温经汤通过减轻雷公藤所致卵巢颗粒细胞凋亡改善卵巢储备功能下降的作用机制OA

Objective:To explore the mechanism of Wenjing(温经)Decoction in ameliorating Leigongteng(雷公藤)-induced diminished ovarian reserve(DOR)based on ovarian granulosa cell apoptosis.Methods:Administration of Leigongteng glycosides(GTW)was used to establish a rat model of DOR by observing the estrous cycle.Then,the rats were assigned into the model control,Femoston(sequential therapy:gavage of 0.2 mg/kg estradiol tablets on days 1-2,and gavage of 1.2 mg/kg estradiol and dydrogesterone tablets on days 3-5),and Wenjing Decoction(4.85,9.7,and 19.4 g/kg)groups.After 4 weeks of treatment,the estrous cycle was observed and the ovarian area was measured.HE staining was employed for ovarian structure observation and follicle counting.Ovarian granulosa cell apoptosis was detec-ted by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay.The protein levels of B-cell lymphoma/leukemia-2(BCL-2)and BCL-2-associated X protein(BAX)in the ovarian tissue were measured by Western blot.The human granulosa-like tumor cell line(KGN)was cultured in vitro and stimulated with GTW.The proliferation of KGN cells was detected by the CCK8 assay.The cells were allocated into normal control,model control,5%blank serum,and 5%Wenjing Decoction-containing serum groups.The apoptosis was detected by flow cytometry,and the protein levels of BAX and BCL-2 were measured by Western blot.Results:In the animal experiment,compared with the blank control group,the model control group presented increased estrous cycle disorder rate,reduced ovarian area,and increased ovari-an granulosa cell apoptosis rate(P<0.01),as well as disrupted follicular structure with granulosa cell disarrangement,reduced number of primordial follicles,primary follicles,secondary follicles,and antral follicles,and increased number of atretic follicles(P<0.01 or P<0.05),up-regulated protein level of BAX,and down-regulated protein level of BCL-2(P<0.01).Compared with the model control group,drug treatment groups showed improved follicular structure and granulosa cell arrangement,and the Femoston and Wenjing Decoction(9.7 g/kg and 19.4 g/kg)groups demonstrated reduced estrous cycle disorder rate,increased ovarian area,and decreased ovarian granulosa cell apoptosis rate(P<0.05 or P<0.01),and reduced number of atretic follicles(P<0.05),and increased number of primordial and antral follicles(P<0.01 or P<0.05).Wenjing Decoction(9.7 g/kg)and Femoston groups showed increased number of primary folli-cles(P<0.01 or P<0.05).The BAX protein level was down-regulated in all treatment groups(P<0.01 or P<0.05),and the BCL-2 protein level was up-regulated in the Wenjing Decoction(19.4 g/kg)group(P<0.05).In the cell ex-periment,compared with the blank control group,the model control group showed reduced cell viability,increased early and overall apoptosis rates(P<0.01),down-regulated BAX expression,and up-regulated BCL-2 expression(P<0.05).Compared with the model control group,no significant changes in the aforementioned parameters were observed in the 5%blank serum group,while the 5%Wenjing Decoction-containing serum group presented increased cell viability,reduced early and overall apoptosis rates(P<0.01 or P<0.05),down-regulated BAX expression,and up-regulated BCL-2 expres-sion(P<0.05).Conclusion:Wenjing Decoction inhibits granulosa cell apoptosis,maintains follicular structural integri-ty,and reduces the number of atretic follicles,thereby restoring ovarian reserve function in DOR rats.