Targeting cannabinoid receptor 2 (CB2) limits collagen production—An in vitro study in a primary culture of human fibroblasts

Abstract

Previous studies showed that cannabinoid 2 (CB2) receptor is involved in skin inflammation, fibrogenesis and re-epithelialization in mice, indicating that this receptor may be implicated in wound healing. Thus, topical use of cannabinoids may have a role in local fibrotic and wound healing diseases such as scars or keloids. We investigate the effect of the CB2 selective receptor agonist (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (JWH133) and the CB2 selective receptor antagonist ( 6, on primary cultures of human fibroblasts. Primary cultures of adult human fibroblasts were obtained from abdominal human skin samples. Fibroblasts pretreated with JWH133 and/or AM630 were stimulated with TGF-β (10 ng/ml). Fibroblast activation into myofibroblasts was quantified by the expression of alpha-smooth muscle actin (α-SMA) using Immunocytochemistry and Western Blot assays. Collagen content was quantified with the Sirius red staining assay. Upon human fibroblasts stimulation with TGF-β, a significant increase on α-SMA and CB2 receptor expression was observed. In these cells, JWH133 decreased α-SMA expression and collagen content. However, this effect was not observed in resting human fibroblasts. AM630 decreased α-SMA expression and collagen content in both resting and activated fibroblasts. This effect was time-and concentration-dependent with an IC 50 value of 11 μM. The CB2 receptor appears to be involved in fibroblast repair during skin wound healing in humans, as TGF-β increases CB2 receptor expression and JWH133 produces an anti-fibrotic effect in human fibroblasts. AM630 also showed an anti-fibrotic effect hypothesizing that other cannabinoid receptors, such as TRPV, may be involved in this response.

The effect of cannabinoids on wound healing: A review

“…When the fibroblasts were not treated with TGF‐β, only AM630 led to decreased collagen deposition and α‐SMA expression. This study showed the complex interplay of CB 2 on the wound healing process 51 …”

“…This study showed the complex interplay of CB 2 on the wound healing process. 51 Styrczewska et al 22 performed scratch test assays with hydrophobic flax fiber extract, which contained CBD, and demonstrated improved wound healing via inhibition of chronic inflammation and promotion of extracellular matrix remodeling and skin cell migration. The authors suggested the improvement is secondary to two components: presence of CBD and the content of phytosterol.…”

Cannabinoid Compounds as a Pharmacotherapeutic Option for the Treatment of Non-Cancer Skin Diseases

“…Another investigation showed that cannabinoid CB 2 receptors are upregulated in neutrophils, macrophages and myofibroblasts in a time-dependent manner during skin wound healing in mice [ 175 ]. Moreover, a recent study using primary cultures of adult human fibroblasts revealed that TGF-β, a profibrotic mediator, significantly increases the expression of CB 1 [ 176 ] and CB 2 receptors [ 177 ], suggesting that both receptors may play a role in fibrotic diseases of the skin and thus represent potential targets for pharmacotherapeutic interventions. Moreover, in mice in the above-mentioned model of streptozotocin-induced type 1 diabetes associated with accelerated skin ageing, the CB 1 receptor has been shown to be downregulated, which was accompanied by higher expression of pro-inflammatory markers [ 166 ].…”

“…Under the same experimental conditions, the authors also found that the CB 2 receptor agonist JWH-133 decreased α-SMA expression and collagen content in TGF-β-stimulated cells, but not in resting cells. On the other hand, the CB 2 receptor antagonist AM-630 also decreased α-SMA expression and collagen content in both resting and TGF-β-stimulated cells, but at higher concentrations (≥3 µM) [ 177 ]. The authors concluded that the effects produced by JWH-133 show potential for the treatment of hypertrophic scars or keloids in humans.…”

Myofibroblasts Are Not Characteristic Features of Keloid Lesions

“…Nevertheless, downregulation of α-SMA continues to be frequently utilized as a surrogate for antifibrotic activity in analysis of novel keloid therapies. [25][26][27][28][29][30][31] Given the emphasis on reduction of α-SMA-expressing myofibroblasts as a therapeutic endpoint, it is important to critically examine the role and presence of myofibroblasts in keloid pathology. We previously performed gene expression profiling of keloid fibroblasts and failed to detect increased expression of ACTA2, the gene encoding α-SMA, compared with normal fibroblasts.…”