Fig. 6.

Effects of EC on the upregulation of intestinal NADPH oxidases and protein oxidation induced by HFD consumption in mice and by TNFα in Caco-2 cells. Total protein levels of NOX4, NOX1, and HNE-protein adducts (MW: 40 kDa) were measured by Western blot in: A- the ileum of mice fed a control diet (empty bars), the control diet supplemented with 20 mg EC/kg body weight (dashed bars), a HFD (HF) (black bars), or the HFD supplemented with 20 mg EC/kg body weight (HFE20) (grey bars); B- Caco-2 cells incubated for 6 h at 37 °C in the absence of additions (control, C) (empty bars); or after addition of 5 ng/ml TNFα in the absence (TNF) (dark blue bars) or the presence of 1 μM EC (TNF + EC) (light blue bars) or 1 μM apocynin (Apo) (grey bars). Bands were quantified and values referred to β-actin or HSC70 levels (loading controls). Results were referred to control group values (C). C- In vitro effects of 1 μM EC and apocynin (Apo) on NADPH oxidase activity were measured in membrane fractions isolated from Caco-2 cells incubated without or with 5 ng/ml TNFα for 6 h. Results are shown as mean ± SE of A- 6–8 animals/treatment, and B,C- 4 independent experiments. #Significantly different from all other groups (p < 0.05, one way ANOVA test).