【摘要】 目的 研究M1型巨噬细胞来源外泌体（M1 macrophage exosomes,M1-Exo）调控血管内皮衰老的作用及其相关分子机制。方法 随机选取年轻组和老年组小鼠各6只，比较两组小鼠肱动脉血流介导的血管舒张功能（flowmediated dilation,FMD）。使用溶剂对照（溶剂组）、100 ng/μL的M1-Exo(M1-Exo 100 ng/μL组)、200 ng/μL的M1-Exo(M1-Exo 200 ng/μL组)分别与内皮细胞共培养，观察细胞衰老相关β半乳糖苷酶（senescence-associated β-galactosidase,SA-β-gal）染色情况，共聚焦成像技术检测细胞内线粒体活性氧水平。设置微小RNA(microRNA,miR)-空白对照组、miR-22-3p模拟物组、miR-阴性对照组、miR-22-3p抑制剂组，比较各组沉默信息调节因子2相关酶1(silent mating type information regulation 2 homolog-1,SIRT1)蛋白表达。短发夹RNA(short hairpin RNA,shRNA)转染shRNA-SIRT1质粒（sh-SIRT1组），并设置sh-空白对照组（sh-NC组）。蛋白免疫印迹检测两组相关蛋白的表达。结果 与年轻组小鼠比较，老年组小鼠FMD明显下降[（48.330±3.807）%vs(28.650±3.209)%,P<0.05]。体外实验显示，与溶剂刺激比较，M1-Exo 100 ng/μL组、M1-Exo 200 ng/μL组SA-β-gal表达升高，线粒体活性氧水平升高（P<0.05）。与miR-空白对照组比较，miR-22-3p模拟物组SIRT1水平降低；与miR-22-3p模拟物组比较，miR-22-3p抑制剂组SIRT1水平升高，差异均有统计学意义（P<0.5）。与sh-NC组比较，sh-SIRT1组核转录因子κB/细胞周期依赖性蛋白激酶抑制因子P21、叉头蛋白O3α/超氧化物歧化酶2信号通路被激活。结论 M1型巨噬细胞外泌体miR-22-3p具有促进血管内皮细胞衰老的作用。更多还原

【Abstract】 Objective To investigate the role of M1 macrophage-derived exosomes(M1-Exo) in regulating vascular endothelial senescence and explore the related molecular mechanism. Methods Six young mice and 6 aged mice were randomly subjected and assigned into a young group and an aged group. The flow-mediated dilation(FMD) of the brachial artery was compared between the two groups. Endothelial cells were co-cultured with solvent control(solvent group), 100 ng/μL M1-Exo(M1-Exo 100 ng/μL group), and 200 ng/μL M1-Exo(M1-Exo 200 ng/μL group) respectively. Senescence-associated β-galactosidase(SA-β-gal) staining was carried out, and confocal imaging was used to detect the production of intracellular mitochondrial reactive oxygen species(ROS). The following groups were established: microRNA(miRNA) blank control group, miR-22-3p mimic group, miRNA negative control group, and miR-22-3p inhibitor group. The protein expression of silent mating type information regulation 2 homolog-1(SIRT1) was compared among these above groups. Short hairpin RNA(shRNA) transfection was performed with shRNA-SIRT1 plasmid to establish a sh-SIRT1 group, and the cells transfected with shRNA blank plasmid served as control group(sh-NC group). Western blotting was used to detect protein expression in both groups. Results The FMD was significantly decreased in the aged group than the young group [(48.330±3.807)% vs(28.650±3.209)%, P<0.05]. In vitro experiments showed that compared with solvent group, the SA-β-gal expression and mitochondrial ROS levels were elevated in both M1-Exo 100 ng/μL and M1-Exo 200 ng/μL groups(P<0.05). The SIRT1 level was decreased in the mi R-22-3p mimic group than the miRNA blank control group, but increased in the miR-22-3p inhibitor group than the miR-22-3p mimic group(P<0.05). Compared with the sh-NC group, the nuclear factor κB/cyclin-dependent kinase inhibitor P21(NF-κB/P21) and Forkhead box O3α/superoxide dismutase 2(FOXO3α/SOD2) signaling pathways were activated in the sh-SIRT1 group. Conclusion M1 macrophage-derived exosomal miR-22-3p promotes vascular endothelial cell senescence.更多还原