复方血栓通胶囊对叔丁基过氧化氢诱导人视网膜色素上皮细胞氧化损伤保护作用  

Protective effect of Fufang Xueshuantong on human retinal pigment epithelium ceils injured by tert-butyl hydroperoxide in vitro

作　　者：陈晓云[1] 肖伟[1] 罗燕[1] 马伟[1] 李涛[1] 陈方[1] 朱晓波[1] 曾美珍[1] 唐仕波[1] 

机构地区：[1]中山大学中山眼科中心,眼科学国家重点实验室,广州510060

出　　处：《中国实用眼科杂志》2010年第10期1144-1150,共7页Chinese Journal of Practical Ophthalmology

基　　金：国家自然科学基金（30872819）;广东省科技计划项目（20098030801024）

摘　　要：目的 探讨复方血栓通胶囊对叔丁基过氧化氢（t-BHP）诱导体外培养的人视网膜色素上皮细胞（hRPE）氧化损伤的保护作用.方法 原代培养hRPE细胞后取第3～5代进行实验.应用MTT法筛选复方血栓通的最佳药物浓度;再分别用MTT及Annexin V/PI双染法检测复方血栓通及其各单方药物对细胞氧化损伤的保护作用.hRPE细胞分为正常对照组、t-BHP模型组和t-BHP＋复方血栓通（0.25mg/ml）组,倒置相差显微镜和Hoechst33258染色观察各组细胞的形态和细胞核;MitoSOX染色检测活细胞线粒体中ROS的产生;JC-1染色检测线粒体膜电位的变化;ELISA法检测细胞分泌的VEGF浓度.结果 500μmol-BHP干预6h后,模型组细胞存活率下降到（49.9±6.3）%,而不同浓度的复方血栓通组细胞存活率均较模型组升高,且以0.25mg/ml的保护作用最佳;细胞存活率、凋亡及坏死率的结果显示复方血栓通较各单方药物的保护作用更为明显;模型组多数细胞肿胀、变圆、漂浮和核固缩,复方血栓通组损伤的细胞明显减少,线粒体内ROS的产生、线粒体膜电位的下降、分泌的VEGF均较模型组明显减少.结论 复方血栓通胶囊对t-BHP诱导hRPE细胞的氧化损伤具有保护作用,且其保护作用较各单方药物更为明显,其作用机制为清除ROS,阻止线粒体膜电位的下降,抑制VEGF的分泌,从而抑制细胞凋亡和坏死,提高细胞存活率.Objective To investigate the protective effect ofFufang Xueshuantong on human retinal pigment epithelium （hRPE） induced by tert-butyl hydroperoxide （t-BHP） in vitro. Methods hRPE cells were isolated from human eyes and cultured. The third to fifth passage were used in the following experiments. hRPE cells were exposed to 500μ M t-BHP and treated with various concentrations of Fufang Xueshuantong simultaneously. MTT assay was used to evaluate the cell viability to determine the optimal concentration of Fufang Xueshuantong. MTT assay and Annexin V/PI staining were used to compare the protective effect of Fufang Xueshuantong and each simple recipe in its composition on t-BHP-injured hRPE cells. hRPE cells were divided into three groups, including normal control group, t-BHP group and t-BHP with Fufang Xueshuantong group.Changes of cells and nuclei morphology were observed under a phase contrast microscope and a fluorescence microscope after Hoechst 33258 staining. The overproduction of ROS and change of mitochondrial membrane potential were assessed by MitoSOX staining and JC-1 staining. The hRPE-secreted VEGF was detected by ELISA kit. Results Exposured to 500μ M t-BHP for 6 hours resulted in the cells viability decreased to 49.9± 6.3%. While the cells viability of Fufang Xueshuantong group at different concentrations was higher than the t-BHP group. 0.25mg/ml Fufang Xueshuantong showed maximally protective effect on hRPE induced by t-BHP. The results of cells viability and apoptosis showed that the protective effect of Fufang Xueshuantong was much better than each simple recipe. Compared to t-BHP group, the application of Fufang Xueshuantong remarkably decreased dead cells and apoptotic nuclei, reduced the level of ROS production in mitochondria,maintained the mitochondrial membrane potential and inhibited the RPE-secreted VEGF induced by t-BHP.Conclusions This study suggests that Fufang Xueshuantong can protect hRPE cells against oxidative injury-induced cell death by reducing ROS in mitochondria, main

关 键 词：复方血栓通胶囊 人视网膜色素上皮细胞 氧化损伤 

分 类 号：R774[医药卫生—眼科]