Protective Effects of Imatinib on a DSS-induced Colitis Model Through Regulation of Apoptosis and Inflammation

Materials and Methods

Animals. Male C57BL/6J mice (6 weeks old, 20-22 g) were obtained from Samtako (Gyeonggi-do, Republic of Korea) and housed in plastic cages under specific pathogen-free conditions (24±2°C, 12 h light/dark cycle). The mice were allowed to adapt to the experimental environment for one week. The animals had free access to sterile water and food throughout the entire experimental period. The animal experiments and protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Kyungpook National University, following standard operating procedures (2022-0206).

Experimental design. To investigate the effects of imatinib on DSS-induced colitis (DSS; 160110; MP Biomedicals, Santa Ana, CA, USA), we randomly assigned 24 mice into four groups: the control group (Control), DSS-induced colitis group (DSS), DSS-induced colitis group treated with 10 mg/kg imatinib (Im10), and the DSS-induced colitis group treated with 20 mg/kg imatinib (Im20).

Imatinib (HY-15463, MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in saline and administered orally for seven days, spanning from day 0 to the seventh day (Figure 1). Acute colitis was induced by providing the mice with 3% DSS (w/v) in their drinking water for five consecutive days, starting from day 0 and continuing until the fifth day. On the 10th day, the physical phenotypes of the colon tissues of mice were examined and the lengths of the large intestines were measured. Additionally, spleen weight was measured, and its percentage of the total body weight was calculated. Colon tissue samples were collected and stored at −150°C until further experimentation.

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Figure 1.

Overview of the study design. Mice were acclimatized for seven days before initiating group-specific treatment. Colitis was induced by administering 3% dextran sulfate sodium (DSS) in drinking water from day 0 to the fifth day. Concurrently, imatinib (at doses of 10 mg/kg or 20 mg/kg) was orally administered once daily from day 0 to the seventh day using oral gavage. All mice were euthanized on the 10^th day.

Disease activity index. Disease Activity Index (DAI) scores were assessed daily during the 10-day period of DSS treatment. The following data were recorded: changes in body weight, stool consistency, and the presence of fecal occult blood. Body weight loss was categorized as follows: 0 (no loss), 1 (1-5% loss), 2 (5-10% loss), 3 (10-20% loss), or 4 (over 20% loss). Stool consistency was assessed as follows: 0 (normally formed), 2 (loose stools), and 4 (diarrhea). The presence of blood in the stool was scored as follows: 0 (negative), 2 (positive), and 4 (gross bleeding). The mean of all the scores obtained for each parameter was calculated to determine the DAI.

Hematoxylin and eosin (H&E) staining. Colon tissue samples were fixed in 4% formaldehyde in 1× phosphate-buffered saline, followed by embedding in paraffin. Afterward, the paraffin-embedded tissue was cut into 5-μm sections. Hematoxylin and eosin staining was conducted to visualize the morphological changes. The stained tissue sections were then examined using a MoticEasyScan One (Motic, Hong Kong, SAR) to check for cellular infiltration, mucosa length and epithelial damage caused by inflammatory cells. Scoring was conducted for all tissue sections.

Histological score. Colonic histological tissues were observed using a MoticEasyScan One (Motic) to assess histological damage. The severity of colonic inflammation, colonic epithelial cell infiltration, crypt destruction, and the extent of cell infiltration were calculated. Each subscore ranged from 0 to 4, representing the absence of changes to maximum tissue damage. Specifically, the scoring criteria were as follows: crypts: 0 (intact crypts), 1 (intact crypts), 2 (variable crypt diameter), 3 (atrophied crypts), and 4 (mucosa devoid of crypts); loss of surface epithelium: 0 (intact surface epithelium), 1 (sloughing off epithelial surface), 2 (patchy loss of surface epithelium), 3 (moderate loss of surface epithelium), and 4 (severe loss/erosion of surface epithelium); cell infiltration: 0 (clear mucosa/submucosa), 1 (mucosal/lamina propria infiltration), 2 (mucosal and submucosal infiltration), 3 (moderate cryptitis/infiltration into crypt epithelial cells), 4 (severe cryptitis). These scores were used to quantify the degree of histological damage.

Quantitative real-time polymerase chain reaction (qRT-PCR) assay. Total RNA was isolated from mouse colon tissues using TRI Solution (TS200-001; Bio Science Technology, Daejeon, Republic of Korea). Next, total RNA was synthesized to cDNA using a PrimeScript™ 1st Strand cDNA Synthesis Kit (6110; Takara Biotechnology Co., Ltd., Shiga, Japan) with oligo-dT primers. The qRT-PCR mixture contained 8 μl of cDNA, 10 μl of Power SYBR^® Green PCR Master Mix (4367659; Thermo Fisher Scientific, Waltham, MA, USA), 1 μl (0.2 pmol) of forward primer, and 1 μl (0.2 pmol) of reverse primer. The qPCR was run on the Applied Biosystems RT-PCR program (StepOnePlus™ Real-Time PCR System, Thermo Fisher Scientific) at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min, and a melt curve stage at 95°C for 15 s. The primer sequences used were as follows: mTNF-α (F: 5′-ATGGGCTCCCTCTCATCAGT-3′, R: 5′-TGGTTTGCTACGACGTGGG-3′); mIL-1β (F: 5′-TGGTTTGCTACGACGTGGG-3′, R: 5′-CCCCAGGGCAT GTTAAGGA-3′); mIL-6 (F: 5′-TGACCCTGAGCGACCTG TCT-3′, R: 5′-RGTTGTGCAATGGCAATTCTGA-3′); and mβ-actin (F: 5′-GGCTCTTTTCCAGCCTTCCT-3′, R: 5′-GT CTTTACGGATGTCAACGTCACA-3′). All data were analyzed using the 2-ΔΔCq method and expressed as the fold change relative to the controls. The relative expression levels of TNF-α, IL-1β, and IL-6 were normalized to those of β-actin.

Western blot analysis. Colon samples were processed using the PRO-PREP for Cell/Tissue Protein Extraction Solution kit (17081; Intron Biotechnology, Kirkland, WA, USA) along with the Protease Inhibitor Cocktail (P3100-001; GenDEPOT, Katy, TX, USA) for lysis. The protein concentrations in the tissue lysates were determined using BCA Protein Assay Reagent A (WF322788; Thermo Fisher Scientific) and Reagent B (23224; PIERCE, Kyiv, Ukraine). Subsequently, 30 μg of protein from each sample was separated on a 10-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% skim milk in 1×Tris-buffered saline (TBS)-0.05% Tween-20 (TBST) at room temperature for 1 h. They were then incubated with primary antibodies against IL-1β (sc-52012; Santa Cruz Biotechnology, Dallas, TX, USA), TNF-α (3707; Cell Signaling Technology, Danvers, MA, USA), IL-6 (sc-57315; Santa Cruz Biotechnology), cleaved caspase-3 (9661; Cell Signaling Technology), p-p53 (377567; Santa Cruz Biotechnology), p53 (sc-126; Santa Cruz Biotechnology), cleaved caspase-8 (D35G2; Cell Signaling Technology), caspase-8 (D35G2; Cell Signaling Technology), ZO-1 (sc-33725; Santa Cruz Biotechnology) and claudin-2 (E1H90; Cell Signaling Technology). β-actin (sc-47778; Santa Cruz Biotechnology) was used as a loading control. After three washes with TBST, the membranes were incubated at room temperature for 1 h with the secondary antibodies normal rat IgG-HRP (sc-2750; Santa Cruz Biotechnology), goat anti-mouse IgG H&L horseradish peroxidase (HRP) (ab6789; Abcam, Cambridge, UK), and anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology). Finally, protein bands were visualized using an Image Quant LAS 500 (Amersham™ ImageQuant™ 500; Cytiva, Uppsala, Sweden), with β-actin as an internal control. Data were quantified using ImageJ (United States National Institutes of Health, Bethesda, MD, USA).

Immunohistochemical analysis. The colon collected from each mouse was directly fixed in 4% buffered paraformaldehyde, dehydrated in 100% ethanol, and embedded in paraffin. Next, 5-μm sections were prepared from the embedded tissue. The colon sections were stained and ZO-1 and occludin were used as tight-junction markers. For ZO-1 and occludin staining, the sections were rehydrated and treated with 3% H₂O₂, soaked and boiled in citrate buffer for 15 min (three times each for 5 min), stored in a dark room with 10% normal serum for 1 h, and incubated with the primary antibody at 4°C overnight. The slides were then incubated overnight with ZO-1 (sc-33725; Santa Cruz Biotechnology) and occludin (E6B4R; Cell Signaling Technology), followed by the secondary antibody for 30 min. Finally, ZO-1– and occludin-positive cells were detected using the VECTASTAIN ABC streptavidin-HRP system (ZG0608; Vector Laboratories, Newark, CA, USA). The sections were then counterstained with hematoxylin before being dehydrated and mounted. All tissue sections were observed under a microscope (MoticEasyScan One; Motic).

Statistical analysis. Data were analyzed using SPSS Statistics for Windows, version 25.0 (IBM, Armonk, NY, USA). One-way analysis of variance (ANOVA) was used to compare the control and DSS groups, as well as the DSS group and each treatment group (n=6). All data are presented as mean±standard error of the mean (SEM). A p-value <0.05 was considered statistically significant.