3-(1H-Benzo[d]imidazol-6-yl)-5-(4-fluorophenyl)-1,2,4-oxadiazole (DDO7232), a Novel Potent Nrf2/ARE Inducer, Ameliorates DSS-Induced Murine Colitis and Protects NCM460 Cells against Oxidative Stress via ERK1/2 Phosphorylation

Figure 1

DDO7232 activated the Nrf2-ARE pathway in NCM460 cells through an Nrf2-dependent mechanism. (a) Nrf2-ARE-inducing activity of DDO7232 in HepG2-ARE-C8 cells assayed by the luciferase reporter gene assay. HepG2-ARE-C8 cells were treated with DDO7232 at gradient concentrations of 2.5, 5, 10, 20, and 40 μM for 12 h. The positive control was 40 μM tBHQ. The ARE-inducing activity of DDO7232 was compared with that of the DMSO control group. (b) Western blot analysis of the induction of Nrf2 and its downstream proteins after DDO7232 treatment for 24 h. β-Actin was used as internal reference. (c) Densitometric analysis was performed to determine the relative ratios of the protein in each fraction. The data were normalized β-actin expression and expressed as the of three individual experiments. The data were analyzed using the ImageJ 1.44p software. (d) The structure of DDO7232. (e) Quantitative real-time PCR analysis of Nrf2, HO-1, NQO1, GCLC, and GCLM in NCM460 cells. Gene transcription levels were determined after treatment with different concentrations (0, 2.5, 5, 10, 20, and 40 μM) of DDO7232 for 8 h. GAPDH was used as a control for the normal transcription of these genes. (f) The mRNA transcription of Nrf2 and its downstream markers including NQO1, GCLM, and HO-1 after treatment with Nrf2 siRNA and DDO7232. NCM460 cells were treated with Nrf2 siRNA (50 nM), DDO7232 (20 μM), and Nrf2 siRNA (50 nM) + DDO7232 (20 μM) for 8 h. GAPDH was used as the control for the normal transcription of these genes. All gene transcription levels were determined by qRT-PCR. The values shown are ( independent assays). , , , statistically significantly different from the nontreated blank control group. (g) Western blot analysis of Nrf2 and the Nrf2-regulated proteins after exposure to Nrf2 siRNA and DDO7232. The NCM460 cells were treated with Nrf2 siRNA (50 nM) or DDO7232 (20 μM) plus Nrf2 siRNA (50 nM) for 8 h. Additional NCM460 cells were treated with DMSO for use as the blank control. β-Actin was used as internal reference.

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