| Objective:To investigate the overactivation of Neutrophil extracellular traps(NETs)in Premature ovarian failure(POF),and provide experimental data for early diagnosis and new treatment targets of POF.Method:1.23 peripheral blood samples of POF patients and healthy women conforming to the exclusion criteria of this study were collected clinically.Neutrophils were extracted and the main component of NETs,Myeloperoxidase,was detected by immunofluorescence double staining.MPO)and citrullinated Histon 3(Cit-H3).2.40 balb/c mice meeting the experimental conditions were selected to construct the animal model of POF mice by intraperitoneal injection of zona pellucida 3(pZP3).The general situation of the mice,changes in body weight and estrous cycle before and after the experiment were recorded.Estradiol(E2)content,follicle-stimulating hormone(FSH)content,ovarian cell apoptosis,Cit-H3 and MPO production were detected.3.20 balb/c mice meeting the experimental conditions were selected and injected with pZP3 to establish the POF mouse model.The mice were randomly divided into POF+inhibitor group(10 mice)and POF+NS group(10 mice).The first group was then intraperitoneally injected with NETs inhibitor Deoxyribonuclease Ⅰ(DNase Ⅰ)at the rate of 5mg/kg,and the second group was intraperitoneally injected with the same amount of NS solution to detect the changes of various indexes of mice.Results:1.Compared with the control group,the average fluorescence intensity of peripheral neutrophils MPO and Cit-H3 in patients with POF was increased(P<0.0001).2.The body weight of POF group(17.53±0.55)g was lower than that of control group(19.01±0.26)g(P<0.001),the estrous cycle of POF group(8.50±1.05)d was longer than that of control group(4.3 5 ± 0.31)d(P<0.05),E2(50.90± 8.59)mIU/mL was higher than that of control group(90.44±14.45)mIU/mL decreased(P<0.05),FSH(22.45±0.96)pg/mL increased compared with control group(17.85±1.03)pg/mL.The ovarian morphology of POF group was consistent with the ovarian characteristics of premature ovarian failure,and the number of developmental follicles at all levels in POF group was significantly increased compared with control group(P<0.05).A large number of green labeled apoptotic cells were observed in the ovarian tissue of the POF group,the contents of MPO and Cit-H3 in the ovarian tissue and bone marrow neutrophils were increased,the average fluorescence intensity was enhanced(P<0.0001),and colocalization phenomenon occurred.3.The body weight of the POF+inhibitor group after modeling was(17.69±0.0.14)g Compared with POF+NS group(17.21 ±0.31)g increased(P<0.001),estrous cycle(6.20±0.53)d decreased(P<0.05),E2(75.18±3.42)mIU/mL ratio POF+NS group(56.96±3.66)mIU/mL increased,FSH(20.78±1.28)mIU/mL decreased than POF+NS group(22.55±1.05)mIU/mL.The ovary morphology of POF+NS group was consistent with the ovary characteristics of POF mice.The number of developmental follicles in POF+NS group was higher than that in POF+NS group,and the number of atretic follicles was significantly lower than that in control group(P<0.05).In the POF+inhibitor group,the number of green labeled apoptotic cells decreased,the contents of MPO and Cit-H3 in the ovarian tissue decreased,the average fluorescence intensity decreased(P<0.0001),and the co-localization phenomenon decreased.Conclusions:1.The co-localization of MPO and Cit H3 in peripheral blood neutrophils of patients with premature ovarian failure indicates the presence of NETs in patients with premature ovarian failure.2.pZP3 can successfully produce a mouse model of immune premature ovarian failure,and the experimental results confirm that there is overactivation of NETs in mice with premature ovarian failure.3.NETs inhibitor DNase I can relieve various indicators of premature ovarian failure mice and effectively reduce ovarian tissue damage. |
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