Peng Anping, Huang Xianzhang, Liu Ruiping, Wang Xiaoyun, Zhuang Junhua
Department of Laboratory Science, Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510120, China.
Department of Gynecology, Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510120, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Jun;30(6):635-8.
To explore the anti-inflammatory effect of triptolide (TPT) by regulating miR-155 in monocytes pre-stimulated by lipopolysaccharide (LPS) from rheumatoid arthritis (RA) patients.
Monocytes were isolated by CD14⁺ magnetic beads from peripheral blood mononuclear cells (PBMCs) of RA and stimulated by LPS for 24 hours. The levels of tumor-necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in monocytes were detected by ELISA and the expression of miR-155 was measured by real-time quantitative PCR (qRT-PCR) in monocytes before and after the treatment of TPT at different concentrations. MiR-155 mimic and negative control were respectively transfected into the LPS-stimulated monocytes by Lipofectamine(TM)2000. Twenty-four hours later, the monocytes were treated with or without TPT for another 24 hours. TNF-α and IL-6 expressions in the cell culture supernatants were detected by ELISA and the expressions of suppressor of cytokine signaling-1 (SOCS1) and Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1) were tested by Western blotting.
TPT suppressed the expressions of TNF-α, IL-6 and miR-155 in LPS-stimulated peripheral blood monocytes from RA patients. Over-expression of miR-155 significantly reversed the down-regulation of TNF-α and IL-6 by TPT in monocytes. TPT up-regulated the expressions of SOCS1 and SHIP-1 in monocytes, but over-expressed miR-155 antagonized the effect of TPT on SHIP-1 while the expression of SOCS1 was not affected.
TPT suppressed the expression of miR-155 and up-regulated the release of SHIP-1, thus inhibiting the inflammatory response in the LPS-stimulated monocytes of RA patients.
通过调节类风湿关节炎（RA）患者脂多糖（LPS）预刺激的单核细胞中的miR-155，探讨雷公藤甲素（TPT）的抗炎作用。
采用CD14⁺磁珠从RA患者外周血单个核细胞（PBMCs）中分离单核细胞，并用LPS刺激24小时。采用酶联免疫吸附测定（ELISA）检测单核细胞中肿瘤坏死因子-α（TNF-α）和白细胞介素6（IL-6）水平，采用实时定量聚合酶链反应（qRT-PCR）检测不同浓度TPT处理前后单核细胞中miR-155的表达。用Lipofectamine™2000分别将miR-155模拟物和阴性对照转染至LPS刺激的单核细胞中。24小时后，单核细胞再分别用或不用TPT处理24小时。采用ELISA检测细胞培养上清液中TNF-α和IL-6的表达，采用蛋白质免疫印迹法检测细胞因子信号转导抑制因子1（SOCS1）和含Src同源2结构域的肌醇5-磷酸酶1（SHIP-1）的表达。
TPT抑制RA患者LPS刺激的外周血单核细胞中TNF-α、IL-6和miR-155的表达。miR-155过表达显著逆转了TPT对单核细胞中TNF-α和IL-6的下调作用。TPT上调单核细胞中SOCS1和SHIP-1的表达，但miR-155过表达拮抗了TPT对SHIP-1的作用，而SOCS1的表达未受影响。
TPT抑制miR-155的表达并上调SHIP-1的释放，从而抑制RA患者LPS刺激的单核细胞中的炎症反应。
