Data_Sheet_1_Cannabinoid Receptor 2 Agonist JWH-015 Inhibits Interleukin-1β-Induced Inflammation in Rheumatoid Arthritis Synovial Fibroblasts and in Adjuvant Induced Arthritis Rat via Glucocorticoid Receptor.docx

Management of pain in the treatment of rheumatoid arthritis (RA) is a priority that is not fully addressed by the conventional therapies. In the present study, we evaluated the efficacy of cannabinoid receptor 2 (CB2) agonist JWH-015 using RA synovial fibroblasts (RASFs) obtained from patients diagnosed with RA and in a rat adjuvant-induced arthritis (AIA) model of RA. Pretreatment of human RASFs with JWH-015 (10–20 μM) markedly inhibited the ability of pro-inflammatory cytokine interleukin-1β (IL-1β) to induce production of IL-6 and IL-8 and cellular expression of inflammatory cyclooxygenase-2 (COX-2). JWH-015 was effective in reducing IL-1β-induced phosphorylation of TAK1 (Thr184/187) and JNK/SAPK in human RASFs. While the knockdown of CB2 in RASFs using siRNA method reduced IL-1β-induced inflammation, JWH-015 was still effective in eliciting its anti-inflammatory effects despite the absence of CB2, suggesting the role of non-canonical or an off-target receptor. Computational studies using molecular docking and molecular dynamics simulations showed that JWH-105 favorably binds to glucocorticoid receptor (GR) with the binding pose and interactions similar to its well-known ligand dexamethasone. Furthermore, knockdown of GR using siRNA abrogated JWH-015's ability to reduce IL-1β-induced IL-6 and IL-8 production. In vivo, administration of JWH-015 (5 mg/kg, daily i.p. for 7 days at the onset of arthritis) significantly ameliorated AIA in rats. Pain assessment studies using von Frey method showed a marked antinociception in AIA rats treated with JWH-015. In addition, JWH-015 treatment inhibited bone destruction as evident from micro-CT scanning and bone analysis on the harvested joints and modulated serum RANKL and OPG levels. Overall, our findings suggest that CB2 agonist JWH-015 elicits anti-inflammatory effects partly through GR. This compound could further be tested as an adjunct therapy for the management of pain and tissue destruction as a non-opioid for RA.

类风湿关节炎（RA）的疼痛管理是当前常规疗法尚未完全攻克的临床重点问题。本研究分别采用确诊RA患者来源的滑膜成纤维细胞（RASFs），以及大鼠佐剂性关节炎（AIA）模型，评估大麻素受体2（CB2）激动剂JWH-015的治疗功效。
用10~20 μM的JWH-015预处理人源RASFs后，可显著抑制促炎细胞因子白细胞介素-1β（IL-1β）诱导的IL-6、IL-8生成，以及炎性环氧合酶-2（COX-2）的细胞表达。
JWH-015还可有效降低IL-1β诱导的人源RASFs中TAK1（Thr184/187位点）与JNK/SAPK的磷酸化水平。
尽管通过小干扰RNA（siRNA）敲低RASFs的CB2表达可减弱IL-1β诱导的炎症反应，但在CB2缺失的情况下，JWH-015仍可发挥抗炎作用，提示其可能通过非经典通路或脱靶受体实现抗炎功效。
采用分子对接与分子动力学模拟的计算研究显示，JWH-105可与糖皮质激素受体（GR）稳定结合，其结合构象与相互作用模式与经典配体地塞米松高度相似。
进一步通过siRNA敲低GR的表达，可完全阻断JWH-015抑制IL-1β诱导的IL-6、IL-8生成的作用。
体内实验中，在关节炎发作时每日给予大鼠5 mg/kg JWH-015腹腔注射（i.p.），持续7天，可显著改善大鼠的AIA症状。采用冯·弗雷纤维丝法进行疼痛评估的结果显示，JWH-015处理的AIA大鼠呈现出显著的镇痛效应。
此外，显微计算机断层扫描（micro-CT）扫描与采集的关节标本骨分析结果证实，JWH-015治疗可抑制骨破坏，同时可调节血清中核因子κB受体活化因子配体（RANKL）与骨保护素（OPG）的水平。
综上，本研究结果表明，CB2激动剂JWH-015可部分通过糖皮质激素受体（GR）发挥抗炎作用。该化合物可作为非阿片类药物，用于RA的疼痛管理与组织损伤防治，有望作为辅助疗法开展进一步研究。