Metformin Improves Premature Ovarian Failure By Alleviating M1 Macrophage-Induced Granulosa Cell Injury

Objective:With the significant increase in survival rates of cancer patients,chemotherapy-induced premature ovarian failure and infertility have become one of the major concerns of young women surviving cancer.Although oocyte and embryo deep cryopreservation can help preserve pregnancy in patients with chemotherapy-induced POF,research to protect female cancer patients from POF caused by chemotherapy drugs and to maximize restoration of ovarian function is of great clinical value and relevance to maintain femininity and enhance their quality of life.Metformin（Met）has been shown to be effective in treating female reproductive disorders such as polycystic ovary syndrome and uterine fibroids in recent years,but whether it can alleviate the toxic damage to the ovaries caused by chemotherapeutic drugs and reshape ovarian function is unclear.The aim of this study was to investigate the specific pathways of ovarian function damage caused by chemotherapeutic drugs through a mouse model with POF,as well as to clarify whether metformin can mitigate the toxic damage of chemotherapeutic drugs to the ovaries during chemotherapy and to elucidate the underlying mechanisms,so as to provide new strategies for the clinical treatment of follicular dysfunction,premature functional failure and infertility.Methods:We used cyclophosphamide and leucovorin to construct a mouse model of POF,treated primary granulosa cells with M1-type macrophage supernatant to simulate changes in the microenvironment of prematurely failing ovaries,analyzed the effects of chemotherapeutic drugs on the ovaries and the protective effects of metformin at the tissue level and cellular level,and explored the potential molecular mechanisms by various techniques such as qPCR,HE,WB,ELISA,IF,etc.1.POF mouse model was constructed by intraperitoneal injection of 120mg/m L Cyclophosphamide（CTX）and 30mg/m L Busulfan（Bu）into 6-week-old female Kunming（KM）mice,with 6-week-old healthy female KM mice injected intraperitoneally with saline as the control group.After 30 days,their ovarian tissues were dissected and separated,and their ovaries were stained through HE.The ovarian tissue structure and follicle count were observed to verify the modeling effect and the damage of chemotherapeutic drugs on the ovaries.2.The expression levels of M1 macrophage marker molecules CD86 and INOS and M2 macrophage markers CD206 and ARG-1,inflammatory cytokines IL-6 and TNF-a,cell senescence marker molecules p53 and p21 and m RNA levels of antioxidant enzymes CAT,GPx and SOD2 were detected by qPCR and WB techniques.3.7 days after the start of modeling,Met was administered by gavage to POF mice at a dose of 260 mg/kg/d for 3 weeks,during which the body weight and estrous cycle of mice were monitored dynamically.At the end of the treatment cycle,ovaries of mice were removed to compare the ovary size between different groups Using HE stain and follicle count to identify the treatment effect.The expression of AMH,a marker of ovarian function,was detected by qPCR technique,and the levels of serum hormones FSH and E2 secretion in mice were detected by ELISA technique.4.After Met gavage treatment,differences in expression of the macrophage marker CD86,CD206,ARG-1,INOS,cytokines IL-6,IL-10,TNF-a,and antioxidant enzymes CAT,GPx,and SOD2 were detected by qPCR and WB techniques.The ability of tissue clearance to scavenge OH-was measured using colorimetric assay;the tissue SOD activity was measured using xanthine oxidase assay.5.RAW264.7 was treated with LPS（1mg/m L）for 24h,while the primary granulosa cells were extracted from ovary,and the cell types were identified by morphological observation and molecular marker expression detection by immunofluorescence and qPCR.6.M1 macrophage supernatant（M1-CM）was collected,and the experimental group used M1-CM to stimulate granulosa cells from mice for 24h to simulate the over-activated state of M1 macrophages in POF ovarian tissues.The treatment group was pretreated with 5m M metformin for 24h in advance,and the expression of inflammatory cytokines TNF-a,IL-6 and senescence marker molecules p53 and p21were detected by WB.Cellular reactive oxygen species level was detected by fluorescent probe method,β-galactosidase staining was used to identify changes in the number of senescent cells.The senescence-associated secretory expression level of senescence-associated secretory phenotype（SASP）by qPCR technique.7.Download gene expression microarray data GSE128240 and analyze differentially expressed genes in control ovaries and chemotherapeutic drug-treated ovaries.Using bioinformatics techniques to enrich for relevant pathways through differential genes.The expression differences of AMPK-related pathway molecules（AMPK and phosphorylated AMPK,SIRT1,PPAR-g）between different treatment groups were detected by WB technique.Results:1.The ovaries of POF mice were reduced in size,the number of primordial follicles and growing follicles at all levels was reduced to different degrees,the number of atretic follicles was significantly increased as well.The ovarian function was significantly impaired,so the modeling was successful.2.The expression of M1 macrophage molecular markers CD86 and INOS was increased in the ovarian tissues of POF and CON groups of mice,and the expression of M2 macrophage markers CD206 and ARG-1 were decreased.The expression of the inflammatory cytokines IL-6 and TNF-awas increased,the expression of senescence marker molecules p53 and p21 were increased,and the expression of antioxidant enzymes CAT,GPx and SOD2 was decreased.3.After Met gavage treatment,the body weight of POF mice increased to some extend,ovarian volume and function were effectively restored,the rate of abnormal estrous cycles,and the number of growing follicles at all levels normalized.The primordial follicle rate decreased from 62.5%to 20.5%,the serum FSH concentration decreased and E₂concentration increased,and the ovarian reserve index AMH expression upegulated.4.After Met gavage treatment,the expression of macrophage marker molecules CD86 and INOS decreased,while CD206 and ARG-1 increased.The expression of pro-inflammatory cytokines IL-6 and TNF-aincreased,while anti-inflammatory cytokine IL-10 expression decreased.At the same time,antioxidant enzymes CAT,GPx,SOD2 m RNA expression was upregulated,tissue OH-scavenging ability increased,total tissue SOD activity and Cu-SOD activity were increased.5.After induced by 1mg/m L LPS for 24h,The cells protrude the pseudopod,and the expression of M1-type macrophage marker molecules m RNA increased,M1polarization was successfully induced.The cytoplasmic staining by FSHR was obvious,and these cells were identified as ovarian granulosa cells indeed.6.The expression of pro-inflammatory cytokines IL-6 and TNF-aincreased significantly in the M1 macrophage supernatant co-culture group,and the expression of senescence marker molecules p53,p21 expression also increased,along with increasing green fluorescence in cells representing reactive oxygen species and blue staining representing senescent cells.5m M metformin pretreatment for 24h significantly downregulated the expression of cellular senescence markers p53,p21and the green fluorescence in cells and the number ofβ-galactosidase positive cells.The expression of SASPs including chemokine CXCL10,inflammatory cytokines IL-6,IL-8,TNF-a,growth factor TGF-βwere significantly reduced.7.The results of bioinformatics analysis showed that PPAR pathway was significantly enriched in ovary samples treated with chemotherapeutic drugs.WB results showed that p-AMPK/AMPK ratio was decreased,SIRT1 expression was downregulated,and PPAR-gexpression was increased in the M1-CM treated group,while the ratio of p-AMPK/AMPK and the expression of SIRT1 increased and the expression of PPAR-decreased in Met pretreatment group.Conclusion:Our research shows that the polarization of macrophages in the ovary of chemotherapy-induced premature ovarian failure is unbalanced,the number of pro-inflammatory macrophages increases,the secretion of inflammatory cytokines increases,and ROS accumulates,which leads to the senescence and death of granulosa cells.Metformin enhances the anti-inflammatory and antioxidant capacity of granulosa cells through AMPK/PPAR-g/SIRT1 pathway and relieves POF.