基于NLRP3通路探讨线粒体自噬在PCOS卵巢组织炎症反应中的作用  

Study on the role of mitochondrial autophagy in ovarian inflammation in PCOS based on NLRP3 pathway

作　　者：李杨[1] 聂岚[1] 罗婷[1] 刘虹璐 罗姣[1] Li Yang;Nie Lan;Luo Ting;Liu Honglu;Luo Jiao(Dept of Infertility and Endocrinology,Hunan Maternal and Child Health Care Hospital,Changsha 410000)

机构地区：[1]湖南省妇幼保健院不孕不育与内分泌专科,长沙410000

出　　处：《安徽医科大学学报》2024年第9期1573-1582,共10页Acta Universitatis Medicinalis Anhui

基　　金：湖南省自然科学基金(编号:S2023JJBMLH1343)。

摘　　要：目的基于NOD样受体热蛋白结构域相关蛋白3(NLRP3)通路探讨线粒体自噬在多囊卵巢综合征(PCOS)卵巢组织炎症反应中的作用。方法人卵巢颗粒细胞(GCs)系SVOG用25 nmol/L双氢睾酮(DHT)处理24 h以建立PCOS细胞模型。SVOG细胞用携带NLRP3(Ad-NLRP3)及阴性载体(Ad-EV)或NLRP3 shRNA(sh-NLRP3)及阴性对照(sh-NC)的腺病毒转染,以过表达或敲低NLRP3表达。使用Mito-Tracker染色和GFP-LC3染色评估细胞中线粒体自噬情况。分别通过TUNEL染色、JC-1染色、Mito-SOX染色分析细胞凋亡、线粒体膜电位、线粒体衍生超氧化物产生情况。32只雌性BALB/c小鼠随机分为对照(Con)组、脱氢表雄酮(DHEA)组、DHEA+sh-NC组、DHEA+sh-NLRP3组,每组8只。除Con组外,其他组用DHEA处理小鼠以建立PCOS模型。DHEA+sh-NLRP3组、DHEA+sh-NC组通过尾静脉注射浓度为1×109 TU/ml的慢病毒包装的sh-NLRP3或sh-NC。透射电子显微镜观察各组小鼠卵巢组织中线粒体的超微结构。结果与DHT+sh-NC组相比,DHT+sh-NLRP3组SVOG细胞的NLRP3水平降低(P<0.05)。DHT+sh-NLRP3组SVOG细胞中GFP-LC3和线粒体的共定位较DHT+sh-NC组增加(P<0.05)。与DHT+sh-NC组相比,DHT+sh-NLRP3组SVOG细胞的TUNEL阳性细胞的数目和Mito-SOX荧光密度降低、多聚体JC-1/单体JC-1比值增加(P<0.05)。与Con+Ad-EV组相比,Con+Ad-NLRP3组SVOG细胞的NLRP3水平、TUNEL阳性细胞数目、Mito-SOX荧光密度增加(P<0.05),GFP-LC3和线粒体的共定位以及多聚体JC-1/单体JC-1比值降低(P<0.05)。与Con组相比,DHEA组小鼠卵巢组织中TUNEL阳性细胞、相对活性氧(ROS)强度和受损线粒体百分比均显著增加(P<0.05)。与DHEA+sh-NC组相比,DHEA+sh-NLRP3组卵巢组织中TUNEL阳性细胞、相对ROS强度和受损线粒体百分比均降低(P<0.05)。结论NLRP3激活诱导的线粒体自噬活性导致线粒体功能障碍并促进GCs中线粒体相关凋亡。NLRP3敲低有利于线粒体稳态,并改善GCs对氧化应激损伤的抵抗,从而促进PCOS的Objective To explore the role of mitochondrial autophagy in ovarian inflammation associated with polycystic ovary syndrome(PCOS)based on the NOD-like receptor thermoprotein domain-related protein 3(NLRP3)pathway.Methods Human ovarian granulosa cell line SVOG was treated with 25 nmol/L dihydrotestosterone(DHT)for 24 h to establish PCOS cell model.SVOG cells were transfected with adenovirus carrying NLRP3(Ad-NLRP3)and negative vector(Ad-EV)or NLRP3 shRNA(sh-NLRP3)and negative control(sh-NC)to overexpress or knockdown NLRP3.Mito-Tracker staining and GFP-LC3 staining were used to evaluate mitochondrial autophagy in cells.TUNEL staining,JC-1 staining and Mito-SOX staining were used to analyze the apoptosis,mitochondrial membrane potential and mitochondrial-derived superoxide production.32 female BALB/c mice were randomly divided into three groups:control(Con)group,DHEA group,DHEA+sh-NC group and DHEA+sh-NLRP3 group,with 8 mice in each group.Except the control group,all other groups treated mice with dehydroepiandrosterone(DHEA)to establish PCOS mouse model.DHEA+sh-NLRP3 group and DHEA+sh-NC group were administrated with sh-NLRP3 or sh-NC encapsulated in lentivirus at a concentration of 1×109 TU/ml via tail vein injection.The ultrastructure of mitochondria in ovarian tissue of mice in each group was observed by transmission electron microscope.Results Compared with DHT+sh-NC group,the level of NLRP3 of SVOG cells in DHT+sh-NLRP3 group decreased(P<0.05).The co-location of GFP-LC3 and mitochondria in SVOG cells in DHT+sh-NLRP3 group was higher than that in DHT+sh-NC group(P<0.05).Compared with DHT+sh-NC group,the number of TUNEL positive cells and Mito-SOX fluorescence density of SVOG cells in DHT+sh-NLRP3 group decreased,and the ratio of polymer JC-1 to monomer JC-1 increased(P<0.05).Compared with Con+Ad-EV group,the level of NLRP3,the number of TUNEL-positive cells and the fluorescence density of mito-SVOG in Con+Ad-NLRP3 group increased(P<0.05),and the co-location level of GFP-LC3 and mitochondria decreased;the ratio

关 键 词：NOD样受体热蛋白结构域相关蛋白3 线粒体自噬 多囊卵巢综合征 氧化应激 线粒体稳态 颗粒细胞 小鼠 

分 类 号：R711.75[医药卫生—妇产科学]