Xu Hongxia, Bao Xiumin, Yang Junya, Kong Hanxin, Li Yan, Sun Zhiwei
Faculty of Environmental Science and Engineering, 47910Kunming University of Science and Technology, Kunming, China.
Department of Reproductive Medical Centre, The First People's Hospital of Yunnan Province, Kunming, China.
Hum Exp Toxicol. 2023 Jan-Dec;42:9603271231152831. doi: 10.1177/09603271231152831.
We investigated the level of Cysteine-rich 61 (CYR61) in premature ovarian failure as well as its regulatory molecular mechanism in this study.
Cyclophosphamide (CTX) was used to induce OGCs (rat ovarian granulosa cells) and rats to establish in vivo and in vitro premature ovarian failure models. H&E staining was used to detect the pathological changes of ovarian histopathology. Si-NLRP3 (NOD-like receptor thermal protein domain associated protein 3, NLRP3) and si-CYR61 were transfected into OGCs using lipofectamine 3000. RT-qPCR and western blot were used to detect the expressions of CYR61 in ovarian tissue and OGCs. It showed that the expression of CYR61 was significantly down-regulated in premature ovarian failure model. Cell viability was detected using a Cell Counting Kit-8 (CCK-8) kit. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling) staining was used to detect the apoptosis. 5-Ethynyl-2'-deoxyuridine (EdU) and SA-β-gal (senescence-associated β-galactosidase) staining were used to assess the proliferation and senescence. The expression of CYR61 in OGCs and ovarian tissues were detected by immunofluorescence and immunohistochemical staining. Overexpression of CYR61 significantly promoted OGCs proliferation and inhibited pyroptosis and apoptosis. Western blot was used to detect the protein expressions of p53 and p21 in OGCs. Flow cytometry was used to detect the pyroptosis. CYR61 overexpression inhibited the expression of NLRP3 and caspase-1 in CTX-induced OGCs according to western blot results. Moreover, we found that CYR61 overexpression down-regulated the protein expressions of p53 and p21 in CTX-induced OGCs.
CYR61 inhibited CTX-induced OGCs senescence, and the mechanism may be related to the regulation of caspase-1/NLRP3-induced pyroptosis.
在本研究中，我们调查了过早卵巢功能衰竭中富含半胱氨酸的61（CYR61）水平及其调控分子机制。
使用环磷酰胺（CTX）诱导大鼠卵巢颗粒细胞（OGCs）和大鼠建立体内和体外过早卵巢功能衰竭模型。采用苏木精-伊红（H&E）染色检测卵巢组织病理学的病理变化。使用脂质体3000将小干扰RNA（Si）-NOD样受体热蛋白结构域相关蛋白3（NLRP3）和Si-CYR61转染到OGCs中。采用逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质印迹法检测卵巢组织和OGCs中CYR61的表达。结果显示，过早卵巢功能衰竭模型中CYR61的表达显著下调。使用细胞计数试剂盒-8（CCK-8）检测细胞活力。采用末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记（TUNEL）染色检测细胞凋亡。使用5-乙炔基-2'-脱氧尿苷（EdU）和衰老相关β-半乳糖苷酶（SA-β-gal）染色评估细胞增殖和衰老。通过免疫荧光和免疫组织化学染色检测OGCs和卵巢组织中CYR61的表达。CYR61的过表达显著促进OGCs增殖并抑制细胞焦亡和凋亡。采用蛋白质印迹法检测OGCs中p53和p2i的蛋白表达。使用流式细胞术检测细胞焦亡。根据蛋白质印迹结果，CYR61的过表达抑制了CTX诱导的OGCs中NLRP3和半胱天冬酶-1的表达。此外，我们发现CYR61的过表达下调了CTX诱导的OGCs中p53和p21的蛋白表达。
CYR61抑制CTX诱导的OGCs衰老，其机制可能与调节半胱天冬酶-1/NLRP3诱导的细胞焦亡有关。
