早发性卵巢功能不全大鼠模型造模方法比较及机制探究OA

Objective This study established rat models of premature ovarian insufficiency(POI)using triptolide(TG)and cyclophosphamide(CTX)and conducted a preliminary investigation into the mechanisms involved to provide a theoretical basis for POI research and clinical treatment.Methods For in vivo experiment,Sprague-Dawley rats with normal estrous cycles were selected and randomly divided into Control,TG,and CTX groups.The estrous cycle was monitored,and tissue samples were collected at day 15 of modeling(day 0 post-modeling)and day 28 post-modeling.The percentages of follicles at various stages were calculated,hormone levels were assessed,and the expression of mitophagy-related proteins,including p-mTOR(phosphorylated mammalian target of rapamycin)/mTOR(mammalian target of rapamycin),Beclin1(coiled-coil myosin-like BCL2-interacting protein),PINK1(PTEN-induced putative kinase 1),Parkin(E3 ubiquitin-protein ligase parkin),p62(sequestosome-1,SQSTM1),and LC3 Ⅱ/Ⅰ(microtubule-associated protein 1 light chain 3-Ⅱ/Ⅰ),was detected.KGN human ovarian granulosa cells were used for in vitro experiments,and the impact of TG and CTX on cell proliferation was determined using the CCK8 assay.Mitochondrial ultrastructure and autophagosomes were observed using transmission electron microscopy.Levels of mitochondrial membrane potential,reactive oxygen species(ROS),and mitochondrial superoxide in KGN cells were measured using high-content analysis.Results Compared with the Control group,rats in the TG and CTX groups exhibited decreased body weight,lethargy,sparse and dull fur,reduced activity,and decreased food and water intake,along with estrous cycle disruption.On day 28 post-modeling,the ovarian index was significantly decreased in both treatment groups(P＜0.05).On day 0 post-modeling,the TG group showed an increased percentage of atretic follicles(P＜0.05),while the CTX group exhibited a decreased percentage of growing follicles(P＜0.01)and an increased percentage of atretic follicles(P＜0.01).On day 28 post-modeling,only the CTX group showed a decreased percentage of growing follicles(P＜0.01)and an increased percentage of atretic follicles(P＜0.01).The TG group showed decreased AMH levels(P＜0.05)on day 0 post-modeling,while the CTX group showed decreased E2 and AMH levels(P＜0.05,P＜0.01)and increased FSH levels(P＜0.01).On day 28 post-modeling,the TG group showed decreased E2 levels(P＜0.05),while the CTX group showed decreased E2 levels(P＜0.01)and increased FSH levels(P＜0.05).In the mechanistic study,the TG group showed increased protein expression of Beclin1 and LC3 Ⅱ/Ⅰ(P＜0.05,P＜0.01)and decreased p-mTOR/mTOR ratio(P＜0.05)on day 0 post-modeling,while the CTX group showed increased expression of Beclin1,PINK1,and LC3 Ⅱ/Ⅰ(P＜0.05,P＜0.01)and a decreased p-mTOR/mTOR ratio(P＜0.01).The TG group showed increased expression of Beclin1,PINK1,and Parkin(P＜0.05,P＜0.01)and decreased p62 expression(P＜0.05)on day 28 post-modeling while the CTX group showed increased expression of Beclin1,PINK1,Parkin,and LC3 Ⅱ/Ⅰ(P＜0.05,P＜0.01)and decreased expression of p-mTOR/mTOR and p62(P＜0.05).At the cellular level,both TG and CTX treatments induced mitochondrial damage accompanied by numerous autophagosomes and autolysosomes,a significant decrease in mitochondrial membrane potential(P＜0.01),and significant increases in ROS and mitochondrial superoxide levels(P＜0.01)compared with the Control group.Conclusions Both TG and CTX successfully established rat models consistent with clinical POI manifestations,although the CTX-induced model was superior and more stable.Both models exhibited excessive mitophagy,suggesting that this may be one of the pathogenic mechanisms underlying POI.