钟基因Bmal1对褪黑素抑制TM3细胞睾酮节律性分泌的作用研究  

The effect of Bmal1 on the testosterone circadian synthesis inhibited by melatonin in mouse leydig cells

作　　者：钱琳 韩静静[1] 郭依晨 严锐[1] 童建[1] 肖卫[1] 张洁[1] QIAN Lin, HAN Jing-jing, GUO Yi-chen, YAN Rui, TONG Jian, XIAO Wei, ZHANG Jie(School of Public Health, Medical College of Soochow University, jiangsu Suzhou 215123, Chin)

机构地区：[1]苏州大学公共卫生学院,江苏苏州215123

出　　处：《毒理学杂志》2018年第1期35-39,40,共6页Journal of Toxicology

摘　　要：目的研究褪黑素对TM3细胞睾酮分泌节律的影响,探讨钟基因Bmal1在褪黑素抑制TM3细胞睾酮节律性分泌的作用。方法运用地塞米松诱导小鼠睾丸间质细胞(TM3细胞)内源性节律的产生,通过ELISA试剂盒和实时定量RT-q PCR检测TM3细胞睾酮及类固醇急性调节基因St AR水平,研究褪黑素对睾酮节律性分泌的作用。运用siRNA干扰技术,下调钟基因Bmal1的表达,研究Bmal1在褪黑素抑制TM3细胞睾酮节律性分泌过程中的作用。结果10-6mol/褪黑素处理后,TM3细胞的睾酮浓度在ZT0、ZT4、ZT8、ZT12、ZT16、ZT20和ZT24各授时时点(zeitgeber time,ZT,光照开始的时间为ZT0)与对照组相比均显著下降,差异有统计学意义(P<0.05),St AR水平在ZT0、ZT4、ZT8和ZT12时点较对照组显著降低(P<0.05)。余玄分析结果显示,地塞米松作用后TM3细胞的睾酮分泌及St AR基因表达具有一定的节律性,峰值时间分别为ZT11:40和ZT8:06。褪黑素处理后,褪黑素组睾酮及St AR的节律均发生变化,表现为中值分别下降36.53%和32.09%,振幅分别减弱18.54%和44.44%。Bmal1干扰后,与对照组相比,褪黑素作用于Bmal1低表达的细胞,睾酮浓度、St AR水平及睾酮分泌节律改变差异均无统计学意义(P>0.05);对St AR节律性表达的影响减小。结论适当剂量的褪黑素不仅可抑制TM3细胞中睾酮水平及St AR的转录水平,而且能改变两者的节律性表达。钟基因Bmal1在褪黑素抑制TM3细胞睾酮节律性分泌过程中发挥了一定的调控作用。Objective To evaluate the effect of melatonin on the circadian synthesis of testosterone and the role of the inhibition of testosterone synthesis by melatonin in TM3 cells. Methods Mouse leydig cells（TM3 cells） were synchronized using dexamethasone.Testosterone concentration and transcriptional level of St AR were evaluated by ELISA and RT-q PCR. Bmal1-specific small interfering RNA（siRNA） was utilized to knockdown Bmal1. Results Compared with the control group,melatonin inhibited the testosterone synthesis at all zeitgeber time（ZT0、ZT4,ZT8,ZT12,ZT16,ZT20 and ZT24,P〈0. 05）,and decreased St AR level at ZT0、ZT4、ZT8 and ZT12（P〈0. 05）. Cosinor analysis revealed the existence of circadian rhythm in the synthesis of testosterone in TM3 cells,and 24 h-rhythmic expression of steroidogenesis genes St AR in TM3 cells. The peak time were ZT11： 40 and ZT8： 06 respectively. Melatonin decreased 24 h oscillations of testosterone and St AR,i. e. decreased mesor 36. 53% and 32. 09%,respectively and amplitude 18. 54% and 44. 44%,respectively. All disturbed parameters were restored by the knockdown of Bmal1 gene（P〉0. 05）. Conclusion Melatonin at certain doses exerted inhibitory actions on circadian rhythm of testosterone synthesis in TM3 cells. Bmal1 might be involved in the inhibiting process of testosterone circadian synthesis by melatonin.

关 键 词：褪黑素 昼夜节律 睾酮 BMAL1 

分 类 号：R99[医药卫生—毒理学]