Hyperandrogenism-mediated ovarian immune microenvironment dysregulation drives granulosa cell apoptosis in PCOS mice and the protective effect of Berberine

WANG You-feng; MO Hui; LI Li

doi:10.13820/j.cnki.gdyx.20251924

Abstract：Objective To investigate the role of hyperandrogenism-mediated inflammatory responses in ovarian dysfunction in polycystic ovary syndrome(PCOS),and to explore the protective effects of Berberine(BBR)on ovarian immune microenvironment and reproductive function in PCOS mice,providing experimental evidence for clinical prevention and treatment.Methods A PCOS mouse model was established by subcutaneous injection of dehydroepiandrosterone[DHEA,6 mg/(100 g·d)]for 21 consecutive days.Body weight and estrous cycles were measured.Ovarian morpholo-gy was examined by H&E staining.Serum testosterone,anti-Müllerian hormone(AMH),and tumor necrosis factor-α(TNF-α)levels were detected using ELISA.In vitro,THP-1 cells were treated with 0,10,50,100 μmol/L testoster-one to induce M1 polarization;TNF-α expression was assessed by RT-qPCR and ELISA.KGN cells were treated with 10 μmol/L testosterone combined with varying concentrations of TNF-α;cell viability was measured by CCK-8 assay,and PI3K phosphorylation was analyzed by Western blot.In vivo,BBR[150 mg/(kg·d),oral]was administered for 14 days,and its effects on ovarian histology,estrous cycles,and serum markers were evaluated.Results Compared with controls,PCOS mice showed significant weight gain(P＜0.05),disrupted estrous cycles,cystic ovarian enlargement,and thinning of granulosa cell layers.Serum testosterone[(3.57±1.27)ng/mL vs.(1.31±0.23)ng/mL],AMH[(1 116.27±109.85)pg/mLrs.(770.54±111.27)pg/mL],and TNF-α[(35.18±6.75)pg/mL vs.(12.37±1.38)pg/mL]levels were significantly elevated(P＜0.05).In vitro,testosterone treatment significantly increased TNF-α mRNA(1.01±0.15 vs.1.24±0.2 vs.5.31±0.21 vs.6.18±0.5)and protein expression(1±0 vs.0.96±0.07 vs.1.21±0.12 vs.1.31±0.08)in M1 macrophages(P＜0.05).Combined testosterone and high TNF-α treatment markedly reduced KGN cell viability(P＜0.05)and inhibited PI3K phosphorylation(1±0 vs.0.54±0.12 vs.0.27±0.08,P＜0.05).BBR intervention improved ovarian histopathology,restored regular estrous cycles,and significantly re-duced serum testosterone[(12.26±1.29)ng/mL vs.(32.1±5.71)ng/mL vs.(17.07±1.47)ng/mL]and TNF-αlevels[(13.08±0.79)pg/mL vs.(28.51±6.3)pg/mL vs.(16.91±0.99)pg/mL](P＜0.05).Conclusion Hy-perandrogenism promotes M1 macrophage polarization and TNF-α release,exacerbating local ovarian inflammation,im-pairing granulosa cell function,and causing ovulatory dysfunction.BBR exhibits dual effects by suppressing inflammation and lowering androgen levels,suggesting its potential as an effective adjunct therapy for PCOS.