基于HA/CD44信号通路研究肝肾同补法对实验性高度近视光感受器细胞凋亡的干预作用及分子机制

In the late period of high myopia, retinal posterior atrophy connected with retinal photoreceptor apoptosis often cause serious visual impairment, and there is no effective treatment currently. Integral and local syndrome differentiation of Traditional Chinese medicine (TCM) considers that such pathologic change relates to the deficiency of liver and kidney. Through clinical observation, we have found that the Bujingyishi tablet which has the effect of tonifying liver and kidney, can regulate and control the visual function of patients having high myopia, and has also the significant intervention effect on the bcl-2 and caspase3 factors causing apoptosis of the retina of animals having myopia. So we put forword hypothesis that“The method of tonifying liver and kidney to regulate and control the apoptosis of experimental high myopia retinal photoreceptor through the signaling pathway of HA/CD44-miR-21-Bcl2/IAP.Using the method of tonifying liver and kidney to intervent experimental high myopia C57BL/6Jmice, HA/CD44-miR-21-Bcl2/IAP signaling pathway as the core, and to carry out mfERG testing, Tunnel staining, western blotting, immunoblotting technique, qPCR, transcriptome sequencing and miRNA sequencing and so on to test the photoreceptor and its adjacent retinal pigmental epithelium(RPE), choroid vascular endothelial cell and sclera fibroblast, and to verify the effect of the target and molecular mechanism of tonifying liver and kidney to regulate and control the apoptosis of retinal photoreceptor through the joint analysis of the function, cell, protein and gene. The purpose of this research is to provide the experimental and theory basis for guiding the clinical early intervention of high myopia, rich the theory of prevention of “preventing disease from exacerbating”.

高度近视晚期常见后极部视网膜萎缩致视力严重受损，与视网膜光感受器凋亡密切相关，目前尚无有效治疗方法。中医整体与局部辨证认为肝肾亏虚为其基本病机，临床观察证实肝肾同补之补精益视片（中医眼科名家陈达夫所创）明显改善高度近视视功能，对近视动物视网膜凋亡因子bcl-2及caspase3显著干预。故提出“肝肾同补通过调控HA/CD44-miR21-Bcl2/IAP信号通路改善实验性高度近视光感受器凋亡”的假说。采用肝肾同补法干预实验性高度近视小鼠，以HA/CD44信号通路为核心，通过mfERG检测、Tunnel染色、免疫组化检测、免疫印迹法、qPCR、转录组及miRNA测序等技术对光感受器及相邻的RPE、脉络膜血管内皮细胞和巩膜成纤维细胞进行检测，从功能、细胞、蛋白到基因来探明肝肾同补调控光感受器凋亡延缓视网膜萎缩的靶点及分子机制，为中医早期干预提供实验支撑和科学依据，丰富“既病防变”的预防学思想。

高度近视晚期常见后极部视网膜萎缩致视力严重受损，与视网膜光感受器凋亡密切相关，目前尚无有效治疗方法。临床观察近视早期就开始出现视网膜变薄，视功能下降，因此应早期干预，肝肾同补之补精益视片能明显改善高度近视患者视功能，研究显示该药对形觉剥夺近视小鼠视功能也有一定改善作用，能显著抑制近视小鼠视网膜厚度降低，维持视网膜光感受器细胞外节盘膜结构有序排列，减少其粗面内质网扩张和线粒体肿胀，减少视网膜细胞凋亡，能显著增加形觉剥夺性近视小鼠视网膜HA、CD44、IAP、Bcl-2的蛋白表达水平和提高HAS2mRNA、CD44 mRNA、BIRC3mRNA和microRNA21的基因表达水平，降低caspase和c-Jun的蛋白含量有关，证实肝肾同补之补精益视片能通过调控HA/CD44-miR21-Bcl2/IAP信号通路改善实验性高度近视光感受器细胞凋亡，对光感受器细胞载体RPE细胞行基因分析发现近视模型组中内质网应激相关因子Insig1、Scd1、凋亡标志蛋白caspase底物Pabpc1及急性细胞磷酸化凋亡相关蛋白PSMD1均在近视模型中显著上调、中药补精益视片干预组后又显著下调，功能分析显示近视小鼠视网膜RPE细胞、巩膜成纤维细胞的功能障碍与糖酵解、氧化磷酸化、内质网应激、神经元凋亡等与神经退行性病变密切相关的生物学功能相关，中药补精益视片可通过减缓近视小鼠视网膜RPE细胞的氧化应激、平衡内质网应激来保护RPE细胞功能，从而维持光感受器细胞正常活动。.本研究揭示肝肾同补之补精益视片防治近视视网膜退变减少视网膜细胞凋亡的疗效机制，为肝肾同补法早期防治近视视网膜退变提供理论依据。