摘要
目的:研究养精种玉汤对环磷酰胺诱导的卵巢储备功能减退(DOR)大鼠沉默信息调节因子1(SIRT1)/过氧化物酶体增殖物激活受体γ共激活因子-1α(PGC-1α)信号通路介导的线粒体生物发生与氧化应激损伤的影响,并探讨其改善卵巢储备功能及卵泡发育的作用机制。方法:选取42只动情周期正常的8周龄雌性SD大鼠,随机分为空白组(7只)和造模组(35只)。造模组大鼠采用腹腔注射环磷酰胺(90 mg·kg^(-1))一次性造模,造模后连续观察7 d,通过动情周期紊乱标准判断造模成功。造模成功后,戊酸雌二醇组(0.09 mg·kg^(-1))及养精种玉汤高、中、低剂量组(19.98、9.99、5.00 g·kg^(-1))药物干预,空白组和模型组给予等体积蒸馏水灌胃,所有组别每日灌胃1次,连续干预4周。实验期间观察并记录各组大鼠的一般状态、体质量及卵巢湿质量,计算卵巢脏器指数。采用酶联免疫吸附测定法(ELISA)检测大鼠血清中促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E_(2))及抗缪勒管激素(AMH)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平;苏木素-伊红(HE)染色观察卵巢组织形态学变化及卵泡发育状态;免疫荧光检测活性氧(ROS)表达水平;化学比色法检测卵巢组织中腺苷三磷酸(ATP)与丙二醛(MDA)含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测线粒体DNA(mtDNA)拷贝数及SIRT1、PGC-1α、核呼吸因子1(NRF1)、线粒体转录因子A(TFAM)等关键基因的mRNA表达水平;蛋白免疫印迹法(Western blot)检测SIRT1、PGC-1α、NRF1、TFAM蛋白表达水平。结果:与空白组比较,模型组大鼠动情周期紊乱,体质量及卵巢指数明显下降(P<0.05);卵巢组织病理学表现为皮质变薄、结构疏松、原始卵泡及生长卵泡数量均显著减少(P<0.01);血清FSH、LH水平显著升高(P<0.01),E_(2)、AMH水平明显降低(P<0.05,P<0.01);卵巢组织中ATP含量及mtDNA拷贝数显著下降(P<0.01),ROS表达增强,MDA水平升高,SOD和GSH-Px活性明显降低(P<0.05,P<0.01);SIRT1、PGC-1α、NRF1、TFAM的mRNA及蛋白表达水平均明显下调(P<0.05,P<0.01)。治疗后,与模型组比较,养精种玉汤各剂量组大鼠体质量及卵巢指数明显回升(P<0.05);血清中E_(2)和AMH水平明显升高,FSH和LH水平明显下降(P<0.05,P<0.01);卵巢组织ATP含量和mtDNA拷贝数明显上调,ROS和MDA水平明显下降,抗氧化酶SOD、GSH-Px活性明显增强(P<0.05,P<0.01);SIRT1/PGC-1α/NRF1/TFAM信号通路相关基因与蛋白表达水平均较模型组明显上调(P<0.05,P<0.01);HE染色显示,各治疗组卵巢结构逐步恢复完整,原始卵泡及生长卵泡数量明显增加(P<0.05,P<0.01),颗粒细胞排列整齐,卵巢功能改善明显。结论:养精种玉汤可能通过激活SIRT1/PGC-1α信号通路,促进线粒体生物发生,提升线粒体功能,减轻氧化应激损伤,从而改善卵巢储备功能减退大鼠的卵巢功能。
Objective:To observe the effects of Yangjing Zhongyutang(YJZYT)on mitochondrial biogenesis and oxidative stress damage mediated by the silent information regulator 1(SIRT1)/peroxisome proliferator-activated receptor gamma coactivator-1alpha(PGC-1α)signaling pathway in cyclophosphamide(CTX)-induced rats with diminished ovarian reserve(DOR),and to explore its mechanism in improving ovarian reserve function and follicular development.Methods:Forty-two 8-week-old female SD rats with normal estrous cycles were randomly divided into a blank control group(n=7)and a model group(n=35).Rats in the model group received a single intraperitoneal injection of CTX(90 mg·kg^(-1))to establish the DOR model.After modeling,estrous cycles were monitored for 7 consecutive days,and model success was confirmed based on criteria for estrous cycle disruption.After successful modeling,rats were divided into groups for intervention:estradiol valerate group(0.09 mg·kg^(-1)),and YJZYT high-,medium-,and low-dose groups(19.98,9.99,5.00 g·kg^(-1)).The blank control group and model group were given an equal volume of distilled water by gavage.All groups received daily gavage once for 4 consecutive weeks.The general state,body weight,and ovarian wet weight of rats were observed and recorded,and the ovarian organ index was calculated.Enzyme-linked immunosorbent assay(ELISA)was used to measure serum levels of follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E_(2)),anti-Müllerian hormone(AMH),superoxide dismutase(SOD),and glutathione peroxidase(GSH-Px).Hematoxylin-eosin(HE)staining was performed to observe ovarian histomorphological changes and follicular development status.Immunofluorescence was used to detect reactive oxygen species(ROS)expression levels.Colorimetric assays were employed to measure adenosine triphosphate(ATP)and malondialdehyde(MDA)content in ovarian tissues.Quantitative Real-time polymerase chain reaction(Real-time PCR)was used to detect mitochondrial DNA(mtDNA)copy number and the mRNA expression levels of key genes including SIRT1,PGC-1α,nuclear respiratory factor 1(NRF1),and mitochondrial transcription factor A(TFAM).Western blot was performed to detect the protein expression levels of SIRT1,PGC-1α,NRF1,and TFAM.Results:Compared with the blank group,rats in the model group exhibited disrupted estrous cycles,obviously reduced body weight,and decreased ovarian index(P<0.05).Ovarian histopathology revealed cortical thinning,loose structure,and a significant reduction in both primordial and growing follicles(P<0.01).Serum FSH and LH levels were significantly elevated(P<0.01),while E_(2) and AMH levels were obviously reduced(P<0.05,P<0.01).ATP content and mtDNA copy number decreased in ovarian tissue(P<0.01),ROS expression increased,MDA levels rose,while SOD and GSH-Px activities obviously decreased(P<0.05,P<0.01),mRNA and protein expression levels of SIRT1,PGC-1α,NRF1,and TFAM were obviously downregulated(P<0.05,P<0.01).After treatment,compared with the model group,body weight and ovarian index obviously recovered in rats administered various doses of YJZYT(P<0.05),serum E_(2) and AMH levels increased,while FSH and LH levels obviously decreased(P<0.05,P<0.01),ovarian tissue ATP content and mtDNA copy number were up-regulated,ROS and MDA levels decreased,and antioxidant enzymes SOD and GSH-Px activity obviously increased(P<0.05,P<0.01),Gene and protein expression levels related to the SIRT1/PGC-1α/NRF1/TFAM signaling pathway were obviously up-regulated compared to the model group(P<0.05,P<0.01),HE staining revealed that ovarian structure gradually recovered to integrity in all treatment groups,with a obviously increase in the number of primordial and growing follicles(P<0.05,P<0.01).Granulosa cells were neatly arranged,indicating marked improvement in ovarian function.Conclusion:YJZYT may improve ovarian function and follicular development in rats with diminished ovarian reserve by activating the SIRT1/PGC-1αsignaling pathway,promoting mitochondrial biogenesis,enhancing mitochondrial function,and alleviating oxidative stress damage.
作者
张萍
杨丽娟
陈胜辉
姚文亮
周玉良
马玲
吴慧颖
徐燕文
周紫嫣
ZHANG Ping;YANG Lijuan;CHEN Shenghui;YAO Wenliang;ZHOU Yuliang;MA Ling;WU Huiying;XU Yanwen;ZHOU Ziyan(Affiliated Reproductive Hospital of Jiangxi University of Traditional Chinese Medicine(TCM),Nanchang 330004,China;Jiangxi University of TCM,Nanchang 330004,China)
出处
《中国实验方剂学杂志》
北大核心
2026年第7期46-55,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
江西省中医药管理局科技计划项目(2024B0581)。
