The circadian clock mediates the response to oxidative stress in a cone photoreceptor‒like (661W) cell line via regulation of glutathione peroxidase activity.
The circadian clock mediates the response to oxidative stress in a cone photoreceptor‒like (661W) cell line via regulation of glutathione peroxidase activity.
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DOI：
10.12688/f1000research.125133.2
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发表时间：
2022
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背景：哺乳动物的视网膜包含一个自主的生物钟，控制着视网膜内的许多生理功能。我们以前的研究表明，通过从视网膜中去除Bmal 1来破坏这种生物钟会影响衰老过程中的视觉功能、视网膜回路和视锥光感受器的活力。在本研究中，我们采用了一个小鼠源性的锥光感受器类神经元细胞，661 W，研究生物钟的分子机制可能会调节锥光感受器的活力在老化过程中。 方法：使用CRISPR/Cas9基因编辑工具从661 W细胞产生Bmal 1敲除（BKO）细胞。通过蛋白质印迹和监测Per 2-luc生物发光昼夜节律来验证661 W中Bmal 1的缺失。为了研究去除Bmal 1对氧化应激攻击的影响，用过氧化氢（H2 O2，1 mM）处理细胞2小时，然后评估细胞活力。还培养并收获细胞用于基因表达分析和抗氧化剂测定。 结果如下：我们的数据表明，661 W细胞含有一个功能性的昼夜节律钟，介导的反应，在体外的氧化应激挑战，这种反应不再存在于BKO细胞。结果表明，661 W细胞的抗氧化防御机制呈现时间依赖性变化，而BKO细胞的抗氧化防御机制总体上减弱，不再呈现时间依赖性变化。 结论：我们的工作支持了这样一种观点，即功能性生物钟的存在及其调节氧化应激反应的能力是在衰老过程中保护视锥细胞的潜在机制。
Background: The mammalian retina contains an autonomous circadian clock that controls many physiological functions within this tissue. Our previous studies have indicated that disruption of this circadian clock by removing Bmal1 from the retina affects the visual function, retinal circuitry, and cone photoreceptor viability during aging. In the present study, we employed a mouse-derived cone photoreceptor‒like cell, 661W, to investigate which molecular mechanisms of the circadian clock may modulate cone photoreceptor viability during aging. Methods: Bmal1 knockout (BKO) cells were generated from 661W cells using the CRISPR/Cas9 gene editing tool. Deletion of Bmal1 from 661W was verified by western blot and monitoring Per2-luc bioluminescence circadian rhythms. To investigate the effect of Bmal1 removal on an oxidative stress challenge, cells were treated with hydrogen peroxide (H 2O 2,1 mM) for two hours and then cell viability was assessed. Cells were also cultured and harvested for gene expression analysis and antioxidant assay. Results: Our data indicated that 661W cells contain a functional circadian clock that mediates the response to an oxidative stress challenge in vitro and that such a response is no longer present in the BKO cell. We also hypothesized that the effect was due to the circadian regulation of the intracellular antioxidant defense mechanism. Our results revealed that in 661W cells, the antioxidant defense mechanism showed time dependent variation , whereas in BKO cells, there was an overall reduction in this antioxidant defense mechanism, and it no longer showed time dependent variation. Conclusions: Our work supported the notion that the presence of a functional circadian clock and its ability to modulate the response to an oxidative stress is the underlying mechanism that may protect cones during aging.