Protective Effects Of Hydroxychloroquine On Ovaries In Mice With Autoimmune Premature Ovarian Insufficiency And Its Mechanisms

Background and aimsPremature ovarian insufficiency(POI),a highly heterogeneous disease with multiple causes,is manifested by infertility,amenorrhea or oligomenorrhea.However,the pathogenesis of POI is very complicated,including genetic factors,immune factors,metabolic factors,iatrogenic factors,infection and so on.Hydroxychloroquine(HCQ)which has been widely used for the treatment of rheumatic diseases possesses various therapeutic properties,such as anti-infection,anti-cancer,anticoagulation and antiinflammatory activity,and it has been found that chloroquine can inhibit ovarian autophagy.However,its anti-inflammatory effects in POI remain unclear.In this study,zona pellucida 3 peptide(pZP3)was used to induce autoimmune POI mouse model,and HCQ was used for intragastric administration,and then 1)to analyze the effect of HCQ on ovarian phenotype and function of autoimmune POI mouse model;2)to study the effect of HCQ on Th17/Treg cells of autoimmune POI mouse model;3)to explore the protective mechanisms of HCQ on ovarian function of autoimmune POI mouse model.Methods1、Sixty female BALB/c mice were divided randomly into four groups:the control group,the model group(POI),the HCQ group and the treatment group(POI+HCQ).The autoimmune POI mice model was induced by injection of pZP3.HCQ was given by intragastric administration into mice.2、Stages of the estrous cycle in each mice were determined by vaginal cytology examination.3、Ovarian structure and follicle counting in each group were viewed via hematoxylin and eosin staining.4、The levels of serum hormones were measured by enzyme-linked immunosorbent assay(ELISA),including FSH、AMH and E2.5、Expressions of CD4 and CD45 in the ovarian follicular fluid of each group were observed by immunohistochemistry.6、The expressions of ZP2 and ZP3 in the ovarian sections of each group were observed by immunohistochemistry.7、The ratios of Th17/Treg in spleen cells of each group were analyzed by flow cytometry analysis.8、Expressions of Foxp3 and IL-17 in ovaries and Toll like receptor(TLR)pathway-related proteins in spleen cells were determined by western blot.Results1、The results showed that the proportion of regular estrous cycle in model groups was significantly lower than that in the control and treatment group.2、Histological examination showed that the connective tissue in the ovaries of the model group was proliferated in large quantities with increased the atretic follicles,and the ovarian interstitial fibrosis was massive.HCQ alleviates the decrease in the number of follicles caused by pZP3.3、We found that HCQ treatment alleviated the decrease of estrogen and the increase of gonadotropin levels due to follicular depletion.4、We demonstrated that applying HCQ following pZP3 attenuated the lymphocytes and white cells infiltration in ovarian follicular fluid.5、HCQ prevents inflammatory factors from interfering with the expressions of ZP2 and ZP3.6、HCQ prevents activated na(?)ve CD4+T cells from differentiating into Th17 cells by affecting the production of cytokines.5、Notably,the TLR pathway may be involved in the potential mechanisms by which HCQ reduces ovarian inflammation to exert protective effect against autoimmune oophoritis.ConclusionsOur results demonstrated that HCQ alleviated ovarian inflammation caused by pZP3,effectively restored ovarian function and folliculogenesis by changing the ratios of Th17/Treg cells through TLR signaling pathway in POI mice,suggesting that HCQ can be a promising therapeutic method for patients with autoimmune POI in the clinic.