E20 mediates the protective effect against cisplatin-induced damage to human ovarian granulosa cells by upregulating Sirt1
NIAN Chunhui1, HUANG Zhicheng1, CUI Wanqing1, GAN Xin1, ZHANG Qiong2, WU Jianzhang1, HU Yue2, HE Wenfei1
1.School of Pharmaceutical, Wenzhou Medical University, Wenzhou 325035, China; 2.Department of Obstetrics and Gynecology, the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China
NIAN Chunhui,HUANG Zhicheng,CUI Wanqing, et al. E20 mediates the protective effect against cisplatin-induced damage to human ovarian granulosa cells by upregulating Sirt1[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2023, 53(8): 637-642，650.
Abstract：Objective: To investigate the protective effect of a novel spirocyclic chalcone-like compound E20 on cisplatin (CDDP)-induced damage in human ovarian granulosa cells (KGN) and to explore its preliminary molecular mechanism. Methods: In the in vitro KGN cell culture system, different concentrations of E20 were added, and the safe concentration of E20 was screened by MTT assay. KGN cells were treated with CDDP alone or in combination with different concentrations of E20 (5 μg/mL), and then changes in cell viability were detected by MTT assay. Different concentrations of CDDP alone or in combination with E20 were used to treat ovarian cancer cells ES-2, and cell viability was detected by MTT assay to calculate the half-maximal inhibitory concentration (IC50). Changes in KGN cell morphology were observed by inverted microscopy, and changes in apoptosis were detected by flow cytometry. Changes in estradiol (E2) levels in KGN cell supernatant were measured by ELISA. Changes in the expression of Sirt1 and apoptosis-related proteins Bax and Bcl-2 in KGN cells were detected by Western blotting. The silencing effect of Sirt1-siRNA transfection on KGN cells was verified by Western blot, and the effect of E20 on KGN cell viability after Sirt1 downregulation was detected by MTT assay. Results: The safe concentration of E20 for KGN cells was below 20 μmol/L. CDDP decreased the viability of KGN and ES-2 cells, while E20 could alleviate CDDP-induced damage to KGN cells without reducing the anti-proliferative ability of CDDP in ES-2 cells. CDDP changed the morphology of KGN cells and increased cell apoptosis (P<0.05), while E20 treatment restored the normal morphology of KGN cells and reduced cell apoptosis (P<0.05). After CDDP stimulation, the protein expression of Sirt1 and Bcl-2 in KGN cells decreased significantly (P<0.05), while the protein expression of Bax increased significantly (P<0.05). E20 treatment could significantly upregulate the expression of Sirt1 and Bcl-2 proteins in cells (P<0.05) and downregulate the expression of Bax protein (P<0.05). Compared with the control (NC-siRNA) group, the Sirt1 protein expression in cells decreased significantly after Sirt1-siRNA transfection (P<0.05). Moreover, after the expression of Sirt1 was silenced, the protective activity of E20 on cells almost disappeared compared with the normal control group. Conclusion: The chalcone derivative E20 can effectively improve the viability and morphology of KGN cells damaged by CDDP, enhance their anti-apoptotic ability, and may play a protective effect by activating the Sirt1 pathway. This study provides a promising new small molecule compound for chemotherapy-induced premature ovarian failure.
