Cannabichromene as a Novel Inhibitor of Th2 Cytokine and JAK/STAT Pathway Activation in Atopic Dermatitis Models

Abstract

Cannabichromene (CBC) is one of the main cannabinoids found in the cannabis plant, and although less well known than tetrahydrocannabinol (THC) and cannabidiol (CBD), it is gaining attention for its potential therapeutic benefits. To date, CBC's known mechanisms of action include anti-inflammatory, analgesic, antidepressant, antimicrobial, neuroprotective, and anti-acne effects through TRP channel activation and the inhibition of inflammatory pathways, suggesting that it may have therapeutic potential in the treatment of inflammatory skin diseases, such as atopic dermatitis (AD), but its exact mechanism of action remains unclear. Therefore, in this study, we investigated the effects of CBC on Th2 cytokines along with the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways involved in AD pathogenesis. We used a 2,4-Dinitrochlorobenzene (DNCB)-induced BALB/c mouse model to topically administer CBC (0.1 mg/kg or 1 mg/kg). The results showed that skin lesion severity, ear thickness, epithelial thickness of dorsal and ear skin, and mast cell infiltration were significantly reduced in the 0.1 mg/kg CBC-treated group compared with the DNCB-treated group (p < 0.001). In addition, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a significant decrease in the mRNA expression of Th2 cytokines (TSLP, IL-4, IL-13) and inflammatory mediators (IFN-γ, IL-1β, IL-6, IL-17, IL-18, and IL-33) (p < 0.05). Western blot analysis also revealed a significant decrease in JAK1, JAK2, STAT1, STAT2, STAT3, and STAT6 protein expression (p < 0.05). These results suggest that CBC is a promising candidate for the treatment of AD and demonstrates the potential to alleviate AD symptoms by suppressing the Th2 immune response.

Keywords: atopic dermatitis; cannabichromene; phytocannabinoid.

Figure 1

Chemical structures of (A) cannabichromene, (B) tetrahydrocannabinol and (C) cannabidiol.

Figure 2

Atopic dermatitis mouse model experiment schedule and clinical experiment results. (A) Animal experiment schedule and group details, (B) Clinical photo on day 7 before the start of the challenge and on day 21 before the sacrifice, (C) Average score of 4 items: erythema, dryness, excoriation, and edema, (D) Average value of both ear thicknesses measured with a Dial thickness Gauge during the challenge period. The experiment was conducted in 5 groups, and a total of 25 animals were used, n = 5 per group. Data shown in the figure are presented as mean ± SEM (### p < 0.001 vs. normal group; *** p < 0.001 vs. DNCB group). DNCB, 1-chloro-2,4-Dinitrochlorobenzene.

Figure 3

Result of staining after treating cannabichromene on atopic dermatitis mouse model dorsal skin. (A) Representative H&E stain and Toluidine blue stain images from each group (200× magnification, arrows indicate mast cells), (B) results of measuring epidermis thickness after H&E stain (total n = 5/group), (C) results of counting mast cell invasion (total n = 5/group), data shown in the figure are provided as mean ± SEM (*** p < 0.001 vs. normal group or DNCB group). DNCB, 1-chloro-2,4-Dinitrochlorobenzene; TAC, 0.03% tacrolimus; H&E, Hematoxylin and Eosin.

Figure 4

Results of confirming the mRNA expression of various cytokines after treating cannabichromene in an atopic dermatitis mouse model. Various cytokines (A) TSLP, (B) IL-4, (C) IL-1β, (D) IL-6, (E) IL-33, (F) IL-18, (G) IFN-γ, (H) IL-13, (I) IL-17 were identified through qRT-PCR (total n = 5/group). All cytokines were normalized to Actb, and expression levels were analyzed using the ΔΔct method. Data shown in the figure are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. normal group or DNCB group). DNCB, 1-chloro-2,4-Dinitrochlorobenzene; TAC, 0.03% tacrolimus.

Figure 5

Results of confirming the expression of proteins related to the JAK/STAT pathway after treating cannabichromene in an atopic dermatitis mouse model. Through Western blot, the expression of JAK/STAT family of proteins (total n = 5/group) was confirmed. The membrane band intensity of all proteins was measured using ImageJ software and normalized to β-actin. Data shown in the figure are presented as mean ± SEM (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. normal group or DNCB group). DNCB, 1-chloro-2,4-Dinitrochlorobenzene; TAC, 0.03% tacrolimus.

Figure 6

The effect of cannabichromene on a mouse model of hapten-induced contact dermatitis. JAK is activated by cytokine binding to various receptors, and when phosphorylated, STAT protein is activated and phosphorylated. Phosphorylated STAT translocates to the nucleus and activates gene transcription. Cannabichromene has been shown to inhibit these pathways, and, based on these results, it is confirmed to have a therapeutic effect on atopic dermatitis.