The mechanism of Bushen Jianpi Formula regulating diminished ovarian reserve in rats based on related apoptosis pathways
Objective To observe the effects of Bushen Jianpi Formula(BSJPF)on ovarian function in rats with cycloph-osphamide-induced decreased ovarian reserve function,and to investigate the mechanism of BSJPF regulating diminished ovarian reserve(DOR)in rats based on tumor necrosis factor-induced apoptosis pathway.Methods A total of 60 healthy female SD rats with normal estrous cycle were randomly divided into normal group(10 rats)and modeling group(50 rats).The modeling group was treated with a single intraperitoneal injection of cyclophosphamide at 75 mg/kg to establish models.The successful modeling rats were then randomly divided into model group,western medicine control group(estradiol valerate 0.103 mg/kg·d+progesterone capsule 2.575 mg/kg·d),and low-(3.21 g/kg·d),medium-(6.43 g/kg·d),and high-dose(12.85 g/kg·d)BSJPF groups,with 10 rats in each group.Normal group and model group were given equal volume of distilled water by gavage once a day,for 14 consecutive days.Estrous cycle,body mass,ovarian organ index,and uterine organ index were checked.The content of tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ)in serum of rats were determined by ELISA.The expressions of B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)in ovarian granulosa cells were measured by immunofluorescence assay.HE staining was used to examine the pathological status of ovarian tissues.Western blot analysis was used to check the ovarian posphorylated-proteinkinase B(p-AKT),p-mammalian target of rapamycin(p-mTOR),and Beclin-1 expressions.Results Compared with the normal group,the estrous cycle of the model group was disturbed,the ovarian structure was disturbed,the number of follicles at all levels was reduced to different degrees,no mature follicles were seen,and obvious inflammatory cell infiltration was seen in the ovarian stroma.There were no significant changes in body mass,ovarian organ index,or uterine organ index.Serum levels of TNF-α and IFN-γ were higher(P<0.05,P<0.01).The protein expressions of Bcl-2,Bax,and Beclin-1 in ovarian granulosa cells were higher(P<0.05,P<0.01),while the protein expressions of p-AKT and p-mTOR were lower(P<0.01).Compared with model group,serum levels of TNF-α and IFN-γ were reduced in western medicine control group,and low-and medium-dose BSJPF groups(P<0.05,P<0.01).The expression level of Bax protein in medium-dose BSJPF group decreased(P<0.05);the expression level of Bcl-2 protein in high-dose BSJPF group increased(P<0.05);Beclin-1 protein expressions in western medicine control group and low-dose BSJPF group decreased(P<0.05,P<0.01),and P-AKT and p-mTOR protein expression levels increased(P<0.01).Compared with western medicine control group,the level of TNF-α in high-dose BSJPF group increased(P<0.01);the levels of IFN-γ in the low-and high-dose BSJPF groups decreased(P<0.05);the expression level of Bax protein in low-dose BSJPF group increased(P<0.05),and the expression levels of Bcl-2 protein in medium-dose BSJPF group increased(P<0.05).In medium-and high-dose BSJPF groups,Beclin-1 protein expression levels increased(P<0.05,P<0.01),and p-AKT and p-mTOR protein expression levels decreased(P<0.01).Conclusion BSJPF can decrease the pathological changes of ovary and strengthen ovarian reserve function.The mechanism may involve inhibiting the expressions of TNF-α and IFN-γ of tumor necrosis factor,increasing the expression of Bcl-2 and lowering the expression of Bax,and activating mTOR pathway to inhibit the autophagy and apoptosis of rat ovarian granulosa cells.The effects of cyclophosphamide modeling on DOR in rats were improved.
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