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