Protective Effect of Uric Acid on ox-LDL-Induced HUVECs Injury via Keap1-Nrf2-ARE Pathway

Figure 5

Nrf2 activation is associated with protective effect of uric acid-mediated on ox-LDL induced HUVECs injury. HUVECs were incubated with ox-LDL (100 μg/mL) for 24 h, with or without uric acid (5 mg/dL) preincubated for 2 h. HUVECs were preincubated with brusatol (300 nM) for 2 h before incubated with UA (5 mg/dL)+ox-LDL (100 μg/mL) for 24 h. (a–d) The cytosolic and nuclear compartments of HUVEC cells were fractionated, cell lysates were analyzed by Western blotting with primary antibodies against Nrf2 and Keap1. Protein levels were quantified by densitometry. α-Tubulin and histone H3 were used as internal controls. (e–h) Cell lysates were analyzed by Western blotting with primary antibodies against Nrf2, NQO1, and HO-1. Protein levels were quantified by densitometry. α-Tubulin was used as internal controls. (i) The effect of brusatol, uric acid, and ox-LDL on ET-1 expression in HUVECs. ET-1 released into the supernatant was measured by ELISA. (j) The effect of brusatol, uric acid, and ox-LDL on NO production in HUVECs. (k–m) Uric acid inhibited Nrf2 ubiquitination. Cells were treated with or without uric acid (5 mg/dL) for 6 h in the presence of MG132 (25 mM). For detecting ubiquitinated Nrf2, samples were subjected to IP with anti-Nrf2, followed by IB with an anti-Ub antibody. Nrf2 ubiquitination protein levels were quantified by densitometry. (l) Representative pictures showing the subcellular distribution of Nrf2 (FITC/green) in HUVECs. Nuclei were stained with DAPI (blue).  μm. The statistical analyses were done using GraphPad Prism V-7.0 software. Data are expressed as the (). One-way ANOVA was used in statistical analyses (/group). ,, , and versus the control. ^#, ^##, ^###, and ^#### versus the ox-LDL+UA group.

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