Participants
This is a case-controlled study performed in the endoscopy centers of Beijing Rehabilitation Hospital, Capital Medical University and the First Affiliated Hospital of Henan University of Science and Technology. Patients with functional constipation were selected using the Rome IV criteria [32]. We enrolled 39 individuals (22 females and 17 males) with functional constipation symptoms for at least 3 months into the study. Their age ranged from 24 to 74 years with a mean age of 53±14 [mean ± standard deviation (SD)] years. Exclusion criteria were patients with other chronic diseases beside constipation, including irritable bowel syndrome with constipation (IBS-C), inflammatory bowel disease, chronic systemic diseases, metabolic diseases, cancer, diseases of the nervous system, a history of antibiotics use within the last 3 months or a history of receiving calcium-channel blockers. Thirty and eight asymptomatic healthy subjects were enrolled into the control group (20 female and 18 male) during routine medical examination. The age of the control group ranged from 30 to 71 years with a mean age of 52±14 years. Both groups had similar composition in terms of age and gender (P>0.05).
Tissue procurement
The colonic biopsies were collected from participants who underwent colonic endoscopy. The samples for RNA extraction were submerged in RNAlater (Qiagen, Hilden, Germany, Cat. no. 76106) overnight to prevent RNA degradation. They were stored at -80oC along with samples for Western blotting. For histological analysis, the biopsies were fixed in 10% formalin for 12 hours, embedded in paraffin wax for staining with hematoxylin & eosin (H&E, Sigma-Aldrich, MO, USA) and immunohistochemistry (IHC) as described below.
Histopathology examination
The 4-μm slices were cut from distal colon tissue samples and stained with H&E for histological examination. The morphological features of the stained biopsies were evaluated under a light microscope to observe tissue structure and to enumerate infiltrating inflammatory cells according to previous description [33].
IHC staining for NADPH oxidases was conducted as described previously [34]. Briefly, tissue samples were stained with primary NOX1, NOX2, NOX4 DUOX2 antibodies at 4˚C overnight. The antigen-antibody complexes were detected with secondary anbodies at 37˚C for 30 minutes and subsequently probed with horseradish peroxidase (HRP)-conjugated streptavidin. A 3,3-diaminobenzidine (DAB) reagent kit was used for color development, after which samples were counterstained with hematoxylin. The IHC staining was used to determine the protein levels of Nox1, Nox2, Nox4 and Duox2 via a semi-quantitative method.
Quantitative real-time PCR assay
PCR assay for NADPH oxidases was done as described previously [33, 34]. Total RNA was extracted using the TRIzol Reagent solution (Invitrogen) according to the manufacturer’s instructions. Two micrograms of total RNA was used for cDNA synthesis using PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara) in a 40μl reaction system following these steps: 37°C for 15 min,85°C for 5s and 4°C for 10 min. The primers were designed by using the Primer3.0 [35] and synthesized Invitrogen, USA (Table 1). Real-time qPCR was done with an ABI7500 Real-Time PCR system (Applied Biosystems). A two-step method was used because of the 60°C annealing temperature. The reaction procedure began with 95°C for 30s, followed by 40 cycles of 95°C denaturation for 5s and 60°C elongation for 40s. The relative quantity of target gene mRNA expression was determined using the comparative Ct (2-ΔΔCt) method and normalized to the quantity of 18srRNA.
Western blotting analysis
Western blotting analysis was conducted as described previously [12, 34]. Protein lysates were prepared from collected tissues in RIPA lysis buffer on ice by homogenization with a grinder. Thirty micrograms of protein from each sample was denatured and resolved by 10% SDS-PAGE then transferred onto polyvinylidene-difluoride membranes. The anti-human antibodies used to detect NOX1, DUOX2, NOX2 and NOX4 proteins. The expression level of the target protein was determined by incubating the membranes with HRP-conjugated anti-rabbit immunoglobulin G and enhanced chemiluminescence reagent. The anti-GAPDH antibody was used to normalize for the protein loading. ChemiDoc XRS (Bio-Rad; USA) was used to capture the images. The intensity of images was quantified with ImageJ 1.48v program (NIH, USA).
Statistical analysis
Student’s t-test and Mann-Whitney U-test were used to assess the different groups. Wilcoxon signed-rank test was used for non-parametric data. Significant difference was defined as P<0.05. Data was reported as means ± standard deviations (SD). All statistical analysis was performed by using the SPSS 22.0 statistics package (SPSS Inc., Chicago, IL, USA).