Sirt1在分子氢保护光诱导体内/体外视网膜感光细胞损伤中的作用与机制研究

Age-related macular degeneration（AMD）is a main reason for the visual impairment in aging population, and an important research field focusing on the prevention and control of AMD is anti-oxidative damage due to that oxidative stress injury is a leading cause of AMD. Recently, some researchers discovered the selective anti-oxidant effect of molecular hydrogen, however, the mechanism remains to be not fully elucidated. Our previous vivo animal experiments indicated that the molecular hydrogen could protect the rat retina from light induced damage, which was closely related to Sirt1. In the present study, the visible light exposure was performed to induce light damage to construct in vitro light-damage cellular model, and then hydrogen-rich culture medium was administrated to investigate its selective effect of free radical as well as the underlying mechanism of anti-oxidant and anti-apoptosis. After that, the protective effect was detected after changing the Sirt1 upstream conditions as inhibit, activate, and inhibit the pathway after activating it. And then the activated level changes of downstream proteins such as FOXO family, Ku70 and NF-κB were analyzed, and the role of Sirt1 in the protective effects of molecular hydrogen through preventing oxidant stress and cell apoptosis were also studied. Lastly, the rat model with light-induced retinal damage was constructed, and then retinal protective effect of molecular hydrogen mediated by Sirt1 and its underlying mechanism were also furtherly investigated. The present study has a potential benefit for the diagnosis and treatment of light-induced related retinal diseases, and provides a useful theoretical reference for the molecular mechanism of hydrogen gas.

年龄相关性黄斑变性是导致老龄人口视力下降的主要原因，其发病机制涉及氧化应激损伤，因此抗氧化损伤措施一直是防治该类型疾病的重要研究领域。最近研究发现分子氢具有选择性抗氧化损伤作用，但因其机制不清而尚未被广泛认可。课题组前期实验显示，分子氢可在大鼠视网膜光损伤模型中发挥感光细胞保护作用，且可能与Sirt1分子密切相关，但其具体机制尚不明确。本研究将首先构建体外感光细胞光损伤模型，分析分子氢的选择性抗氧化作用。之后，改变Sirt1上游条件即抑制、激活、激活后抑制，分析其在分子氢细胞保护作用中的调控规律，并对Sirt1下游蛋白FOXO家族、Ku70、NF-κB等分子定量分析，探究分子氢保护作用所涉及的Sirt1信号通路。最后，在体内大鼠视网膜光损伤模型，对Sirt1信号通路进一步验证及评价。本研究将有望为视网膜退行性变相关疾病的预防和临床治疗新靶点提供方向，并为氢气的分子学机制提供有益的理论依据。

本研究在前期研究基础上，进一步深入探讨了氧化应激在视网膜光损化中的作用规律与机制，发现过度的氧化应激反应是引起光感受器细胞减少的重要原因，包括ROS的过度产生和LDH的过度堆积，进而引起光感受器细胞的凋亡增加，分子机制包括促凋亡基因caspase9和Bax的表达增加和抑凋亡基因Bcl-2的表达降低。应用富氢培养基和富氢生理盐水均可减轻光诱导的视网膜感光细胞损伤，表现为LDH细胞毒性的降低和抑制感光细胞数的减少，其分子机制主要是抗氧化和抗凋亡。应用富氢能够抑制ROS的累积，提高抑凋亡基因Bcl-2的表达，降低促凋亡基因caspase9和Bax的表达，进而使感光细胞凋亡率降低。分子氢的这一生物学作用能够被Sirt1的抑制剂反转，此外，应用Sirt1的激动剂能够模拟富氢对视网膜光损伤的保护作用，提示Sirt1分子可能是分子氢发挥抗氧化和抗凋亡作用的重要分子机制。此外，本研究进一步还发现，光诱导的感光细胞损伤后同时引起Sirt1分子的表达降低，应用富氢可使Sirt1分子的表达增高，同时还可提高光诱导后FoxO1及FoxO3的表达，这一结果同样可被Sirt1抑制剂所拮抗，而Sirt1的激动剂同样能够模拟分子氢引起的Sirt1-FoxO1/FoxO3通路变化。以上这些结果提示，分子氢的保护作用可能与Sirt1-FoxO1/FoxO3通路介导的抗氧化应激途径和抗凋亡途径密切相关。

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