滋阴疏肝汤对卵巢储备功能不全模型小鼠卵巢颗粒细胞凋亡的影响  

Effect of Ziyin Shugan Tang on apoptosis of ovarian granular cells in ovarian reserve insufficiency model mice

作　　者：郭锐利 赵新伟 王琰[1] 马建新[1] GUO Ruili;ZHAO Xinwei;WANG Yan;MA Jianxin(Department of Reproductive Medicine,Cangzhou Hospital of Integrated TCM-WM·Hebei,Cangzhou,Hebei 061000,China)

机构地区：[1]河北省沧州中西医结合医院生殖医学科,河北沧州061000

出　　处：《安徽医药》2024年第10期2087-2093,I0007,共8页Anhui Medical and Pharmaceutical Journal

基　　金：河北省中医药管理局项目(2020487)。

摘　　要：目的探讨滋阴疏肝汤对卵巢储备功能不全模型小鼠卵巢颗粒细胞(GCs)凋亡的影响。方法研究起止时间为2020年1月至2022年12月。将30只雌性C57BL/6小鼠按随机数字表法分为对照(NC)组、环磷酰胺模型(CTX)组、低剂量(ZY-L)组及高剂量(ZY-H)组滋阴疏肝汤组、辅酶Q10(Q10)组,实验结束前,抽取小鼠外周血,利用酶联免疫吸附测定(ELISA)检测相关激素及氧化应激相关指标水平。实验结束后处死小鼠,提取卵巢原代颗粒细胞。采用化学发光法测定ATP水平,利用DNA断裂的原位末端标记(TUNEL)法检测细胞凋亡情况。采用实时荧光定量逆转录PCR(RT-qPCR)及蛋白质印迹法检测线粒体功能相关基因及细胞凋亡信号调节激酶1抗体(ASK1)/应激活化蛋白激酶(JNK)信号通路相关基因的表达水平。结果滋阴疏肝汤干预可显著降低小鼠外周血促卵泡生成素(FSH)和促黄体生成素(LH)水平(P<0.05),提高抗米勒管激素(AMH)、雌二醇(E2)、总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSHPx)水平(P<0.05)。与CTX组相比,ZY-H组及Q10组GCs中神经萎缩蛋白1(OPA1)(NC:1.00±0.05;ZY-H:0.78±0.41;Q10:0.89±0.46)、线粒体融合蛋白1(Mfn1)(NC:1.00±0.04;ZY-H:0.65±0.23;Q10:0.81±0.51)、线粒体融合蛋白2(Mfn2)(NC:1.00±0.08;ZY-H:0.43±0.16;Q10:0.72±0.23)的信使RNA(mRNA)/蛋白表达水平明显升高(P<0.05),而靶向线粒体分裂蛋白(Drp1)(NC:1.00±0.03;ZY-H:2.03±0.53;Q10:1.52±0.41)和线粒体裂变1蛋白抗体(Fis1)(NC:1.00±0.03;ZY-H:1.32±0.13;Q10:1.44±0.26)的mRNA/蛋白表达水平明显降低(P<0.05)。RT-qPCR及蛋白质印迹法检测显示,经滋阴疏肝汤和辅酶Q10处理后,GCs中胱天蛋白酶3/9(caspase-3/9)、ASK1、JNK、细胞色素C(Cyt-c)的mRNA及蛋白水平均下降(P<0.05)。TUNEL检测显示,滋阴疏肝汤干预可明显减少卵巢凋亡细胞的数量。结论滋阴疏肝汤可降低小鼠体内氧化应激水平,保护线粒体功能,使卵巢储备�Objective To explore the effect of Ziyin Shugan Tang on apoptosis of ovarian granulosa cells(GCs)in mice with ovarian reserve dysfunction.Methods The duration of this study was from January 2020 to December 2022.Thirty-six female mice were randomly assigned into control(NC)group,cyclophosphamide model(CTX)group,low-dose Ziyin Shugan Tang(ZY-L)group,high-dose Ziyin Shugan Tang(ZY-H)group,and Coenzyme Q10(Q10)group.Primary ovarian GCs were extracted and the levels of relevant indicators were detected using Enzyme-linked immunosorbent(ELISA)assay.ATP levels were measured using chemiluminescence assay,and cell apoptosis was detected using TUNEL assay.RT qPCR and Western blot were used to detect the expression levels of mitochondrial function related genes and apoptotic signaling regulates kinase I(ASK1)/Jun N-terminal kinase(JNK)signaling pathway related genes.Results The intervention of Ziyin Shugan Tang significantly reduced the levels of Follicle-stimulating hormone(FSH)and Luteinizing Hormone(LH)in the peripheral blood of mice(P<0.05),and increased the levels of Anti-Müllerian Hormone(AMH),Estradiol(E2),Total Antioxidant Capacity(T-AOC),Superoxide dismutase(SOD),Glutathione peroxidase(GSHPx)(P<0.05).Compared with the CTX group,while the levels of Optic Atrophy 1(OPA1)(NC:1.00±0.05;ZY-H:0.78±0.41;Q10:0.89±0.46),Mitofusin-1(Mfn1),(NC:1.00±0.04;ZY-H:0.65±0.23;Q10:0.81±0.51)and Mitofusin-2(Mfn2)(NC:1.00±0.08;ZY-H:0.43±0.16;Q10:0.72±0.23)Messenger RNA(mRNA)/protein in ZY-H and Q10 groups increased(P<0.05).Compared with the NC group,the CTX group showed an increase in Data Reduction Package 1(Drp1)(NC:1.00±0.03;ZY-H:2.03±0.53;Q10:1.52±0.41)and Mitochondrial Fission 1 Protein(Fis1)(NC:1.00±0.03;ZY-H:1.32±0.13;Q10:1.44±0.26)mRNA/protein levels(P<0.05),while the After treatment with Ziyin Shugan Tang and coenzyme Q10,the mRNA and protein levels of cysteineasparaticacid-specificproteases-3/9(caspase-3/9),ASK1,JNK,and Cytochrome c(Cyt-c)all decreased(P<0.05).TUNEL detection showed that Ziyin Shugan Tang and

关 键 词：滋阴疏肝 卵巢储备功能 小鼠 近交C57BL 颗粒细胞 环磷酰胺 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]