GSK4112对尼罗罗非鱼肝脏Nr1d1和Ulk1b基因昼夜节律性表达的影响

Effects of GSK4112 on Circadian Rhythmicity Expression of Nr1d1 and Ulk1b in Liver of Nile Tilapia(Oreochromis niloticus)

为了探究GSK4112对生物钟以及自噬基因在尼罗罗非鱼(Oreochromis niloticus)肝脏的昼夜表达规律的作用。本试验分别对尼罗罗非鱼注射等量生理盐水和GSK4112,并在注射24 h后的06:00、09:00、12:00、15:00、18:00、21:00、24:00时以及翌日03:00、06:00时采样,采样在一昼夜内完成。每组随机取3尾鱼进行解剖,取其肝脏样品采用RT-qPCR对mRNA表达水平,进行Matlab余弦分析,确定其节律性表达,并将注射前后肝脏样品用透射电镜检测自噬小体数量变化。结果表明:生物钟基因Nr1d1与自噬基因Ulk1b在对照组的尼罗罗非鱼肝脏中表达均具有显著性时间差异(P<0.05),且均呈现节律性振荡(P<0.3);但基因表达趋势不同,且达到峰值的时间亦不同。注射Nr1d1激动剂GSK4112后Nr1d1与Ulk1b在尼罗罗非鱼肝脏中均无昼夜节律性,虽然达到峰值的时间不同,但基因表达趋势相同,mRNA表达水平均表达量受到抑制,振幅和中值减小,且自噬小体数量也减少。这些结论表明了GSK4112在尼罗罗非鱼肝脏中对生物钟基因Nr1d1的表达起到抑制作用,同时影响其生物节律性。通过对下游自噬基因Ulk1b的昼夜节律调控,抑制自噬小体的数量,降低自噬的发生。本研究对鱼类由昼夜节律失调引起的自噬失调可能导致常见的代谢紊乱具有指导作用。

To investigate the effects of GSK4112 on circadian expression of clock and autophagy genes in the liver of Nile Tilapia(Oreochromis niloticus).Nile tilapia were injected with the same amount of saline and GSK4112 respectively,and sampled at 06:00,09:00,12:00,15:00,18:00,21:00,24:00,and 03:00,06:00 the next day after24 hours of injection.Three fish in each group were randomly dissected,and the mRNA level of liver tissue were determined by RT-qPCR.The rhythmic expression was determined by MATLAB cosine analysis,while the number of autophagosomes were detected in liver before and after injection of GSK4112 by transmission electron microscopy.The results showed that the expression of clock gene Nr1 d1 and autophagy gene Ulk1 b mRNA in control group had significant time difference(P<0.05),and both showed rhythmicity(P<0.3),but the trend of gene expression was different,and the peak time were also different in the liver of Nile tilapia.After the injection of Nr1 d1 agonist GSK4112,both Nr1 d1 and Ulk1 b showed no circadian rhythm.Although the time to reaching the peak time was different,but the trend of gene expression was similar.The expression level of mRNA were inhibited,the amplitude and median values were decreased,and the number of autophagosomes was also decreased.These results found that GSK4112 inhibits the expression of clock gene Nr1 d1 and affects its biological rhythm in the liver of Nile tilapia,which by regulating the circadian rhythm of downstream autophagy gene Ulk1 b,the number of autophagosomes was inhibited and the occurrence of autophagy was reduced.This study may provide guidance for the autophagy disorders caused by circadian rhythm disorders in fish,which may lead to common metabolic disorders.