低浓度硝酸盐对人血管内皮细胞缺氧复氧损伤保护作用的实验研究

The experimental study of the protective effect of low concentration nitrate on the hypoxia reoxygenation injury of vascular endothelial cells

目的以血管内皮细胞缺氧复氧损伤模型作为缺血再灌注损伤的体外研究模型,探究硝酸盐对血管内皮缺血再灌注损伤的保护作用及可能机制。方法以人脐静脉内皮细胞(HUVECs)建立人血管内皮细胞缺氧复氧损伤模型,缺氧时间为3 h,复氧时间为2 h。以CCK-8法验证不同浓度的硝酸钠对血管内皮细胞活性的影响,确定适宜浓度。以10μmol/L作为硝酸钠保护血管内皮细胞缺氧复氧损伤的适宜浓度,以Annexin V-PE/7AAD染色流式细胞法验证细胞凋亡水平的变化,以实时定量PCR和免疫印迹法验证Sialin蛋白及其编码基因SLC17A5的表达水平变化。结果 CCK-8实验证实了在较低浓度的硝酸钠作用下,缺氧复氧损伤后血管内皮细胞活性较对照组(未加硝酸盐组)有显著升高(P<0.05),以10μmol/L作用最为明显。极低浓度(1μmol/L)及较高浓度(50μmol/L,100μmol/L)硝酸钠作用下,细胞活性较对照组无明显差异。10μmol/L硝酸钠作用下,缺氧复氧损伤后血管内皮细胞细胞凋亡水平显著下降(P<0.05),Sialin蛋白及其编码基因SLC17A5的表达水平均有明显升高(P<0.05)。结论低浓度的硝酸盐能够提高血管内皮细胞缺氧复氧损伤后细胞活性,降低其凋亡水平,提示硝酸盐对组织瓣缺血再灌注损伤可能具有保护作用,该作用可能与硝酸盐转运蛋白Sialin有关。

Objective To investigate the protective effects of nitrate on vascular endothelial ischemia reperfusion injury of vascular endothelial cells.Methods Human umbilical vein endothelial cells(HUVECs)were used to establish the cell hypoxia-reoxygenation injury model.CCK-8 assay was used to detect the effects of sodium nitrate at different concentrations on hypoxia reoxygenation injury of vascular endothelial cells.Annexin V-PE/7 AAD flowcytometry was used to examine the apoptosis and the expression of nitrate transporter(Sialin)was detected by Real-time PCR and Western blot.Results The viability of vascular endothelial cells with sodium nitrate at a low concentration was significantly increased(P<0.05).Sodium nitrate at extremely low concentration(1μmol/L)and relatively high concentration(50μmol/L,100μmol/L)showed no significant differences in cell viability.With 10μmol/L sodium nitrate,the apoptosis ratio of vascular endothelial cells after hypoxia-reoxygenated injury significantly decreased(P<0.05),and the expression of sialin protein and coding gene SLC17 A5 were significantly increased(P<0.05).Conclusions Low concentration of nitrate can improve the cell viability of human vascular endothelial cells after hypoxia-reoxygenation injury and reduce cell apoptosis ratio,suggesting that nitrate may have a protective effect on tissue flap ischemia-reperfusion injury,which may be related to nitrate transporter sialin.