The rat paw edema model induced by carrageenan injection is one of the most well-validated models of acute inflammation [15]. In our experiment, we observed the classical biphasic response in the control group (rapeseed oil). The first phase (0–2.5 h), mediated by the release of biogenic amines such as histamine, serotonin, and bradykinin [16], was characterized by progressive edema of the right paw following carrageenan administration in all groups. The second phase (3–6 h), characterized by COX-2-induced prostaglandin overproduction and the release of pro-inflammatory cytokines such as TNFα and IL-1β [16], occurred at 3 h (increase in paw volume, p = 0.047 vs. 0 h), consistent with literature describing this model in Wistar rats [17].

The difference in volume between the inflamed and control paws served as a key parameter for assessing the inflammatory response, reducing inter-individual variability and enabling direct evaluation of the effects of the tested substances. In our study, no significant differences were observed between groups at 0 h and 1 h, indicating comparable baseline conditions and the absence of a substance effect during the early phase of inflammation. A key observation in the control group was a statistically significant decrease in edema at 10 h (p = 0.001 vs. 3 h), suggesting a spontaneous resolution phase. The statistically significant increase in edema at 3 h relative to baseline (p = 0.024) and its decrease at 10 h compared to the peak (p = 0.019) confirm that the carrageenan model relied on prostaglandin synthesis during the studied time window [18].

Pearson correlation analysis in the control group showed a positive correlation between paw volume at 3 h and TNFα mRNA expression, in line with reports identifying TNFα as a key cytokine driving edema and neutrophil recruitment in this model [19]. Additionally, a positive Spearman correlation was observed between COX-2 and NFκB, known for directly inducing PTGS2 (prostaglandin-endoperoxide synthase 2, a synonym to COX2) gene transcription in response to inflammatory stimuli [20].

At the molecular level, inflammation was further confirmed by increased mRNA transcription of CB1 and CB2 receptor genes in the inflamed right paw of animals receiving Extract D, compared to controls, reflecting a protective mechanism aimed at limiting hyperalgesia and edema [21]. CB2 activation in macrophages and neutrophils typically inhibits the release of pro-inflammatory mediators [22]. Extract D more effectively stimulated ECS receptor overexpression than external CBD, suggesting an entourage effect in which other phytocannabinoids and, possibly, terpenes synergistically enhance the endocannabinoid response [23]. In the CBD treated group, a negative Spearman correlation was observed between paw volume at 3 h and CB2 mRNA level, supporting the protective role of CB2 component in peripheral tissues, especially when considering the fact that the CB2 agonism inhibits leukocyte infiltration and cytokine release, directly reducing inflammatory exudate [22].

A similar inflammatory pattern to the control group was observed in the ASA group. Edema increased until 3 h and then significantly decreased at 10 h, confirming the validity of the model. ASA results align with classical irreversible COX inhibitor pharmacodynamics, reflecting its ability to reduce prostaglandin synthesis and attenuate edema in the carrageenan model [24]. Positive Pearson correlations between paw volume and COX-2 mRNA expression at 6 h were observed, along with a strong positive Spearman correlation between CB2 and NFκB transcript levels. The high COX-2 transcription in ASA-treated animals may appear paradoxical but can be explained by compensatory upregulation of COX-2 mRNA following enzymatic inhibition [25]. The positive CB2–NFκB correlation suggests that increased levels of CB2 transcripts (expression) may buffer NFκB activity, representing a protective mechanism despite ASA treatment [22,26]. Paradoxically, ASA also induced a significant increase in COX-2 mRNA compared to controls, consistent with feedback-driven compensatory transcription [27]. Nevertheless, ASA effectively reduced NFκB mRNA levels, confirming its anti-inflammatory genomic effect.

While the literature emphasizes the anti-inflammatory effects of CBD and other phytocannabinoids, including reductions in TNF, IL1, and IL6 [28], and oral CBD has been shown to reduce carrageenan edema in both phases [17], our results revealed more complex dynamics. In the CBD group, a negative Spearman correlation was found between paw volume at 0 h and NFκB transcripts, suggesting early pathway suppression consistent with the results presented by Nicholas and Kaplan [29]. A positive Pearson correlation between CB1 and COX-1 mRNA levels in this group may indicate nociceptive interactions, in which CB1 modulates inflammatory pain via constitutive prostanoids [30]. Significantly lower COX-2 mRNA expression in the inflamed paw compared to the contralateral paw in the CBD and Extract D groups supports local anti-inflammatory action via suppression of COX-2 transcription below physiological levels, consistent with cannabinoid-mediated MAPK/p38 inhibition [7]. An interesting observation in our study was the significantly lower median paw volume in the left (control) paw in the CBD group at the 10 h time point compared with the control group. Although saline injection does not induce the acute inflammatory cascade characteristic of carrageenan, the injection procedure itself and the introduction of fluid into the plantar space elicit local tissue microtrauma and transient mechanical edema. The accelerated return to baseline volume observed in the CBD-treated group suggests a potent systemic anti-edematous effect of this compound. This finding aligns with the existing literature, which demonstrates that systemically acting CBD effectively expedites the resolution of inflammation and facilitates tissue fluid resorption, even following mild mechanical injuries [7,17].

Extract D reduced TNFα mRNA level in the inflamed paw relative to the healthy paw, highlighting that plant chemotype (THC: CBD ratio, terpene profile) can determine whether a preparation acts suppressively or immunostimulatory on cytokine pathways [31]. In contrast, Extract B showed pro-inflammatory activity, increasing TNFα mRNA in the right paw compared to all other groups. This may reflect pro-inflammatory or irritant components, or a terpene profile that enhances the early immune response. The pro-inflammatory effects were evident from sharp edema increases at 3 h, maintained at 6 h, and persistently elevated edema at 10 h following Extract D. Spearman correlations in the Extract B group revealed links between COX-2 mRNA profile and paw volume at 3 h and changes in both paws, suggesting a possible shift toward mediators characteristic of the resolution phase (e.g., 15d-PGJ2 prostaglandins) [32]. Negative Pearson correlations between paw volume changes and COX-1 transcripts further underscore the complexity of extract-mediated modulation, distinct from separately administered CBD, consistent with an entourage effect [33]. At 3 h, the Extract B group exhibited significantly greater edema than ASA-treated animals, indicating a lack of anti-inflammatory activity during the acute phase.

Paradoxically, at 10 h, all cannabinoid-receiving groups showed greater paw volume differences than controls, suggesting that these substances did not reduce edema and may have contributed to its maintenance or exacerbation in the late phase. Discrepancies with the literature may reflect differences in experimental conditions, extract formulation, administration route, or interactions with other extract components. It is possible that terpenes or fatty acids in the extracts enhance pro-inflammatory responses, outweighing CBD’s anti-inflammatory effects [34]. These findings raise the hypothesis that extract components may modulate the resolution phase of inflammation or partially sustain it, warranting further compositional and mechanistic analyses. Although preclinical literature generally attributes immunomodulatory or anti-inflammatory properties to cannabinoids and terpenes [35], other evidence suggests that terpenes may exert only transient suppression or complex modulation of inflammatory responses [36].

The paradoxical increase in paw edema observed across three groups (CBD-administered group, Extract B-administered group and Extract D-administered group) at 10 h post-carrageenan warrants further explanation of the mechanism of action. One potential explanation lies in the well-documented bell-shaped dose–response curve characteristic of CBD pharmacology. Gallily et al. [36,37] demonstrated that CBD exhibits maximal anti-inflammatory efficacy at intermediate doses (approximately 5 mg/kg intraperitoneally), with diminished effects at both lower and higher doses [37]. Given the low oral bioavailability of CBD (approximately 6% in the fasted state) [38], the effective systemic CBD concentration achieved after intragastric administration of 20 mg/kg may have placed the response on the subtherapeutic limb of this inverted U-shaped curve [38]. Alternatively, it cannot be ruled out that the achieved concentration was sufficient to activate pro-inflammatory pathways (e.g., via TRPV1/TRPV2 agonism) without reaching the threshold required for anti-inflammatory CB2-mediated effects. Qin et al. demonstrated that CBD acts as a potent TRPV2 agonist (EC50 = 3.7 µM), triggering dose-dependent CGRP release from dorsal root ganglion neurons [39], while McKenna and McDougall showed that cannabinoid-mediated activation of TRPV channels can promote neurogenic inflammation via substance P and CGRP release [40].

Furthermore, the divergent inflammatory profiles of Extract B and Extract D underscore the importance of the entourage effect hypothesis in cannabinoid pharmacology. While the concept posits that whole-plant preparations may exert enhanced therapeutic effects through synergistic interactions among cannabinoids, terpenes, and flavonoids [33], recent systematic reviews have revealed that evidence for a consistent, predictable entourage effect remains limited and context-dependent [41,42]. In fact, the term “contra-entourage effect” [42] has been proposed to describe situations in which extract components interact antagonistically, potentially diminishing or reversing the beneficial effects of individual cannabinoids [42,43,44]. Our finding that full-spectrum extracts did not outperform pure CBD, and in fact tended to sustain edema, is consistent with this emerging paradigm. Blevins et al. demonstrated that individual Cannabis-derived terpenes exert differential immunomodulatory effects on human primary leukocytes, with α-pinene showing anti-inflammatory properties while limonene had no effect [41]. Downer et al. further noted that certain Cannabis terpenes may produce transient immunostimulatory rather than immunosuppressive effects, depending on concentration and cellular context [42].

The question of causality in the observed correlations between paw volume and gene expression represents a fundamental interpretive challenge. The positive Pearson correlation between paw volume at 3 h and TNFα mRNA in the control group may reflect either TNFα-driven edema formation through vascular permeability enhancement and neutrophil recruitment (primary driver), or tissue-injury-induced transcriptional upregulation secondary to tissue swelling and cellular stress (secondary response) [45]. Both mechanisms likely operate simultaneously in a positive feedback loop. Similarly, the negative correlation between paw volume and CB2 expression in the CBD group at 3 h may reflect either a protective CB2 response limiting edema severity or, alternatively, suppression of CB2 transcription by advanced inflammation. Resolving this directionality requires time-resolved gene expression studies (sampling at 1, 3, 6, and 10 h), pharmacological intervention with selective antagonists, and protein-level validation approaches planned for forthcoming studies.

The authors plan to conduct a long-term observational study extending the monitoring period to 24–72 h post-carrageenan to determine whether cannabinoid preparations delay the resolution phase or actively sustain inflammation. Additionally, the interaction between pro-resolving lipid mediators (lipoxins, resolvins) and the endocannabinoid system, as demonstrated by Pamplona et al., who showed that lipoxin A4 acts as an allosteric enhancer of CB1 [46], warrants investigation in the context of cannabinoid-mediated modulation of inflammation resolution.