基于p53/Nrf2信号通路探讨新加苁蓉菟丝子汤对早发性卵巢功能不全大鼠颗粒细胞铁死亡的影响

Xinjia Congrong Tusizi Decoction Regulates Ferroptosis of Granulosa Cells in Rat Model of Premature Ovarian Insufficiency via p53/Nrf2 Signaling Pathway

目的:探究新加苁蓉菟丝子汤(中药)对早发性卵巢功能不全(POI)模型大鼠卵巢功能的影响及调控肿瘤抑制蛋白p53(p53)/核因子E_(2)相关因子2(Nrf2)通路减轻颗粒细胞铁死亡的潜在机制。方法:将48只SPF级雌性SD大鼠随机分为6组:正常组、模型组、辅酶Q_(10)组(0.002 7 g·kg^(-1))和中药低、中、高剂量组(1.1、2.2、4.4 g·kg^(-1)),每组8只。以灌胃雷公藤甲素混悬液建立POI模型大鼠,造模成功后,各组予相应药物干预14 d。称量大鼠体质量、卵巢湿重并计算卵巢指数,苏木素-伊红(HE)染色观察卵巢组织形态学并计算生长卵泡及闭锁卵泡比例,酶联免疫吸附测定法检测血清卵泡刺激素(FSH)、雌二醇(E_(2))和抗缪勒管激素(AMH)水平,DCFH-DA荧光探针检测颗粒细胞活性氧(ROS)含量,比色法检测细胞亚铁离子(Fe^(2+))、脂质过氧化物(LPO)、丙二醛(MDA)、还原型谷胱甘肽(GSH)、超氧化物岐化酶(SOD)水平,免疫组化法和蛋白免疫印迹法(Western blot)检测肿瘤抑制蛋白p53、Nrf2、溶质载体家族7成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)蛋白表达情况。结果:与正常组比较,模型组大鼠卵巢湿重、体质量及卵巢指数降低(P<0.01),卵巢组织体积缩小、生长卵泡占比减少(P<0.01),闭锁卵泡占比增多(P<0.01),血清AMH和E_(2)水平降低(P<0.01),FSH水平升高(P<0.01),颗粒细胞内Fe^(2+)、ROS、LPO及MDA含量显著增加(P<0.01),GSH和SOD含量显著降低(P<0.01),免疫组化和Western blot检测结果提示卵巢组织内p53蛋白表达增多(P<0.01),Nrf2、SLC7A11及GPX4蛋白表达降低(P<0.05,P<0.01)。与模型组比较,中药各剂量组大鼠体质量、卵巢湿重及卵巢指数显著升高(P<0.01),卵巢组织形态均不同程度改善,生长卵泡占比增加(P<0.01),闭锁卵泡占比减少(P<0.01),颗粒细胞内ROS含量明显降低(P<0.05,P<0.01);中药中、高剂量组血清FSH水平降低(P<0.01),E_(2)、AMH水平升高(P<0.01),颗粒细胞内Fe^(2+)含量下降(P<0.01),GSH含量上升(P<0.01);中药高剂量组颗粒细胞内LPO和MDA含量显著降低(P<0.01),SOD含量显著升高(P<0.01),免疫组化和Western blot检测结果提示卵巢组织内p53蛋白表达减少(P<0.05),Nrf2、SLC7A11及GPX4蛋白表达增加(P<0.05,P<0.01)。结论:新加苁蓉菟丝子汤能保护POI模型大鼠卵巢功能,其作用机制可能与调控p53/Nrf2信号通路减轻卵巢颗粒细胞的铁死亡有关。

Objective:To investigate the effects of Xinjia Congrong Tusizi decoction (XJCTD) on ovarian functions in the rat model of premature ovarian insufficiency (POI) and decipher the mechanism of regulating the tumor suppressor protein (p53)/nuclear factor E_2-related factor 2 (Nrf2) pathway to attenuate granulosa cell ferroptosis.Method:Forty-eight SPF-grade female SD rats were randomized into control,model,low-,medium-,and high-dose (1.1,2.2,4.4 g·kg^(-1)) XJCTD,and Western medicine (coenzyme Q_(10),0.002 7 g·kg^(-1)) groups,with eight rats in each group.The rat model of POI was established by gavage of triptolide (TP),and after successful modeling,each group was administrated with the corresponding drugs by gavage for 14 d.The body weight and ovarian weight of each rat were weighed and the ovarian index was calculated.The morphology of the ovarian tissue was observed by hematoxylin-eosin staining,and the proportions of growing follicles and atretic follicles were calculated.The serum levels of anti-Müllerian hormone(AMM),estradiol (E_2),and follicle-stimulating hormone (FSH) were measured by enzyme-linked immunosorbent assay (ELISA).The DCFH-DA fluorescent probe was used to measure the reactive oxygen species (ROS) content in granulosa cells.The content of cellular Ferrous ion (Fe^(2+)),lipid peroxide (LPO),malondialdehyde (MDA),glutathione (GSH),and superoxide dismutase (SOD) was detected by colorimetry.The expression of the tumor suppressor protein p53,Nrf2,solute carrier family 7 member 11 (SLC7A11),and glutathione peroxidase 4 (GPX4) was determined by immunohistochemistry and Western blot.Result:Compared with the control group,the model group showed decreased ovarian weight,body weight,and ovarian index (P<0.01),reduced ovarian tissue volume and proportion of growing follicles (P<0.01),increased proportion of atretic follicles (P<0.01),lowered AMH and E_(2) levels and elevated FSH level in the serum(P<0.01),and elevated levels of Fe^(2+),ROS,LPO,and MDA (P<0.01) and lowered levels of GSH and SOD in granulosa cells (P<0.01).Moreover,the modeling up-regulated the expression of p53 (P<0.01) and downregulated the expression of Nrf2,SLC7A11,and GPX4 (P<0.05,P<0.01) in the ovarian tissue.Compared with the model group,XJCTD increased the body weight,ovarian weight,and ovarian index (P<0.01),alleviated the pathological changes in the ovarian tissue,increased the proportion of growing follicles (P<0.01),decreased the proportion of atretic follicles (P<0.01),and reduced the content of ROS in granulosa cells (P<0.05,P<0.01).In addition,medium-and high-dose XJCTD lowered the FSH level (P<0.01) and raised E_(2) and AMH levels (P<0.01) in the serum,reduced the Fe^(2+)content (P<0.05,P<0.01),and increased the SOD content(P<0.01) in granulosa cells.High-dose XJCTD reduced the LPO and MDA content (P<0.01) and increased the SOD content (P<0.01) in the granulosa cells,down-regulated the expression of p53 (P<0.05),and up-regulated the expression of Nrf2,SLC7A11,and GPX4 in the ovarian tissue (P<0.05,P<0.01).Conclusion:XJCTD may protect the ovarian function in the rat model of POI by regulating the p53/Nrf2 signaling pathway to attenuate the ferroptosis of ovarian granulosa cells.