高雄激素通过诱导细胞焦亡影响卵巢颗粒细胞增殖  

Hyperandrogenism affects ovarian granulosa cell proliferation by inducing pyroptosis

作　　者：项雨 丁慧敏 陈田 徐天月 葛红山 XIANG Yu;DING Hui-min;CHEN Tian;XU Tian-yue;GE Hong-shan(Nanjing University of Chinese Medicine,Nanjing 210023;Taizhou People’s Hospital,Taizhou 225300;Zhangjiagang Fist People’s Hospital,Zhangjiagang 215600)

机构地区：[1]南京中医药大学,南京210023 [2]泰州市人民医院,泰州225300 [3]张家港第一人民医院,张家港215600

出　　处：《生殖医学杂志》2023年第9期1399-1406,共8页Journal of Reproductive Medicine

摘　　要：目的探讨多囊卵巢综合征(PCOS)中高雄激素对卵巢颗粒细胞焦亡、增殖的影响。方法3周龄的ICR小鼠随机分为对照组(5只)和PCOS组(5只),PCOS组小鼠连续注射脱氢表雄酮(DHEA,6 mg/100 g体重)21 d,对照组小鼠则注射等体积不含DHEA的芝麻油。通过HE染色观察两组小鼠卵巢形态的变化;运用实时定量PCR和蛋白免疫印迹(WB)观察炎症及焦亡相关基因和蛋白在小鼠卵巢中的表达情况。通过睾酮(10μmol/L)诱导KGN细胞建立高雄诱导的PCOS体外细胞模型,对照组为不添加睾酮的培养基处理的KGN细胞,PCR法检测对照组和睾酮组细胞中炎症(NLRP3、NF-κB、IL-1β)及焦亡(ASC、Caspase-1)相关基因的表达情况,运用WB和免疫荧光技术观察焦亡相关蛋白(Caspase-1、N-GSDMD/GSDMD)的表达情况,CCK8实验和LDH释放实验观察睾酮对KGN细胞生长的影响。结果HE染色显示PCOS组小鼠卵巢未成熟小卵泡及闭锁卵泡明显增多,提示PCOS模型构建成功。实时定量PCR结果显示,PCOS小鼠卵巢组织中NLRP3、NF-κB、IL-1β、ASC和Caspase-1的mRNA水平显著升高(P<0.05);WB的结果也显示炎症相关蛋白(NLRP3、IL-1β、IL-18)和焦亡相关蛋白(Caspase-1、N-GSDMD/GSDMD)的水平显著高于对照组(P<0.05)。在睾酮诱导的细胞模型中,睾酮组细胞观察到明显的焦亡征象,且炎症及焦亡相关基因表达均显著升高(P<0.05);睾酮处理后焦亡相关蛋白(Caspase-1、N-GSDMD/GSDMD)水平也显著高于对照组(P<0.05)。另外,随着睾酮浓度的增加,KGN细胞存活率逐渐下降;LDH释放实验结果显示,睾酮处理后KGN细胞的活性降低,且在10μmol/L时LDH的释放量最大。结论高雄诱导的PCOS模型中存在异常的炎症激活和异常的焦亡,这可能是高雄激素影响卵巢颗粒细胞增殖生长的基础,为探讨高雄激素促进PCOS维持和发展的机制提供了新思路。Objective:To investigate the effect of hyperandrogenism on pyroptosis and proliferation of ovarian granulosa in polycystic ovary syndrome(PCOS).Methods:The 3-week-old ICR mice were randomly divided into control(n=5)and PCOS groups(n=5).The mice in the PCOS group were continuously injected with DHEA(6 mg/100 g)for 21 days,while the mice in the control group were injected with the equal volume of sesame oil without DHEA.The morphological changes of the ovaries in both groups were observed by hematoxylin-eosin staining(HE).The expression of genes and proteins related with inflammation(NLRP3,NF-KB,IL-1β)and pyroptosis(ASC,Caspase-1)in the two groups were detected by polymerase chain reaction(PCR).KGN cells were induced by testosterone(10μmol/L)to establish in vitro cell model of PCOS,while the KGN cells in the control group were treated with culture medium without testosterone.The expression of the genes related with inflammation(NLRP3,NF-κB,IL-1β)and pyroptosis(ASC,Caspase-1)in cells were detected by PCR,and the expression of proteins related with pyroptosis(Caspase-1,N-GSDMD/GSDMD)were detected by Western blot.Finally,the effects of testosterone on growth of KGN cells were observed by the CCK8 assay and LDH release assay.Results:HE staining showed that immature small follicles and atretic follicles significantly increased in the ovary of mice in the PCOS group,suggesting that the PCOS model was successfully established.Real time quantitative PCR results showed that the mRNA levels of NLRP3,NF-κB,IL-1β,ASC and Caspase-1 significantly increased in in ovarian tissue of PCOS mice(P<0.05).The Western blot results also showed the proteins levels related with inflammation related proteins(NLRP3,IL-1β,IL-18)and pyroptosis(Caspase-1,N-GSDMD/GSDMD)were significantly higher than those of the control group(P<0.05).In the testosterone-induced cell model,obvious pyroptosis signs were observed in the testosterone group,and the expressions of inflammation and pyroptosis-related genes were significantly increased(P<0.05).The

关 键 词：多囊卵巢综合征 高雄激素 慢性炎症 焦亡 

分 类 号：R363[医药卫生—病理学]