氯化锂对雄性小鼠睾酮生成障碍的影响及槲皮素保护作用的体内外研究  

Effect of lithium chloride on testosterone production dysfunction in male mice and protective effect of quercetin in vivo and in vitro

作　　者：王立宏 万梅 凌曦 曹佳[1] 敖琳[1] 邹鹏 Wang Lihong;Wan Mei;Ling Xi;Cao Jia;Ao Lin;Zou Peng(College of Preventive Medicine,Institute of Toxicology,Army Medical University(Third Military Medical University),Chongqing 400038,China)

机构地区：[1]陆军军医大学(第三军医大学)军事预防医学系军事毒理学教研室,重庆400038

出　　处：《中华生殖与避孕杂志》2024年第12期1265-1276,共12页Chinese Journal of Reproduction and Contraception

基　　金：国家自然科学基金青年项目(82003429);国家自然科学基金重点项目(82130097);陆军军医大学红医苗圃人才项目(2022)。

摘　　要：目的研究氯化锂(lithium chloride,LiCl)对雄性小鼠生殖毒性的作用,探讨槲皮素对睾酮生成障碍保护效应的分子机制。方法25只4~5周龄的雄性C57BL/6小鼠按照随机数字表法分为5组,分别为对照组、LiCl染毒组[38.4 mg/(kg·d)LiCl+玉米油,记为LiCl组]、槲皮素对照组[50 mg/(kg·d)槲皮素,记为High-Quer组]、低剂量槲皮素联合LiCl染毒组[38.4 mg/(kg·d)LiCl+10 mg/(kg·d)槲皮素,记为Low-Quer+LiCl组]和高剂量槲皮素联合LiCl染毒组[38.4 mg/(kg·d)LiCl+50 mg/(kg·d)槲皮素,记为High-Quer+LiCl组]。HE染色观察睾丸组织结构,计算机辅助精子分析(computer-aided sperm analysis,CASA)系统检测精液参数,透射电子显微镜观察小鼠睾丸间质细胞超微结构。采用0 mmol/L、5 mmol/L、10 mmol/L、20 mmol/L的LiCl染毒小鼠睾丸间质细胞株TM3细胞24 h后,测定细胞总超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性,TMRE探针检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),Image-iTTM脂质过氧化探针检测细胞脂质过氧化水平,分别使用Fe^(2+)含量检测试剂盒和FerroOrange探针检测细胞内Fe^(2+)含量,酶联免疫吸附法测定睾酮、孕酮和雌二醇水平,Western blotting测定睾酮等相关蛋白表达水平。结果LiCl组小鼠精子总数[(36.78±1.81)×10^(6)]、精子浓度[(18.39±0.90)×10^(6)/mL]、精子活力[(25.70±3.32)%]和血清睾酮水平[(7.26±0.29)μg/L]均比对照组[(51.60±4.96)×10^(6)、(25.80±2.48)×10^(6)/mL、(41.47±2.83)%、(7.87±0.29)μg/L]低(P=0.002、P=0.002、P=0.001、P=0.013);LiCl组小鼠睾丸间质细胞出现线粒体空泡化、肿胀,睾丸组织睾酮合成关键酶胆固醇侧链裂解酶(cholesterol side-chain cleavage enzyme,Cyp11a1)、类固醇生成急性调节蛋白(steroidogenic acute regulatory,StAR)和细胞色素P45017α-羟化酶(cytochrome P45017α-hydroxylase,Cyp17a1)、睾丸间质细胞生物标志物(3β-HSD1和17β-HSD3)�Objective To study the reproductive toxicity of lithium chloride(LiCl)in male mice and to explore the molecular mechanism of the protective effect of quercetin on testosterone production dysfunction.Methods Twenty-five male C57BL/6 mice aged 4-5 weeks were randomly divided into five groups according to the random number table method:control group,LiCl infected group[38.4 mg/(kg·d)LiCl+corn oil,noted as LiCl group],quercetin control group[50 mg/(kg·d)quercetin,noted as High-Quer group],low-dose quercetin combined with LiCl infection group[38.4 mg/(kg·d)LiCl+10 mg/(kg·d)quercetin,noted as Low-Quer+LiCl group]and high dose quercetin combined with LiCl infected group[38.4 mg/(kg·d)LiCl+50 mg/(kg·d)quercetin,noted as High-Quer+LiCl group].The structure of testicular tissue,semen parameters,and the ultrastructure of Leydig cells in mice were detected by HE staining,computer-aided sperm analysis system(CASA),and transmission electron microscopy,respectively.TM3 mouse Leydig cells were treated with 0 mmol/L,5 mmol/L,10 mmol/L,20 mmol/L LiCl for 24 h.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),mitochondrial membrane potential(MMP),lipid peroxidation levels,and expression levels of testosterone-related protein were measured by SOD and GSH-Px kits,TMRE probe,Image-iT TM lipid peroxidation probe,and Western blotting,respectively.Intracellular Fe^(2+)concentration was detected by Fe^(2+)detection kit and FerroOrange probe.The levels of testosterone,progesterone,and estradiol were measured using enzyme-linked immunosorbent assay kits.Results In the LiCl group,the total sperm count[(36.78±1.81)×10^(6)],sperm concentration[(18.39±0.90)×10^(6)/mL],sperm motility[(25.70±3.32)%]and serum testosterone level[(7.26±0.29)μg/L]were lower than those of control group[(51.60±4.96)×10^(6),P=0.002;(25.80±2.48)×10^(6)/mL,P=0.002;(41.47±2.83)%,P=0.001;(7.87±0.29)μg/L,P=0.013],the expression levels of Cyp11a1,StAR and Cyp17a1,Leydig cell biomarkers(3β-HSD1,17β-HSD3)and ferroptosis regulato

关 键 词：氯化锂 槲皮素 睾丸间质细胞 睾酮 铁死亡 

分 类 号：R73[医药卫生—肿瘤]