ABSTRACT

As the circadian pacemaker of birds, the retina possesses the ability to receive light information, generate circadian oscillation, and secrete melatonin. Previous studies have confirmed that monochromatic green light can accelerate the circadian rhythmic expression of clock genes in the chick retina, thereby increasing cAanat mRNA level and melatonin secretion. However, as the core components of the transcriptional-translational negative feedback loop, the role that cBmal1 and cClock plays in the regulation of the retinal molecular clock system and melatonin secretion under monochromatic green light is unknown. To explore their in these processes, embryonic chick retinal cells at six embryo ages were isolated and cultured under light-dark (LD) 12:12 monochromatic green light with, and the role of cBmal1 and cClock in the regulation of the retinal molecular clock and melatonin secretion in the chick retina was explored by siRNA interference and overexpression. The results showed siRNA interference and overexpression of cBmal1 obliterated the circadian rhythm of cCry1, cPer2, cPer3, cAanat, and melatonin secretion. Moreover, the siRNA interference of cBmal1 significantly reduced the average expression levels of the positive clock genes cBmal2 and cClock, positive clock protein CLOCK, negative clock genes cCry1, cCry2, cPer2, cPer3, as well as cAanat and retinal melatonin. The over-expression of cBmal1 increased the average levels of the above-detected targets. However, siRNA interference and overexpression of cClock did not change the rhythm of all of the clock genes, clock proteins, cAanat, and melatonin secretion, while it only affected the circadian mesors (24 h time series means), amplitudes, and acrophases (peak times) of cCry1, cPer2, cPer3, cAanat, and melatonin, as well as the average levels of arrhythmic cBmal2 and cCry2. Moreover, interference and overexpression of cClock did not affect cBmal1 mRNA level and BMAL1 protein expression. The above results reveal interference and overexpression of cBmal1 completely abolished the molecular circadian oscillation and the rhythm of melatonin output signal of chick retinal cells, indicating that cBmal1 is on the top of the avian retinal molecular clock feedback loop and regulates the downstream molecular clock oscillation and output under monochromatic green light. cClock plays a subordinate role in maintaining the circadian oscillation of the molecular clock and melatonin secretion in retinal cells, and it has a stabilizing and amplifying effect on molecular clock oscillation.

摘要

作为鸟类的昼夜节律起搏器，视网膜具有接收光信息，产生昼夜节律振荡和分泌褪黑激素的能力。先前的研究已经证实，单色绿光可以加速小鸡视网膜中时钟基因的昼夜节律性表达，从而增加cAanat mRNA水平和褪黑激素分泌。但是，作为转录-翻译负反馈回路的核心组成部分，cBmal1和cClock的作用在视网膜分子时钟系统的调节中起着作用，单色绿光下褪黑激素的分泌尚不清楚。为了探索它们在这些过程中的作用，分离了六个胚胎年龄的雏鸡视网膜细胞，并在光暗（LD）12:12单色绿光下培养，其中cBmal1和cClock在调节视网膜分子时钟和通过siRNA干扰和过表达来探索雏鸡视网膜中的褪黑激素分泌。结果表明，siRNA干扰和cBmal1的过表达消除了cCry1，cPer2，cPer3，cAanat和褪黑激素分泌的昼夜节律。此外，cBmal1的siRNA干扰显着降低了正时钟基因cBmal2和cClock，正时钟蛋白质CLOCK，负时钟基因cCry1，cCry2，cPer2，cPer3以及cAanat和视网膜褪黑激素的平均表达水平。cBmal1的过表达增加了上述检测到的靶标的平均水平。但是，siRNA干扰和cClock的过表达并没有改变所有时钟基因，时钟蛋白，cAanat和褪黑激素分泌的节律，而只影响了昼夜节律（24小时时间序列平均值），振幅和顶峰（峰值）。次）的cCry1，cPer2，cPer3，cAanat和褪黑激素，以及心律不齐cBmal2和cCry2的平均水平。此外，cClock的干扰和过表达不影响cBmal1 mRNA水平和BMAL1蛋白表达。以上结果表明cBmal1的干扰和过度表达完全消除了雏鸡视网膜细胞的分子昼夜节律振荡和褪黑激素输出信号的节律，表明cBmal1位于禽视网膜分子时钟反馈回路的顶部，并调节下游分子时钟振荡和在单色绿光下输出。时钟 在维持视网膜细胞中分子时钟的昼夜节律振荡和褪黑激素分泌中起次要作用，并且对分子时钟振荡具有稳定和放大作用。