用双向电泳技术探讨益生注射液抗缺血再灌注损伤的分子机制

Molecular Mechanism of Yisheng Injection against Anoxia-Reoxygenation Injury to Endothelial Cells by Two-Dimensional Gel Electrophoresis

目的 :用双向电泳技术探讨天然成分组方药物益生注射液抗移植物缺血再灌注损伤的分子机制。方法 :利用血管内皮细胞缺氧损伤模型模拟移植器官的缺血再灌注损伤过程 ,通过双向电泳技术观察受损细胞用药修复前后蛋白质组的变化 ,探索药物作用的靶标及分子机制。结果 :发现缺血再灌注损伤可以导致内皮细胞大量蛋白质含量发生变化 ,但只有 8种蛋白质在用药前后含量发生明显改变 (其中 6种蛋白质在细胞受损后含量下降 ,益生药物干预后上调 ;另 2种受损后含量增高 ,药物作用后下调 )。结论 :双向电泳技术为分析药物作用的分子机制提供了新的有效手段 ,益生注射液可能将血管内皮细胞的 8种蛋白质作为靶标 ,通过调节其含量而发挥抗缺血再灌注 (缺氧再给氧 )

Objective: To explore the molecular mechanism of Yisheng Injection against ischemia reperfusion injury (IRI) of graft with an application of two dimensional gel electrophoresis (2 DE). Methods: Human umbilical veins endothelial cell (EC) line ECV304 was cultured in RPMI medium 1640 without glucose under the condition with 100% N2 for 1.5 hours, then in RPMI medium 1640 with glucose under normal conditions for 5 hours, i.e. to use anoxia reoxygenation injury (ARI) model to mimic endothelial cells' post transplantation IRI. A natural medicine, Yisheng Injection, was added into the medium in experimental group; meanwhile 2 DE was used to analyze the change of ECs' protein content in all groups before and after the injection of the medicine and to explore the target of the natural drug and its molecular mechanism. Result: The results showed that IRI (or ARI) could change the content of different proteins. Only 8 of the proteins significantly changed, among which 6 proteins were down regulated and 2 proteins were up in ECs treated with ARI, and then became normal after being intervened with Yisheng Injection. Conclusion: 2 DE was a new effective measure to analyze the molecular mechanism of medicine's function. These 8 proteins might be the targets of Yisheng Injection that acts against ARI (or IRI) to endothelial cells with a regulation of the proteins' contents.