摘要
目的糖尿病视网膜病变(diabetic retinopathy,DR)中糖基化终产物(advanced glycosylation end products,AGE)可引起细胞内产生过氧化物和炎症因子,导致视网膜微血管内皮细胞(retinal microvascular endothelial cell,RMEC)凋亡,视功能破坏。文中观察藏红花素对在AGE作用下RMEC活性、凋亡比例、活性氧簇(reactive oxygen species,ROS)产物、细胞内和上清中肿瘤坏死因子(tumor necrosis factor,TNF)-α含量的干预,探讨藏红花素对AGE引起RMEC凋亡的保护机制。方法采用免疫磁珠法分离培养RMEC,浓度为10、50和100μmol/L藏红花素培养液作用于RMEC,观察4d内细胞增生变化并记录生长曲线。不同浓度藏红花素溶液作用于AGE处理的RMEC 12 h,锥虫蓝染色计数活细胞率,流式细胞术检测凋亡细胞比例,荧光光度计测定细胞内ROS含量,采用蛋白电泳和ELISA测定RMEC内和上清中TNF-α含量。结果 RMEC在10、50μmol/L藏红花素的培养液中可以继续增生。培养至3 d和4 d时,100μmol/L藏红花素可使细胞增生受到抑制。与对照组相比,100 mg/L AGE处理12 h和24 h后RMEC活细胞比例明显下降。10、50和100μmol/L藏红花素可不同程度保护AGE处理后RMEC活细胞比例。与对照组相比,100 mg/L AGE处理12 h后凋亡细胞比例明显升高。10、50和100μmol/L藏红花素处理12h后凋亡细胞比例均有下降。AGE处理12h后RMEC内ROS水平明显升高。10、50和100μmol/L藏红花素可不同程度抑制AGE处理后RMEC内ROS水平。AGE处理后RMEC内和上清液中TNF-α水平明显升高。10、50和100μmol/L藏红花素可明显抑制AGE处理后RMEC内和上清液中TNF-α水平。结论藏红花素可能通过抑制AGE诱导RMEC中ROS产生并减少TNF-α合成途径,防止细胞凋亡。
Objective Advanced glycosylation end products（AGE） in diabetic retinopathy may result in the production of reactive oxygen species（ROS） and inflammation cytokines,which cause apoptosis of retinal microvascular endothelial cells（RMEC） and loss of visual function.The authors investigated the effects of crocin on the viability,apoptosis,and ROS production,intracellular and supernatant tumor necrosis factor（TNF）-α level of AGE-induced RMECs in rats,as well as the protective mechanism of crocin in AGE-induced RMEC apoptosis.Methods Rat RMECs were isolated using magnetic beads and cultured in the crocin medium at the concentrations of 0,10,50 and 100 μmol/L,respectively.The proliferation curves of the RMECs were recorded within 4 days.The RMECs treated with 100 mg/L AGE were exposed to various concentrations of crocin.The viability of the RMECs was measured after trypan blue staining,their apoptosis rate detected by flow cytometry,the intracellular ROS products determined by fluorescence enzyme-labeled meter,and the levels of intercellular and supernatant TNF-α measured by protein electrophoresis and ELISA.Results The proliferation of the RMECs continued in the 10 and 50 μmol/L crocin media,but was inhibited in the 100 μmol/L crocin medium at 3 and 4 days.Compared with the normal control group,the viability of the RMECs was decreased after treated with 100 mg/L AGE for 12 and 24 hours.Crocin at 10,50 and 100 μmol/L protected the viability of the RMECs.The apoptosis rate of the RMECs was significantly increased after treated with 100 mg/L AGE for 12 hours,but remarkably decreased after treated with crocin at 10,50 and 100 μmol/L for 12 hours.AGE markedly elevated the level of ROS in the RMECs（P0.01）,and 10,50 and 100 μmol/L of crocin variably reduced the production of ROS in the AGE-induced RMECs.The TNF-α levels in the RMECs and supernatant were obviously raised after treated with AGE,but significantly suppressed by crocin at 10,50 and 100 μmol/L.Conclusion Crocin can prevent the apoptosis RMECs inhibiting AGE-induced ROS production and reducing the synthesis of TNF-α.
出处
《医学研究生学报》
CAS
北大核心
2012年第11期1141-1145,共5页
Journal of Medical Postgraduates
基金
南京军区科技创新项目(10MA040)
关键词
视网膜微血管内皮细胞
糖基化终产物
藏红花素
凋亡
活性氧簇
肿瘤坏死因子
Retinal microvascular endothelial cell
Advanced glycosylation end product
Crocin
Apoptosis
Reactive oxygen species
Tumor necrosis factor
