Gugliandolo, P. Licata, A. F. Peritore, R. Siracusa, R. D’Amico, M. Cordaro, R.Fusco, D. Impellizzeri, R. Di Paola, S. Cuzzocrea, R. Crupi and C. D. Interlandi
- TNF-α is a proinflammatory molecule with increased expression and present in high pathological levels in canine atopic dermatitis
- IL-6 (proinflammatory molecule) production is boosted in atopic dermatitis and its blockage can help improving this disease
- Both TNF-α and IL-6 are associated with the pathogenesis of arthritis in dogs
- CBD reduced the secretion of TNF-α and IL-6 (molecules that drive the inflammatory response) in an ex vivo model of inflammation in canine blood cells
- The TNF-α to IL-10 (pro-inflammatory molecule) ratio is a marker of the balance between key pro- and anti-inflammatory levels
- Treatment with CBD restored the TNF-α to IL-10 ratio to a healthy balance
- CBD also reduced NF-κB activation (NF-κB is a regulator of the inflammatory response and of many pro-inflammatory cytokines)
- Finally, administration of CBD to the inflamed cells resulted in a lower COX-2 (which is upregulated in inflammation) expression
OBJECTIVES & HYPOTHESIS → The aim of the study was to investigate the anti-inflammatory and immunomodulatory properties of CBD directly in dogs.
METHODS → The study used an ex vivo model where whole blood from healthy dogs was stimulated with LPS (evokes a pro-inflammatory environment, thus allowing the characterization of the effect of CBD on canine inflammatory response). Whole blood samples were obtained from 6 healthy dogs and added directly to different medias:
- Control: whole blood + vehicle (DMSO 0.1%)
- LPS: whole blood + LPS 100 ng/mL + vehicle (DMSO 0.1%)
- CBD 50 μg: whole blood + LPS 100 ng/mL + CBD (kanarescue 5% in DMSO 0.1%) 50 μg/mL
- CBD 100 μg: whole blood + LPS 100 ng/mL CBD (kanarescue 5% in DMSO 0.1%) 100 μg/mL
The study then focused on the levels of the major regulating molecules. IL-6 and TNF-α are the major pro-inflammatory cytokines (molecules/factors) driving inflammatory response and immune cell activation; they play a key role in the physiological inflammatory response such as during infection, but also in several diseases where elevated levels of these cytokines are associated with a poor clinical outcome. IL-10 is an anti-inflammatory cytokine and immune-regulating mediator. The balance between TNF-α and IL-10 is important for immune homeostasis maintenance; commonly, an increase in TNF-α is counterbalanced by a simultaneous increase in the anti-inflammatory cytokine IL-10, which suppresses the production of many activating and regulatory mediators. Furthermore, the study evaluated the effect of CBD on Nf-κB (key regulator of inflammatory response that regulates the expression of several pro-inflammatory mediators) and COX-2 expression. COX-2 plays a fundamental role in the inflammatory cascade, and thus in acute and chronic inflammation.
RESULTS → When stimulation with LPS was performed in the presence of CBD (50 and 100 μg/mL) there was a significant reduction in cytokines secretion. The study observed a dose dependence effect of CBD; when compared to CBD 50 μg/mL, the dose of CBD 100 μg/mL produced a bigger inhibitory effect on IL-6 and TNFα release in whole blood supernatant.
Graphic 1: IL-6 and TNF-α levels after LPS 100ng/mL stimulation and with concomitant use of CBD at different doses (50 and 100 ng/mL)
When evaluating the TNF-α to IL-10 ratio as a cytokine marker of the balance between key pro and anti-inflammatory levels, the results show that LPS stimulation produces a significant increase in the TNF-α to IL-10 ratio in whole dog blood, while treatment with CBD is able to restore the TNF-α to IL-10 ratio compared to the control groups. Finally, the LPS stimuli induced an increase in Nf-κB p-65 activation, while there was a reduction in Nf-κB expression for the groups incubated with LPS and CBD. Moreover, levels for COX-2, known to be upregulated during inflammation, were increased in the LPS group. The CBD treatment (50 and 100 μg/mL) group showed significantly lower COX-2 expression compared to LPS.