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
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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 m RNA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 m RNA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 m RNA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。
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 m RNAs in RAW264.7 macrophages and flow cytometry was used to detect the average fluorescence intensity of Dectin-1and TLR2 proteins. TNF-α level was measured by enzyme-linked immunosorbent assay. Results The expressions of Dectin-1 and TLR2 m RNAs 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 m RNAs 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 andCyclosporin A cause refractory fungal infection.
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 m RNAs in RAW264.7 macrophages and flow cytometry was used to detect the average fluorescence intensity of Dectin-1and TLR2 proteins. TNF-α level was measured by enzyme-linked immunosorbent assay. Results The expressions of Dectin-1 and TLR2 m RNAs 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 m RNAs 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 andCyclosporin A cause refractory fungal infection.
引文
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[2]PINKE K H,LIMA H G,CUNHA F Q,et al.Mast cells phagocyte Candida albicans and produce nitric oxide by mechanisms involving TLR2 and Dectin-1[J].Immunobiology,2015,221(2):220-227.
[3]YANG Z,MARSHALL J S.Zymosan treatment of mouse mast cells enhances dectin-1 expression and induces dectin-1-dependent reactive oxygen species(ROS)generation[J].Immunobiology,2009,214(4):321-330.
[4]VINCENT J L,RELLO J,MARSHALL J,et al.EPICⅡGroup of Investigators.International study of the prevalence and outcomes of infection in intensive care units[J].Jama the Journal of the American Medical Association,2009,302(21):2323-2329.
[5]LEMOINE S,JARON B,TABKA S,et al.Dectin-1 activation unlocks IL-12A expression and reveals the TH1 potency of neonatal dendritic cells[J].Journal of Allergy Clinical Immunology,2015,136(5):1355-1368.
[6]ROSENTUL D C,PLANTINGA T S,MARIJE O,et al.Genetic variation in the dectin-1/CARD9 recognition pathway and susceptibility to candidemia[J].Journal of Infectious Diseases,2011,204(7):1138-1145.
[7]GERSUK G M,UNDERHILL D M,LIQUN Z,et al.Dectin-1and TLRs permit macrophages to distinguish between different Aspergillus fumigatus cellular states[J].Journal of Immunology,2006,176(6):3717-3724.
[8]DILLON S,AGRAWAL S,BANERJEE K,et al.Yeast zymosan,a stimulus for TLR2 and dectin-1,induces regulatory antigen-presenting cells and immunological tolerance[J].Journal of Clinical Investigation,2006,116(4):916-928.
[9]GREENBLATT M B,ANTONIOS A,BELLA H,et al.Calcineurin regulates innate antifungal immunity in neutrophils[J].Journal of Experimental Medicine,2010,207(5):923-931.
[10]洪英礼,金英顺,崔镇花,等.TLR活化参与环孢素A诱发的慢性肾小管间质损伤[J].中国病理生理杂志,2011,27(3):555-559.
[11]潘春树,何雪菲,钱华.常用免疫抑制剂的作用机制及应用[J].安徽医药,2008,12(10):892-894.