Cannabinol modulates the endocannabinoid system and shows TRPV1-mediated anti-inflammatory properties in human keratinocytes

Abstract

Cannabinol (CBN) is a secondary metabolite of cannabis whose beneficial activity on inflammatory diseases of human skin has attracted increasing attention. Here, we sought to investigate the possible modulation by CBN of the major elements of the endocannabinoid system (ECS), in both normal and lipopolysaccharide-inflamed human keratinocytes (HaCaT cells). CBN was found to increase the expression of cannabinoid receptor 1 (CB₁) at gene level and that of vanilloid receptor 1 (TRPV1) at protein level, as well as their functional activity. In addition, CBN modulated the metabolism of anandamide (AEA) and 2-arachidonoylglicerol (2-AG), by increasing the activities of N-acyl phosphatidylethanolamines-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH)-the biosynthetic and degradative enzyme of AEA-and that of monoacylglycerol lipase (MAGL), the hydrolytic enzyme of 2-AG. CBN also affected keratinocyte inflammation by reducing the release of pro-inflammatory interleukin (IL)-8, IL-12, and IL-31 and increasing the release of anti-inflammatory IL-10. Of note, the release of IL-31 was mediated by TRPV1. Finally, the mitogen-activated protein kinases (MAPK) signaling pathway was investigated in inflamed keratinocytes, demonstrating a specific modulation of glycogen synthase kinase 3β (GSK3β) upon treatment with CBN, in the presence or not of distinct ECS-directed drugs. Overall, these results demonstrate that CBN modulates distinct ECS elements and exerts anti-inflammatory effects-remarkably via TRPV1-in human keratinocytes, thus holding potential for both therapeutic and cosmetic purposes.

Keywords: cannabinol; cytokines; endocannabinoid system; keratinocytes; skin inflammation.

FIGURE 1

Gene expression of (A) eCB‐binding receptors (CB₁, CB₂, GPR55, TRPV1, and PPARα/γ/δ) and (B) eCBs metabolic enzymes (NAPE‐PLD, FAAH, DAGLα/β, and MAGL) in HaCaT cells following 24 h treatment with 2.5 μM CBN. The values were expressed as 2^(−ΔΔCt) and normalized to β‐actin and GAPDH as housekeeping genes. Data are the mean ± SEM of three independent experiments (n = 3). Statistical analysis was performed using a multiple t‐test, followed by the correction with the Bonferroni–Dunn method (**p < 0.01 vs. control).

FIGURE 2

Protein expression of (A) eCBs‐binding receptors (CB₁, CB₂, GPR55, TRPV1, and PPARα/γ/δ) and (B) metabolic enzymes (NAPE‐PLD, FAAH, DAGLα/β, and MAGL) in HaCaT cells following 24 h treatment with 2.5 μM CBN. Densitometric analyses of immunoreactive bands were normalized to β‐actin as housekeeping protein. Data are the mean ± SEM of three independent experiments (n = 3). Statistical analysis was performed using a multiple t‐test, followed by the correction with the Bonferroni–Dunn method (**p < 0.01 vs. control). Representative bands of the main ECS components are shown in the lower panels.

FIGURE 3

(A) Intracellular Ca²⁺ release triggered by capsaicin (a selective agonist of TRPV1) in HaCaT cells following 24 h treatment with 2.5 μM CBN. Data are means ± SEM of three independent experiments (n = 3). Statistical analysis was performed using an unpaired t‐test (*p < 0.05 vs. control). (B) CB₁ binding activity in HaCaT cells following 24 h treatment with 2.5 μM CBN. Data are the mean ± SEM of three independent experiments (n = 3). Statistical analysis was performed using an unpaired t‐test (***p < 0.001 vs. control).

FIGURE 4

Activity of eCBs‐metabolic enzymes: (A) NAPE‐PLD, (B) FAAH, (C) DAGLα/β, and (D) MAGL, in HaCaT cells following 24 h treatment with 2.5 μM CBN. Data are the mean ± SEM of three independent experiments (n = 3). Statistical analysis was performed using an unpaired t‐test (*p < 0.05, **p < 0.01, and ***p < 0.001 vs. control).

FIGURE 5

Release of ILs after HaCaT cells exposure to 2.5 μM CBN alone, 5.0 μg/mL LPS, 10 μM HC, and 2.5 μM CBN in the presence of LPS for 24 and 48h. (A) IL‐1β expression (pg/mL); (B) IL‐8 expression (ng/mL); (C) IL‐12 expression (pg/mL); (D) IL‐31 expression (pg/mL); and (E) IL‐10 expression (pg/mL). Data are presented as the mean ± SEM of three independent experiments (n = 3). Statistical analysis was performed using a one‐way ANOVA test followed by a Bonferroni post hoc test (**p < 0.01, ***p < 0.001, ****p < 0.0001 vs. CTRL; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. LPS).

FIGURE 6

Release of ILs after HaCaT cells exposure to 5.0 μg/mL LPS, 10 μM HC, 2.5 μM CBN alone or in combination with the two selective antagonists for CB₁ (SR1 at 0.1 μM) and TRPV1 (CPZ at 5.0 μM). (A) IL‐31 expression (pg/mL) after 24 h treatment and (B) IL‐12 expression (pg/mL) after 48 h treatment. Data are presented as the mean ± SEM of three independent experiments (n = 3). Statistical analysis was performed using a one‐way ANOVA test followed by a Bonferroni post hoc test (***p < 0.001, ****p < 0.0001 vs. CTRL; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. LPS; ^$ p < 0.05 vs. CBN).

FIGURE 7

Heat map of the most relevant 17 proteins of the MAPK signaling pathway in HaCaT cells following 24 h treatment with LPS (5.0 μg/mL) in the presence of 2.5 μM CBN, alone or in combination with the two selective antagonists for CB₁ (SR1 at 0.1 μM) and TRPV1 (CPZ at 5.0 μM). Samples were analyzed through the human phosphorylation array, and data are expressed as means of three sets of experiments for each condition (n = 3).