经后增殖方改善卵巢功能不全小鼠卵巢储备及凋亡状态的研究

Effect of Jinghouzengzhi Recipe on ovarian reserve and apoptosis in cyclophosphamide-induced ovarian insufficiency mice

目的探讨中药经后增殖方对环磷酰胺诱导的卵巢功能不全小鼠卵巢储备功能与细胞凋亡状态的改善作用。方法将30只雌性小鼠随机分为环磷酰胺(cyclophosphamide,Cy)组、环磷酰胺+经后增殖方(JHZZR)组和空白对照组,经颈椎脱臼处死后取血清及卵巢组织待测。采用ELISA方法检测血清抗苗勒氏管激素(AMH)、促卵泡生成激素(FSH)和雌二醇(E2)水平,卵巢组织HE染色后计数各级卵泡数目,同时采用实时荧光定量PCR(quantitative real-time PCR,qPCR)方法检测卵巢组织凋亡因子Caspase3 mRNA的表达水平,用TUNEL法检测卵巢颗粒细胞的凋亡状态。结果与空白对照组相比,Cy组和Cy+JHZZR组小鼠卵巢内各级发育中卵泡数量均明显减少,闭锁卵泡增多。Cy+JHZZR组窦前卵泡与窦状卵泡数量较Cy组有所增加,闭锁卵泡数量减少,差异均有统计学意义(P<0.05)。与Cy组比较,Cy+JHZZR组小鼠血清中AMH水平明显升高[(6.12±1.66)ng/mL vs.(4.48±1.77)ng/mL,P<0.05],而FSH和E2水平差异无统计学意义(P>0.05)。Cy组和Cy+JHZZR组小鼠卵巢组织Caspase3 mRNA相对表达量分别为5.86±1.33和4.45±1.02,均高于空白对照组(2.89±1.10,P<0.01),而两组间差异也有统计学意义(P<0.05)。空白对照组、Cy组和Cy+JHZZR组的凋亡指数(apoptosis index,AI)分别为(8.37±3.17)%、(16.85±5.46)%和(12.80±3.34)%,Cy组和Cy+JHZZR组的AI均较空白对照组明显升高,且Cy+JHZZR组的AI低于Cy组,差异均有统计学意义(P<0.05)。结论经后增殖方可以改善环磷酰胺诱导的卵巢功能不全小鼠卵巢储备功能,并降低卵巢细胞的凋亡状态。

Objective To investigate the effect of the Chinese herbal formula Jinghouzengzhi Recipe(JHZZR)on ovarian reserve and apoptotic status in cyclophosphamide-induced ovarian dysfunction mice.Methods Thirty female mice were randomly divided into cyclophosphamide(Cy)group,cyclophosphamide+JHZZR(Cy+JHZZR)group,and blank control group.Serum and ovarian tissues were collected after cervical dislocation.ELISA was used to measure serum levels of anti-Müllerian hormone(AMH),follicle-stimulating hormone(FSH),and estradiol(E2).The number of follicles at each stage in ovarian tissues was counted after HE staining.Real-time quantitative PCR was performed to detect the expression level of Caspase3 mRNA in ovarian tissues,and TUNEL assay was used to detect the apoptotic status of granulosa cells.Results Compared with the blank control group,the number of developing follicles in the ovaries of mice in the Cy group and Cy+JHZZR group significantly decreased,while the number of atretic follicles increased.The number of primordial follicles and primary follicles in the Cy+JHZZR group increased compared to the Cy group,while the number of atretic follicles decreased,with statistical significance(P<0.05).The AMH level in the serum of mice in the Cy+JHZZR group was higher than that in the Cy group[(6.12±1.66)ng/mL vs.(4.48±1.77)ng/mL,P<0.05],while there was no significant difference in FSH and E2 levels.The relative expression levels of Caspase3 mRNA in the ovarian tissues of mice in the Cy group and Cy+JHZZR group were 5.86±1.33 and 4.45±1.02,respectively,both were higher than those in the blank control group(2.89±1.10,P<0.01),with statistical significance between the two groups(P<0.05).The apoptosis index(AI)of the blank control group,Cy group,and Cy+JHZZR group were(8.37±3.17)%,(16.85±5.46)%,and(12.80±3.34)%,respectively.The AI of the Cy group and Cy+JHZZR group were significantly higher than that of the control group,and the AI of the Cy+JHZZR group was lower than that of the Cy group,with statistical significance(P<0.05).Conclusion JHZZR can improve ovarian reserve and reduce apoptotic status of ovarian cells in cyclophosphamide-induced ovarian dysfunction mice.