加减益经汤对大鼠卵巢储备功能下降的影响及机制研究

Study on the effect and mechanism of modified Yijing decoction on the decrease of ovarian reserve function in rats

目的研究加减益经汤对大鼠卵巢储备功能下降(DOR)的影响及可能机制。方法取140只SPF级雌性SD大鼠,使用随机数字表法分为对照组、模型组、阳性对照组(戊酸雌二醇片0.09 mg/kg)、抑制剂组[加减益经汤23.6 g/kg+低氧诱导因子1α(HIF-1α)转录抑制剂YC-12 mg/kg]和加减益经汤低、中、高剂量组(5.9、11.8、23.6 g/kg)。除对照组外,其余各组大鼠股静脉注射环磷酰胺复制DOR模型;造模成功2 h后,各组大鼠灌胃生理盐水或相应药物,每日1次,连续8周。记录各组大鼠动情周期,计算卵巢指数,观察卵巢组织病理学变化和自噬情况;检测血清中促卵泡素(FSH)、促黄体生成激素(LH)、抗苗勒氏管激素(AMH)、雌激素(E2)水平;检测卵巢组织中HIF-1α、B淋巴细胞瘤2基因/腺病毒E1B相互作用蛋白3重组蛋白(Bnip3)、自噬关键分子酵母Atg6同系物(Beclin1)蛋白及mRNA水平。结果相较于对照组大鼠,模型组和各给药组大鼠卵巢指数均显著降低(P<0.05);模型组大鼠动情周期紊乱,卵巢组织呈病理形态,卵巢细胞出现自噬溶酶体,AMH及E2水平均显著降低(P<0.05),FSH、LH水平和HIF-1α、Bnip3、Beclin1蛋白及mRNA相对表达量均显著升高(P<0.05)。相较于模型组大鼠,阳性对照组和加减益经汤各剂量组大鼠动情周期恢复正常,卵巢组织病理形态和卵巢细胞自噬均有所改善,卵巢指数、AMH及E2水平均显著升高(P<0.05),FSH、LH水平和HIF-1α、Bnip3、Beclin1蛋白及mRNA相对表达量(除加减益经汤低剂量组HIF-1α蛋白、Bnip3蛋白及mRNA外)均显著降低(P<0.05);抑制剂组大鼠HIF-1α、Bnip3、Beclin1蛋白和HIF-1α、Beclin1 mRNA相对表达量均显著降低(P<0.05)。结论加减益经汤能够改善DOR模型大鼠卵巢功能、调节性激素分泌、保护卵巢组织,其作用机制可能与抑制HIF-1α/Bnip3/Beclin1通路相关。

OBJECTIVE To study the effect and mechanism of modified Yijing decoction on the decrease of ovarian reserve function(DOR)in rats.METHODS A total of 140 SPF female SD rats were randomly divided into control group,model group,positive control group(Estradiol valerate tablets 0.09 mg/kg),inhibitor group[modified Yijing decoction 23.6 g/kg+transcriptional inhibitor for hypoxia inducible factor-1α(HIF-1α)YC-12 mg/kg]and modified Yijing decoction low-dose,medium-dose and high-dose groups(5.9,11.8,23.6 g/kg).Except for control group,rats in other groups were given cyclophosphamide via femoral vein to induce DOR model;after successful modeling for 2 h,rats in each group were given normal saline or relevant medicine intragastrically,once a day,for 8 consecutive weeks.The estrous cycle of rats in each group was recorded,and the ovarian index was calculated to observe the histopathological changes and autophagy in the ovary;serum levels of follicle stimulating hormone(FSH),luteinizing hormone(LH),anti-Müllerian hormone(AMH)and estrogen(E2)were measured;protein and mRNA levels of HIF-1α,B lymphoblastoma 2 gene/adenovirus E1B interacting protein 3 recombinant protein(Bnip3)and yeast Atg6 homolog of autophagy key molecule(Beclin1)in ovarian tissues of rats were all detected.RESULTS Compared with control group,the ovarian indexes of model group and administration groups were decreased significantly(P<0.05).Model group possessed disturbed estrous cycle and the ovary tissues showed pathological morphology,the autophagolysosomes appeared in ovary cells,AMH and E2 levels were significantly decreased(P<0.05);FSH and LH levels,protein and mRNA expressions of HIF-1α,Bnip3 and Beclin1 were significantly increased(P<0.05).Compared with model group,the estrous cycle of rats in positive control group and modified Yijing decoction groups gradually returned to normal,the pathological morphology of ovarian tissue and the autophagy of ovarian cells were improved;the ovarian index,AMH and E2 levels were significantly increased(P<0.05);the levels of FSH and LH,protein and mRNA expressions of HIF-1α,Bnip3 and Beclin1(except for protein of HIF-1α,protein and mRNA of Bnip3 in modified Yijing decoction low-dose group)were decreased significantly(P<0.05).The protein expressions of HIF-1α,Bnip3 and Beclin1,mRNA expressions of HIF-1α and Beclin1 were decreased significantly in the inhibitor group (P<0.05). CONCLUSIONS Modified Yijing decoction can improve ovarian function,regulate the secretion of sex hormone,and protect ovarian tissue in DOR model rats,and the mechanism of which may be related to the inhibition of HIF-1α/ Bnip3/Beclin1 pathway.