Anti-Inflammatory Effects of Cannabigerol In Vitro and In Vivo Are Mediated Through the JAK/STAT/NFκB Signaling Pathway

Abstract

Cannabinoid compounds have potential as treatments for a variety of conditions, with cannabigerol (CBG) being known for its anti-inflammatory properties. In this study, we investigated the effects of CBG in a cellular model of 1-chloro-2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD). In the cellular model, we confirmed the cytotoxicity of CBG and downregulated the expression of inflammatory markers CCL26, IL1B, IL6, and TNF (p < 0.001). In the mouse model, clinical, histological, and immunological changes were analyzed. The results showed that CBG improved dermatitis severity score, epidermal thickness, and mast cell count and reduced inflammatory cytokines (Tslp, Il1b, Il4, Il6, Il13, Il17, Il18, Il22, and Il33) by qRT-PCR (p < 0.001). Western blot results showed modulated changes in JAK1, JAK2, TYK2, STAT1, STAT2, STAT3, p-STAT3, STAT6, and p-STAT6 (p < 0.05). Subsequently, p-IκBα, NF-κB, and p-NF-κB signaling factors were also reduced (p < 0.05), with corresponding changes in skin barrier factors. The results of this study indicate that CBG effectively alleviates AD-like symptoms and suggest the potential of CBG as a therapeutic agent.

Keywords: Janus kinase-signal transducer and activator of transcription; atopic dermatitis; cannabigerol.

Figure 1

Effect of CBG treatment against IL-4 and IL-13 induction. (a) CBG toxicity in HaCaT keratinocytes. assessed by MTT assay and expressed as a percentage relative to Control cells. Data represents the mean ± SEM from three independent experiments. ** p < 0.01 *** p < 0.001 compared with Control cells. (b–e) HaCaT cells were treated with 50 ng each of IL-4 and IL-13 for 18 h, followed by CBG treatment at indicated concentrations for 6 h. Cytokine mRNA levels were measured and expressed as relative fold increases compared to CBG controls. Data represents the mean ± SEM from three independent experiments. Data compared among multiple groups were analyzed using one-way ANOVA. * p < 0.5, ** p < 0.01, *** p < 0.001 compared with BSA control or IL-4, IL-13 treated cells. CBG, cannabigerol; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 2

CBG alleviated AD on clinical evaluation. (a) To evaluate the efficacy of topical CBG treatment in an AD-like environment, an experimental schedule was established as shown in the accompanying figure. The treatment phases and their frequency are indicated by arrows. The experiment includes abdominal sensitization with 1% DNCB (black), maintenance of the AD environment during the challenge period with 0.4% DNCB (red), and application of vehicles, tacrolimus, and CBG (blue). (b) Clinical results at the end of a 2-week challenge in an animal model of AD. Each image shows normal skin or skin treated with vehicle (acetone: olive oil suspension), tacrolimus, 0.1 mg/kg, and 1 mg/kg CBG on days 7, 13, and 21. (c) Skin severity score, encompassing erythema/hemorrhage, scaling/dryness, and edema, is shown for Days 7–21. (d) Ear thickness assessed results on Day 7–21. Data represents the mean ± SEM (n = 5). Data compared among multiple groups were analyzed using one-way ANOVA. ### p < 0.001 compared to Normal group, *** p < 0.001 compared to DNCB group. CBG, cannabigerol; AD, atopic dermatitis; DNCB, 1-chloro-2,4-dinitrobenzene; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 3

CBG reduced skin thickness and mast cell count. (a) Representative images from H&E and toluidine blue staining of dorsal skin tissues from mice after the challenge period. Histological features of dorsal skin in BALB/c mice are depicted. Mast cells (purple) in the dermis are indicated and marked with black arrows. Original magnification = ×200, scale bar = 100 μm. (b) Epidermal thickness changes for each group. (c) The number of mast cells counted and compared across each group. Data represents the mean ± SEM. Data compared among multiple groups were analyzed using one-way ANOVA. *** p < 0.001 compared to Normal or DNCB group. CBG, cannabigerol; H&E, Hematoxylin and Eosin; DNCB, 1-chloro-2,4-dinitrobenzene; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 4

Effects of CBG on cytokine expression in a DNCB-induced AD mouse model. Changes in mRNA expression levels of (a) Tslp, (b) Il1b, (c) Il4, (d) Il6, (e) Il13, (f) Il17, (g) Il18, (h) Il22, (i) Il33 in dorsal skin tissue. Total RNA was extracted and analyzed using qRT-PCR (n = 5/group). Gene expression levels were normalized to Actb. The qRT-PCR was duplicated, and data were analyzed by CFX Manager software (version 3.0) (Bio-Rad Laboratories, Hercules, CA, USA). Data represents the mean ± SEM. Data compared among multiple groups were analyzed using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Normal or DNCB group. CBG, cannabigerol; DNCB, 1-chloro-2,4-dinitrobenzene; AD, atopic dermatitis; SEM, standard error of the mean.

Figure 5

Effects of CBG on JAK/STAT, NF-κB signaling pathway, and skin barrier proteins in a DNCB-induced AD mouse model. Changes in protein expression levels of (a) JAK/STAT pathway family proteins. (b) NF-κB pathway family proteins (c) skin barrier protein in dorsal skin tissue. Original magnification = ×200, scale bar = 100 μm Immunoblotting intensities were calculated with ImageJ software (version 1.54f). Data represents the mean ± SEM. Data compared among multiple groups were analyzed using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to normal or DNCB group. CBG, cannabigerol; DNCB, 1-chloro-2,4-dinitrobenzene; AD, atopic dermatitis; SEM, standard error of the mean; ANOVA, analysis of variance.

Figure 6

Effects of CBG on AD models. In the DNCB-induced AD model, the JAK/STAT signaling and NF-κB signaling pathways are activated by the combination of various inflammatory cytokines that have been found to have therapeutic effects on AD by modulating the signaling of these pathways. CBG, cannabigerol; AD, atopic dermatitis; DNCB, 1-chloro-2,4-dinitrobenzene.