Role of BMAL1 and CLOCK in regulating the secretion of melatonin in chick retina under monochromatic green light
Role of BMAL1 and CLOCK in regulating the secretion of melatonin in chick retina under monochromatic green light
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单色绿光下BMAL1和CLOCK在调节鸡视网膜褪黑素分泌中的作用
DOI：
10.1080/07420528.2020.1830790
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发表时间：
2020-10
影响因子：
2.8
通讯作者：
Yaoxing Chen
中科院分区：
文献类型：
--
作者：
Jiang Bian;Zixu Wang;Yulan Dong;Jing Cao;Yaoxing Chen
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。先前的研究证实，单色绿光可以加速鸡视网膜生物钟基因的昼夜节律表达，从而增加cAanat mRNA水平和褪黑激素分泌。然而，作为转录-翻译负反馈回路的核心组成部分，cBmal1和clock在单色绿光下调控视网膜分子时钟系统和褪黑激素分泌中的作用尚不清楚。为了探究其在这一过程中的作用，本研究分离了6个胎龄的鸡胚视网膜细胞，在光暗（LD） 12:12单色绿光下培养，通过siRNA干扰和过表达cBmal1和clock在鸡视网膜分子钟和褪黑激素分泌调控中的作用。结果显示，siRNA干扰和cBmal1的过表达会破坏cCry1、cPer2、cPer3、cAanat和褪黑激素分泌的昼夜节律。此外，cBmal1的siRNA干扰显著降低了阳性时钟基因cBmal2和cClock、阳性时钟蛋白clock、阴性时钟基因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。然而，siRNA干扰和过表达cClock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，而仅影响cCry1、cPer2、cPer3、cAanat和褪黑激素的昼夜节律因子（24小时时间序列均值）、振幅和峰期（峰值时间），以及心律失常cBmal2和cCry2的平均水平。干扰和过表达cClock不影响cBmal1 mRNA水平和BMAL1蛋白表达。上述结果表明，干扰和过表达cBmal1完全消除了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位于禽类视网膜分子时钟反馈回路的顶端，调节单色绿光下下游分子时钟振荡和输出。时钟在维持视网膜细胞内分子钟的昼夜振荡和褪黑激素分泌中起从属作用，对分子钟振荡具有稳定和放大作用。
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.
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