摘要
目的探讨红霉素对过氧化氢(H2O2)诱导的支气管上皮细胞表达炎症介质白细胞介素8(IL8)及抗氧化剂谷胱甘肽(GSH)的影响和可能的作用机制。方法培养16HBE株的人支气管上皮细胞,应用四甲基偶氮唑蓝(MTT)法观察过氧化氢及红霉素对细胞生长的影响。将传代后的细胞按24、36、48h3个时间段分组,每个组再分为对照组、H2O2组、红霉素+H2O2组。通过酶联免疫吸附实验检测细胞上清中IL8的浓度;通过凝胶迁移率阻滞实验(EMSA)来检测转录因子核因子κB(NFκB)和激活蛋白1(AP1)的活性;通过酶联免疫检测仪检测细胞内GSH、谷氨酰半胱氨酸合成酶(γGCS)的含量;应用Western印迹检测谷氨酰半胱氨酸合成酶重链亚单位(γGCSHS)蛋白表达。结果红霉素(5μg/ml)预孵育36、48h后可以抑制H2O2(0.01mmol/L)诱导的HBE上清中IL8的含量;红霉素(5μg/ml)预孵育36、48h后可以下调H2O2(0.01mmol/L)诱导的支气管上皮细胞转录因子NFkB、AP1的活性;红霉素(5μg/ml)预孵育48h抑制H2O2(0.01mmol/L)诱导的HBE细胞GSH和γGCS的含量(3.46±0.41)以及γGCSHS蛋白表达、γGCSHS启动子区域AP1转录活性。结论红霉素通过下调NFκB、AP1的活性而抑制H2O2诱导的炎症介质IL8的释放,进而影响GSH及γGCS的合成调控。
Objective To study the effects of erythromycin on Hydrogen peroxide(H_2O_2)-induced interleukin-8 synthesis and regulation of glutathione in human bronchial epithelial cells.Methods Human bronchial epithelial (16-HBE) growth curve was recorded by MTT, cells were divided into three groups: (1)control(incubation for 24,36,48 h),(2)H_2O_2 pre-incubation for 24,36,48 h before adding H_2O_2 ,(3)H_2O_2+EM(Pre-incubation EM for 24,36,48 h before adding H_2O_2). IL-8 levels were measured in culture supernatants by ELISA, activation of transcription factor NF-κB and AP-1 in HBE was evaluated by Electrophoretic mobility shift assay(EMSA). Intracellular GSH and Gamma-GCS concentrations were measured by spectrophotometric assay,γ-GCS-HS protein were determined by Western blot.Results Erythromycin(1,10 μg/ml)and H_2O_2(0.01,0.1 mmol/L)have no effects on cell growth, Preincubation with EM(5 μg/ml ) for 36 h and 48 h significantly inhibit H_2O_2(0.01 mmol/L)induced increase of IL-8 levels in HBE supernatants, in the mean time decrease the expression of NF-κB and AP-1.Preincubation with EM(5 μg/ml ) for 48 h significantly inhibit H_2O_2(0.01 mmol/L)induced increase of γ-GCS levels,γ-GCS-HS protein expression and AP-1 binding of γ-GCS-HS promoter in HBE. However GSH and γ-GCS-HS protein expression in EM+H_2O_2 group significantly higher than those in control group.Conclusion Erythromycin inhibits oxidant-mediated IL-8 levels through down-regulation of NF-κB and AP-1 binding in HBE ,which can further influence the synthesis of GSH and expression γ-GCS in HBE.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第14期976-980,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目(30170401)
广东省自然科学基金资助项目(000321)
