Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China; Tongji University Cancer Center, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai 200072, China.
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China.
Phytomedicine. 2020 Feb;67:153160. doi: 10.1016/j.phymed.2019.153160. Epub 2019 Dec 26.
Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis.
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.
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.
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.
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.
越来越多的证据表明，大麻素受体参与了器官纤维化的发病机制。
本研究旨在发现新型大麻素受体 2（CB2）激动剂，并评估 CB2 激活在治疗系统性硬化症中的潜力。
开发了基于海肾萤光素酶的分裂萤光素酶互补测定法（SLCA）来检测 CB2 和β-arrestin2 之间的相互作用。然后，使用 SLCA 方法筛选了 366 种天然产物文库，以寻找潜在的 CB2 激动剂。还利用几种 GPCR 功能测定法，包括基于 HTRF 的 cAMP 测定法和钙动员，评估 CB2 的激活。使用博来霉素诱导的实验性系统性硬化症来评估体内抗纤维化作用。通过 H&E 和天狼猩红染色评估真皮厚度和胶原含量。
通过使用 SLCA，发现了雷公藤红素是 CB2 的一种新激动剂。此外，雷公藤红素以剂量依赖的方式触发几种 CB2 介导的下游信号通路，包括钙动员、抑制 cAMP 积累和受体脱敏，并且对 CB1 具有中等选择性。此外，雷公藤红素在脂多糖（LPS）处理的鼠 Raw 264.7 巨噬细胞和原代巨噬细胞中表现出抗炎特性。最后，我们发现雷公藤红素在博来霉素诱导的系统性硬化症小鼠模型中发挥抗纤维化作用，同时伴有炎症状态的减轻。
总之，使用新开发的基于 arrestin 的 SLCA 鉴定出雷公藤红素是一种新型选择性 CB2 激动剂，CB2 的激活通过雷公藤红素减少了炎症反应，并防止了博来霉素诱导的系统性硬化症小鼠模型中真皮纤维化的发展。
