Han Xiao, Zhou Dao-Bin, Xu Cai-Min, Yang Yang, Duan Ming-Hui, Wang Xuan, Zhang Jie-Ping, Zhao Yong-Qiang, Shen Ti, Wu Yong-Ji
Department of Hematology, Peking Union Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2011 Jun;19(3):738-43.
Erythropoietin (EPO) is the major means of treating anemia of chronic disease (ACD) through stimulating hematopoiesis, inhibiting hepcidin and decreasing proinflammatory factors. Recently, it has been found that monocytes are another source of hepcidin. EPO can reduce the hepcidin stimulated by IL-6 in monocytes, it is assumed that EPO can reduce hepcidin indirectly by reducing IL-6. However, the specific mechanism of EPO inhibiting the proinflammatory cytokines in monocytes is unclear now. This study was purposed to investigate the effect of EPO on monocyte proinflammatory factors and its molecular mechanism. IL-6 mRNA and TNF-α mRNA were detected by real time PCR, level of signaling molecule PARP-1 protein was detected by Western blot. THP-1 monocytes were stimulated by 1 µg/ml lipopolysaccharide (LPS) to observe the impact of EPO at different concentrations (0.5, 1, 2, 5, 10 U/ml) for different time (0, 3, 6, 12, 24 hours) on the expression of IL-6 mRNA, TNF-α mRNA and PARP-1 protein. 1 µg/ml or 5 µg/ml EPO receptor (EPOR) antibody and/or 3-aminobenzamide (3-AB, PARP-1 inhibitor) were added to observe the antagonistic effect on EPO and the impact on PARP-1. The results showed that LPS could stimulate the THP-1 cells. EPO could decrease the levels of IL-6 and TNF-α stimulated by LPS in a dose- and time-dependent manners. The most significant decrease in IL-6 mRNA expression was observed in 2 U/ml EPO for 6 hours. And down-regulation of TNF-α mRNA expression was pronounced at 10 U/ml EPO for 3 hours. IL-6 mRNA expression could be stimulated by LPS, PARP-1 protein was induced at the same time. EPO inhibited the expression of IL-6 mRNA, while PARP-1 protein also decreased. Down-regulation of IL-6 mRNA and PARP-1 protein level was pronounced at 2 U/ml EPO for 6 hours. 3AB is a direct inhibitor of PARP-1. Similar to 3AB, EPO receptor antibody could antagonize the decline of IL-6 induced by EPO. It is concluded that EPO can inhibit the expression of IL-6 and TNF-α in monocytes, and the inhibition of IL-6 expression may be associated with decrease of PARP level.
促红细胞生成素（EPO）是治疗慢性病贫血（ACD）的主要手段，它通过刺激造血、抑制铁调素并减少促炎因子来发挥作用。最近，人们发现单核细胞是铁调素的另一个来源。EPO可以降低单核细胞中由白细胞介素-6（IL-6）刺激产生的铁调素，据推测EPO可通过降低IL-6间接降低铁调素。然而，目前EPO抑制单核细胞中促炎细胞因子的具体机制尚不清楚。本研究旨在探讨EPO对单核细胞促炎因子的影响及其分子机制。通过实时定量聚合酶链反应（PCR）检测IL-6信使核糖核酸（mRNA）和肿瘤坏死因子-α（TNF-α）mRNA，采用蛋白质免疫印迹法检测信号分子聚（ADP-核糖）聚合酶-1（PARP-1）蛋白水平。用1微克/毫升脂多糖（LPS）刺激THP-1单核细胞，观察不同浓度（0.5、1、2、5、10单位/毫升）的EPO在不同时间（0、3、6、12、24小时）对IL-6 mRNA、TNF-α mRNA和PARP-1蛋白表达的影响。加入1微克/毫升或5微克/毫升的EPO受体（EPOR）抗体和/或3-氨基苯甲酰胺（3-AB，PARP-1抑制剂），观察其对EPO的拮抗作用以及对PARP-1的影响。结果显示，LPS可刺激THP-1细胞。EPO能以剂量和时间依赖性方式降低LPS刺激产生的IL-6和TNF-α水平。在2单位/毫升EPO作用6小时时，IL-6 mRNA表达下降最为显著。在10单位/毫升EPO作用3小时时，TNF-α mRNA表达下调明显。LPS可刺激IL-6 mRNA表达，同时诱导PARP-1蛋白产生。EPO抑制IL-6 mRNA表达，同时PARP-1蛋白也减少。在2单位/毫升EPO作用6小时时，IL-6 mRNA和PARP-1蛋白水平下调明显。3AB是PARP-1的直接抑制剂。与3AB类似，EPO受体抗体可拮抗EPO诱导的IL-6下降。结论是，EPO可抑制单核细胞中IL-6和TNF-α的表达，对IL-6表达的抑制可能与PARP水平降低有关。
