绿茶提取物对缺血再灌注损伤视网膜神经节细胞的保护作用及机制研究

Purpose: To investigate the mechanism of protective effects of green tea extract and purified catechins on the retinal ganglion cells (RGCs) in retinal ischemia reperfusion model induced by acute elevation of intraocular pressure (IOP)..Methods: Confocal scanning laser ophthalmology (CSLO) will be used to visualize the number of the RGCs, cell body size, dendritic field, axonal diameter, ending branch number, and total dendritic branch length of the Thy1-CFP23Jrs, Thy1-YFP16Jrs transgenic mice in vivo. PhNR、VEP、ERG will estimate the function of the RGCs and the retinas. Real-time PCR and Western Blot to measure the expression of the inflammation related factors, include TLR-4, NF-kBp65, IL-1β, TNF-a, IL-6, MCP-1, NLRP1/NLRP3 and caspase-1. The expression of the cytochrome b, Tfam, OPA1, ND6, ATpase8 reflect the mitochondrial function. HPLC measure the Cmax and AUC of the individual catechins in the vitreous and retnia..Results expected: We have previously demonstrated protective effects of catechins against oxidative stress in the retina. Results of this study will directly reveal the biological effects of catechins in the retinal ganglion cells during ischemic insults by acute IOP elevation.

研究绿茶提取物对缺血再灌注(I/R)损伤后RGCs保护作用机制。Thy1-CFP23Jrs,Thy1-YFP16Jrs小鼠应用蓝光共焦激光扫描检眼镜(CSLO)观察治疗后RGCs的形态数目，胞体面积，轴突直径，树突面积，树突终末分支数量。PhNR、VEP、ERG研究RGCs及视网膜功能学变化。Real-time PCR,测量炎症相关因子TLR-4,NF-kBp65,IL-1β, TNF-a, IL-6, MCP-1, NLRP1/NLRP3,caspase-1的表达。Western Blot检测与RGCs存活相关通路JAK,STAT,P13K,AKT,ERK1/2,p38MAPK,JNK表达；线粒体复合物表达cytochrome b,Tfam,OPA1,ND6,ATpase8表达。HPLC检测玻璃体视网膜中各组药物到达的浓度、吸收峰值。为发掘潜在药物治疗靶点，为RGCs保护剂提供理论依据。

目的：氧化应激是引起青光眼患者视网膜神经节细胞损伤的一个关键因素，我们之前用SD大鼠实验证明绿茶提取物可以有效缓解碘酸钠诱导引起的视网膜变性。目前，我们假设绿茶提取物可以有效改善缺血再灌注损伤诱导的视网膜神经节细胞凋亡。..方法：采用缺血再灌注损伤法诱导成年大鼠（Fisher F344）视网膜神经节细胞损伤。绿茶提取物（Theaphenon-E）在缺血再灌注损伤后灌胃4次，每次浓度为275mg/kg。为了评估绿茶提取物的作用，我们检测了视网膜神经节细胞存活率以及细胞凋亡、氧化应激和炎症相关蛋白的表达。..结果：与未经处理的正常大鼠相比，缺血再灌注损伤能显著诱导视网膜神经节细胞的丢失。同时，caspase-8、氧化应激和炎症相关蛋白的表达在缺血再灌注损伤大鼠中显著上调。而且，较生理盐水喂养的缺血再灌注损伤大鼠的视网膜神经节细胞数目相比，绿茶提取物喂养的细胞存活率更高。绿茶提取物治疗还降低了缺血引起的caspase-8、氧化应激和炎症相关蛋白的表达增加。然而，绿茶提取物对未经处理的正常大鼠的RGC存活率没有影响。..结论：总的来说，绿茶提取物对缺血损伤的视网膜神经节细胞具有保护作用，揭示了其对青光眼和视神经病变的潜在治疗策略。