CAJ | 학술논문

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.
목적 탐토복방혈전통효낭대숙정기과양화경(t-BHP)유도체외배양적인시망막색소상피세포(hRPE)양화손상적보호작용.방법 원대배양hRPE세포후취제3～5대진행실험.응용MTT법사선복방혈전통적최가약물농도;재분별용MTT급Annexin V/PI쌍염법검측복방혈전통급기각단방약물대세포양화손상적보호작용.hRPE세포분위정상대조조、t-BHP모형조화t-BHP+복방혈전통(0.25mg/ml)조,도치상차현미경화Hoechst33258염색관찰각조세포적형태화세포핵;MitoSOX염색검측활세포선립체중ROS적산생;JC-1염색검측선립체막전위적변화;ELISA법검측세포분비적VEGF농도.결과 500μmol-BHP간예6h후,모형조세포존활솔하강도(49.9±6.3)%,이불동농도적복방혈전통조세포존활솔균교모형조승고,차이0.25mg/ml적보호작용최가;세포존활솔、조망급배사솔적결과현시복방혈전통교각단방약물적보호작용경위명현;모형조다수세포종창、변원、표부화핵고축,복방혈전통조손상적세포명현감소,선립체내ROS적산생、선립체막전위적하강、분비적VEGF균교모형조명현감소.결론 복방혈전통효낭대t-BHP유도hRPE세포적양화손상구유보호작용,차기보호작용교각단방약물경위명현,기작용궤제위청제ROS,조지선립체막전위적하강,억제VEGF적분비,종이억제세포조망화배사,제고세포존활솔.
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, maintaining the mitochondrial membrane potential,inhibiting RPE from secreting VEGF.