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 4

DDO7232 protected NCM460 cells from DSS-induced injury and downregulated inflammatory factors by activating Nrf2. (a) DDO7232 protected NCM460 cells from DSS-induced damage at different concentrations. The NCM460 cells were cotreated with different concentrations of DSS (from 2.5 mg/mL to 80 mg/mL) and 20 μM of DDO7232 for 12 h. Cell viability was measured using the MTT assay. (b) DDO7232 protected NCM460 cells in a concentration-dependent manner to reduce DSS-induced damage. The NCM460 cells were treated with different concentrations of DDO7232 (from 1 μM to 20 μM) and 20 mg/mL of DSS with each group for 12 h. The cells of control group were not treated with DDO7232 or DSS. Cell viability was measured by the MTT assay. (c) DDO7232 downregulated inflammatory factors, including IL-6, IL-1β, and TNF-α, as measured in the supernatant from DSS-induced NCM460 cells. Quantification of the inflammatory factors, including IL-6, IL-1β, and TNF-α, was determined by ELISA. (d) NCM460 cells were transiently transfected with siRNA Nrf2 (50 nM) or control siRNA and then treated with DDO7232 (20 μM). (d) Western blot analysis of Nrf2 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). Additional NCM460 cells were treated with DMSO for use as the blank control. β-Actin was used as internal reference. (e) IL-6, IL-1β, and TNF-α mRNA transcription after knockdown by Nrf2 siRNA (50 nM) and with DDO7232 (20 μM) or without DDO7232 treatment in DSS-treated NCM460 cells. DSS, DSS + DDO7232 (20 μM), and DSS + DDO7232 (20 μM) + siRNA Nrf2 groups were evaluated via qRT-PCR. (f) Living cell microscopy. NCM460 cells were pretreated with 20 μM or 5 μM DDO7232 for 12 h and then exposed to 40 mg/mL DSS for another 12 h. After these treatments, 10 μM DCFH-DA was used to stain the NCM460 cells for 20 min at 37°C, and living cell fluorescence microscopy was performed. Data are expressed as (). Differences are statistically significant at , , and .

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