CAJ | 학술논문

目的探讨红霉素对香烟烟雾刺激的人巨噬细胞组蛋白去乙酰化酶2(H DAC2)表达的影响及其机制研究.方法体外培养人类单核细胞系U937细胞,用佛波脂将其诱导分化为人巨噬细胞.按传统方法制备好香烟烟雾提取物(CSE)用于实验.将传代后的细胞分组:对照组、CSE组、红霉素+CSE组、HDAC的特异性抑制剂曲古霉素A(TSA)组.应用流式细胞仪检测人巨噬细胞活性氧(ROS)的含量;应用蛋白印迹实验(Western blot)检测HDAC2和核因子κB(NF-κB)蛋白表达;通过酶联免疫吸附试验检测细胞上清液中肿瘤坏死因子α(TNF-α)的浓度.结果 CSE可刺激人巨噬细胞释放ROS,具有浓度依赖性和时间依赖性;红霉素(1 mg/L)预孵育24 h可以增强1％ CSE抑制的人巨噬细胞HDAC2蛋白表达;红霉素(1 mg/L)预孵育24 h可以下调1％ CSE诱导的NF κB的活性;红霉素(1 mg/L)预孵育24 h可以抑制1％CSE诱导的TNF-α的合成和释放.结论红霉素通过提高HDAC2蛋白表达进而抑制香烟烟雾诱导氧化应激导致的NF-κB活性增强和炎症介质TNF-α的合成和释放.
목적 탐토홍매소대향연연무자격적인거서세포조단백거을선화매2(H DAC2)표체적영향급기궤제연구.방법 체외배양인류단핵세포계U937세포,용불파지장기유도분화위인거서세포.안전통방법제비호향연연무제취물(CSE)용우실험.장전대후적세포분조:대조조、CSE조、홍매소+CSE조、HDAC적특이성억제제곡고매소A(TSA)조.응용류식세포의검측인거서세포활성양(ROS)적함량;응용단백인적실험(Western blot)검측HDAC2화핵인자κB(NF-κB)단백표체;통과매련면역흡부시험검측세포상청액중종류배사인자α(TNF-α)적농도.결과 CSE가자격인거서세포석방ROS,구유농도의뢰성화시간의뢰성;홍매소(1 mg/L)예부육24 h가이증강1％ CSE억제적인거서세포HDAC2단백표체;홍매소(1 mg/L)예부육24 h가이하조1％ CSE유도적NF κB적활성;홍매소(1 mg/L)예부육24 h가이억제1％CSE유도적TNF-α적합성화석방.결론 홍매소통과제고HDAC2단백표체진이억제향연연무유도양화응격도치적NF-κB활성증강화염증개질TNF-α적합성화석방.
Objective To study the effect of erythromycin (EM) on cigarette smoke induced histone deacetylase-2 (HDAC2) protein expression in human macrophage cells in vitro.Methods The aqueous cigarette smoke extract (CSE) was always prepared fresh on the day of the experiment.The U937 monocytic cells were differentiated into macrophages by using phorbol 12 myristate 13-acetate according to standard procedures.The U937 differentiated cells were treated with either CSE (1％) or EM (1 mg/L),and HDAC inhibitor trichostatin A (100 μg/L) for 24 hours.Reactive oxygen species (ROS) was measured by flow cytometry.Western blotting was used for HDAC2 and nuclear factor-κB (NF-κB) protein assays.The level of tumor necrosis factor-α (TNF-α) in the supernatant was determined by enzyme linked immunosorbent assay.Results CSE (1％) increased ROS production in a dose-and time dependent manner.Preincubation with EM (1 mg/L) for 24 hours significantly inhibited CSE (1％)induced decrease of HDAC2 protein expression.Futhermore,Preincubation with EM inhibited the activation of NF κB and the release of TNF α in human macrophages.Conclusions EM is able to restore HDAC2 level decreased by oxidative stress and inhibit NF-κB activity resulting in decreasing TNF-α release,which has shown an important explanation that EM possesses the anti-inflammatory effect induced by cigarette smoke.