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Fe2+含量检测试剂盒和FerroOrange探针检测细胞内Fe2+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)×106]、精子浓度[(18.39±0.90)×106/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)×106、(25.80±2.48)×106/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P450 17α-羟化酶(cytochrome P450 17α-hydroxylase,Cyp17a1)、睾丸间质细胞生物标志物(3β-HSD1和17β-HSD3)和铁死亡调控蛋白(GPX4、SLC7A11和Nrf2)的表达水平均较对照组显著下调(均P<0.001),而低剂量槲皮素干预可显著改善LiCl诱导的上述损伤(精子总数:P<0.001,精子浓度:P<0.001,精子活力:P=0.015,血清睾酮:P=0.026,Cyp11a1、StAR、Cyp17a1、3β-HSD1、17β-HSD3、GPX4、SLC7A11 和 Nrf2:均 P<0.001).20 mmol/L LiCl 组 TM3 细 胞 睾 酮 水 平[(7.28±0.24)μg/L]比0 mmol/L LiCl组[(12.50±0.38)μg/L]低(P<0.001),睾酮合成关键酶Cyp11a1、StAR和Cyp17a1蛋白水平均较0 mmol/L LiCl组显著下调(均P<0.001),SOD[(2.42±0.11)U/mg]、GSH-Px活性[(1.29±0.03)mU/mg]和MMP[(57.24±1.69)%]均比0 mmol/L LiCl组[(3.11±0.09)U/mg、(1.54±0.01)mU/mg、(100.00±0)%]低(均P<0.001),脂质过氧化水平[(211.18±3.60)%]和Fe2+含量[(26.44±0.94)μmol/L]均比 0 mmol/L LiCl组[(100.00±0)%、(7.12±0.29)μmol/L]高(均P<0.001),GPX4、SLC7A11和Nrf2蛋白表达水平均较0 mmol/L LiCl组显著下调(均P<0.001).与20 mmol/L LiCl染毒组[脂质过氧化:(194.46±3.16)%、(194.70±3.93)%;MMP:(78.74±0.52)%、(75.32±1.29)%]比较,铁死亡抑制剂Fer-1和槲皮素均显著降低细胞脂质过氧化水平[(181.71±3.80)%,P=0.004;(166.88±3.22)%,P<0.001],提高细胞MMP[(86.26±0.79)%,P=0.040;(81.09±1.32)%,P=0.001],并显著上调睾酮合成关键酶以及铁死亡调控蛋白的表达水平(均P<0.05).结论 槲皮素可能通过抑制细胞铁死亡保护LiCl诱导的睾丸间质细胞损伤和睾酮生成障碍.
Abstract
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-iTTM lipid peroxidation probe,and Western blotting,respectively.Intracellular Fe2+concentration was detected by Fe2+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)×106],sperm concentration[(18.39±0.90)×106/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)×106,P=0.002;(25.80±2.48)×106/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 regulatory proteins(GPX4,SLC7A11 and Nrf2)in testis were significantly down-regulated compared with control group(all P<0.001).LiCl also induced mitochondrial vacuoles and swelling of Leydig cells.However,low-dose quercetin intervention could significantly ameliorate the above LiCl-induced damage(total sperm count:P<0.001,sperm concentration:P<0.001,sperm motility:P=0.015,serum testosterone level:P=0.026,Cyp11a1,StAR,Cyp17a1,3β-HSD1,17β-HSD3,GPX4,SLC7A11,and Nrf2:all P<0.001).In 20 mmol/L LiCl group,the testosterone level[(7.28±0.24)μg/L]in TM3 cells was lower than that of 0 mmol/L LiCl group[(12.50±0.38)μg/L,P<0.001],the protein levels of Cyp11a1,StAR and Cyp17a1 were significantly down-regulated compared with 0 mmol/L LiCl group(all P<0.001),the activities of SOD[(2.42±0.11)U/mg],GSH-Px[(1.29±0.03)mU/mg]and MMP[(57.24±1.69)%]were lower than those of 0 mmol/L LiCl group[(3.11±0.09)U/mg,(1.54±0.01)mU/mg,(100.00±0)%,all P<0.001],the level of lipid peroxidation[(211.18±3.60)%]and the concentration of Fe2+[(26.44±0.94)μmol/L]were higher than those of 0 mmol/L LiCl group[(100.00±0)%,(7.12±0.29)μmol/L,all P<0.001],and the expression of ferroptosis regulatory proteins was significantly down-regulated compared with 0 mmol/L LiCl group(all P<0.001).Compared with 20 mmol/L LiCl group cells[lipid peroxidation:(194.46±3.16)%,(194.70±3.93)%;MMP:(78.74±0.52)%,(75.32±1.29)%],ferroptosis inhibitors Fer-1 and quercetin significantly decreased the level of lipid peroxidation[(181.71±3.80)%,P=0.004;(166.88±3.22)%,P<0.001],increased the level of MMP[(86.26±0.79)%,P=0.040;(81.09±1.32)%,P=0.001],and significantly up-regulated the expression of key enzymes in testosterone synthesis and ferroptosis regulatory proteins(all P<0.05).Conclusion Quercetin may protect LiCl-induced Leydig cells injury and testosterone production dysfunction by inhibiting cell ferroptosis.
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