活性氧在氯化镉诱导的小鼠睾丸间质细胞凋亡中的作用

Effect of reactive oxygen species in cadmium chloride induced apoptosis of mouse Leydig cells

目的探究活性氧(reactive oxygen species,ROS)在氯化镉(cadmium chloride,CdCl_(2))诱导的睾丸间质细胞毒性中的潜在作用及机制。方法以不同浓度CdCl_(2)(0、5和10μmol/L)染毒小鼠睾丸间质TM3细胞24 h。CCK-8法检测CdCl_(2)对TM3细胞活性的影响;Hoechst33342染色法检测凋亡小体;DCFH-DA探针法检测TM3细胞ROS水平;1 mmol/L N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)预处理1 h后用10μmol/L CdCl_(2)染毒TM3细胞24 h,Western blot检测细胞内促凋亡蛋白Caspase-9和cleaved Caspase-3的表达水平;RT-qPCR检测细胞内抗凋亡基因Bcl-2以及促凋亡基因Caspase-9和Caspase-3 mRNA表达水平。结果CdCl_(2)染毒细胞24 h后,TM3细胞活力降低且凋亡小体数量增加。10μmol/L CdCl_(2)染毒组细胞Caspase-9蛋白表达水平为0.86±0.10,较对照组(0.56±0.07)升高(P<0.05);5和10μmol/L CdCl_(2)染毒组细胞cleaved Caspase-3蛋白表达水平分别为0.65±0.03和1.05±0.13,较对照组(0.37±0.11)升高(P<0.05);5和10μmol/L CdCl_(2)染毒组细胞内ROS含量分别为60.47±1.39和80.63±1.34,亦较对照组(46.80±1.24)增加(P<0.05)。与镉染毒组相比,NAC抑制了Caspase-9(CdCl_(2)组:0.89±0.07;CdCl_(2)+NAC组:0.28±0.02)和cleaved Caspase-3(CdCl_(2)组:1.53±0.21;CdCl_(2)+NAC组:0.66±0.07)蛋白的表达,差异有统计学意义(P<0.05);同时CdCl_(2)+NAC组ROS水平(62.64±0.93)也较CdCl_(2)染毒组(80.13±0.94)降低(P<0.05)。5和10μmol/L CdCl_(2)染毒组细胞Caspase-9 mRNA水平与对照组(0.97±0.10)相比分别升高为1.40±0.14和1.90±0.12(P<0.05),cleaved Caspase-3 mRNA水平与对照组(0.88±0.08)相比分别升高为1.42±0.11和1.59±0.12(P<0.05),但Bcl-2 mRNA水平与对照组(0.94±0.02)相比分别降低为0.60±0.02和0.50±0.09(P<0.05)。与镉染毒组相比(0.57±0.06),CdCl_(2)+NAC组镉诱导的Bcl-2 mRNA表达水平显著改善(0.92±0.03),Caspase-9(CdCl_(2)组:1.96±0.07;CdCl_(2)+NAC组:1.04±0.02)和Caspase-3(CdCl_(2)组:1.65±0.02;CdCl_(2)+NAC组:0.66±0.04)的表达水平降低(P<0.05)。结论小鼠睾丸间质细胞TM3中的Caspase级联反应可被CdCl_(2)诱导的过量ROS激活,抑制ROS可显著降低CdCl_(2)诱导的TM3细胞凋亡。

OBJECTIVE To study the effect of reactive oxygen species(ROS)in cadmium chloride-induced apoptosis of mouse Leydig cells(TM3 cells)and explore the underlying molecular mechanisms.METHODS TM3 cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure.The cells were treated with different concentrations of CdCl_(2)(0,5 and 10μmol/L)for 24 h.CCK-8 assay was used to detect the effect of CdCl_(2)on TM3 cell activity.Hoechst33342 staining was performed to explore the formation of apoptotic bodies.DCFH-DA probe was used to detect the level of ROS in the cells.TM3 cells were pretreated with 1 mmol/L NAC for 1 h and then treated with 10μmol/L CdCl_(2)for 24 h.The protein expression levels of pro-apoptotic proteins Caspase-9 and cleaved Caspase-3 were detected by Western blot;RT-qPCR was used to measure the expression of anti-apoptotic gene Bcl-2 and pro-apoptotic genes Caspase-9 and Caspase-3.RESULTS After exposure to CdCl_(2)for 24 h,viability of TM3 cells decreased and the number of apoptotic bodies increased.Western blot result showed that the protein level of Caspase-9 in the 10μmol/L CdCl_(2)treatment group was increased to 0.86±0.10(P<0.05)compared with the control group(0.56±0.07).Compared with the control group(0.37±0.11),the protein level of cleaved Caspase-3 in the 5 and 10μmol/L CdCl_(2)treatment groups were increased to 0.65±0.03 and 1.05±0.13(P<0.05).Compared with the control group(46.80±1.24),the intracellular ROS content in the 5 and 10μmol/L treatment groups increased to 60.47±1.39 and 80.63±1.34(P<0.05).Compared with the cadmium-treated group,NAC inhibited Caspase-9(CdCl_(2)group:0.89±0.07;CdCl_(2)+NAC group:0.28±0.02)and cleaved Caspase-3(CdCl_(2)group:1.53±0.21;CdCl_(2)+NAC group:0.66±0.07),the difference was statistically significant(P<0.05).At the same time,NAC decreased the ROS level(62.64±0.93)in the CdCl_(2)exposure group(80.13±0.94)(P<0.05).In addition,RT-qPCR result showed that the Caspase-9 mRNA levels in the 5 and 10μmol/L CdCl_(2)treatment groups were 1.40±0.14 and 1.90±0.12(P<0.05),compared with the control group(0.97±0.10).Compared with the control group(0.88±0.08),the cleaved Caspase-3 mRNA levels in the 5 and 10μmol/L CdCl_(2)treatment groups were increased to 1.42±0.11 and 1.59±0.12(P<0.05).While in the 5 and 10μmol/L CdCl_(2)-treated group,compared with the control group(0.94±0.02),the Bcl-2 mRNA level were decreased to 0.60±0.02 and 0.50±0.09(P<0.05).Compared with the cadmium treatment group(0.57±0.06),NAC could significantly improve the cadmium-induced Bcl-2 mRNA expression level(0.92±0.03),and Caspase-9(CdCl_(2)group:1.96±0.07;CdCl_(2)+NAC group:1.04±0.02)and Caspase-3(CdCl_(2)group:1.65±0.02;CdCl_(2)+NAC group:0.66±0.04)were decreased(P<0.05).CONCLUSION The Caspase cascade in mouse Leydig cells can be activated by excessive ROS induced by CdCl_(2),and inhibition of ROS production can significantly reduce the CdCl_(2)-induced apoptosis of TM3 cells.