【摘要】 目的 研究恩格列净(EMP)对氧化低密度脂蛋白(ox-LDL)诱导内皮祖细胞(EPCs)损伤的保护作用及机制。方法 通过密度梯度离心法提取、分离并培养小鼠骨髓来源的的EPCs。采用Dil标记乙酰化低密度脂蛋白(Dil-ac-LDL)联合FITC标记荆豆凝集素-1(FITC-UEA-1)双摄取法鉴定。将EPCs分为正常对照组，ox-LDL组以及ox-LDL联合不同浓度恩格列净实验组。CCK-8检测细胞活力，Transwell检测细胞迁移，FITC-Annexin V/PI检测细胞凋亡，ELISA检测细胞上清液中血管内皮细胞生长因子(VEGF)、基质细胞衍生因子-1α(SDF-1α)含量；流式细胞术检测一氧化氮(NO)的合成情况。Western blot检测AMPK、p-AMPK、eNOS、p-eNOS的蛋白表达。结果 提取的EPCs诱导培养至第7天经鉴定为小鼠骨髓EPCs。与对照组比较，ox-LDL组细胞活力降低，迁移细胞减少，凋亡增加，VEGF、SDF-1α含量降低，NO合成减少(P<0.05);与ox-LDL组相比，不同浓度恩格列净组细胞活力有所提高，迁移细胞增多，凋亡减少，VEGF、SDF-1α含量升高，NO合成增加(P<0.05)。ox-LDL处理可明显抑制AMPK及eNOS磷酸化(P<0.05),恩格列净处理可以改善AMPK及eNOS磷酸化水平(P<0.05),而AMPK抑制剂Compound C可使恩格列净改善EPCs功能活性的作用受到明显的抑制(P<0.05)。结论 恩格列净可改善ox-LDL诱导的EPCs功能障碍，其机制与调控AMPK/eNOS信号通路有关。更多还原

【Abstract】 Objective To investigate the protective effects of Empagliflozin(EMP) on the functional damage of mouse endothelial progenitor cells(EPCs)induced by oxidizedlow-densitylipoprotein(ox-LDL) and the related mechanisms.Methods Mouse bone marrow derived EPCs were extracted, separated, and cultured by density gradient centrifugation. Then, the cells were identified by the uptake of Dil-acetylated low density lipoprotein(Dil-ac-LDL) combined with FITC labeled ulex europaeus agglutinin-1(FITC-UEA-1) binding. EPCs were divided into control group, ox-LDL group and ox-LDL combined with different concentrations of EMP experimental groups, and simultaneously cultured for 72 hours. Cell viability, migration were analyzed by CCK-8 assay and Transwell assay, respectively. The cell apoptosis were measured by FITC-Annexin V/PI staining. The levels of vascular endothelial growth factor(VEGF), stromal-Derived factor-1 alpha(SDF-1α) in the medium were detected by enzyme linked immunosorbent assay(ELISA). The synthesis of nitric oxide(NO) was measured by flow cytometry. The protein expression of AMPK, p-AMPK, eNOS, p-eNOS in the EPCs were determined by Western blot. Results The extracted EPCs were cultured and identified as mouse bone marrow EPCs on day 7. Compared with control group, the cell viability of the ox-LDL group decreased, the migratory cell number decreased, the cell apoptosis rate increased, the VEGF and SDF-1αcontents decreased and the NO synthesis decreased(P<0.05). Compared with ox-LDL group, the cell viability increased, the migratory cell number increased, the cell apoptosis rate decreased, the VEGF and SDF-1αcontents increased and the NO synthesis increased in EMP groups at different concentrations(P<0.05).Besides, ox-LDL treatment could significantly inhibit the phosphorylation of AMPK and eNOS(P<0.05), EMP treatment could improve the phosphorylation levels of AMPK and eNOS(P<0.05). However, the protective effects of EMP could be significantly blocked by AMPK inhibitor(Compound C)(P<0.05). Conclusion Empagliflozin can improve ox-LDL-induced EPCs dysfunction by regulating AMPK/eNOS signaling pathway.更多还原