This study reveals that cannabidiol (CBD) exerts conditional and context-dependent protection against deoxynivalenol (DON)-induced intestinal toxicity, with its efficacy modulated by the toxin dose, exposure duration, and cellular differentiation state. While CBD partially mitigates DON damage under specific conditions, its benefits are counterbalanced by paradoxical effects that challenge its utility as a universal therapeutic agent.

Swine are highly susceptible to DON exposure, making IPEC-J2 cells an ideal model for the study of DON toxicity [24]. Consistent with previous findings [25,26], DON reduced IPEC-J2 viability in a dose- and time-dependent manner, with significant cytotoxicity observed at 0.5 μM—a lower threshold than previously documented. CBD pre-treatment restored viability at low DON concentrations (0.5–2 μM), but this protection was diminished at higher doses (≥8 μM) (Figure 2A,B). Notably, CBD alone induced cytotoxicity at ≥10 μM (Figure 1B), emphasizing the need for precise dosing to avoid adverse effects.

DON-induced apoptosis is primarily driven by Bax/Bcl-2 dysregulation and caspase activation [27]. CBD’s modulation of apoptosis was strikingly dependent on cellular differentiation. In growing IPEC-J2 cells, CBD increased the Bax/Bcl-2 ratio at 2 μM DON (Figure 3A), suggesting a pro-apoptotic effect under subtoxic stress. This contrasts with its anti-apoptotic activity in differentiated cells, where CBD reduced the Bax/Bcl-2 ratio across all DON concentrations (Figure 3B). Such divergence may stem from differences in the stress response pathways between proliferating and mature epithelia. For instance, growing cells prioritize survival signaling to maintain their proliferative capacity, whereas differentiated cells activate repair mechanisms [28]. These findings caution against extrapolating CBD’s effects across heterogeneous intestinal cell populations.

In growing cells, it amplified NFκB, IL-6, and COX-2 expression at 2 μM DON (Figure 4A), potentially exacerbating early inflammatory responses. Conversely, in differentiated cells, CBD suppressed IL-6 and COX-2 at 50 μM DON but enhanced inflammation at lower doses (Figure 4B). This biphasic behavior mirrors the immunomodulatory properties of cannabinoids, which can activate or inhibit NFκB depending on the ligand concentration and cellular context [29]. These results underscore the fact that CBD’s anti-inflammatory efficacy is contingent on the toxin intensity and epithelial maturity.

DON-induced toxicity is closely linked to oxidative stress, as evidenced by increased TXNIP expression and decreased SOD1 and CAT levels [15]. CBD’s antioxidative effects were similarly context-dependent. In growing cells, it exacerbated TXNIP expression—a marker of oxidative damage—at low DON levels but suppressed it at high doses (Figure 5A). While CBD reduced TXNIP expression at 8 μM DON in growing cells (p < 0.001), the levels remained 200-fold higher than those of controls, indicating limited biological relevance. In contrast, differentiated cells exhibited consistent reductions in TXNIP alongside enhanced expression levels of SOD1 and CAT genes (Figure 5B), suggesting that CBD stabilizes the antioxidant defenses in mature epithelia. However, even in differentiated cells, CBD’s suppression of TXNIP at 50 μM DON (p < 0.001) did not restore the baseline oxidative stress markers, underscoring its restricted efficacy under severe toxin exposure. Given previous reports that antioxidants such as selenium nanoparticles and ferulic acid mitigate DON toxicity, CBD may serve as a valuable addition to oxidative stress-targeted interventions [30,31]. However, CBD’s failure to restore CAT activity at ≥2 μM DON in growing cells highlights limitations under sustained oxidative stress.

CBD’s modulation of oxidative stress pathways is dependent on the cell state and toxin concentration. In growing cells, CBD paradoxically amplifies oxidative damage markers at low DON levels but mitigates them at high doses. In contrast, differentiated cells exhibit consistent antioxidant benefits, suggesting that cellular maturity critically determines CBD’s efficacy. These findings underscore the importance of the toxin exposure intensity and epithelial differentiation status in optimizing CBD-based interventions.

DON disrupts tight junction proteins, leading to increased intestinal permeability [13]. CBD reinforced tight junction gene expression (Claudin-1, Occludin) and preserved barrier integrity at 5 μM DON (Figure 6 and Figure 7C), likely by amplifying the compensatory upregulation triggered by moderate toxin exposure. However, at 50 μM DON, CBD failed to prevent permeability disruption (Figure 7D), indicating an inability to counteract severe barrier damage. Similar limitations have been reported for other barrier-stabilizing agents, where very high toxin concentrations overwhelm repair mechanisms [32].

In conclusion, CBD exhibits limited and conditional efficacy against DON-induced intestinal damage, with its benefits restricted to moderate toxin exposure in swine intestinal cells. Its paradoxical effects under subtoxic conditions and cytotoxicity at higher doses underscore the need for context-specific application. While CBD holds potential as part of a multifaceted mycotoxin mitigation strategy, its role as a standalone therapeutic remains uncertain. However, translating these findings into practical applications requires further research to optimize the CBD dosages, explore its synergistic effects with other compounds, and validate its protective effects in vivo [33]. Future studies should also investigate the molecular mechanisms of CBD within the endogenous cannabinoid system, particularly the roles of CB1 and CB2 receptors. These efforts will pave the way for the development of innovative strategies to mitigate mycotoxin-related hazards and promote animal health and welfare.