Protective action of NADPH oxidase inhibitors and role of NADPH oxidase in pathogenesis of colon inflammation in mice

Figure 1 Effect of dextran sulphate sodium treatment on histological parameters in Balb/c mice. Analysis of histological parameters was performed as described in Materials and Methods (original magnification × 10). A: Untreated mice (n = 12); B: Acute dextran sulphate sodium (DSS)-induced colitis group (oral administration of 3.5% DSS solution for 7 d; n = 8); C: Chronic DSS-induced colitis group (oral administration of 3.5% DSS for 44 d; n = 8).

Figure 2 Expression of Cd68, Cd3e, Ptprc, and Vill in the colonic epithelial cells of male Balb/c mice. Cd68, Cd3e, Ptprc, and Vill were specific cell type markers (Ptprc - a hematopoietic cell marker, Cd3e - a T cell marker, Cd68 - a monocyte/macrophage marker, Vill - an epithelial cells marker). The expression level of genes in the cells was determined by real-time PCR. ^aStatistically significant differences in expression levels of inflammatory cell markers (Cd68, Cd3e, Ptprc) and epithelial cell specific marker Vill in all the experimental groups. Values are presented as mean ± SE. Control group: n = 12. Both DSS-induced colitis groups: n = 8. ^aP < 0.05, both DSS-induced colitis groups vs control. DSS: Dextran sulphate sodium.

Figure 3 Assessment of viability of mouse colonic epithelial cells. Cells were incubated with 20 μg/mL LPS, 1 mmol/L apocynin (Apoc), 20 μg/mL LPS + 1 mmol/L Apoc, and without stimulation for 24 h. A: Untreated mice; B: Acute DSS-induced colitis group; C: Chronic DSS-induced colitis group. ^aP < 0.05, DSS-induced colitis groups untreated vs one of the stimulated subgroups in the control; ^cP < 0.05, DSS-induced colitis groups vs LPS and LPS + Apoc treated subgroups in the control; ^eP < 0.05, chronic DSS-induced colitis groups in the untreated subgroup vs control; ^gP < 0.05, chronic DSS-induced colitis groups in the LPS subgroup vs control; Values are presented as mean ± SE. Untreated mouse group: n = 12. Both DSS-induced colitis groups: n = 8. LPS: Lipopolysaccharide.

Figure 4 Assessment of hydrogen peroxide production. A: Mouse colonic epithelial cells were incubated for 30 min with 10 μg/mL LPS, 10 μmol/L DPI, 10 μg/mL LPS + 10 μmol/L DPI or without stimulation; B: Colonic biopsies (10 mg) of DSS-induced colitis mice and control subjects were incubated without stimulation for 30 min. ^aP < 0.05, DSS-induced colitis groups vs stimulated and un-stimulated cell subgroups within control. ^cP < 0.05, untreated vs DPI treated subgroups in the chronic DSS-induced colitis group. ^eP < 0.05, LPS vs LPS + DPI treated cell subgroups in both DSS-induced colitis groups. ^gP < 0.05, untreated vs LPS treated cell subgroups in the acute DSS-induced colitis group. ⁱP < 0.05, DSS-induced colitis groups in the mouse colonic biopsies vs control. Values are mean ± SE. Untreated mouse group: n = 12. Both DSS-induced colitis groups: n = 8.

Figure 5 Assessment of TNF-α production by mouse colonic epithelial cells. TNF-α concentration was measured in cell media after 24 h incubation with 20 μg/mL of LPS, 1 mmol/L of apocynin (Apoc), 20 μg/mL LPS + 1 mmol/L Apoc or without stimulation; ^aP < 0.05, DSS-induced colitis groups in all subgroups vs control; ^cP < 0.05, untreated vs LPS treated subgroup in the chronic DSS-induced colitis group; ^eP < 0.05, LPS vs LPS + apocynin treated subgroup in both DSS-induced colitis groups. Values are presented as mean ± SE. Untreated mouse group: n = 12. Both DSS-induced colitis groups: n = 8.

Figure 6 Expression of Nox1 in the colonic epithelial cells of male Balb/c mice. The expression levels of genes in these cells were determined by real-time PCR. ^aP < 0.05 chronic DSS-induced colitis group vs both other groups. Values are presented as mean ± SE. Control group: n = 12. Both DSS-induced colitis groups: n = 8. DSS: Dextran sulphate sodium.