麒麟丸对卵巢储备功能减退模型小鼠生育能力及卵巢组织HIF-1α/Bnip3/Beclin1信号通路的影响

Effects of Qilin Pills(麒麟丸) on Fertility and HIF-1α/Bnip3/Beclin1 Signaling Pathway in Ovarian Tissues in Diminished Ovarian Reserve Model Mice

目的探讨麒麟丸对卵巢储备功减退(DOR)小鼠生育能力的影响及可能作用机制。方法36只C57BL/6雌性小鼠随机分为空白组、模型组、麒麟丸组,每组12只。麒麟丸组以麒麟丸混悬液5.46g/(kg·d)灌胃,空白组及模型组予0.5%羧甲基纤维素钠溶液20ml/(kg·d)灌胃,共5周。灌胃1周后,模型组和麒麟丸组腹腔注射环磷酰胺溶液75mg/kg诱导DOR小鼠模型,空白组腹腔注射生理盐水10ml/kg,均每周1次,共4周。用药期间每日观察动情周期,用药结束后每组随机处死6只小鼠,Western Blot法检测卵巢组织缺氧诱导因子1α(HIF-1α)、B淋巴细胞瘤-2基因/腺病毒E1B相互作用蛋白3(Bnip3)、自噬关键分子酵母Atg6同系物(Beclin1)蛋白表达。其余雌鼠正常饲养7周后,与雄鼠合笼4周,观察受孕时间、受孕数、吸收胎数及健康仔鼠数。结果与空白组相比,模型组雌鼠动情周期紊乱,长期停滞于动情间期,受孕时间延长,吸收胎数量增加,卵巢组织HIF-1α、Bnip3、Beclin1蛋白表达显著升高(P<0.05或P<0.01);与模型组相比,麒麟丸组动情周期有所恢复,受孕时间缩短,卵巢组织HIF-1α、Bnip3、Beclin1蛋白表达均显著降低(P<0.05或P<0.01)。结论麒麟丸可调节DOR模型小鼠动情周期,保护生育能力,其作用机制可能与调控卵巢组织HIF-1α/Bnip3/Beclin1信号通路有关。

Objective To investigate the effect of Qilin Pills(麒麟丸)on fertility in diminished ovarian reserve(DOR)model mice as well as the related mechanism.Methods Thirty-six female C57 BL/6 mice were randomly divided into 3 groups:blank group,model group and Qilin Pills group,with 12 mice in each group.Qilin Pills group was gavaged with 5.46 g/(kg·d)of Qilin Pills suspension,while the blank group and model group were gavaged with 20 ml/(kg·d)of 0.5% sodium carboxymethyl cellulose solution for 5 weeks.One week after the gavage,75 mg/kg of cyclophosphamide solution was intraperitoneally injected into the mice in model group and Qilin Pills group to induce the model of DOR,while 10 ml/kg of normal saline was intraperitoneally injected into the blank group,once per week for four weeks.The mice were tested daily for vaginal smears during treatment;after the medication,six mice in each group were randomly selected to be killed;Western blotting was used to detect hypoxia-inducible factor 1α(HIF-1α),B-cell lymphoma-2/adenovirus E1 B interacting protein 3(Bnip3)and the homolog of autophagy key molecule yeast Atg6(Beclin1)protein expression.After seven-week normal feeding,the remaining mice were caged with male mice for 4 weeks,and the time of conception,number of conceptions,number of absorbed fetus and number of healthy offspring were observed.Results Compared to the blank group,the model group's estrous cycle was disordered and stagnated at the estrous period;the conception time was prolonged,and the number of absorbed embryos increased;the protein expression of HIF-1α,Bnip3 and Beclin1 in the ovarian tissue was significantly increased(P<0.05 or P<0.01).Compared to those of the model group,the estrous cycle of Qilin Pills group was restored,and the conception time was shortened;the HIF-1α,Bnip3 and Beclin1 protein expression was significantly decreased(P<0.05 or P<0.01).Conclusion Qilin Pills can regulate the estrous cycle of DOR model mice and protect its fertility,which may be related to the regulation of HIF-1α/Bnip3/Beclin1 signaling pathway.