Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis

Background: Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. Purpose: The purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. Methods: A gaussia princeps luciferase–based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and β-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. Results: Celastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. Conclusion: Taken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.