槲皮素改善大鼠骨髓来源内皮祖细胞生物学功能及其机制研究

Quercetin improves biological functions of rat bone marrow-derived EPCs

目的:探讨槲皮素(quercetin,QUE)对大鼠骨髓来源内皮祖细胞(endothelial progenitor cells,EPCs)生物学功能的影响与机制。方法:密度梯度离心法分离大鼠骨髓单个核细胞,条件培养基EGM-2诱导分化后,进行双荧光染色及免疫表型鉴定。将培养14 d的细胞用PI3K抑制剂BYL719(3μmol/L)和ERK抑制剂FR180204(15μmol/L)预处理2 h后,再加入不同浓度QUE(0、10、20、40、80和100μmol/L)处理。MTT法检测细胞活力,Transwell法检测细胞迁移,Western blot法检测AKT、内皮型一氧化氮合酶(eNOS)和ERK蛋白表达及磷酸化水平。结果:QUE呈浓度依赖性提高EPCs活力,促进EPCs迁移;PI3K抑制剂BYL719能抑制QUE诱导的EPCs活力和迁移能力,ERK抑制剂FR180204能抑制QUE诱导的EPCs活力,而对EPCs迁移能力并没有影响。QUE能激活AKT、eNOS和ERK蛋白;BYL719能同时抑制AKT和ERK蛋白的激活,FR180204仅能抑制ERK的激活,而未能抑制AKT的激活,但两者对QUE诱导的eNOS蛋白表达均有抑制作用。结论:QUE能部分通过PI3K/AKT/eNOS和ERK/eNOS信号转导通路,提高EPCs活力及迁移能力,促进EPCs发挥心血管保护作用。

AIM： To investigate the effect of quercetin on the biological functions of rat bone marrow-derived endothelial progenitor cells （EPCs） and its potential mechanisms.METHODS： The bone marrow-derived mononuclear cells of Sprague-Dawley rats were isolated by density gradient centrifugation. The differentiated EPCs were cultured specially and stained with DiI-Ac-LDL and FITC-UEA-1. CD133＋ and FLK-1＋ were detected on the cell surfaces. After 14 d, the EPCs were incubated with a PI3K inhibitor BYL719 （3 μmol/L） and an ERK inhibitor FR180204 （15 μmol/L）. After incubation of the inhibitors for 2 h, the cells were treated with quercetin at different concentrations （0, 10, 20, 40, 80 and 100 μmol/L）. MTT assay and Transwell assay were used to detect cell viability and the number of migratory cells. The protein levels of AKT, eNOS, ERK and their phosphorylated status were determined by Western blot.RESULTS： Quercetin enhanced the viability and migration of the EPCs at a dose-dependent manner. However, the PI3K inhibitor BYL719 suppressed the QUE-induced cell viability and migration. Moreover, ERK inhibitor FR180204 exerted the similar inhibitory effect on the cell viability but had no effect on cell migration. Quercetin activated the phosphorylation of AKT, eNOS and ERK. On the other hand, BYL719 was observed to inhibit the phosphorylation of AKT and ERK. FR180204, however, was showed to inhibit the phosphorylation of ERK only. On the contrast, the stimulatory effects that quercetin exerted on the expression of eNOS and its phosphorylation were suppressed by BYL719 and FR180204.CONCLUSION： Quercetin stimulates the viability and migration of EPCs via PI3K/AKT/eNOS and ERK/eNOS signaling pathway, which would be beneficial for cardiovascular health.