基于CFH上下游调控通路探讨补肾活血方对干性年龄相关性黄斑变性的干预机制

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly worldwide. Dry AMD affects 85–90% of all AMD patients. The pathogenesis of dry AMD is unclear,so there are no effective therapies. Recent reports suggest complement factor H(CFH) and para-inflammation plays crucial roles in the pathology of dry AMD，and miRNA-146a is an important upstream regulatory factor of CFH，C3a andC5a are downstream products. Our previous work focus on the role of complement in the pathogenesis of AMD and the intervention mechanism of BuShenHuoXueFang. The results suggested that CFH gene mutations associated with dry AMD, BuShenHuoXueFang helps to improve visual function and up-regulate amounts of CFH，so it is proved that BuShenHuoXueFang is effective for dry AMD. Thus，In this study,we planned to detect the protein content and mRNA level of miRNA-146a,CFH,C3a and C5a by electron microscopy,real-time PCR, RT-PCR, Western blot, ELISA and immunofluorescence Technology,.thoroughly study the regulatory role of drugs on CFH upstream and downstream pathway and the effect of drugs on inflammatory reaction by in vitro and in vivo. We hope to find the intervention effect and drug targets，in order to from multi-angle clarify the intervention mechanism of Bushen Huoxue Fang on dry AMD.

年龄相关性黄斑变性（AMD）是老年人首要致盲性眼病，干性AMD是最主要的临床类型，目前发病机制不明，尚无有效治疗。近年发现视网膜亚炎症是其病理基础，补体H因子（CFH）表达下调是其重要危险因素。在CFH上下游通路中，miRNA-146a是重要的上游调控因子，C3a、C5a是重要的下游产物。我们前期研究发现CFH基因突变与干性AMD相关，补肾活血方对CFH有上调作用，可改善干性AMD的视功能。在此基础上，本项目拟以ApoE4小鼠及RPE细胞为研究对象，通过电镜、realtime-PCR、Western blot及免疫荧光等技术，观察病理改变，检测miRNA-146a、CFH、C3a、C5a蛋白表达及mRNA水平变化，从体内、体外双重探讨补肾活血方对视网膜亚炎症的影响及对CFH上下游通路调控的分子机制，多角度阐明中药对干性AMD的干预机制,为补肾活血方治疗干性AMD提供更多的理论支撑。

老年性黄斑变性是老年性首要致盲性眼病之一。干性AMD是最主要的临床类型，目前发病机制不明，尚无有效治疗,因此干性AMD的防治一直是近年来眼科的 研究重点、热点及难点。本项目以C57BL/6雄性小鼠尾静脉注射碘酸钠法建立干性AMD动物模型及ARPE-19细胞株用H2O2诱导法建立干性AMD细胞模型。通过Masson染色、电镜观察、RT-PCR、Western blot、ELISA，观察视网膜组织病理改变，miRNA-146a、CFH、C3a、C5a蛋白表达水平及mRNA表达水平的变化。本项目的目的是揭示杞黄颗粒对干性AMD的补体通路的影响。结果显示杞黄颗粒①从形态学方面可明显改善小鼠视网膜色素上皮细胞的凋亡及光感受器外节损伤。②杞黄颗粒明显改善H2O2损伤后的ARPE-19细胞的凋亡情况。③杞黄颗粒可升高小鼠视网膜组织CFH的基因表达及蛋白表达，同时对H2O2损伤后ARPE-19细胞的CFH的基因表达及细胞外蛋白表达也同样有升高作用。④杞黄颗粒可降低小鼠视网膜组织C3a的蛋白表达，同时对H2O2损伤后ARPE-19细胞的C3a的基因表达及细胞外蛋白表达也同样有降低作用。⑤杞黄颗粒可降低小鼠视网膜组织C5a的基因表达及蛋白表达，同时对H2O2损伤后ARPE-19细胞的C5a的基因表达及细胞外蛋白表达也同样有降低作用。这些研究为杞黄颗粒治疗干性 AMD 提供理论依据，为进一步深入探讨杞黄颗粒对AMD视网膜的保护作用提供前期基础，为进一步推广补肾活血法用于治疗干性AMD提供科学依据。