摘要
目的探讨邻苯二甲酸二丁酯(dibutyl phthalate,DBP)通过腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白(AMP activated protein kinase/mammalian rapamycin target protein,AMPK/mTOR)信号通路对大鼠睾丸间质细胞(Leydig)凋亡的影响。方法生殖功能损伤大鼠模型按照体质量排序分组法分为模型(DBP)组17只、模型+AMPK抑制剂[DBP+化合物C(compound C,CC)]组17只、模型+AMPK激动剂[DBP+二甲双胍(metformin,MF)]组17只、DBP+AMPK抑制剂+激活剂(DBP+CC+MF)组17只。另取11只大鼠作为空白组。空白组和DBP组腹腔注射等量生理盐水,DBP+CC组、DBP+MF组分别腹腔注射20 mg/kg CC、200 mg/kg MF,DBP+CC+MF组腹腔注射20 mg/kg CC+200 mg/kg MF,qd,连续4周。放射性免疫分析法检测各组血清黄体生成素(luteinizing hormone,LH)、卵泡刺激素(follicle-stimulating hormone,FSH)、睾酮水平;全自动精子质量分析系统分析精子质量;HE染色法观察睾丸生精小管上皮细胞病变情况;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling,TUNEL)法和流式细胞术检测Leydig细胞凋亡情况;RT-PCR法和Western blotting法检测AMPK、mTOR、半胱氨酸天冬氨酸蛋白酶3(Caspase 3)mRNA和蛋白及p-AMPK、p-mTOR蛋白的表达。结果与DBP组的血清FSH水平[(9.07±0.52)U/L]相比,DBP+MF组血清FSH水平[(9.88±0.67)U/L]升高,DBP+CC组[(6.82±0.60)U/L]降低,差异均有统计学意义(均P<0.001);与DBP组血清LH、睾酮水平、精子浓度及(a+b)级精子占比[(4.51±0.75)U/L、(3.25±0.11)mg/L、(16.46±3.40)×10^(6)/mL、(25.43±4.36)%]相比,DBP+MF组血清LH、睾酮水平及精子浓度、(a+b)级精子占比[(3.97±0.70)U/L、(2.96±0.11)mg/L、(13.15±2.63)×10^(6)mL、(22.20±4.13)%]降低,DBP+CC组[(6.52±0.71)U/L、(4.48±0.15)mg/L、(25.47±2.18)×10^(6)/mL、(45.60±4.78)%]升高,差异均有统计学意义(DBP组比DBP+MF组PLH=0.038,其余均P<0.001)。HE染色显示,空白组睾丸组
Objective To explore the effect of dibutyl phthalate(DBP)on the rat testis Leydig cell apoptosis by AMP activated protein kinase/mammalian rapamycin target protein(AMPK/mTOR)signaling pathway.Methods Rats with reproductive function impairment were divided into model(DBP)group of 17 rats,model+AMPK inhibitor[DBP+compound C(CC)]group of 17 rats,model+AMPK agonist[DBP+metformin(MF)]group of 17 rats,DBP+AMPK inhibitor+agonist(DBP+CC+MF)group of 17 rats by body mass ranking grouping method.Another 11 rats were taken as the blank group.The blank group and DBP group were intraperitoneally injected with the same amount of normal saline,while DBP+CC group and DBP+MF group were intraperitoneally injected with 20 mg/kg CC and 200 mg/kg MF respectively,and DBP+CC+MF group was intraperitoneally injected with CC and MF once a day for 4 weeks.Luteinizing hormone(LH),follicle-stimulating hormone(FSH)and testosterone(T)were measured by radioimmunoassay.Sperm quality was analyzed by automatic sperm quality analysis system.Leydig cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)and flow cytometry.The expressions of AMPK,mTOR,Caspase 3 mRNA and protein,p-AMPK and p-mTOR protein were detected by RT-PCR and Western blotting.Results The serum level of FSH in DBP+MF group[(9.88±0.67)U/L]increased,while that in DBP+CC group[(6.82±0.60)U/L]decreased compared with DBP group[(9.07±0.52)U/L](all P<0.001).The serum LH,T levels and sperm concentration,percentage of(a+b)grade sperm in DBP+MF group[(3.97±0.70)U/L,(2.96±0.11)mg/L,(13.15±2.63)×10^(6)/mL,(22.20±4.13)%],DBP+CC group[(6.52±0.71)U/L,(4.48±0.15)mg/L,(25.47±2.18)×10^(6)/mL,(45.60±4.78)%]increased compared with DBP group[(4.51±0.75)U/L,(3.25±0.11)mg/L,(16.46±3.40)×106/mL,(25.43±4.36)%](DBP group vs.DBP+MF group PLH=0.038,the other all P<0.001).HE staining showed that the structure of testis in blank group was normal.In DBP group and DBP+CC+MF group,the epithelial cells of seminiferous tubules atrophied and
作者
曲晓伟
郭海彬
冯科
夏彦清
万锋
李俊涛
Qu Xiaowei;Guo Haibin;Feng Ke;Xia Yanqing;Wan Feng;Li Juntao(Henan Provincial People's Hospital Reproductive Medicine Center,Zhengzhou 450003,China;Department of Andrology,the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,China)
出处
《中华生殖与避孕杂志》
CSCD
北大核心
2022年第3期268-276,共9页
Chinese Journal of Reproduction and Contraception
基金
河南省科技攻关计划项目(142102310393)。
关键词
生殖
睾丸
间质细胞
凋亡
邻苯二甲酸二丁酯
Reproduction
Testis
Interstitial cells
Apoptosis
Dibutyl phthalate
