Patients and specimens

A total of 66 patients diagnosed with Crohn's disease (CD), 33 patients diagnosed with ulcerative colitis (UC), and 10 healthy volunteers were prospectively recruited for this study. All patients with inflammatory bowel disease (IBD) were enrolled during the active phase of the disease. Exclusion criteria included periods of remission in IBD, as well as comorbidities such as diabetes, asthma, chronic obstructive pulmonary disease, heart failure, renal dysfunction, rhinitis, and infectious diseases. The diagnosis of IBD was made according to the recommended standards set by the World Gastroenterology Organization (WGO) [16]. The protocol received approval from the Ethics Committee of the First Affiliated Hospital of Anhui Medical University, and informed consent was obtained from all participating subjects. Urine samples were collected and the levels of LTE4, PGE2, and Hcy were quantified using the enzyme-linked immunosorbent assay (ELISA) technique provided by Wuhan Xinqidi Biological Technology Co., Ltd., China. The study was reported in accordance with Declaration of Helsinki.

This study was approved by the Institutional Review Board (IRB) at The First Affiliated Hospital of Anhui Medical University. Every patients were consented by an informed consent process that was reviewed by the IRB.

Induction of hyperhomocysteinemia in rat’s colitis

A colitis model was established in Sprague–Dawley male rats weighing 200 ± 20 g by intracolonic administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS). The control group received an enema of normal saline of equal volume instead of TNBS (Sigma-Aldrich Chemical Co., USA). Specific pathogen-free (SPF)-grade Sprague–Dawley (SD) male rats were obtained from the Experimental Animal Center in Anhui Province. The rats were randomly divided into four groups (n = 8 for each group) and were maintained for 1 week before the experiments. Group A served as the normal group, Group B as the TNBS/ethanol model group, Group C as the normal + Hcy injection group, and Group D as the TNBS/ethanol + Hcy injection group. Hcy (Sigma-Aldrich Chemical Co., USA) was dissolved in 0.9% normal saline to achieve a pH value of 7.4. Following our previous study, Hcy (0.03 μmol/g) was administered subcutaneously on the first day after the preparation of the TNBS/ethanol model, with an 8-h interval for 30 consecutive days. This dosage regimen was found to be sufficient in inducing hyperhomocysteinemia in rats [9]. Throughout the experiment, the body weight and stool properties of rats in each group were observed and recorded to assess the activity and severity of colon inflammation using the disease activity index (DAI). The distal colon samples of the rats were preserved in a 10% formaldehyde solution, subsequently embedded in paraffin, and subjected to histological index (HI) evaluation using HE staining. The protocols employed in this study were ethically approved by the Ethics Committee of Animal Experiments at Anhui Medical University. All invasive procedures were performed under pentobarbital sodium anesthesia, and utmost efforts were made to minimize any potential distress experienced by the animals. All methods are reported in accordance with ARRIVE guidelines.

All animal studies and experimental procedures were approved by the Animal Care and Use Committee of the animal facility at The First Affiliated Hospital of Anhui Medical University. All methods were performed in accordance with the relevant guidelines and regulations.

Culture of Caco-2 cells

Caco-2 cells, a well-established human colorectal adenocarcinoma cell strain, were procured from the cell bank of the Chinese Academy of Sciences. These cells were chosen due to their extensive utilization in investigating the permeability of the human digestive tract mucosa. Following a specific duration of cultivation, typically lasting 21 days, the brush border and intercellular connections of Caco-2 cells undergo complete differentiation, exhibiting morphological, functional, and enzymatic similarities to the human intestinal barrier. The cultivation of Caco-2 cells was conducted in DMEM medium supplemented with high glucose, 1% non-essential amino acids, L-glutamine, 10% FBS, and antibiotics (10000 U/ml penicillin, 10000 μg/ml streptomycin).

Detection of 5-LOX, COX-2 and NF-κB(p65) mRNA expression in rats colonic mucosa by real-time quantitative polymerase chain reaction (RT-qPCR)

Tissue samples were collected and promptly frozen in liquid nitrogen. Total RNA was extracted from 100 mg of colonic tissue following the guidelines provided by the manufacturer (Invitrogen, USA). This involved homogenizing the tissue in a guanidine thiocyanate solution, extracting it with acid phenol/chloroform, and precipitating it with isopropanol. The resulting RNA pellets were washed with 70% ethanol, dissolved in DEPC water, and stored at – 80 ℃. To generate single stranded cDNA, aliquots of each RNA sample (1 μg) were subjected to reverse transcription using a kit (Thermo, USA) along with specific primers and reverse transcriptase. Samples of cDNA were then analysed by PCR amplification using pairs of oligonucleotide primers specific for 5-LOX, COX-2 and NF-κB(p65). 5-LOX primer sequences were: Forward primer:5′-AGTTGCCTAAGAAGCTCCCA-3′, Reverse primer: 5′-GCATCAATGCCATCCAGTAG -3′, amplifying a 133 bp cDNA fragment; COX-2 primer sequences were: Forward primer: 5′-AATCGCTGTACAAGCAGTGG-3′, Reverse primer: 5′-GCAGCCATTTCTTTCTCTCC-3′, amplifying a 144 bp cDNA fragment; NF-κB(p65) primer sequences were: Forward primer: 5′-GCCTCATCCACATGAACTTG-3′, Reverse primer: 5′-CGCTTCTTCACACACTGGAT-3′, amplifying a 120 bp cDNA fragment; β-actin primer sequences were: Forward primer: 5′-CCCATCTATGAGGGTTACGC-3′, Reverse primer: 5′-TTTAATGTCACGCACGATTTC-3′, amplifying a 150 bp cDNA fragment; Each PCR reaction contained 2 × SYBR Green mixture, 10 μM Forward primer, 10 μM Reverse primer, cDNA, RNase Free water. Following 5 min of denaturation at 95 ℃, hot star reactions were initiated (annealing at 60 ℃ for 30 s, extension at 72 ℃ for 30 s, and denaturation at 95 ℃ for 10 s) and run for up to 40 cycles. The relative mRNA levels were determined using the 2ˆ−ΔΔCt method.

Electrophoretic mobility shift assay (EMSA)

Nuclear proteins were extracted from Caco-2 cells and subjected to electrophoretic mobility shift assay (EMSA) in order to assess the binding activity between NF-κB and DNA. The concentration of nuclear proteins was determined using the BCA kit (Pierce, USA). Subsequently, nuclear proteins (ranging from 1 to 5 μl, depending on the concentration of nucleoprotein) were incubated with the reaction buffer for 10 min, followed by incubation with a biotin-labeled NF-κB probe (32P end-labeled oligonucleotide). The resulting reaction mixture was separated on a nondenaturing 6.5% polyacrylamide gel, which was then exposed to radiograph film at a low temperature. The binding of labeled oligonucleotide to nuclear proteins was blocked by adding unlabeled oligonucleotide to the reaction mixture to confirm that binding of 32P end-labeled oligonucleotide to NF-κB was specific sequence.

Detection of LTE4 and PGE2 levels in rat colonic homogenate and the supernatant of Caco-2 cells by ELISA

Colonic tissue specimens measuring 8 cm were obtained and processed for colonic homogenate. A total of 5*10^5 cells were cultured on 6-well cell culture plates using DMEM medium supplemented with varying concentrations of Hcy (0, 10, 20, 50, 100 μmol/L). Following incubation for 1 h, 3 h, and 6 h, the supernatant of Caco-2 cells was collected. The levels of LTE4 and PGE2 in both the colonic homogenate and the supernatant of Caco-2 cells were determined using the ELISA method.

Statistical analysis

The data were presented as mean ± standard deviation (SD). Between groups or paired data were analyzed using a t test. Multiple group comparisons were conducted using variance analysis. Quantitative data were analyzed using Pearson correlation analysis. Statistical analysis was performed using SPSS 20.0 software. The charts were generated using the Origin 9 Software System. A significance level of P < 0.05 was deemed statistically significant.