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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 investigate the effect and possible mechanism of erythromycin on expression of interleukin 8 (interleukin 8) and antioxidant glutathione (GSH) in bronchial epithelial cells induced by hydrogen peroxide (H2O2). Methods Human bronchial epithelial cells of 16HBE strain were cultured and the effects of hydrogen peroxide and erythromycin on the cell growth were observed by MTT assay. The passaged cells were divided into 24, 36, 48h3 time periods, and each group was further divided into control group, H2O2 group, erythromycin + H2O2 group. The concentration of IL8 in the supernatant of the cells was detected by enzyme-linked immunosorbent assay. The activity of nuclear factor-kappa B (NFκB) and activator of transcription 1 (AP1) was detected by gel permeation-shift assay (EMSA) The content of glutathione synthetase (γGCS) and GSH in the cells were detected by detector. The expression of γGCSHS was detected by Western blotting. Results Erythromycin (5μg / ml) pretreated for 36,48h inhibited the level of IL8 in HBE supernatant induced by H2O2 (0.01mmol / L). Erythromycin (5μg / ml) H2O2 (0.01mmol / L) induced bronchial epithelial cells transcription factor NFkB, AP1; Pretreatment with erythromycin (5μg / ml) 48h inhibited H2O2 (0.01mmol / L) induced GSH and γGCS levels in HBE cells (3.46 ± 0.41) and γGCSHS protein expression, γGCSHS promoter region AP1 transcriptional activity. Conclusion Erythromycin can inhibit the release of IL8 induced by H2O2 by down-regulating the activity of NFκB and AP1, thereby affecting the synthesis and regulation of GSH and γGCS.