资癸益冲方对卵巢储备功能下降模型大鼠卵巢氧化损伤的影响及其作用机制

Effects of Zigui Yichong Formula on ovarian oxidative damage in rats with diminished ovarian reserve and its mechanism

目的探讨资癸益冲方对卵巢储备功能下降(DOR)模型大鼠卵巢氧化损伤的影响及其作用机制。方法采用环磷酰胺腹腔注射诱导DOR大鼠模型,将诱导造模成功的大鼠随机分为模型组、资癸益冲方高剂量组(28 g/kg)、资癸益冲方低剂量组(7 g/kg),另选10只动情周期规律的大鼠作为正常组。各组分别给予相应处理因素28 d后,选动情前期处死大鼠,检测血清雌二醇(E2)、抗苗勒管激素(AMH)、卵泡刺激素(FSH)、黄体生成素(LH)水平;HE染色观察卵巢组织病理形态学改变;检测卵巢组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活力和丙二醛(MDA)含量;免疫组化和Western Blot法检测肿瘤抑制因子p53、周期蛋白依赖激酶抑制因子p21和核因子相关因子-2(Nrf2)的蛋白表达。结果与正常组比较,模型组大鼠血清E2、AMH水平降低,FSH、LH水平升高(均P<0.05);卵巢组织中各级卵泡数减少,闭锁卵泡增多,颗粒细胞层数减少;卵巢组织SOD、GSH-Px、CAT活力降低,MDA含量升高(均P<0.05);p53、p21、Nrf2蛋白表达增强(均P<0.05)。与模型组比较,资癸益冲方高、低剂量组大鼠血清E2、AMH水平升高,FSH、LH水平降低(均P<0.05);卵巢组织中闭锁卵泡减少,颗粒细胞层数增多;卵巢组织SOD、GSH-Px、CAT活力升高,MDA含量降低(均P<0.05);p53、p21和Nrf2蛋白表达下降(均P<0.05)。结论资癸益冲方能改善DOR大鼠的卵巢储备功能,减轻卵巢氧化损伤,降低卵巢组织中p53、p21和Nrf2的蛋白表达。

Objective To investigate the effects of Zigui Yichong Formula(Gui-nourishing and Chong-benefiting Formula,ZGYCF)on ovarian oxidative damage in rats with diminished ovarian reserve(DOR)and its mechanism.Methods The rat models of DOR were established by administering intraperitoneal injection of cyclophosphamide.30 rats with successful modeling were randomly divided into model group,high-dose ZGYCF group(28 g/kg),and low-dose ZGYCF group(7g/kg)(n=10 in each group).10 rats with regular estrous cycle were set as the normal control group.The rats were treated with corresponding drugs except those in the normal group for 28 days and all were sacrificed before estrous cycle.Then the levels of serum estradiol(E2),anti-mullerian hormone(AMH),follicle stimulating hormone(FSH)and luteinizing hormone(LH)were determined.The morphological changes of ovarian tissue were observed with HE staining.Theactivity of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT)and the content of malonicdialdehyde(MDA)were determined.The protein expression of p53,p21 and Nrf2 in ovarian tissue were detected with immunochemistry technology and Western Blot assay.Results Compared with the normal group,the levels of E2,AMH were decreased and the levels of FSH and LH were increased(P<0.05);the number of follicles in the ovarian tissue was decreased,the number of atresia follicles was increased and the number of granulosa cells was decreased;the activity of SOD,GSH-Px,CAT was decreased and the content of MDA was increased(P<0.05);the protein expression of p53,p21 and Nrf2 in ovarian tissue was increased(P<0.05)in the model group.Compared with the model group,the levels of E2,AMH were increased and the levels of FSH,LH were decreased(P<0.05);the number of atresia follicles was decreased and the number of granulosa cells was increased;the activity of SOD,GSH-Px,CAT was increased and the content of MDA was decreased(P<0.05);the protein expression of p53,p21 and Nrf2 in ovarian tissue was decreased(P<0.05)in high-dose ZGYCF group and low-dose ZGYCF group.ConclusionZigui Yichong decoction can improve the ovarian reserve function of DOR rats,reduce ovarian oxidative damage and protein expression of p53,p21 and Nrf2 in ovarian tissue.