Current pharmacological treatments for inflammatory bowel disease often rely on high doses of anti-inflammatory or immunosuppressive agents, which, while effective, are frequently associated with significant limitations including increased risk of infections, systemic toxicity, loss of response, and difficulties in managing acute disease flares [28,44,45]. These adverse effects can compromise patient safety and limit long-term treatment adherence [46]. Cyclosporine A is primarily reserved for severe or steroid-refractory ulcerative colitis due to its potent immunosuppressive properties. However, its use is restricted by serious adverse effects, including nephrotoxicity, hypertension, and increased infection risk, making it unsuitable for long-term maintenance therapy [47,48]. Olsalazine and other 5-aminosalicylic acid (5-ASA) derivatives are generally better tolerated, yet high-dose regimens often provoke headaches, nausea, and rare hepatotoxicity, and many patients fail to achieve sustained remission on monotherapy [49,50]. Targeted therapies such as monoclonal antibodies and small molecules, while effective in managing moderate to severely active IBD, are expensive and not effective in all patients [51,52].

To address this gap, we hypothesized that low-dose combination therapies targeting complementary mechanisms could provide enhanced therapeutic outcomes while minimizing adverse effects. In this context, the combination of cannabidiol with conventional agents like cyclosporine A or olsalazine offers a promising alternative. CBD acts on multiple pharmacological targets, including CB1, CB2, GPR35, and peroxisome proliferator-activated receptors (PPARs), all of which are involved in inflammation and intestinal homeostasis, thereby exerting analgesic, anti-inflammatory, immunomodulatory, antipsychotic, and antimicrobial properties [18,19,53,54]. Notably, CBD has been shown to exert antimicrobial activity, particularly against gram-negative bacteria, through membrane disruption, as well as reduce biofilm formation and antibiotic resistance [55]. While CSA is associated with infection risk, and olsalazine requires high doses, the combination of sub-therapeutic doses of these agents with low-dose CBD would increase the effectiveness while also minimizing the chance of CSA-induced infection. Olsalazine also influences overlapping pathways with CBD, including NF-κB inhibition, oxidative stress modulation, and immune regulation [19,56,57]. Moreover, they may offer greater therapeutic flexibility for both maintenance and flare-phase treatment in IBD.

In this study, we comprehensively evaluated the therapeutic efficacy and safety of sub-therapeutic dose CBD in combination with cyclosporine A or olsalazine across both acute and chronic settings. Our findings consistently demonstrate that co-administration of sub-therapeutic doses of CBD enhances the effects of CSA or olsalazine in ameliorating clinical disease activity, preserving intestinal integrity, suppressing inflammatory mediators, and correcting metabolic and haematological disturbances. In the acute DSS model, CBD alone had a negligible impact on DAI or visceral pain, whereas CSA monotherapy reduced disease severity in a dose-dependent manner. Strikingly, the 10 mg/kg CBD + 5 mg/kg CSA combination significantly reduced DAI and grimace scores (Figure 1). A similar effect was observed with olsalazine: while monotherapies were not effective, the combination of 10 mg/kg CBD + 50 mg/kg olsalazine produced significant suppression of both DAI and visceral pain (Figure 1). Extending these combination regimens into a chronic colitis paradigm, both combinations maintained these benefits over repeated DSS cycles—sustaining low DAI scores, preventing weight loss, and reducing diarrhoea and faecal bleeding (Figure 2). The consistency between acute and chronic outcomes underscores the robustness of CBD’s potential to enhance the efficacy of CSA or olsalazine. These results suggest that CBD’s immunomodulatory and antioxidant properties synergize with CSA’s T-cell inhibition and olsalazine’s free-radical scavenging to amplify anti-inflammatory outcomes. These findings are in line with our previous findings wherein we reported enhanced effects of low-dose CBD (10 mg/kg) in combination with tetrahydrocannabinol (THC) or ZCZ011, a CB1 receptor allosteric modulator [43]. CBD on its own has also shown beneficial effects, but at a higher dose (60 mg/kg) [16].

The improvement in colon morphology and molecular markers readouts further confirmed clinical observations. CBD combinations fully prevented DSS-induced colon shortening and strongly suppressed MPO activity in colon tissue and spleen (Figure 3 and Figure 4). This suggests that CBD enhances mucosal protection and limits neutrophil infiltration to the colon and spleen. These findings are again in line with our previous findings, where CBD combination with THC and ZCZ011 restored colon length and MPO activity [43]. CBD alone also resulted in a similar response, but required higher doses [16,26]. CBD was also shown to directly reduce pain and neutrophil infiltration in an animal model of ocular pain and inflammation through modulation of the 5-hydroxytryptamine 1A (5-HT1A) receptor [58].

To further validate the efficacy of the combination treatments, we assessed the expression of key colonic inflammatory markers. In both acute and chronic colitis models, vehicle-treated mice showed a significant increase in pro-inflammatory cytokines, IL-6, IL-1β, MCP-1, and TNF-α, compared to healthy controls, which was markedly attenuated by the combination therapies (Figure 5 and Figure 6). Additionally, we investigated the expression of IL-17, a cytokine known for its strong pro-inflammatory properties and its pivotal role in the pathogenesis and persistence of IBD, in the chronic model [59]. Elevated IL-17 levels are a well-documented feature in individuals with active colitis [60]. Consistent with clinical observations, our chronic colitis model exhibited significantly increased IL-17 expression compared to healthy controls. Importantly, both combination treatments markedly reduced this elevation, underscoring their therapeutic potential in modulating key inflammatory pathways associated with chronic intestinal inflammation. While monotherapies like olsalazine often necessitate dose escalation to sustain therapeutic benefits—frequently accompanied by an increased risk of adverse effects [30,61]—our low-dose combination strategy achieved similar levels of mucosal healing and cytokine suppression to those observed with high-dose cyclosporine A, without the associated toxicity. Mechanistically, CBD likely enhances the efficacy of cyclosporine A and olsalazine through complementary modulation of overlapping anti-inflammatory pathways. By acting as a PPARγ agonist, CBD suppresses NF-κB activity and downstream cytokine production, mechanisms also engaged by 5-ASA compounds, thereby amplifying mucosal anti-inflammatory responses [62,63,64]. Concurrently, CBD’s activation of CB₂ receptors on immune cells limits leukocyte infiltration and cytokine release, synergizing with CSA’s calcineurin/NFAT inhibition of T-cell activation [18,47,65,66]. CBD additionally inhibits NLRP3 inflammasome assembly and inducible nitric oxide synthase (iNOS) expression, reducing IL-1β and TNF-α levels [67,68], and promotes epithelial barrier integrity via PKA/AMPK signalling [69]. These convergent receptor-mediated actions allow for dose reductions of each drug while maintaining or improving therapeutic outcomes in colitis models. This broad control of both innate and adaptive inflammatory mediators aligns with the growing recognition that effective IBD management demands multi-targeted interventions rather than single-pathway blockade [22]. The current findings suggest that CBD’s immunomodulatory properties complement the established anti-inflammatory actions of CSA and olsalazine, converging on both innate and adaptive immune pathways to halt the cytokine cascade that drives mucosal injury.

Beyond classical colitis parameters, DSS exposure elicited pronounced metabolic disturbances, characterised by dysregulated GLP-1 signalling. This was further evidenced by hypoglycaemia, weight loss, and altered plasma and colonic GLP-1 concentrations (Figure 7 and Figure 8). GLP-1, an incretin hormone, despite its glucose regulatory and anti-inflammatory actions, is also implicated in several intestinal diseases, including IBD [41,70]. In acute colitis, we observed increased plasma and decreased colonic GLP-1 levels, consistent with our previous findings [16,43]. The loss of colonic L-cells with DSS likely explains reduced mucosal GLP-1, whereas systemic elevation may represent a compensatory mechanism to preserve gut integrity and limit inflammation [70]. Both combination therapies—cyclosporine A (CSA) + CBD and olsalazine + CBD—normalized blood glucose and mitigated weight loss. Notably, CSA + CBD selectively reduced plasma GLP-1 without altering colonic levels, while olsalazine + CBD robustly restored both plasma and colonic GLP-1, even exceeding healthy control values. In the chronic colitis model, olsalazine + CBD again improved glycaemic control and plasma GLP-1 but had no effect on colonic GLP-1; conversely, CSA + CBD increased colonic GLP-1 without impacting systemic levels or glucose. These differential patterns suggest that CSA + CBD may enhance local enteroendocrine function, whereas olsalazine + CBD exerts broader metabolic actions. Importantly, both regimens realigned gut hormonal signalling and improved overall metabolic homeostasis, highlighting a novel GLP-1–mediated axis for cannabinoid-based modulation in IBD. In addition to its role in glucose regulation, GLP-1 has been demonstrated to alleviate colitis in preclinical models [42,71].

A comprehensive safety evaluation (Table 1 and Table 2) demonstrated that the CSA + CBD combination was well tolerated, with no significant alterations observed in hepatic enzymes, renal function markers, or haematological parameters. Moreover, this regimen significantly ameliorated colitis-associated hyperammonemia—a systemic complication often linked to neurological disorders such as Alzheimer’s disease, depression, and anxiety, as well as hepatic dysfunction and gut microbiota dysbiosis, all of which represent recognized extraintestinal manifestations (EIMs) of inflammatory bowel disease (IBD) [22,72,73]. These findings suggest that, in addition to mitigating classical intestinal inflammation, the CSA + CBD combination may also confer therapeutic benefits for EIMs. In comparison, the olsalazine + CBD combination corrected anaemia and thrombocytosis but was associated with mild elevations in ALT, AST, and creatinine levels. Granulocyte count was slightly elevated in the vehicle group compared to the healthy control, although not statistically significant; this was significantly reduced by the olsalazine and CBD combination. Oral dosing shortly before euthanasia may explain the modest elevations in liver and kidney parameters observed with the cannabidiol–olsalazine combination, in contrast to the intraperitoneal (i.p.) CSA + CBD arm. In our prior work, i.p. CBD at 10 mg/kg did not adversely affect hepatic or renal function [16]. Although clinical olsalazine is generally well tolerated at doses up to 3 g/day, rare cases of interstitial nephritis and mild transaminase elevations have been reported with 5-aminosalicylates, suggesting the need for caution with higher doses [74,75]. It is therefore possible that a final oral dose on Day 24 induced transient hepatic or renal stress. To fully characterize safety, future studies should include alternative routes of administration, extended dosing regimens, comprehensive liver and kidney function panels, and histopathological evaluation of these organs. Nevertheless, the absence of overt haematological toxicity and the normalization of spleen and kidney weight indices in both treatment groups support the overall systemic safety of these therapeutic regimens.

While this study focused on female BALB/c mice, the therapeutic effects of CBD and its combinations have been documented in both sexes and across various mouse strains. For example, in male mice, CBD has been shown to ameliorate experimental colitis via immunomodulatory and antioxidant mechanisms [76]. Similarly, studies in male Swiss mice have demonstrated significant anti-inflammatory effects of CBD in models of intestinal and systemic inflammation [24]. Nonetheless, we acknowledge that sex and genetic background may influence pharmacodynamics and immune responses in the combination treatments. Future studies including both sexes, multiple strains, and alternative colitis models are warranted to fully explore the generalizability and translational relevance of the findings. Mechanistically, validation of CBD’s enhancement of cyclosporine A and olsalazine will require targeted interventions, such as receptor antagonists, RNA interference, or use of CB1-/CB2-, GPR35-, or PPAR-deficient models, to define the specific pathways involved. Although we demonstrated significant reductions in colonic cytokine levels, parallel analyses of circulating inflammatory markers would clarify systemic effects. Detailed characterization of immune cell subsets (e.g., Th1/Th17 cells and macrophages), alongside histopathological assessment of colonic crypt architecture, goblet cell preservation, and immune infiltration, will further elucidate the tissue-protective mechanisms. Future studies incorporating GLP-1 receptor (GLP-1R) antagonists or genetic knockout models will be critical to delineate the mechanistic contribution of GLP-1 signalling in mediating the observed therapeutic effects. These studies will be critical for translating our preclinical insights into safe, effective IBD therapies.