Mechanism Of Metformin Attenuates PM2.5-Induced Premature Ovarian Failure By Regulating Nrf2 Signaling Pathway

Objective: Premature ovarian failure(POF)is a clinical syndrome affecting the reproductive health of women of childbearing age.In recent years,epidemiological studies have found that the incidence of POF is higher and younger,but its pathogenesis has not been fully understood.In this study,PM2.5 was used to establish mouse and cell premature ovarian failure models to explore the pathogenesis of premature ovarian failure,and metformin was used to correct PM2.5-induced mouse premature ovarian failure and cell premature ovarian failure models to explore its protective mechanism and related pathways.Methods: 1.Animal experiment:(1)Construction of premature aging mouse model : C57BL/6J female mice aged 6-8 weeks were randomly divided into 4 groups: A.PM2.5-induced senescence group: PM2.5 8mg/kg/2d,nasal drip for 28 days;b.Metformin(Met)monotherapy group: Met 300mg/kg/d by gavage for 28 days;c.Metformin(Met)pretreatment group: PM2.5 8mg/kg/2d by nasal drip,Met 300mg/kg/d by gavage for 28 days;d.Control group: the same amount of normal saline was given to the first three groups by intranasal and gavage for 28 days.After gavage on day 28,all mice were sacrificed for subsequent experiments.(2)Monitoring of the estrous cycle of mice: vaginal smears from day 1 to day 9 were used to screen mice with obvious regular estrous cycle changes for the following experiments,and vaginal smears from day 16 to day 28 were used to observe and record the estrous cycle of mice.Vaginal smears were vaginal exfoliated cells,and the estrous cycle of mice in different groups was monitored after HE staining.(3)The body weight and ovary weight of mice in different groups were recorded after sacrifice.(4)After eye blood collection,the serum levels of anti-Mullerian test tube hormone(AMH),estrogen(E2),follicle stimulating hormone(FSH)and progesterone(P4)were measured by enzyme-linked immunosorbent assay(Elisa).(5)The ovaries of mice were subjected to Nrf2 immunohistochemical staining.(6)Western blotting was used to study the regulatory effects of Nrf2,NQO1,HO-1,PARP,Bcl2 and Bax protein expression in different groups of mice.2.Cell experiment:(1)Construction of cell model: KGN cells were pretreated with or without Met(1m M)for 12 h,and then treated with PM2.5(0,100,200,400μg/ml)for 48 h.The cells were collected for following experiments.(2)The changes in the number and morphology of KGN cells stimulated by different concentrations of PM2.5 were observed by inverted microscope.(3)CCK-8 assay was applied to measure the toxic effects of different concentrations of PM2.5 on KGN cells.(4)ROS detection reagent was utilized to detect the effect of metformin on PM2.5-induced ROS production level in KGN cells after metformin pretreatment.Results: 1.Animal experiment:(1)PM2.5 can cause slow weight gain,disordered estrous cycle and prolonged luteal phase in mice.(2)PM2.5 could cause endocrine dysfunction in mice.The levels of AMH and E2 in serum of PM2.5 group were significantly decreased,the level of FSH was increased,and there was no significant difference in P4 level compared with the control group(P > 0.05).(3)Western Blot results showed that PM2.5 reduced the levels of antioxidant proteins Nrf2,NQO1 and HO-1 in mouse ovaries,increased the levels of apoptosisrelated proteins PARP and Bax,and decreased the level of Bcl2,indicating that PM2.5 caused excessive oxidation and apoptosis in mouse ovaries.(4)Immunohistochemistry results showed that PM2.5 significantly reduced the content of Nrf2 in mice ovaries.(5)Metformin pretreatment could obviously correct the disordered estrous cycle of mice,the weight of mice and the weight of ovaries.Serum hormone levels,antioxidant and apoptosis-related proteins were also corrected.Nrf2 was restored in the ovary.2.Cell experiment:(1)PM2.5 aggravated the cytotoxicity of KGN cells in a concentration-dependent manner.(2)Under the stimulation of different concentrations of PM2.5,the number of KGN cells gradually decreased,and the cells showed the performance of senescence,such as cell shrinkage,smaller volume,the overall shape became narrow,and the nucleus became relatively large.(3)After PM2.5 stimulation,KGN cells produced high levels of ROS,and pretreatment with metformin was able to reduce ROS production in cells.Conclusion: 1.PM2.5 can cause ovarian dysfunction in mice,including estrous cycle disorder(prolonged luteal phase),impaired luteal function,abnormal hormone secretion(decreased levels of AMH and E2,increased levels of FSH),and lead to premature ovarian failure.2.PM2.5 causes excessive oxidative stress in the ovary through Nrf2 signaling pathway,leading to premature ovarian failure in mice.3.Metformin attenuates premature ovarian failure induced by excessive oxidative stress by regulating the Nrf2 signaling pathway.4.Metformin has a certain clinical potential for the prevention and treatment of premature ovarian failure in the future.