ox-LDL-Induced Endothelial Progenitor Cell Oxidative Stress via p38/Keap1/Nrf2 Pathway

Figure 2

ox-LDL inhibited Nrf2 nuclear translocation via MAPK-p38 pathway. (a, b) EPCs were exposed to different concentration of ox-LDL (5-20 μg/mL) for 24hours. The ratio of the control group was assigned a value of 1. Quantification of relative quantity of n-Nrf2/Histone H3 and c-Nrf2/GAPDH showed that ox-LDL concentration dependently decreased n-Nrf2/Histone H3, but not significantly influenced the c-Nrf2/GAPDH levels. (c, d) EPCs were transfected with specific siRNA against Keap1 or silencer select negative control (control group) for 24 hours. Western blot detection showed that Keap1 expression level was significantly inhibited by Keap1 siRNA transfection. (e, f) The inhibition of Nrf2 nuclear translocation in response to ox-LDL was reversed by pretreatment with the Keap1 siRNA, as shown by Western blot. (g, h) ox-LDL time- and dose-dependently decreased NQO1 mRNA transcription. (i) Pretreatment with Keap1 siRNA reversed ox-LDL-induced downregulation of NQO1 mRNA transcription. (j, k) Pretreatment with Keap1 siRNA reversed ox-LDL-induced downregulation of NQO1 protein expression. (l, m) ox-LDL significantly increased p38 phosphorylation. (n, o) Treatment with 1 μM SB203580, a specific p38 inhibitor, reversed the ox-LDL-induced downregulation of Nrf2 nuclear translocation. (p) Pretreatment with SB203580 reversed ox-LDL-induced downregulation of NQO1 mRNA transcription. vs. control. ^# vs. ox-LDL group.

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