制。
采用 CDDP 构建 POI 小鼠模型。收集卵巢组织样本，进行等重同位素标记相对和绝对定量分析（iTRAQ）筛选出感兴趣的蛋白 HSP90AB1 和 HSPA5，并通过实时定量 PCR 和 Western blot 验证其表达水平的下降。体外培养人颗粒细胞、KGN 和 COV434 细胞，转染靶向 HSPA5 和 HSP90AB1 的 siRNA，然后用 CDDP 处理，或用 CDDP 加/不加 4-苯丁酸（4-PBA）和 3-甲基腺嘌呤（3-MA）处理。通过 Western blot、DALGreen 染色和流式细胞术评估 ERS、自噬和凋亡水平。体内实验中，对接受腹腔注射生理盐水、CDDP、CDDP+4-PBA 和 CDDP+3-MA 的小鼠的卵巢进行免疫荧光、苏木精和伊红（H&E）染色计数卵泡和末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）染色检测细胞凋亡。采用酶联免疫吸附试验（ELISA）试剂盒测定血浆激素水平。
本研究在体外和体内阐明了 CDDP 诱导的颗粒细胞凋亡中 ERS、自噬和凋亡之间的关系。通过抑制 HSPA5 和 HSP90AB1 减轻 ERS 可减轻 CDDP 诱导的自噬和凋亡。4-PBA 处理可显著减轻培养的 KGN 和 COV434 细胞中 CDDP 诱导的细胞自噬和凋亡。然而，用 3-MA 抑制自噬对 ERS 和凋亡的 CDDP 诱导变化几乎没有恢复作用。体内实验也表明，用 4-PBA 处理可防止 CDDP 引起的卵巢损伤和激素失调，但用 3-MA 处理则不然。
CDDP 诱导的 ERS 可促进颗粒细胞发生自噬和凋亡，导致过多的卵泡丢失和内分泌紊乱。用 4-PBA 减轻 ERS，而不是用 3-MA 减轻自噬，可防止 CDDP 诱导的颗粒细胞凋亡和卵巢损伤。因此，4-PBA 可用于保护女性化疗期间的卵巢。
