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红细胞生成素(erythropoietin,EPO)是治疗慢性病贫血(anemia of chronic disease,ACD)的主要药物,通过刺激造血、抑制hepcidin和前炎症因子而改善贫血。近来发现单核细胞是hepcidin的另一来源,EPO能降低IL-6诱导的hepcidin,推测EPO可能通过降低IL-6进而抑制hepcidin的间接途径。然而,EPO降低单核细胞IL-6相应的分子生物学机制还不清楚。本研究探讨EPO对单核细胞前炎症因子的作用及其分子学机制。采用实时定量PCR检测IL-6 mRNA和TNF-αmRNA表达,Western blot方法检测PARP-1信号分子的蛋白水平。采用1μg/ml脂多糖(lipopolysaccharide,LPS)刺激THP-1单核细胞,观察EPO不同浓度(0.5,1,2,5,10 U/ml)和作用时间(0,3,6,12,24小时)对THP-1单核细胞IL-6 mRNA、TNF-αmRNA以及PARP-1蛋白的抑制作用。加用1μg/ml或5μg/mlEPO受体(EPOR)抗体和/或3-氨基苯甲酰胺(3AB,PARP-1抑制剂),观察其对EPO的拮抗作用以及对PARP-1信号分子的影响。结果表明,1μg/ml LPS可以明显促进THP-1单核细胞IL-6和TNF-αmRNA表达。EPO可抑制LPS诱导的IL-6和TNF-αmRNA表达,且呈浓度和时间依赖性:对于IL-6 mRNA,2 U/ml EPO作用6小时的抑制作用最明显;对于TNF-αmRNA,10 U/ml EPO作用3小时的抑制作用最明显。研究发现,LPS诱导IL-6 mRNA表达升高的同时PARP-1蛋白水平也明显增加,EPO抑制IL-6 mRNA表达的同时PARP-1蛋白也下降,且2 U/ml EPO作用6小时对IL-6和PARP-1蛋白均有明显抑制。3AB是PARP-1的直接抑制剂,EPO受体的抗体与3AB相似,可拮抗EPO对IL-6的抑制作用。结论 :EPO能够抑制单核细胞IL-6和TNF-α表达,EPO可能通过降低PARP-1水平抑制IL-6的表达。
Erythropoietin (EPO), the primary drug used to treat anemia of chronic disease (ACD), ameliorates anemia by stimulating hematopoiesis, inhibiting hepcidin and proinflammatory cytokines. Recently found that monocytes are another source of hepcidin, EPO can reduce IL-6-induced hepcidin speculated that EPO may be through the lower IL-6 and thus inhibit hepcidin indirect pathway. However, the molecular mechanism of EPO that reduces monocyte IL-6 is unclear. This study was to investigate the effect of EPO on proinflammatory cytokines and its molecular mechanisms. The expression of IL-6 mRNA and TNF-αmRNA was detected by real-time quantitative PCR, and the protein level of PARP-1 was detected by Western blot. THP-1 monocytes were stimulated with 1μg / ml lipopolysaccharide (LPS) to observe the effects of different concentrations of EPO (0.5,1,2,5,10 U / ml) and action time (0,3,6,12,24 H) on the THP-1 monocytes IL-6 mRNA, TNF-α mRNA and PARP-1 protein inhibition. The antagonism of EPO and its effect on PARP-1 signaling molecules were observed by adding 1 μg / ml or 5 μg / ml EPO receptor antibody and / or 3-aminobenzamide (3AB, PARP-1 inhibitor) . The results showed that 1μg / ml LPS can significantly promote THP-1 monocytes IL-6 and TNF-αmRNA expression. EPO inhibited LPS-induced IL-6 and TNF-αmRNA expression in a concentration- and time-dependent manner: IL-6 mRNA had the most obvious inhibitory effect at 2 U / ml EPO for 6 hours; for TNF-αmRNA, 10 U / ml EPO inhibition of 3 hours the most obvious. The study found that LPS induced IL-6 mRNA expression increased while PARP-1 protein levels were significantly increased, EPO inhibition of IL-6 mRNA expression while PARP-1 protein also decreased, and 2 U / ml EPO effect 6 hours IL -6 and PARP-1 protein were significantly inhibited. 3AB is a direct inhibitor of PARP-1, EPO receptor antibodies similar to 3AB, EPO can antagonize the inhibition of IL-6. CONCLUSION: EPO can inhibit the expression of IL-6 and TNF-α in monocytes. EPO may inhibit the expression of IL-6 by decreasing the level of PARP-1.