补肾益气和血方对大鼠卵巢储备功能的影响与作用机制

Effect and mechanism of BSYQHX prescription on ovarian reserve function of rats

目的:观察补肾益气和血方对卵巢储备功能下降(DOR)模型大鼠卵巢储备功能影响的作用与其机制。方法:构建DOR模型大鼠,随机分为空白组(生理盐水,15 ml/kg),模型组(雷公藤多苷,50 mg/kg)、西药组(戊酸雌二醇,0.2mg/kg)与中药(补肾益气和血方)低剂量(4.31g/kg)、中剂量(8.62g/kg)、高剂量(17.25g/kg)组,每组8只。阴道涂片显微镜下观察大鼠动情周期及卵巢组织形态学变化,酶联免疫吸附试验法测定促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E2)、抗苗勒氏管激素(AMH)水平,免疫组织化学法测定血管内皮生长因子(VEGF)及其受体蛋白;实时荧光定量(RT-PCR)法检测卵巢组织VEGF mRNA表达。结果:①造模后模型组大鼠动情周期显著延长,卵巢、子宫指数均显著下降,血清FSH水平和FSH/LH值显著升高,AMH、E2水平显著降低(均P<0.01)。②与模型组比较,补肾益气和血方高剂量组能显著改善卵巢、子宫指数,中、高剂量组能显著降调高FSH和FSH/LH比值,提高E2、AMH水平(P<0.01)。与西药组比较,中药中、高剂量组FSH水平低,中药低、中剂量组E2水平高(P<0.01)。③与空白组比较,模型组VEGF及其受体flt-1、kdr蛋白量显著降低(P<0.05);与模型组比较,不同浓度的中药干预后,flt-1、kdr蛋白和其相应的mRNA表达量均显著增高(P<0.01),但与西药组比较无差异(P>0.05)。结论:补肾益气和血方可有效改善大鼠卵巢储备功能减退,降低血清FSH和FSH/LH比值,提高E2、AMH水平,促进卵巢组织血管生成,其机制可能与上调VEGF/VEGFR蛋白及其相应的mRNA表达有关。

Objective:To observe the effect and mechanism of BSYQHX prescription on ovarian reserve function of model rats with declined ovarian reserve(DOR).Methods:The DOR model rats were developed,and they were randomly divided into six groups(8 rats in each group).The rats in group A were given normal 15 ml/kg,the rats in group B were given tripterygium glycosides 50 mg/kg,the rats in group C were given estradiol valerate 0.2mg/kg,and the rats in group D1 were given BSYQHX prescription 4.31g/kg,the rats in group D2 were given BSYQHX prescription 8.62g/kg,and the rats in group D3 were given BSYQHX prescription 17.25g/kg.The vaginal smear in estrous cycle and the change of ovarian histomorphology of the rats in the 6 groups were observed under microscope.The follicle stimulating hormone(FSH),luteinizing hormone(LH),levadiol(E2),and anti mullerian hormone(AMH)of the rats in the 6 groups were measured by ELLels,the expression of VEG and its receptor protein levels of the rats in the 6 groups were measured by immunohistochemistry,and the expression of VEGFmRNA in ovarian tissue of the rats in the 6 groups was detected by RT-PCR.Results:The duration of estrus cycle of the rats in group B had prolonged significantly,and the levels of ovarian and uterine indexes of the rats in group B had decreased significantly(P<0.01).The serum levels of FSH and FSH/LH value of the rats in group B had increased significantly(P<0.01),but the levels of AMH and E2 of the rats in group B had decreased significantly(P<0.01).The levels of the ovarian and uterine indexes of the rats in group D3 were significant higher than those of the rats in group in group B(P<0.01).The FSH level and FSH/LH value of the rats in group D2 and group D3 had been decreased significantly,and the E2 and AMH levels had been increased significantly(P<0.01).The level of FSH of the rats in group D2 and group D3 was significant lower than that of the rats in group C group(P<0.01).The level of E2 in D1 and D2 groups was significant higher than that of the rats in C group(P<0.01).The levels of VEGF,its receptor Flt-1,and KDR protein of the rats in group B were significant lower than those of the rats in group A(P<0.05).The expression levels of Flt-1 and KDR protein levels and their corresponding mRNA expressions of the rats in groups D1,D2 and D3 were significant higher than those of the rats in group B(P<0.01),but which had no statistical significant different from those of the rats in group C(P>0.05).Conclusion:BSYQHX prescription can effectively improve the DOR of rats,reduce their FSH serum level and FSH/LH value,increase the levels of their E2 and AMH,and promote the neovascularization of their ovarian tissue.The mechanism may be related to the up regulation of VEGF/VEGFR protein and related to its corresponding mRNA expression.