Inflammatory bowel disease (IBD) remains challenging to manage due to the limitations of current therapies, including biologics and immunosuppressive agents, which often provide incomplete remission, cause adverse effects and have limited accessibility due to high cost [30,31]. Cannabis and cannabinoids have been utilized globally as alternative therapies for managing gastrointestinal symptoms such as diarrhoea, pain and inflammation [18]. This study highlights a novel therapeutic approach targeting the endocannabinoid system (ECS)—an endogenous lipid signalling network that regulates pain, inflammation and metabolism [5]—using sub-therapeutic doses of THC combined with ZCZ011 or CBD. By leveraging the ECS’s ability to modulate immune responses, this strategy overcomes the psychoactive concerns associated with high-dose THC while delivering potent anti-inflammatory and metabolic benefits [20].

Using well-established DSS-induced acute and chronic colitis models, our study demonstrates that low-dose THC (2.5 mg/kg) in combination with ZCZ011 (20 mg/kg) or CBD (10 mg/kg) significantly attenuates disease severity, restores tissue integrity and improves systemic markers of inflammation (Figure 1 and Figure 2). In both colitis models, the combination treatments effectively reduced disease activity index (DAI), body weight loss, diarrhoea severity and faecal blood, achieving comparable efficacy to a therapeutic dose of THC (10 mg/kg). This is in line with the previous findings where the modulation of ECS by CBD or HU308, a CB2 receptor agonist, significantly reduced colitis in DSS-induced colitis [23]. Similarly, the combination of THC and CBD has been shown to reduce colitis in a rat model of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats, suggesting that the effects observed in our study are not model-specific [14]. These findings align with earlier evidence on cannabinoid-based therapies, reinforcing the ECS as a viable therapeutic target in IBD [32,33]. Importantly, our results highlight the consistency of these therapeutic effects across acute and chronic disease states, further supporting their translational relevance.

After observing significant improvements in clinical markers, we examined the health of the colon and spleen, as both organs are known to be inflamed in DSS-induced colitis [23,34]. Combination treatments effectively mitigated colonic shortening, reduced neutrophil infiltration (measured via MPO activity) and normalised spleen weight in both acute and chronic colitis models (Figure 3 and Figure 4). This dual effect on local and systemic inflammation highlights the anti-inflammatory synergy of THC with ZCZ011 or CBD and suggests ECS modulation as a critical mechanism underlying these benefits. Consistent reductions in colon shortening, spleen inflammation and MPO activity across both models emphasize the potential of cannabinoid therapies to target multiple inflammatory pathways [23,35]. Targeting ECS was also shown to be beneficial in other models of pain and inflammatory conditions [19,32,36].

One of the novel insights of this study is the impact of combination therapies on pro-inflammatory cytokines and metabolic health. Pro-inflammatory cytokines, including IL-6, TNF-α, IL-1β, IL-17, IFN-γ and chemokine MCP-1, are key drivers of IBD pathology, contributing to immune cell recruitment, tissue damage, mucosal injury and systemic inflammation [37,38]. Our previous study demonstrated a significant elevation of IL-6, TNF-α, IL-1β, IL-17, IFN-γ and MCP-1 in colitis and showed that activation of the CB2 receptor by HU308 significantly reduces these cytokine levels [23]. Interestingly, combination treatments in this study significantly reduced these cytokines and chemokines (Figure 5), which correlated with improvements in clinical markers and tissue integrity. These results are consistent with the established role of cannabinoids in suppressing cytokine storms, primarily through CB2 receptor activation [23,39,40].

We further explored the effects of these therapies on GLP-1, an incretin hormone known for its anti-inflammatory and glucose-regulatory properties [28]. Colitis is often associated with metabolic disturbances, including impaired glucose homeostasis, body weight regulation and the development of non-alcoholic fatty liver disease (NAFLD) [41,42]. Elevated levels of cytokines have been also linked to abnormal glucose and lipid metabolism [43]. Beyond glucose regulation, GLP-1 has been also shown to reduce inflammation [27,28,44] and the expression of cytokines at local and systemic levels [45,46]. Recent findings also highlight the pivotal role of GLP-1 in maintaining metabolic health, which is often disrupted during colitis [47]. In our models, we observed increased plasma GLP-1 levels but decreased colonic levels, which is likely due to inflammation-induced dysregulation of enteroendocrine L-cells, the primary source of GLP-1 [48]. DSS-induced epithelial damage and inflammation may impair local GLP-1 synthesis or retention in the colon. Meanwhile, systemic inflammation and cytokine signalling (e.g., IL-6, TNF-α) could stimulate L-cells in unaffected gut regions to release GLP-1 into the bloodstream as a compensatory response [49]. Additionally, GLP-1’s systemic increase may serve as a protective mechanism to maintain gut integrity and modulate inflammation, despite its reduced colonic levels due to degradation by dipeptidyl peptidase-4 (DPP-4) or local retention issues. Interestingly, a key novel finding of this study is the normalization of both plasmatic and colonic GLP-1 levels following the combination treatments (Figure 6 and Figure 7). This restoration was accompanied by improvements in blood glucose homeostasis and body weight stability, indicating that these therapies not only ameliorate inflammation but also address colitis-associated metabolic dysregulation. This observation aligns with prior evidence indicating that chronic colonic inflammation can impair glucose utilization, leading to hypoglycaemia and metabolic stress [50]. Emerging evidence suggests that cannabinoids can modulate GLP-1 pathways to restore metabolic balance [51,52,53], and our findings reinforce this dual therapeutic potential. We reported similar findings in our previous study, where the cannabinoids CBD and HU308 normalised both plasmatic and colonic GLP-1 levels [23]. Future studies should aim to elucidate the precise mechanisms underlying these effects, focusing on downstream signalling pathways in GLP-1 regulation and glucose metabolism, quantification of L-cells and (DPP-4) enzyme activity in the colon.

Consistent with the beneficial effects of the combination treatments in colitis markers, the safety profile of combination therapies was assessed through haematological, liver and kidney profiles. Acute colitis led to modest platelet and procalcitonin dysregulation, likely reflecting systemic inflammation, which was effectively restored to control levels by a combination of THC and ZCZ011 treatment (Table 1). No haematological dysregulation was observed in chronic colitis (Table 2). Liver biochemistry remained stable across all groups, with no hepatotoxicity observed with the treatments (Figure 8 and Figure 9), indicating no long-term toxicity. Additionally, the treatments significantly reduced plasma ammonia levels, which were elevated in vehicle-treated groups. Elevated ammonia levels are associated with various diseases, including neuropsychiatric disorders such as depression, anxiety and Alzheimer’s disease [54,55,56,57,58], as well as liver pathologies, which may manifest as extraintestinal complications of IBD [58,59,60]. By mitigating ammonia dysregulation, combination therapies may offer broader protective effects against IBD-associated systemic complications [60]. Cannabinoid therapies are increasingly recognized for their hepatoprotective [61] and renoprotective activity [62,63,64], primarily through their antioxidative and anti-inflammatory properties [65,66,67,68]. The DSS-induced colitis model is widely used for studying IBD; however, its relevance to human disease is limited by differences in aetiology, including genetic and environmental factors. Despite these limitations, the model effectively replicates key pathological features of IBD, such as clinical symptoms, epithelial barrier disruption, immune cell infiltration and pro-inflammatory cytokine production, making it a valuable tool for preclinical research. Furthermore, the chronic model employed in this study captures the phases of relapse and remission, closely resembling the clinical progression of human IBD. Nevertheless, incorporating complementary models or clinical studies to validate the findings of this study would enhance their translational relevance.