Screening And Anti-fibrosis Activity Of New Selective Agonists For Cannabinoid Receptor CB2

Systemic sclerosis(SSc),as known as scleroderma,is a systemic autoimmune connective tissue disease that characterized by skin fibrosis and a variety fibrosis of internal organs such as liver,lungs,heart and vasculopathy.Although SSc is an uncommon disease,its prognosis is severe with approximately 60% survival rate for ten years.Conventional immunosuppression is the main therapeutic strategy for SSc patients by targeting the inflammation and immune response dysregulation in SSc pathogenesis.However,specific targeted therapies are still unavailable now.Therefore,development of novel therapeutic approaches is an urgent need to improve the clinical benefit and quality of life for SSc patients.Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis,especially the CB2 receptor.The purpose of this study was to discover novel cannabinoid receptor 2(CB2)agonist and assess their potential to activate CB2 in treating systemic sclerosis.A gaussia princeps luciferase–based split luciferase complementation assay(SLCA)was developed for detection of the interaction between CB2 and ?-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.Bleomycininduced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects.Dermal thickness and collagen content were examined via H&E and sirius red staining.Celastrol was identified as a new agonist of CB2 by using SLCA.Furthermore,celastrol triggered several CB2-mediated downstream signaling pathways,including calcium mobilization,inhibition of cAMP accumulation,and receptor desensitization in a dose-dependent manner,and it had 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 exerted anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions.Taken together,celastrol was identified a novel selective CB2 agonist using a new developed arrestin-based SLCA,and CB2 activation by celastrol reduced the inflammatory response,and prevented the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.Celastrol was a potential therapeutic agent for SSc.