Phytomedicine ( IF 6.7 ) Pub Date : 2023-05-03 , DOI: 10.1016/j.phymed.2023.154857 Fang-Yuan Liu 1 , Meng-Qing Wang 1 , Ming-Ming Liu 1 , Tao Li 1 , Xiao-Hua Wang 1 , Fei Jiang 1 , Xin-Jie Wu 2 , Juan Cheng 3 , Li Cai 4 , Rong Li 5
|
Background
Shikonin (SKN), the main bioactive component isolated from Lithospermum erythrorhizon Sieb et Zucc, has multiple activities including anti-rheumatic effect, but its specific roles and the precise mechanisms in regulating biological properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are unclear and need further clarification.
Purpose
This study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-α-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms.
Methods
SKN was intraperitoneally given to AIA rats and its therapeutic role was valued. The effects of SKN in vivo and in vitro on the production of pro-inflammatory factors were examined by ELISA and western blot. Wound-healing, transwell and phalloidin staining assay were carried out to evaluate the effects of SKN on TNF-α-induced migration and invasion in RA FLS. The involvement of Wnt/β-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for β-catenin and western blot for pathway-related proteins.
Results
SKN treatment in AIA rats reduced paw swelling, arthritis index and pathological damage of ankle joints, indicating its anti-arthritic effect in vivo. SKN had anti-inflammatory roles in vivo and in vitro, evidenced by inhibiting the production of pro-inflammatory factors (like IL-1β, IL-6, IL-8, TNF-α, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-α-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-α-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-α-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/β-catenin signaling in AIA rat synovium and in TNF-α-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3β (Ser9) and β-catenin, the raised protein level of GSK-3β and the decreased nuclear translocation of β-catenin. Interestingly, the combination of LiCl (Wnt/β-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-α-induced MH7A cells, whereas XAV939 (Wnt/β-catenin inhibitor) enhanced the therapeutic roles of SKN.
Conclusion
SKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-α-stimulated RA FLS via interrupting Wnt/β-catenin pathway.
中文翻译：
紫草素通过阻断Wnt/β-catenin通路激活对大鼠佐剂性关节炎及类风湿成纤维细胞样滑膜细胞炎症、迁移和侵袭的治疗作用
背景
紫草素 (SKN) 是从紫草中分离出来的主要生物活性成分，具有抗风湿等多种活性，但其在调节类风湿关节炎 (RA ) 成纤维细胞样滑膜细胞 (FLS)生物学特性方面的具体作用和确切机制) 不清楚，需要进一步澄清。
目的
本研究探讨了 SKN 对大鼠佐剂性关节炎 (AIA) 和 TNF-α 诱导的 RA FLS（MH7A 细胞）的细胞炎症、迁移和侵袭的治疗作用，并进一步证明了相关机制。
方法
SKN 被腹膜内给予 AIA 大鼠，其治疗作用受到重视。通过 ELISA 和蛋白质印迹检查 SKN 在体内和体外对促炎因子产生的影响。进行伤口愈合、transwell 和鬼笔环肽染色试验以评估 SKN 对 TNF-α 诱导的 RA FLS 迁移和侵袭的影响。Wnt/β-连环蛋白通路的参与通过β-连环蛋白的免疫组织化学或免疫荧光测定和通路相关蛋白的蛋白质印迹来检查。
结果
AIA 大鼠的 SKN 治疗减少了爪肿胀、关节炎指数和踝关节的病理损伤，表明其在体内具有抗关节炎作用。SKN 在体内外均具有抗炎作用，可通过抑制血清中促炎因子（如 IL-1β、IL-6、IL-8、TNF-α、MMP-2 和 MMP-9）的产生来证明AIA 大鼠的和滑膜，以及 TNF-α 诱导的 MH7A 细胞。明胶酶谱结果表明 SKN 在体外抑制 TNF-α 诱导的 MMP-2 活性。此外，SKN 抑制 MH7A 细胞中 TNF-α 诱导的迁移、侵袭和细胞骨架重组。从机制上讲，SKN 抑制 AIA 大鼠滑膜和 TNF-α 诱导的 MH7A 细胞中 Wnt/β-catenin 信号的激活，表现为 Wnt1、p-GSK-3β (Ser9) 和 β-catenin 的蛋白质水平降低，提高 GSK-3β 的蛋白水平和减少 β-catenin 的核转位。有趣的是，LiCl（Wnt/β-连环蛋白激动剂）的组合取消了 SKN 对 TNF-α 诱导的 MH7A 细胞炎症、迁移和侵袭的治疗作用，而 XAV939（Wnt/β-连环蛋白抑制剂）增强了治疗作用SKN。
结论
SKN 通过阻断 Wnt/β-连环蛋白通路对大鼠 AIA 和 TNF-α 刺激的 RA FLS 的细胞炎症、迁移和侵袭显示出治疗作用。
京公网安备 11010802027423号