石斛夜光丸调控Gq-GPCR/PLC/氧化应激通路干预视网膜光损伤的效应机制研究

Death and degeneration of retinal pigment epithelial cells is the central mechanism underling the pathogenesis of a variety of retinal degenerative disorders, including age-related macular degeneration (AMD) and retinitis pigmentosa. Light-induced retinopathy not only shares the similar pathological changes with these retinal degenerative disorders in terms of photoreceptor cell death and RPE degeneration, it is also mechanistically implicated in the etiology of these disorders. Therefore, research aiming at effective intervention of light-induced retinopathy will offer theoretical and practical insight into the clinical management of AMD and related retinal degenerative disorders. Using AMD as an example, effective therapeutic measures are still lacking as a result of unclarified mechanisms in its pathogenesis. ShiHuYeGuangWan (SHYGW) has long been clinically applied in the treatment of AMD and other retinal degenerative disorders. Although AHYGW is recognized as therapeutically effective, the mechanisms underlining its therapeutic effects remain to be explored. Our previous studies have uncovered that light-induced retinopathy is mechanistically caused by altered functionality Gq-GPCR and NADPH oxidase-mediated ROS overproduction. Enhanced functionality of Gq-GPCR, increased PLC activity and NADPH oxidase-mediated ROS production forms a novel signaling cascade contributing to the photoreceptor cell death during light-induced retinopathy. Therefore, our current study propose to evaluate and compare the protective effects of SHYGW and major formula derived from SHYGW, namely BuShen and QinGan composition, on photoreceptor cells and RPE. Further mechanistic studies will be focused on elucidating the impact of each of herbal formulation on Gq-GPCR/PLC/NADPH oxidase/ROS. In the end, we aim to delineate the cellular target and molecular element that mediate the effect of SHYGW, which will ultimately provide scientific and experimental evidence supporting the clinical application of SHYGW in the treatment of retinal degenerative disorders.

光感受器细胞死亡及色素上皮细胞变性是多种视网膜退行性病变的共同病理环节，包括年龄相关性黄斑变性、视网膜色素变性等。视网膜光损伤病理表现与上述退行性病变一致，同时也是这些疾病的发病机制之一。因此，针对光损伤光感受器细胞死亡和色素上皮细胞变性的有效干预对多种视网膜退行性病变的治疗研究有着积极的理论和临床指导意义。石斛夜光丸是临床治疗视网膜退行性病变的经典有效方剂，然而其效应机制研究存在大量空白。申请人前期工作提示"Gq-GPCR/PLC/氧化应激"通路是光感受器细胞及色素上皮细胞病理改变的关键调控机制。本项目将以光感受器及色素上皮细胞为效应靶点，对石斛夜光丸及其补肾明目、清肝明目拆方的视网膜保护作用进行比较研究，并深入探讨各中药组方对"Gq-GPCR/PLC/氧化应激"信号级联反应的影响, 明确其各自明目效应的细胞靶点及分子效应环节，为其在视网膜退行性病变治疗中的应用提供科学的理论和实验依据。

光感受器细胞的退行性改变是多种视网膜退行性疾病发生视觉损害甚至致盲的核心及终端病理环节之一，目前现代医学尚缺乏有效的光感受器细胞保护手段。本项目围绕光感受器细胞死亡，对经典明目复方石斛夜光丸的视网膜保护效应进行系统的研究，主要的研究结果如下：1、石斛夜光丸具有显著的光感受器细胞结构及功能保护效应。2、石斛夜光丸具有显著的抗光感受器细胞凋亡的效应，可能是其发挥光感受器细胞保护效应的重要细胞生物学基础之一；石斛夜光丸对光感受器细胞凋亡相关基因的表达起到显著的调节效应，是其发挥抗光感受器细胞凋亡的重要分子生物学机制之一。3、研究结果提示光感受器细胞死亡进程中伴随视网膜二级神经元双极细胞、水平细胞的退行性改变，石斛夜光丸对双极细胞及水平细胞可起到显著的保护效应，可能是其发挥视网膜功能保护的重要细胞生物学基础之一，同时提示石斛夜光丸对视觉信号传导通路完整性的维持具有重要的意义。4、石斛夜光丸显著下调光损伤视网膜cAMP的水平，提示其具有下调Gs偶联的GPCR通路功能性的效应，为其光感受器细胞保护效应提供了新的机制认识。6、石斛夜光丸对光感受器细胞死亡相关反应性神经胶质增生、小胶质细胞激活等病理改变具有显著的抑制效应，提示其有助于损伤视网膜结构和功能的重塑。7、石斛夜光丸具有显著的抗视网膜炎症反应、改善视网膜能量代谢及线粒体保护效应，是其视网膜保护效应的可能途径之一。8、石斛夜光丸中滋补肝肾药物、白蒺藜及人参皂苷等药物组成或组分具有显著的光感受器细胞保护及抗氧化、抗炎效应，可能是其发挥视网膜保护效应的物质基础之一。综上所述，本研究阐明了经典明目方药石斛夜光丸的光感受器细胞保护效应及其相关细胞、分子机制，并提示了其光感受器细胞保护效应可能的物质基础，不仅为更好地理解石斛夜光丸的视网膜保护效应提供了新的实验依据，同时为石斛夜光丸临床应用于以光感受器细胞为核心病理改变的视网膜退行性疾病的防治提供了科学的实验和理论依据。