Abstract

Ulcerative Colitis (UC) is a chronic inflammatory bowel disease affecting the colon mucosa. The origin of UC is not completely clarified, but genetic and autoimmune reactions causing intestinal inflammation and injury were considered the main pathological manifestations. UC-caused injury is a risk factor for colon cancer. Inflammatory response inhibition, along with wound healing of colonic epithelial cells, is critical for UC treatment. The present short report examines the effects of opium tincture on wound-healing molecular pathways.

The rat model of UC was produced using Dextran Sulfate Sodium (DSS)-Induced acute colitis. The UC model rats were treated with taper up-off of opium tincture protocol called Dezhakam-Step-Time (DST) in four different dosages. The colon tissues were collected and RNA extraction and cDNA synthesis were performed. The expression level of fifteen genes related to wound healing was evaluated using the quantitative Realtime PCR.

Fourteen genes were significantly altered in UC models vs. normal controls. Twelve genes were changed after the taperup-off treatment of opium tincture. The wound healing-related genes were up-regulated after the opium tincture treatment, especially at higher dosages.

The early findings of the gene expression study regarding UC model rats treated with opium tincture suggest that the wound healing pathways activity has been increased after the treatment. It seems that the opium tincture may induce the activation of wound healing with upregulation of related genes including growth factors which may lead to a potential repair of injured tissues such as intestinal mucus that in turn could be a hope for treatment of UC and reduction of the risk of UC induced colon cancer.

Keywords: Ulcerative colitis; Opium tincture; Taper up-off; Wound healing; Quantitative real-time PCR.

Citation: Dezhakam H, Dezhakam A, Dezhakam A, Dezhakam S, Haghighatfard A. Short report of ulcerative colitis rodent model treatment with taper up- off protocol of opium tincture revealed an expression alteration in wound healing genes. Open J Clin Med Images. 2025; 5(2): 1207.

Introduction

Ulcerative Colitis (UC) is a chronic inflammatory bowel disease characterized by inflammation of the colon’s mucosa with unclear origins. Epidemiologically, UC exhibits a bimodal age of onset, with peaks typically occurring between 15-25 years and again between 55-65 years of age [1]. The incidence and prevalence of UC vary geographically, with higher rates observed in North America and Europe compared to Asia and Africa, although incidence is rising globally [2]. The etiology of UC is multifactorial and not fully understood, involving a complex interplay of genetic predisposition, environmental factors, and immune dysregulation [3]. Genetic studies have identified numerous susceptibility genes, particularly those involved in immune responses, such as the HLA region [4]. Environmental factors, including alterations in the gut microbiome, and lifestyle factors like diet and smoking, are also implicated in disease development. It is hypothesized that in genetically susceptible individuals, an aberrant immune response to luminal antigens triggers chronic inflammation and damage to the colonic mucosa [5].

Wound healing is a multifaceted process; starts with a series of cellular and molecular procedures and consists of several stages: Hemostasis, inflammation, Proliferation, and remodeling [6]. Growth factors are essential proteins that act as mediators and receptors in the complex process of wound healing, orchestrating the necessary cellular interactions, and are involved in tissue regeneration like fibroblasts, platelets, and epithelial, etc. reduction of growth factors functions may lead to impaired healing process [7].

IL-22, a cytokine, has been identified as playing a role in UC, and animal model studies suggest that IL-22 may protect against colitis by promoting epithelial cell proliferation, mucus production, and antimicrobial peptide production, all of which contribute to intestinal barrier repair and reduced inflammation [8]. IL-22 plays a significant role in this process by promoting the proliferation of epithelial cells, the production of mucus, and the expression of antimicrobial peptides, all of which contribute to the repair of the intestinal barrier [9]. However, IL-22’s role is complex and controversial, and chronic overexpression could lead to increased inflammation, colonic neutrophil infiltration, and resistance to ustekinumab therapy [10]. The activity of IL22 is influenced by various factors including AhR, STAT3, CARD9, Musculin, Treg cells, retinoic acid, and the gut microbiota [11].

In ulcerative colitis, the intestinal mucosal barrier is damaged, and the gut microbiota is dysfunctional. This damage leads to inflammation, with immune cells infiltrating the mucosa and submucosa. Effective wound healing is crucial in UC to repair the damaged tissue and restore normal function [12]. Several factors influence wound healing in UC. STAT3 activation, often triggered by IL-22, is essential for mucosal wound healing. CARD9 also promotes intestinal wound healing, possibly through its regulation of IL-22 [13]. On the other hand, a balanced gut microbiota is important; a decrease in beneficial bacteria and an increase in pathogenic bacteria can impair the healing process [14].

While opium-based medicines are widely used as painkillers, potential side effects, and dependence concerns related to opium, careful considerations regarding these compounds are required. Opium tincture, also known as Laudanum is an opioid-based analgesic medication that also has been used as a treatment for diarrhea, control of neonatal opioid withdrawal syndrome, and used as a cough suppressant [15]. The effects of opium and opiate alkaloids on the gene expression and epigenetic patterns of mammals are not clarified. Gene expression analysis may shed light on the polyhedral of the role of opioid compounds on the whole body including basic molecular pathways such as the immune system, wound healing, and melanogenesis [16].

The present article is the report of early findings of the study aimed to evaluate the transcriptomic pattern of the ulcerative colitis rat model treated with a novel protocol of taper up-off of opium tincture, to understand the potential effects of opium on ulcerative colitis. This early report focuses on the expression level of wound healing-related genes in opium tincture-treated, untreated, and control groups.

Materials and methods

Animal modeling protocol

This study utilized Sprague-Dawley rats, with twelve males and twelve females aged 7-8 weeks in each group. At the start of the study, male body weights ranged from 220 grams to 245 grams, and female body weights ranged from 192 grams to 202 grams. The Dextran Sulfate Sodium (DSS)-Induced acute Colitis used in this study closely mimics the pathology of human UC [17]. Acute colitis was induced by administering 3% DSS in drinking water for a week (Days 0-7). Mice were then given normal drinking water for 5 days before starting the treatment on Day 12. Animals were housed in groups of three per cage with access to ad libitum food. The daily body weights and morbidity were assessed each morning and fecal samples were collected from all rats on sacrifice day. Disease Activity Index (DAI) scores were calculated based on body weight recovery at sacrifice, stool consistency, and the presence of blood in stool. Histopathological damage was scored from H&E-stained distal colon cross-sections, evaluating crypt architecture, inflammatory cell infiltrates, muscle thickening, goblet cell depletion, and crypt abscesses, as previously described [18].

Opium tincture treatment protocol

The rats received opium tincture according to a laboratory designed taper-up-off protocol, the Dezhakam-Step-Time (DST) method. This method consisted of an 18-step administration schedule, with doses given twice daily for 9 days to taper up, and then twice daily for another 9 days to taper down. Treatment began with the lowest dose, which was then increased by 20% (multiplied by a coefficient of 0.8) at each administration. After 18 administrations (9 days), the process was reversed, with the dose reduced by 20% at each of the following 18 administrations. Consequently, the first and last doses were the same. The experimental groups and their details are outlined in Table 1. Four different dosage regimens were used. As an example, the first regimen started at 2.35 mg/kg, reaching 65.58 mg/kg by the 18th dose. The starting doses for the second, third, and fourth regimens were 3.52 mg/kg, 4.7 mg/kg, and 5.87 mg/ kg, respectively.

Scarification and tissue collection

At the end of the experiment, rodents were euthanized by sodium pentobarbital overdose and administered intravenously based on protocols published in previous pharmacology and toxicity studies. Almost a 2-centimeter portion of the distal colon of euthanized rats was collected for molecular analysis. Housing and euthanasia procedures are conducted based on the ARRIVE55 Guidelines checklist and protocols of previous studies [19,20].

Molecular evaluations

The RNA extraction mini kit (Qiagen, Hilden, Germany) was used for total RNA extraction from the rat colon samples according to the manufacturer’s instructions. To remove any contaminating genomic DNA, the extracted RNA was treated with DNase I, RNase-free (Fermentas, Latvia; #EN0521) following the manufacturer’s protocol. The quantity of the extracted RNA was determined using a Nanodrop-1000 spectrophotometer, and RNA quality was assessed using the BioRad Experion automated electrophoresis system (BioRad Laboratories Inc.). All extracted RNA samples were stored at -80°C until further processing. The cDNA was synthesized using the RevertAid Premium First Strand cDNA Synthesis Kit (Thermo Scientific Fermentas, Latvia; #K1652), following the manufacturer’s protocol.

The candidate genes (Table 2) were quantified using RealTime PCR. Specific primers and probes were designed using the GenScript Real-time PCR (TaqMan) Primer Design software and their specificity was confirmed by BLAST analysis against the NCBI database. Standard curves were generated using serial four-fold dilutions of pooled cDNA from randomly selected control rat RNA samples. Quantitative Real-Time PCR was performed in triplicate using the CFX96 Touch Real-Time PCR Detection System (BIO-RAD, California, United States). Quality control criteria included the absence of signal in no-template control samples and a standard curve R² value greater than 0.99. PCR reaction efficiency was calculated using the Lin-Reg PCR online software (Amsterdam, Netherlands). The TaqMan® PCR Starter Kit (Thermo Scientific Fermentas, Latvia) was used for all samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the housekeeping gene for normalization, and relative gene expression ratios were determined using the Livak (2-ΔΔCt) method.

Statistical assessments

Statistical analyses were conducted using SPSS version 25. The normality of data distribution for all variables was assessed using the Kolmogorov-Smirnov test. One-way ANOVA was employed to determine statistical differences between multiple group comparisons. To control for potential confounding factors, ANCOVA was used to evaluate the persistence of significant differences between groups, with RNA integrity number, cDNA synthesis quality, qPCR plates/runs, and primer and probe efficiency defined as covariates. Bonferroni correction was applied for multiple comparisons. Descriptive data are presented as mean ± Standard Deviation (SD).

Results

Clinical observations

Body weights of all rodents were measured before the first dose administration of DSS-induced colitis and after the UC modeling before the treatment and after the treatment. Results showed that the weight of DSS-induced colitis was significantly lower compared with normal controls (p-value=0.003) which was consequential to DSS treatment and was expected in the colitis animals. During the treatment period, one female rat from G3, and two male and one female rat from the untreated DSS-induced colitis group died before the scarification day.

Gene expression analysis

RNA quality analysis showed all samples had RNA Quality Indicator (RQI) values higher than 9.5. Findings of gene expression comparisons between groups compared with normal control rats have been presented in Table 3. The most significant change in expression level has been detected in G3 and G4. From 15 genes three genes (VEGF165, FGF2, and F3) showed no significant difference between the UC model and treatment groups. All other twelve genes were significantly altered in the G3 and G4 treatment groups compared with the UC model. No significant difference was observed in mRNA levels of any genes between female and male rats in any group.

Table 1: Groups’ demographic data.