原花青素对高糖环境下大鼠内皮祖细胞血管内皮生长因子受体2及其传导通路的影响

Oligomeric Proanthocyanidin Exert a Great Effect on Expression of VEGFR-2 and Signaling Pathways Downstream in Endothelial Progenitor Cell Cultivated in High Concentration of Glucose

目的探讨高糖环境下原花青素(OPC)对体外培养的大鼠内皮祖细胞血管内皮生长因子受体2(VEGFR-2)及其下游相关通路的影响。方法采用Ficoll密度梯度离心法分离大鼠骨髓内皮祖细胞(EPC);分别检测正常糖组(5.5 mmol/L葡萄糖+25 mmol/L甘露醇)、高糖组(30 mmol/L葡萄糖)培养的EPC增殖和氧化应激产物产生情况,再通过在不同原花青素浓度下检测各时间点EPC增殖的方法,筛选出最佳原花青素浓度(30 mg/L);分别用Matrigel基质胶检测正常糖+OPC组、正常糖组、高糖+OPC组、高糖组EPC成管能力;最后在1、3、5、7天分别检测正常糖+OPC组、正常糖组、高糖+OPC组、高糖组EPC的丙二醛水平以及VEGFR-2、p-AKT、核因子κB(NF-κB)和核因子κB抑制蛋白α(IKB-α)的相对表达量。结果随时间推移,高糖组中EPC细胞凋亡和氧化应激产物均较正常糖组逐渐增多;正常糖+OPC组、正常糖组EPC成管数目无明显差异,高糖+OPC组成管数目较高糖组明显增多;在1、3、5、7天时OPC在正常糖浓度下对EPC产生的氧化应激产物及VEGFR-2、p-AKT、NF-κB和IKB-α蛋白的相对表达量没有显著影响,而在高糖环境下可以明显降低氧化应激产物的生成及上调VEGFR-2、p-AKT和NF-κB蛋白相对表达量,下调IKB-α蛋白相对表达量。结论原花青素可能通过缓解高糖环境对EPC的氧化损伤作用,上调内皮祖细胞VEGFR-2及其下游通路蛋白表达,进而促进细胞增殖。

Aim To investigate the expression of vascular endothelial growth factor receptor-2( VEGFR-2) and signaling pathways downstream in endothelial progenitor cell( EPC) treated with procyanidine under the environment of high glucose. Methods To isolate rat bone marrow EPC with Ficoll density gradient centrifugation,the proliferation and oxidative stress production was respectively detected using spectrophotometer and enzyme standard instrument in control glucose group( 5. 5 mmol / L glucose + 25 mmol / L mannitol) and high glucose group( 30 mmol / L glucose). To detect the proliferation of the EPC treated with various concentration of anthocyanins in different time points,in order to select optimum concentration of oligomeric proanthocyanidin( OPC)( 30 mg / L). Tube formation capacity was respectively detected using Matrigel matrix in four groups,including control glucose + OPC group,control glucose group,high glucose + OPC group and high glucose group. Finally malondialdehyde( MDA) value and protein expression of VEGFR-2,p-AKT,nuclear factor-kappa B( NF-κB) and inhibitor kappa B-α( IKB-α) was respectively detected in the 1,3,5,7 days. Results Compared with control glucose group,the number of cell apoptosis and oxidative stress production were more in high glucose group. Tube formation capacity had no obvious difference between control glucose + OPC group and control glucose group. The number of tube formation in high glucose + OPC group was more than high glucose group. In control glucose group,the protein expression of VEGFR-2,p-AKT,NF-κB,IKB-α and oxidative stress production had no statistical differences in EPC treated with OPC in 1,3,5,7 days. In high glucose group,oxidative stress production significantly reduced,while the protein expression of VEGFR-2,p-AKT,NF-κB obviously increase in EPC treated with OPC in1,3,5,7 days,excluding the protein expression of IKB-α. Conclusion OPC can alleviate oxidative damage of EPC affected on high glucose,and improve expression of VEGFR-2,and activate its downstream pathway to promote EPC proliferation.