Fig 2: Histology and IHC staining of colonic biopsies. Sections from patients (A) with constipation and healthy subjects (B) showed normal tissue structure without infiltration of inflammatory cells (n = 15 for each group, H&E, 400×). Representative immunostainings for NOX1 (C and D), DUOX2 (E and F), NOX2 (G and H), and NOX4 (I and J). The expression of NOX1 was increased in the brush surface and cytoplasm of epithelial cell of crypts of colonic biopsies in constipation subjects (C) compared to control subjects (D). DUOX2 had a similar distribution pattern as NOX1, that is, the increased expression in the brush surface and cytoplasm of colon epithelial cells of constipation subjects (D), in control subjects, there was slight expression of DUOX2 in the brush surface of colon epithelial cells (E); NOX2 was mainly expressed in unidentified cells of the submucosa. There was no difference of NOX2 expression between constipation subjects (G) and control subjects (H); NOX4 had similar expression pattern to NOX2, that is, positive staining was seen in unidentified cells of the submucosa, there was no different the subjects in 2 groups (I and J). *P < .05, vs control group, n = 17 for constipation group, n = 18 for control group. DUOX = dual oxidase, H&E = hematoxylin & eosin, IHC = immunohistochemistry, NOX = nicotinamide adenine dinucleotide phosphate oxidase.

Fig 3: Western analysis of the protein levels of NADPH oxidases in colonic biopsies. NOX1, DUOX2, NOX2, and NOX4 protein levels were analyzed in the colonic mucosa of constipation patients and control subjects. Eight samples were analyzed for each protein from both groups except 7 samples were done for NOX4. *P < .05 compared to control subjects. DUOX = dual oxidase, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, NADPH = nicotinamide adenine dinucleotide phosphate oxidase, NOX = nicotinamide adenine dinucleotide phosphate oxidase.