ManLAM诱导结核性关节炎FLS细胞中IL-37表达的机制

Mechanism of ManLAM Inducing IL-37 Expression in FLS Cells in Tuberculous Arthritis

目的使用人成纤维样滑膜细胞(FLS细胞)系探究甘露糖修饰的脂阿拉伯甘露聚糖(Man LAM)对人结核性关节炎中白介素-37 (IL-37)产生的作用及相关分子机制。方法从H37RV结核杆菌中提取并纯化Man LAM,提取人FLS细胞中的总RNA,将RNA反转录为c DNA。蛋白质免疫印迹法(Western Blot)对p38丝裂原活化蛋白激酶类/拮抗剂和抑制剂(p38和ERK1/2 MAPKs)进行检测。ELISA法和流式细胞术检测FLS细胞中表达出来的IL-37水平,用以评估Man LAM诱导FLS细胞中IL-37表达的能力以及观察p38和ERK1/2等抑制剂对Man LAM诱导产生的IL-37表达的影响。结果发现ManLAM诱导IL-37 mRNA及蛋白表达具有时间和剂量依赖性,然而这种活性几乎可以全部被ERK1/2 (U0126)和p38(SB203580)抑制剂所消除。TLR2表达受到干扰后,ERK1/2和p38磷酸化水平也显著下降,同时IL-37的产物也大大减少。但是干扰TLR4对Man LAM诱导IL-37的表达几乎没有影响。结论 Man LAM通过上调TLR2/p38或ERK1/2信号通路来诱导人FLS细胞中IL-37的产生,而且,这为解释Man LAM促进结核杆菌持久性的病理学作用提供了一个重要的依据。

Objective Human fibroblast-like synovial cells( FLS cells) were used to investigate the effect of mannose-modified lipoarabinomannan( ManLAM) on interleukin-37( IL-37) production in human tuberculosis arthritis and its molecular mechanism.Methods ManLAM was extracted and purified from tuberculosis bacillus H37 RV. Total RNA was extracted from FLS cells and RNA was reversely transcribed into c DNA. P38 mitogen-activated protein kinases/antagonists and inhibitors( p38 and erk1/2 MAPKs)were detected by Western blotting. The expression of IL-37 in FLS cells was detected by ELISA and flow cytometry to evaluate the ability of ManLAM to induce IL-37 expression in FLS cells and to observe the effect of inhibitors such as p38 and erk1/2 on the expression of IL-37 induced by ManLAM. Results It was found that the expression of IL-37 gene and protein induced by ManLAM was time-and dose-dependent,while this activity was almost totally abolished by the ERK1/2( U0126) and p38( SB203580) inhibitors. ManLAM stimulation significantly induced ERK1/2 and p38 phosphorylation in FLS cells,as well as cell surface TLR2 expression. After interfering TLR2 expression,ERK1/2 and p38 phosphorylation levels were markedly decreased and also IL-37 production.TLR4 interference had little effect on ManLAM-induced IL-37 production. Conclusion Our results indicate that ManLAM induces IL-37 production of human type II alveolar epithelial cells via up-regulating TLR2/p38 or ERK1/2 pathway,and this provides an important basis for explaining the pathological role of ManLAM in promoting the persistence of tuberculosis bacillus.