Jiang Qijun, Chen Qiao, Li Chengpeng, Gong Zhigang, Li Zhigang, Ding Shifang
Department of Cardiology, Affiliated Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Yanhu Road, 39 Wuhan, Hubei Province, China.
Department of Cardiology, General Hospital of Central Theater Command, Wuluo Road 627, Wuhan, 430070 Hubei Province, China.
Stem Cells Int. 2022 Jan 31;2022:5897194. doi: 10.1155/2022/5897194. eCollection 2022.
Nrf2 which was recently reported to regulate the antioxidant genes and cellular redox regulators was highly expressed in EPCs. However, its role in ox-LDL-induced EPC oxidative stress and apoptosis has not been fully illustrated.
EPCs isolated from human peripheral blood mononuclear cells were treated with different concentrations of ox-LDL, Keap1 siRNA, and a specific p38 MAPK inhibitor SB203580 and then used to assay the cytoplasmic Nrf2, nuclear Nrf2, NAD(P) H:quinone oxidoreductase 1 (NQO1) and Bax/Bcl-2 levels with Western blot, NQO1 mRNA levels with RT-PCR, ROS levels with H2DCF-DA, loss/disruption of mitochondrial membrane potential with JC-1, apoptosis with Annexin V and PI, migration with transwell chambers, and tube formation with Matrigel.
ox-LDL decreased the nuclear Nrf2/Histone H3 to cytoplasmic Nrf2/GAPDH ratio, NQO1 mRNA, and protein levels. ox-LDL enhanced ROS production, induced the loss of membrane potential, and increased the cell shrinkage, pyknotic nuclei, and apoptosis of EPCs. Keap1 siRNA increased Nrf2 nuclear translocation, NQO1 mRNA transcription, and protein expression and prevented ROS generation and formation of JC-1 monomers. ox-LDL increased the activation of p38. SB203580 significantly eliminated ox-LDL induced inhibition of Nrf2 nuclear translocation, depression of NQO1 mRNA transcription, generation of ROS, and formation of JC-1 monomers in EPCs. Keap1 siRNA decreased the Bax/Bcl-2 ratio which was increased by ox-LDL in EPCs. ox-LDL decreased EPC migration and tube formation. Keap1 siRNA preserved the migration and tube formation of EPCs.
ox-LDL activated EPCs p38/Keap1/Nrf2 pathway and induced oxidative stress, dysfunction, and apoptosis of EPCs.
近期报道的可调节抗氧化基因和细胞氧化还原调节因子的Nrf2在血管内皮祖细胞（EPCs）中高表达。然而，其在氧化型低密度脂蛋白（ox-LDL）诱导的EPC氧化应激和凋亡中的作用尚未完全阐明。
从人外周血单个核细胞中分离出的EPCs用不同浓度的ox-LDL、Keap1小干扰RNA（siRNA）和特异性p38丝裂原活化蛋白激酶（MAPK）抑制剂SB203580处理，然后用蛋白质免疫印迹法检测细胞质Nrf2、细胞核Nrf2、烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）：醌氧化还原酶1（NQO1）和Bax/Bcl-2水平，逆转录聚合酶链反应（RT-PCR）检测NQO1信使核糖核酸（mRNA）水平，2′,7′-二氯二氢荧光素二乙酸酯（H2DCF-DA）检测活性氧（ROS）水平，JC-1检测线粒体膜电位的丧失/破坏，膜联蛋白V和碘化丙啶检测凋亡，Transwell小室检测迁移，基质胶检测管腔形成。
ox-LDL降低了细胞核Nrf2/组蛋白H3与细胞质Nrf2/甘油醛-3-磷酸脱氢酶（GAPDH）的比值、NQO1 mRNA及蛋白水平。ox-LDL增强了ROS生成，诱导了膜电位丧失，并增加了EPCs的细胞皱缩、核固缩和凋亡。Keap1 siRNA增加了Nrf2核转位、NQO转录及蛋白表达，并阻止了ROS生成和JC-1单体形成。ox-LDL增加了p38的活化。SB203580显著消除了ox-LDL诱导的EPCs中Nrf2核转位抑制、NQO1 mRNA转录降低、ROS生成及JC-1单体形成。Keap1 siRNA降低了ox-LDL诱导的EPCs中升高的Bax/Bcl-2比值。ox-LDL降低了EPCs的迁移和管腔形成。Keap1 siRNA保留了EPCs的迁移和管腔形成。
ox-LDL激活了EPCs的p38/Keap1/Nrf2通路，并诱导了EPCs的氧化应激、功能障碍和凋亡。
