背景:类风湿性关节炎(RA)是一种以滑膜炎和关节炎疼痛为特征的慢性自身免疫性疾病。青藤碱(SIN),源自中草药青藤的根茎(学名:青藤(Thunb。)Rehd。EtWils),长期用于治疗类风湿性关节炎的中药。它已被证明具有抗炎作用,镇痛药,和免疫抑制作用,临床上副作用最小。然而,控制其在关节病理学治疗中的作用的机制,特别是成纤维细胞样滑膜细胞(FLSs)功能障碍,关节炎疼痛仍不清楚。
目的:本研究旨在探讨SIN对关节炎性关节炎症和关节FLS功能障碍的影响及其机制。
方法:在大鼠中诱发胶原诱导性关节炎(CIA),并通过组织病理学评估SIN对关节病理的治疗作用。接下来,我们使用LPS诱导的FLSs进行了一系列实验,分为五组(天真,LPS,SIN10、20、50μg/ml)。采用qPCR和ELISA检测炎症因子的表达。通过改良的Transwell测定和qPCR检测细胞的侵袭能力。使用Transwell迁移和细胞划痕测定来评估细胞的迁移能力。用免疫荧光和激光共聚焦显微镜观察相关蛋白的分布和含量,以及WesternBlot和qPCR。用质粒(CRMP2T514A/D)转染FLS以直接调节CRMP2蛋白的翻译后修饰,并监测对FLS功能的下游影响。
结果:SIN减轻CIA大鼠关节炎症,如滑膜增生的改善所证明,炎性细胞浸润和软骨损伤,以及抑制LPS诱导的FLS释放促炎细胞因子。体外研究表明,SIN对LPS诱导的FLS的侵袭和迁移具有浓度依赖性。此外,SIN下调LPS诱导的FLSs细胞CRMP2的表达,但在残基T514处增加其磷酸化。此外,质粒转染(CRMP2T514A/D)对pCRMP2T514的调节显着影响了FLSs的迁移和侵袭。最后,SIN促进FLS中pCRMP2T514的核易位。
结论:SIN可能通过调节CRMP2T514磷酸化及其FLSs的核转位发挥抗炎镇痛作用,抑制促炎细胞因子释放,抑制异常入侵和迁移。FLS中T514位点CRMP2的磷酸化可能为治疗RA的炎性关节破坏和关节炎性疼痛提供新的治疗靶点。
BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and arthritic pain. Sinomenine (SIN), derived from the rhizome of Chinese medical herb Qing Teng (scientific name: Sinomenium acutum (Thunb.) Rehd. Et Wils), has a longstanding use in Chinese traditional medicine for treating rheumatoid arthritis. It has been shown to possess anti-inflammatory, analgesic, and immunosuppressive effects with minimal side-effects clinically. However, the mechanisms governing its effects in treatment of joint pathology, especially on fibroblast-like synoviocytes (FLSs) dysfunction, and arthritic pain remains unclear.
OBJECTIVE: This study aimed to investigate the effect and underlying mechanism of SIN on arthritic joint inflammation and joint FLSs dysfunctions.
METHODS: Collagen-induced arthritis (CIA) was induced in rats and the therapeutic effects of SIN on joint pathology were evaluated histopathologically. Next, we conducted a series of experiments using LPS-induced FLSs, which were divided into five groups (Naïve, LPS, SIN 10, 20, 50 μg/ml). The expression of inflammatory factors was measured by qPCR and ELISA. The invasive ability of cells was detected by modified Transwell assay and qPCR. Transwell migration and cell scratch assays were used to assess the migration ability of cells. The distribution and content of relevant proteins were observed by immunofluorescence and laser confocal microscopy, as well as Western Blot and qPCR. FLSs were transfected with plasmids (CRMP2 T514A/D) to directly modulate the post-translational modification of CRMP2 protein and downstream effects on FLSs function was monitored.
RESULTS: SIN alleviated joint inflammation in rats with CIA, as evidenced by improvement of synovial hyperplasia, inflammatory cell infiltration and cartilage damage, as well as inhibition of pro-inflammatory cytokines release from FLSs induced by LPS. In vitro studies revealed a concentration-dependent suppression of SIN on the invasion and migration of FLSs induced by LPS. In addition, SIN downregulated the expression of cellular CRMP2 that was induced by LPS in FLSs, but increased its phosphorylation at residue T514. Moreover, regulation of pCRMP2 T514 by plasmids transfection (CRMP2 T514A/D) significantly influenced the migration and invasion of FLSs. Finally, SIN promoted nuclear translocation of pCRMP2 T514 in FLSs.
CONCLUSIONS: SIN may exert its anti-inflammatory and analgesic effects by modulating CRMP2 T514 phosphorylation and its nuclear translocation of FLSs, inhibiting pro-inflammatory cytokine release, and suppressing abnormal invasion and migration. Phosphorylation of CRMP2 at the T514 site in FLSs may present a new therapeutic target for treating inflammatory joint\'s destruction and arthritic pain in RA.