摘要
目的:研究新型螺杂环查尔酮衍生物E20对顺铂(CDDP)所致人卵巢颗粒细胞(KGN)损伤的保护作用及其分子机制。方法:体外培养KGN细胞系中加入不同浓度E20后通过MTT法筛选出E20的安全浓度;选择浓度为5μg/mL的CDDP单独作用或与不同浓度的E20联合作用KGN细胞,MTT法检测细胞活力的变化;选择不同浓度的CDDP单独作用或与E20联合作用卵巢癌细胞ES-2,MTT法检测细胞活力并计算出半数抑制率(IC50);倒置显微镜观察不同处理后KGN细胞形态变化;流式细胞术测定KGN细胞凋亡变化;ELISA法测定KGN细胞上清液中雌二醇(E2)水平变化;蛋白质印迹(Western blot)法检测KGN细胞Sirt1和凋亡分子Bax、Bcl-2蛋白表达变化;Sirt1-siRNA转染KGN细胞后用Western blot法验证沉默效果,并用MTT法检测Sirt1下调后E20对KGN细胞活力的影响。结果:E20对KGN细胞的安全浓度在20μmol/L以下;CDDP能使KGN细胞和ES-2细胞活力下降,E20能减轻CDDP对KGN细胞的损伤且不会降低CDDP对ES-2细胞的抗增殖能力。CDDP能使KGN细胞形态改变、细胞凋亡增加(P<0.05),而经E20处理后KGN细胞形态恢复正常、细胞凋亡减少(P<0.05)。经CDDP刺激后,KGN细胞的Sirt1、Bcl-2蛋白表达量明显减少(P<0.05),Bax蛋白表达量明显增加(P<0.05),而E20处理可显著上调细胞中的Sirt1、Bcl-2蛋白表达(P<0.05),下调Bax蛋白表达(P<0.05)。与NC-siRNA组相比,Sirt1-siRNA转染后细胞Sirt1蛋白表达量明显减少(P<0.05),且与Control组相比,沉默Sirt1表达后,E20对细胞的保护活性几乎消失。结论:查尔酮衍生物E20可以很好地改善CDDP损伤KGN细胞的活力、形态,提高抗凋亡能力,其可能通过激活Sirt1途径的机制起保护作用。
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(5μg/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 20μmol/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 wit
作者
粘春惠
黄志成
崔婉晴
甘欣
张琼
吴建章
胡越
何文斐
NIAN Chunhui;HUANG Zhicheng;CUI Wanqing;GAN Xin;ZHANG Qiong;WU Jianzhang;HU Yue;HE Wenfei(School of Pharmaceutical,Wenzhou Medical University,Wenzhou 325035,China;Department of Obstetrics and Gynecology,the Second Affiliated Hospital&Yuying Children’s Hospital of Wenzhou Medical University,Wenzhou 325027,China)
出处
《温州医科大学学报》
CAS
2023年第8期637-642,650,共7页
Journal of Wenzhou Medical University
基金
浙江省自然科学基金项目(LY23H040003)
温州市基础性科研项目(Y2020089)
浙江省医药卫生临床研究应用项目(2022494299)
浙江省中医药科技计划科研基金项目(2022ZB219)。
关键词
查尔酮衍生物
化疗
卵巢早衰
卵巢颗粒细胞
顺铂
chalcone derivatives
chemotherapy
premature ovarian failure
ovarian granulosa cells
cisplatin
