甲氨蝶呤联合环磷酰胺对类风湿关节炎滑膜细胞IL-6介导的RANKL表达的影响及其可能机制的研究

Rheumatoid arthritis (RA) is a systemic autoimmune disease that causes irreversible joint damage and significant disability. Receptor activator of nuclear factor-κB ligand (RANKL) is a essential factor for osteoclastogenesis that finally causes bone resorption and joint damage in RA. Interleukin-6 (IL-6), as a key inflammatory cytokine, can directly induce RANKL expression through janus kinase 1/2 (JAK1/2)-signal transducer and activators of transcription 3 (STAT3) pathway. Therefore IL-6-initiated JAK1/2-STAT3 pathway plays a critical role for joint destruction in RA and maybe a good target for therapeutic intervention. Disease-modifying anti-rheumatic drugs (DMARDs), such as mathotrexate (MTX), can decrease the level of serum IL-6 and suppress RANKL expression in a dose-dependent manner, ultimately retard the progression of joint damage. Through over ten years clinical research, we observed that the therapeutic alliance of MTX and cyclophosphamide (CTX) had a better curative effect in treating RA. In our previous research, combination MTX with CTX can more significantly relieve inflammatory symptoms and delay joint damage in RA. The alliance of MTX and CTX had synergistic effect in decreasing the level of serum cytokines, such as TNF-α, IL-1β, IL-6 and IL-17. Through animal studies, we observed combination MTX with CTX can obviously induce apoptosis of synovicytes and retard pathological progression of synovitis. But it is indefinite whether the therapeutic alliance of MTX and CTX synergistically suppress RANKL expression of RA synovial cells. Which component of trans-signaling pathway is a key site of its synergistic effect? This study will give the answer.

类风湿关节炎（RA）是一种以残毁性关节炎为主要特征的系统性自身免疫病，破骨细胞活化在RA患者关节破坏中起主要作用，而滑膜细胞RANKL分泌增加是破骨细胞分化的必要条件。研究表明，IL-6经JAK1/2-STAT3信号途径在滑膜细胞RANKL表达中起决定性作用，而以MTX为代表的DMARDs可降低RA患者血清IL-6的水平，并呈剂量依赖性的抑制RANKL表达，从而延缓骨破坏。我科采用MTX联合CTX小剂量间歇给药治疗RA，经十余年的临床检验，取得显著疗效，大量基础及临床研究证实，MTX联合CTX能更有效的改善RA关节炎症状，延缓骨关节破坏，降低血清TNF-α、IL-1β、IL-6、IL-17等细胞因子水平，动物实验亦证明联合组能更有效的诱导滑膜细胞凋亡，改善滑膜病理改变，但其能否协同抑制RA滑膜细胞RANKL的表达及其在信号传导通路的具体作用位点尚不明确，本研究将给出答案。

类风湿关节炎（RA）是一种以残毁性关节炎为主要特征的自身免疫病，破骨细胞活化在RA关节破坏中起主要作用，而滑膜细胞RANKL分泌增加是破骨细胞分化的必要条件。我科采用MTX联合CTX小剂量间歇给药治疗RA，取得显著疗效。大量基础及临床研究证实，MTX联合CTX能更有效地诱导滑膜细胞凋亡，改善滑膜病理改变，延缓骨关节破坏，但其能否协同抑制RA滑膜细胞RANKL的表达及其在信号传导通路的具体作用位点尚不明确。本研究采用RA成纤维滑膜细胞（RA-FLS）和CIA小鼠分别进行体外和体内实验，研究了MTX联合CTX对RANKL和OPG表达的影响，以及STAT3和AKT两条通路在其中的作用。实验采用了RT-PCR、Western-blot和Cell-Based ELISA方法进行实验。结果显示，IL-6/sIL-6R可明显诱导RA-FLS上RANKL的表达。低剂量的MTX(10nM)联合低剂量的CTX（1ug/ml）后作用RA-FLS，可明显降低RANKL，升高OPG，与单用10倍剂量MTX（100nM）组相比，差异有统计学意义。STAT3和AKT的磷酸化被分别抑制后，不同剂量的MTX和CTX的联合对RANKL的表达产生不同的作用。在MTX(10nM)和CTX联合中，STAT3磷酸化被抑制后，RANKL表达降低；而AKT磷酸化被抑制后，RANKL表达升高；在MTX(100nM)和CTX联合中，STAT3和AKT磷酸化被抑制后，RANKL表达均降低；在MTX(1000nM)和CTX联合中，STAT3磷酸化被抑制后，RANKL表达升高，AKT磷酸化被抑制后，RANKL表达无变化。结果表明MTX(10nM)和CTX（1ug/ul）的联合能很好地降低RANKL的表达，不同剂量的MTX与CTX的联合对RANKL的作用受不同通路和作用位点的影响。