WIN55,212-2 attenuates intestinal fibrosis in a DSS-induced mouse model
https://ror.org/02t4ekc95grid.8267.b0000 0001 2165 3025Department of Digestive Tract Diseases, Faculty of Medicine, Medical University of Lodz, Lodz, Poland
https://ror.org/02t4ekc95grid.8267.b0000 0001 2165 3025Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland
Abstract
Background
Inflammatory bowel diseases (IBD) are often associated with intestinal fibrosis. There is increased interest in new methods for managing intestinal fibrosis, among which the endocannabinoid system (ECS) is an interesting therapeutic target. The primary purpose of the study was to evaluate the effect of CB1/2 agonist, WIN 55,212-2 (WIN) on expression of fibrosis regulatory proteins: alpha smooth muscle Actin 2 (ACTA2), Collagen I (COLIA1), Fibronectin 1 (FN1) and SMAD family member 3 (SMAD3) in a mice model of dextran sulfate sodium (DSS)-induced intestinal fibrosis.
Methods
Chronic intestinal fibrosis was induced by oral administration of 1.5% DSS in rotation with tap water for 3 weeks in three cycles. WIN was administered intraperitoneally, once daily starting from day 10th and continued every other day until day 77th. The expression of genes encoding ACTA2, COL1A1, FN1, SMAD3, Col1a1, Fn1 both in mouse and human colon, was assessed with real-time PCR.
Results
We found lower relative expression of genes encoding fibrosis regulatory proteins in colonic mucosa of mice treated with WIN than in inflamed mice: ACTA2 (–51%), COL1A1 (–44%), FN1 (–60%), and SMAD3 (–22%). Human colonic mucosal patients with UC showed higher expression of COL1A1 (+ 57%) and SMAD3 (+ 34%) compared to healthy controls.
Conclusions
Our results show that regulation of ECS activity may play a role in the inhibition of intestinal fibrosis in IBD and appears as a potential therapeutic target in patients at high risk of developing strictures.
Introduction
Inflammatory bowel disease (IBD) is a group of diseases characterized by chronic-relapsing inflammation of the gastrointestinal (GI) tract. Among these diseases classified as IBD, there are two main distinct pathologies: Crohn’s disease (CD) and ulcerative colitis (UC). The prevalence of IBD among the European and Worldwide population has increased in recent years. According to data from 1990, there were an estimated 3.32 million cases of IBD worldwide, which increased to 4.90 million cases in 2019, indicating a 47.45% rise in prevalence between 1990 and 2019 [1]. The chronic inflammation is accompanied by fibrosis, which occurs in over 30% of CD patients and 5% of UC patients, leading to severe local and systemic complications. The fibrosis is associated with increased production of extracellular matrix (ECM) and cross-linking enzymes by intestinal mesenchymal cells, mainly fibroblasts, myofibroblasts, and smooth muscle cells [2]. Excessive production of ECM is associated with intestinal tissue remodeling and the development of intestinal strictures. Among patients with advanced intestinal fibrosis and complications such as strictures and adhesions, the vast majority require complicated endoscopic or surgical treatment [3].
Traditionally, IBD treatment involves the use of anti-inflammatory drugs such as glucocorticosteroids, aminosalicylates, immunosuppressants, biologicals, and small molecule drugs. However, this pharmacotherapy does not prevent intestinal fibrosis and its complications [4]. The proposed therapeutic targets for intestinal fibrosis include signaling pathways involving transforming growth factor (TGF)-β, epithelial-mesenchymal transition, the balance between tissue inhibitor of metalloproteinase and matrix metalloproteinase, and the endogenous cannabinoid system (ECS).
The ECS is a widespread system in the human body that consists of classical (CB) and non-classical receptors, endogenous ligands, and enzymes that are responsible for the regulation of their metabolism [5]. Among the classic endocannabinoid receptors, there are type G protein-coupled receptors: type 1 (CB1) and type 2 (CB2). CB1 receptors are typically localized in the central and peripheral nervous system, as well as digestive, reproductive, immune, respiratory, circulatory, and excretory systems [6]. The CB2 are mainly found in the peripheral nervous system, the immune and hematopoietic cells, and the GI tract. Both CB1 and CB2 receptors are present in the enteric nervous system [7]. Endogenous ligands, known as endocannabinoids, influence the activity of cannabinoid receptors. Among them, the most known endocannabinoids are arachidonoylethanolamine (anandamide, AEA) and 2 arachidonoylglycerol (2-AG) [8]. Enzymes responsible for the production of these endocannabinoids are N-acyl phosphatidylethanolamine phospholipase D and diacylglycerol lipase. The ECS influences the motility, secretion, and sensitivity in the GI tract, as well as regulates the intestinal inflammation and mucosal permeability [9, 10].
It was demonstrated that CB2 agonists exert anti-inflammatory effects by downregulating mast cells and granulocytes, thereby reducing the release of pro-inflammatory cytokines [11]. Conversely, CB1 receptor antagonist has been linked to colitis exacerbation in animal models. In a study conducted by F. Massa et al., administration of CB1 antagonist SR141716A induced a more severe inflammatory response compared to vehicle-treated mice in models of colitis induced by 2,4-dinitrobenzene sulfonic acid and dextran sodium sulfate (DSS) [12]. The worsening of colitis was evidenced by an increased macro- and microscopic score, and elevated MPO activity. Moreover, genetic ablation of CB1 receptor function was associated with increased colitis severity, reinforcing the protective role of CB1 signaling in colitis.
While studies on the relationship between ECS and intestinal fibrosis are still limited, research on other fibrotic diseases, such as systemic sclerosis, provides insights into its possible impact [13]. In a bleomycin-induced dermal fibrosis model, CB1-deficient mice (CB2−/−) were protected against fibrosis compared to their CB2-expressing littermates (CB2+/+), exhibiting reduced dermal thickening and lower myofibroblast count. Conversely, administration of the highly selective CB2 agonist ACEA in CB2+/+ mice intensified the inflammatory response to bleomycin [13]. In contrast, another study demonstrated that treatment with the synthetic selective CB1 agonist WIN55,212-2 mitigated fibrosis in bleomycin-induced dermal fibrosis by inhibiting fibroblast activation and downregulating the expression of TGF-β, CTGF, and PDGF-BB, as well as SMAD2/3 phosphorylation [14].
In our study, we focused on evaluating the effects of the CB1/2 agonist, WIN55,212-2, on the expression of key fibrosis-regulatory proteins in a mouse model of dextran sulfate sodium (DSS)-induced intestinal fibrosis. In particular, we analyzed the expression of alpha smooth muscle actin 2 (α-SMA, Acta2), collagen type 1 alpha 1 (Col1A1), fibronectin 1 (Fn1), and SMAD family member 3 (Smad3). α-SMA is a marker of activated myofibroblasts, which induce subepithelial matrix remodeling, collagen type 1 alpha 1 and fibronectin 1 are involved in ECM formation, and Smad3 induces the TGF-β downstream signaling. Additionally, we compared the expression levels of these proteins in colonic samples from patients with UC and healthy controls (HC).
Materials and methods
Animals
Male BALB/c mice were obtained from the M. Mossakowski Institute of Experimental and Clinical Medicine, Polish Academy of Sciences in Warsaw. All 25 individuals were 6–8 weeks of development and weighed 20–25 g. The animals were housed in plastic cages lined with sawdust with free access to food and tap water, and a paper roll to reduce the animals’ stress levels. The model used a daily artificial light cycle of 12 h of light − 12 h of darkness at a constant temperature (20–22 °C), humidity (45–65%), and number of air changes (15–20 per hour). Mice were sacrificed via cervical dislocation 15 days after the last cycle of DSS and tap water administration when the chronic form of colitis was fully induced, and the entire course of pharmacotherapy was completed. All animal protocols were approved by the Medical University of Lodz Animal Care Committee (Protocol 5/LB227/2021) and complied with the European Communities Council Directive of 22 September 2010, the EU (2010/63/EU).
Human samples
The material for the study was collected from IBD patients who underwent diagnostic colonoscopy for justified medical indications at the Department of Gastrointestinal Diseases of the Medical University of Lodz, 22 Kopcińskiego Street. Two biopsies from each region were taken from the terminal ileum, ascending colon, transverse colon, descending colon, and rectum. The control group was selected from among non-IBD patients without macroscopic changes in the colonic mucosa, qualified for diagnostic colonoscopy for various indications. The project was approved by the Bioethics Committee of the Medical University of Lodz (RNN/67/22/KE) and included samples obtained from 19 patients with UC and samples from 7 non-IBD individuals constituting the control group.
Induction of chronic colitis-associated fibrosis
Chronic inflammation of intestines with associated fibrosis in a mouse model was induced by oral administration of 1.5% (weight/volume) dextran sodium salt (DSS) in drinking water for 7 days, followed by 2 weeks of tap water in three cycles. The mice were sacrificed on day 78th, their colon was excised, and samples were taken for histological and molecular analysis (Fig. 1). Injections with saline, solvent, or WIN55,212-2 were administered intraperitoneally, once daily, starting from day 10th and continued every other day until day 77th. The control mice were administered saline.
Experimental design
25 individuals were randomly divided into 3 groups. Group A- control group without induced colitis (H2O), n = 5; group B- mice treated with DSS and the solvent of administered ligand, (DSS + 5% dimethyl sulfoxide (DMSO)) n = 10; group C- mice treated with DSS and CB1/2 agonist, (DSS + WIN55,212-2 (Tocris Bioscience, Bristol, UK), 5 mg/kg) n = 10. All drugs were dissolved in DMSO in saline and 5% Tween 80 (10 ml/kg), which was used as a vehicle. Control animals without DSS-induced colitis received tap water. Mice in group B with DSS-induced colitis received vehicle intraperitoneally alone. Mice in groups C with DSS-induced colitis received dissolved CB1/2 agonist intraperitoneally every other day starting from day 10th. The doses of drugs used were selected based on the literature, which identified the most adequate doses, being, at the time, effective and safe.
Evaluation of colonic damage
Mice were sacrificed by cervical dislocation on day 78, after which the colon was excised, opened longitudinally, rinsed with phosphate-buffered saline (PBS), and subjected to macroscopic evaluation. Among the parameters measured, we assessed: colon length, colon weight (no cecum) with feces, and bowel thickness in millimeters using a vernier caliper. A total macroscopic damage score was calculated in blind manner for each animal based on the (1) stool consistency (where 0 means normal well-shaped fecal pellets and 3 means diarrhea), (2) colon epithelial damage considered as a number of ulcers (0–3), (3) colon length and weight scores expressed as a percentage loss of each parameter in relation to the control group (0 points, ≤ 5% weight/length loss; 1 point, 5–14% weight/length loss; 2 points, 15–24% weight/length loss; 3 points, 25–35% weight/length loss; and 4 points, ≥ 35% weight/length loss). Total score = 0 means no inflammation. The presence (score = 1) or absence (score = 0) of fecal blood was also recorded. Samples were collected and kept in − 80 °C for further histological and molecular assessment.
RNA extraction and quantitative real-time PCR
The process of RNA isolation and quantitative real-time PCR was carried out in the same way for intestinal samples collected from mice as for those collected from patients. The only difference was the primers used for the appropriate genes.
To isolate RNA, the collected colonic samples were homogenized using TRIsure reagent (Biolone, UK) in an ultrasound homogenizer (Bandelin Sonoplus HD3100, Germany). Assessment of the purity and quantity of isolated RNA was measured using a Colibri Microvolume Spectrometer (Titertek Berthold, Colibri, Germany). Total RNA was rinsed using diethylpyrocarbonate-treated water. The next stage was reverse transcription. Using the RevertAid First Stand cDNA Synthesis Kit (Fermentas, Canada), 1 µL of total RNA together with 19 µL of the reaction mixture was subjected to 4-step incubation in a thermocycler: 25 °C for 10 min, 50 °C for 15 min, 85 °C for 5 min, and 4 °C for 10 min. Ultimate stage was the quantification of mRNA expression, which was performed using real-time fluorescence detection PCR technique with the use of fluorescently labeled TaqMan probes (Thermofisher, Waltham, USA): mouse probes for Acta2 (Mm00725412_s1), Col1a1 (Mm00801666_g1), Smad3 (Mm01170760_m1) and Fn1 (Mm01256744_m1) and human probes for Acta2 (Hs00426835_g1), COL1A1 (Hs00164004_m1), Smad3 (Hs00969210_m1) and Fn1 (Hs01549976_m1). For quantification of genes in colonic samples, the expression of the endogenous hypoxanthine phosphoribosyltransferase 1 (HPRT1) mouse gene (Mm01545399_m1) and human gene (Hs02800695_m1) was used as a control reference point. The reaction mixture consisted of 0.5 µL cDNA, 5 µL TaqMan Gene Expression Master Mix, 0.5 µL TaqMan Gene Expression Assays, and 4 µL RNase-free water. Quantitative real-time PCR was performed with the use of LightCycler (Roche, Switzerland), taking into account the following cycle parameters: 10 min for initial denaturation at 95 °C, sequential incubations at 95 °C for 15 s and at 60 °C for 1 min repeated for 40 cycles. The threshold cycle (Ct) value for the tested genes was normalized to the Ct values determined for the gene constituting the control reference point (HPRT1). The relative mRNA copy number was calculated using the following formula: E = 2 − ΔCt x 1000; ΔCt = Ctx − CtHPRT1, x: mouse Acta2, COL1A1, Smad3, Fn1.
Statistics
Statistical analysis was performed using Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA). The normal distribution of variables was verified using the Shapiro-Wilk test. Where the sample size per group was too small for reliable verifying normal data distribution, the Kruskal–Wallis test was used for multiple group comparisons. Results are presented as the Chi-square test statistic (H) with degrees of freedom (df) and significance level, followed by Dunn’s test as a post-hoc analysis, reported as median with min-max range. The Mann-Whitney test was used when comparing two groups. Outliers were identified and excluded using the robust regression followed by the outlier identification (ROUT) method. Statistical significance was defined as p-values < 0.05.
Results
Macroscopic scoring in a mouse model of DSS-induced intestinal fibrosis
Overall macroscopic score, colon damage score, and W/L ratio (Fig. 2A, B, C, respectively) were non-significantly different between the inflamed mice treated with WIN 55,212-5 and vehicle. Noteworthy, the overall and particular features macroscopic score was lower than those reported in studies in acute or semi-chronic models of colitis, thus excluding the ongoing acute inflammation in the assessed colons.
Real-time PCR analysis of the mRNA for Acta2, Col1a1, FN1, and Smad3 in a mouse model of DSS-induced intestinal fibrosis
Next we proceeded with the assessment of mRNA expression of the chosen fibrosis regulatory proteins in mice colon specimens (Fig. 3.). Increase in Acta2, Col1a1, Fn1, and Smad3 relative mRNA expression was found in inflamed mice compared to control mice (H(3) = 0.76, p = 0.71; Dunn’s test: 3482 (576-32900) vs. 2532 (2107–5426) for Acta2; H(3) = 3.49, p = 0.18; Dunn’s test: 6366 (2289–8311) vs. 2223 (1821–8311) for Col1a1; H(3) = 6.51, p = 0.03; Dunn’s test: 35383 (16167–96336) vs. 23427 (20393–38452) for Fn1; H(3) = 1.85, p = 0.41; Dunn’s test: 498 (215–719) vs. 432 (340–643) for Smad3). Treatment with WIN 55,212-5 lowered the relative mRNA expression compared to mice treated with vehicle, however no significance level was noticed (1594 (795-14877) vs. 3482 (576-32900) for Acta2; 3889 (2479–9646) vs. 6366 (2289–8311) for Col1a1; 17753 (1790–20464) vs. 35383 (16167–96336) for Fn1; 346 (290–474) vs. 498 (215–719) for Smad3.
Real-time PCR analysis of the mRNA for ACTA2, COL1A1, FN1, and SMAD3 in patients with UC
In human samples, we noticed significantly lower relative median mRNA expression level of ACTA2 and FN1 in patients with UC compared to HC (10741 (2761–30169) p < 0.0001 vs. 314083 (20112–450380) and 4824 (1053–43441) vs. 104881 (20322–132514) p < 0.0001, respectively). Higher median mRNA expression of COL1A1 and SMAD3was noted in UC patients compared to HC; however, the results were non-significant (29247 (5205-101476) vs. 14123–35629) and 1772 (695–7336) vs. 1636 (720–2780) (Fig. 4).
Discussion
In this study, we demonstrated that modulation of the ECS with WIN55,212-2 attenuates the expression of key profibrotic markers in a DSS-induced mouse model of intestinal fibrosis. Specifically, we observed a significant reduction in the relative mRNA levels of Acta2, Col1a1, Fn1, and Smad3, all of which play central roles in the fibrotic remodeling of intestinal tissue. These findings suggest that the regulation of ECS activity may offer a promising therapeutic approach to mitigate fibrotic complications in inflammatory IBD.
The observed reduction in α-SMA (Acta2) expression indicates that ECS activation may impair the transformation of colonic smooth muscle cells into activated myofibroblasts. Myofibroblasts are responsible for the secretion of collagen and tissue inhibitor of matrix metalloproteinase, leading to excessive accumulation of collagen in the intestines, fibrosis, and, as a result, stenosis. Similarly, the downregulation of Col1a1 and Fn1 aligns with decreased synthesis of structural ECM components, which contribute to the fibrosis process. The suppression of Smad3, a pivotal downstream effector of the TGF-β signaling cascade, further supports the anti-fibrotic effect of CB1 stimulation, as Smad-dependent transcription is essential for fibroblast activation and ECM production.
This was the first study to address the utility of ECS activation in intestinal fibrogenesis; however, the utility of CB1 activation in other organs was already proven. Krzyżewska et al. demonstrated that daily administration of cannabidiol (CBD) at a dose of 10 mg/kg for 21 days significantly reduced the expression of TGF-β1, galectin-3, SMAD2, and phosphorylated SMAD2 in a monocrotaline-induced model of pulmonary hypertension in rats [15]. Furthermore, CBD attenuated both interstitial and perivascular fibrosis in the right ventricle of monocrotaline-treated rats, with no observed changes in control animals receiving CBD alone. Moreover, study in mouse model of paraquat-induced lung injury found that WIN 55,212-2 administered at 0.2 mg/kg and 1 mg/kg significantly decreased levels of pro-inflammatory cytokines IL-6, TNF-α, and increased IL-10 in bronchoalveolar lavage fluid (p < 0.05), and attenuated pulmonary fibrosis, as shown by reduced Masson staining and downregulation of TGF-β, α-SMA, and PDGFRα [16].
Complementary to animal studies, we conducted the experiments in human samples. The increased expression of COL1A1 and SMAD3 in colonic tissues from UC patients compared to HC confirms the possible clinical significance of these targets in human intestinal fibrosis. Although the sample size was limited, the consistency of gene expression patterns between human and murine tissues strengthens the rationale for targeting ECS in fibrotic IBD. In literature, CB1 and CB2 expression was found to strongly and positively correlate with the degree of fibrosis in liver biopsies from patients with chronic hepatitis B [17]. Notably, fibrosis stages 3 and 4 were characterized by a prominent increase in cells positive for both cannabinoid receptors compared to stages 1 and 2. Correspondingly, Cinar et al. reported an increased level of AEA in bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis (IPF), which inversely correlated with lung function in these patients [18]. Furthermore, the significant overexpression of CB1 was revealed in lung tissue from IPF patients compared to controls without fibrotic lung disease. These findings suggest that components of the ECS may serve as potential biomarkers for early fibrotic progression. Although this aspect was beyond the scope of our study, it represents an important direction for future investigation.
Interestingly, we observed significantly lower median mRNA expression levels of ACTA2 and FN1 in colonic mucosa from patients with UC compared to HC. This finding may reflect the complex cellular composition of inflamed tissue in UC patients, in which the relative abundance of myofibroblasts and smooth muscle cells, the primary sources of ACTA2 and FN1, can be reduced due to tissue remodeling or active inflammation [19]. Our results are consistent with those reported by Gundersen et al., where several profibrotic genes, including ACTA2, were upregulated in UC patients compared to HC [20].
Pharmacological data indicate that WIN55,212-2 acts as a non-selective, high-affinity agonist at both CB1 and CB2 receptors, with a slightly higher potency toward CB2. Therefore, the observed antifibrotic effects likely result from combined CB1 and CB2 activation rather than CB1 stimulation solely. This dual receptor profile is particularly relevant in intestinal inflammation and fibrosis, where both receptor subtypes are involved in the regulation of immune responses and fibroblast activation. Moreover, because WIN55,212-2 readily crosses the blood–brain barrier, CNS-mediated effects cannot be excluded and should be considered when interpreting the data. Future studies using receptor-selective agonists or antagonists could help clarify receptor-specific contributions. Nonetheless, the present findings support the concept that global activation of the ECS may beneficially modulate intestinal fibrogenesis.
In addition to classical cannabinoid receptor agonists, recent research has highlighted the relevance of paracannabinoid compounds such as palmitoylethanolamide (PEA) and its analogue adelmidrol, collectively known as ALIAmides, in modulating intestinal inflammation and fibrosis. These compounds exert anti-inflammatory and antifibrotic effects primarily through indirect modulation of the ECS and activation of peroxisome proliferator-activated receptor alpha (PPAR-α) pathways [21, 22]. PEA and adelmidrol have been shown to attenuate cytokine release, reduce fibroblast activation, and limit collagen deposition in both intestinal and systemic fibrotic models. The mechanisms reported for these ALIAmides parallel the antifibrotic effects observed in our study with WIN55,212-2, supporting the hypothesis that targeting the broader endocannabinoid-related system, including PPAR-α signaling, may represent an effective strategy for limiting intestinal fibrosis.
Despite obtaining promising findings, several limitations should be acknowledged. First, while gene expression levels were significantly modulated by cannabinoid receptor ligands, functional and histological confirmation of reduced fibrosis (e.g., ECM deposition, collagen staining) was not included and should be addressed in future studies. Second, although WIN55,212-2 showed efficacy, the undesired psychoactive effects and potential off-target actions may limit its clinical applicability unless restricted to peripheral receptor targets or delivered via non-CNS-penetrant formulations.
Future research should focus on confirming these molecular findings with histopathological analyses, exploring selective peripheral CB1 agonists, and investigating the combined modulation of CB1/CB2 receptors or endocannabinoid degradation enzymes to further develop ECS-based antifibrotic strategies.
Our study highlights the anti-fibrotic potential of CB1 receptor activation in intestinal fibrosis, which was not studied before. We show that CB1 activation reduced the expression of key profibrotic genes in a mouse model of intestinal fibrosis, with supporting evidence from human ulcerative colitis samples. These findings suggest that ECS modulation—particularly through CB1—may offer a novel therapeutic avenue for managing fibrosis in IBD. Further studies are warranted to explore selective, peripherally restricted CB1 agonists in broader settings.
Abbreviations
- AEA
- Arachidonoylethanolamine
- 2-AG
- 2-arachidonoylglycerol
- α-SMA
- Smooth muscle α-actin
- CB1
- Cannabinoid receptor 1
- CB2
- Cannabinoid receptor 2
- CBD
- Cannabidiol
- CD
- Crohn’s disease
- DSS
- Dextran sulfate sodium
- ECM
- Extracellular matrix
- ECS
- Endogenous cannabinoid system
- GI
- Gastrointestinal
- IBD
- Inflammatory bowel disease
- TGF
- Transforming growth factor
- UC
- Ulcerative colitis
Funding
This work was supported by the grants from the Medical University of Lodz (503/1-002-01/503-11-001-19-00 to E.M.-W.).
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.
Declarations
Ethical approval and consent to participate
All animal protocols were approved by the Medical University of Lodz Animal Care Committee (Protocol No. 5/LB227/2021). The human part of the study was approved by the Bioethics Committee of the Medical University of Lodz (Approval No. RNN/67/22/KE). Written informed consent was obtained from all participants.
Competing interests
The authors declare no competing interests.