Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Keywords: 4-PBA; Cisplatin; Endoplasmic reticulum stress; Granulosa cell apoptosis; Ovarian damage.