基于Keap1/Nrf2/HO-1信号通路探讨滋肾调肝方对卵巢储备功能下降模型大鼠的影响  

Zishen Tiaogan Prescription Treats Diminished Ovarian Reserve in Rats via Keap1/Nrf2/HO-1 Signaling Pathway

作　　者：李钟彤 张亚萍[1] 游琛 李青青 王颖杰 区思雯 薛桃妹 张楚琪 LI Zhongtong;ZHANG Yaping;YOU Chen;LI Qingqing;WANG Yingjie;OU Siwen;XUE Taomei;ZHANG Chuqi(Guangxi University of Chinese Medicine,Nanning 530000,China;Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning 530000,China)

机构地区：[1]广西中医药大学,南宁530000 [2]广西中医药大学附属瑞康医院,南宁530000

出　　处：《中国实验方剂学杂志》2025年第7期72-80,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：广西自然科学基金项目(2024GXNSFAA010238);桂派杏林青年英才计划项目(2022C029);“西部之光”访问学者接收培养项目(教人司〔2022〕325号);第五批全国中医临床优秀人才研修项目(国中医药人教函〔2022〕239号);全国名老中医药专家刘燕平传承工作室建设项目(国中医药人教函〔2022〕75号);桂派中医药传承创新团队项目(2022B002)。

摘　　要：目的:观察滋肾调肝方改善卵巢储备功能下降(DOR)模型大鼠氧化应激损伤的作用,并探讨Kelch样ECH关联蛋白1(Keap1)/核因子E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)信号通路在其中的作用机制。方法:将48只雌性SD大鼠随机分为正常组12只,造模组36只,造模组大鼠连续每日皮下注射半乳糖(350 mg·kg^(-1))联合制动应激法建立模型,造模28 d后将正常组和造模组各处死6只验证模型。模型验证成功后,将造模组分为模型组,戊酸雌二醇(0.09 mg·kg^(-1))组和滋肾调肝方低、中、高剂量(6.39、12.78、25.56 g·kg^(-1))组,以6只/组进行相应干预,持续4周。苏木素-伊红(HE)染色法观察大鼠动情周期及卵巢病理形态学变化,计算大鼠卵巢脏器指数;酶联免疫吸附测定法(ELISA)检测血清中性激素及氧化应激相关指标的变化;蛋白免疫印迹法(Western blot)和实时荧光定量聚合酶链式反应(Real-time PCR)检测卵巢组织中Nrf2、Keap1、HO-1蛋白及mRNA表达情况;免疫组化(IHC)检测卵巢组织中超氧化物歧化酶2(SOD2)蛋白的阳性表达。结果:与正常组比较,模型组大鼠卵巢中生长卵泡明显减少、卵泡颗粒层排列松散;大鼠卵巢指数和血清抗缪勒管激素(AMH)、雌二醇(E2)水平显著下降,黄体生成素(LH)、促卵泡生成素(FSH)水平均显著升高(P<0.01);超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和还原型谷胱甘肽(GSH)水平显著下降(P<0.01),丙二醛(MDA)水平显著升高(P<0.01),卵巢组织中Keap1蛋白及mRNA表达显著增加(P<0.01),Nrf2、HO-1蛋白表达显著减少(P<0.01),Nrf2 mRNA表达明显降低(P<0.05),HO-1 mRNA表达显著下降(P<0.01),SOD2蛋白阳性表达显著减少(P<0.01);与模型组比较,戊酸雌二醇组及滋肾调肝方低、中、高剂量组卵巢指数均明显增加(P<0.05,P<0.01),血清中AMH、E2水平显著升高(P<0.01),FSH、LH水平显著降低(P<0.01);卵巢内生长卵泡数量增多,血清中SOD活性增强,CAT活力升高,GSH�Objective:To observe the effect of Zishen Tiaogan prescription on the oxidative stress injury in the rat model of diminished ovarian reserve(DOR)and explore the role of the Kelch-like ECH-associated protein 1(Keap1)/nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway.Methods:Forty-eight female SD rats were randomly assigned into a normal group(n=12)and a modeling group(n=36).The rats in the modeling group received subcutaneous injection of galactose(350 mg·kg^(-1))combined with immobilization stress daily.After 28 days of modeling,6 rats in the normal group and 6 rats in the modeling group were sacrificed to examine the modeling results.The successfully modeled rats were assigned into model,estradiol valerate(0.09 mg·kg^(-1)),and low-,medium-,and high-dose(6.39,12.78,25.56 g·kg^(-1),respectively)Zishen Tiaogan prescription groups.The intervention lasted for 4 weeks with 6 animals per group.Hematoxylin-eosin staining was used to observe the estrous cycle and the pathological changes in the ovarian tissue.The ovarian index was calculated.Enzyme-linked immunosorbent assay was employed to measure the serum levels of sex hormones and oxidative stress-related indexes.Western blot and real-time PCR were employed to determine the protein and mRNA levels,respectively,of Nrf2,Keap1 and HO-1 in the ovarian tissue.The positive expression of superoxide dismutase 2(SOD2)in the ovarian tissue was detected by immunohistochemistry(IHC).Results:Compared with the normal group,the model group showed reduced follicles in the ovary,loose arrangement of the follicle granule layer,declined levels of anti-Mullerian hormone(AMH)and estradiol(E2)in the serum,elevated levels of luteinizing hormone(LH)and follicle-stimulating hormone(FSH)(P<0.01),lowered levels of superoxide dismutase(SOD),catalase(CAT),and glutathione(GSH)(P<0.01),and increased accumulation of malondialdehyde(MDA)(P<0.01).In addition,the modeling led to up-regulated protein and mRNA levels of Keap1(P<0.01),the expression of Nrf2 and HO-1 prot

关 键 词：滋肾调肝方 卵巢储备功能下降 氧化应激 Kelch样ECH关联蛋白1(Keap1)/核因子E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)信号通路 

分 类 号：R271[医药卫生—中医妇科学] R289[医药卫生—中医学] R242