免疫抑制剂对酵母聚糖诱导巨噬细胞Dectin-1、Toll样受体2及肿瘤坏死因子-α表达的影响

Effect of immunosuppressants on Zymosan A-induced expressions of Dectin-1, TLR2 and TNF-alpha in macrophages

摘要:

目的  研究霉酚酸酯和环孢素A对酵母聚糖（Zymosan A）诱导小鼠RAW264.7巨噬细胞模式识别受体Dectin-1、Toll样受体2（TLR2）表达及细胞因子肿瘤坏死因子-α（TNF-α）释放的影响。方法  体外培养RAW264.7巨噬细胞，分别给予不同浓度的霉酚酸酯、环孢素A预处理细胞24 h，再利用100μg/ml Zymosan A单独刺激细胞，逆转录聚合酶链反应和流式细胞术检测细胞Dectin-1、TLR2 mRNA和蛋白水平的变化，酶联免疫吸附试验检测上清液中TNF-α浓度的变化。结果  Zymosan A单独作用巨噬细胞Dectin-1、TLR2 mRNA和蛋白水平明显上调，TNF-α浓度升高（P <0.05）。Zymosan A作用于霉酚酸酯或环孢素A预处理24 h的巨噬细胞Dectin-1、TLR2 mRNA和蛋白水平较Zymosan A单独作用组明显下调，TNF-α分泌量减少（P <0.05）。结论  霉酚酸酯和环孢素A抑制巨噬细胞Dectin-1和TLR2的转录和翻译，并下调TNF-α的释放，降低机体对真菌病原体的清除能力，这可能是应用霉酚酸酯和环孢素A引起难控性真菌感染的机制之一。

Abstract:

Objective To study the effects of Mycophenolate mofetil and Cyclosporin A on Zymosan A-induced expressions of Dectin-1, Toll-like receptor 2 (TLR2) and tumor necrosis factor alpha (TNF-α) in RAW264.7 macrophages. Methods During in vitro culture, RAW264.7 macrophages were pre-treated with   different dosages of Mycophenolate mofetil and Cyclosporine A for 24 h, and then stimulated with 100 μg/ml of Zymosan A alone. RT-PCR was used to detect the expressions of Dectin-1 and TLR2 mRNAs in RAW264.7 macrophages and flow cytometry was used to detect the average fluorescence intensity of Dectin-1 and TLR2 proteins. TNF-α level was measured by enzyme-linked immunosorbent assay. Results The expressions of Dectin-1 and TLR2 mRNAs and proteins, as well as the concentration of TNF-α increased significantly in the Zymosan A+ macrophage group (P < 0.05). Mycophenolate mofetil and Cyclosporin A inhibited the expressions of Zymosan A-induced Dectin-1 and TLR2 mRNAs and proteins in various degrees and reduced the secretion of cytokine TNF-α (P < 0.05). Conclusions Mycophenolate mofetil and Cyclosporin A reduce the transcription and translation of Dectin-1 and TLR2 and the secretion of the inflammatory cytokine TNF-α induced by Zymosan A, aggravating infection by inhibiting the body's ability to recognize and remove the fungal pathogens. It is probably one of the key mechanisms that Mycophenolate mofetil and Cyclosporin A cause refractory fungal infection.