摘要
目的 研究肺炎支原体(MP)脂质相关膜蛋白(LAMPs)刺激人支气管上皮细胞(HBEC)表达黏蛋白5AC(MUC5AC)的作用机制.方法 大量培养MP,提取LAMPs,采用Westerm blot检测LAMPs作用HBEC下MUC5AC蛋白水平,并用特异性siRNA分别沉默和过表达HBEC细胞白细胞介素17(IL-17),设立空白对照组、LAMPs组、LAMPs+si-IL17 组、IL-17 组、LAMPs+IL-17 组.Westernblot 分析 LAMPs 对细胞核转录因子 κB(NF-κB)相关信号分子(NF-κB p65、p-NF-κB p65、IKK-β)和MUC5AC蛋白表达的影响.用NF-κB抑制剂预处理HBEC细胞后,Western blot检测LAMPs刺激后MUC5AC的分泌水平.结果 与对照组相比,提取的LAMPs能刺激HBEC细胞表达MUC5AC蛋白(1.68±0.31)明显升高,差异有统计学意义(t=6.38,P＜0.05).与LAMPs组相比,LAMPs+si-IL17组NF-κB p65、p-NF-κB p65、IKK-β 和 MUC5AC 蛋白表达水平(分别为 1.12±0.37、1.67±0.27、7.53±0.31、1.26±0.17)均明显降低,差异有统计学意义(P均＜0.01).LAMPs+过表达IL-17后,NF-κB信号分子NF-κB p65、p-NF-κB p65、IKK-β和MUC5AC 蛋白表达水平(分别为 1.87±0.19、3.07±0.33、3.94±0.22、2.93±0.15)较对照组、IL-17 组、LAMPs 组均显著增加,差异均有统计学意义(F=6.72、12.84、21.19、11.31,P均＜0.01).而NF-κB抑制剂处理后,LAMPs诱导细胞MUC5AC表达(1.21±0.10)较前(1.59±0.12)显著下降,差异有统计学意义(t=2.51,P=0.012).结论 MP-LAMPs可经IL-17诱导NF-κB信号通路刺激HBEC细胞MUC5AC表达.
Abstract
Objective To investigate the mechanism of mucin 5AC(MUC5AC)expression stimulated by lipid-associated membrane protein(LAMPs)of Mycoplasma pneumoniae(MP)in human bronchial epithelial cells(HBEC).Methods MP was cultured in large quantities,LAMPs were extracted,and Western blot was used to detect the MUC5AC protein level of LAMPs induced HBEC.Specific siRNA was used to silence and overexpress interleukin 17(1L-17)in HBEC cells,respectively.Blank control group,LAMPs group,LAMPs+si-IL17 group,IL-17 group,and LAMPs+IL-17 group were set up.Western blot was used to analyze the effect of LAMPs on nuclear factor-κB(NF-κB)-related signaling molecules(NF-κB p65,p-NF-κB p65,IKK-β)and MUC5AC protein expression.After HBEC cells were pretreated with NF-κB inhibitors,Western blot was used to detect the secretion level of MUC5AC after LAMPs stimulation.Results Compared with the control group,LAMPs could stimulate a significant increase in the expression of MUC5AC protein(1.68±0.31)in HBEC cells,the difference was statistically significant(t=6.38,P＜0.05).Compared with the LAMPs group,the protein expression levels of NF-κB p65,p-NF-κB p65,IKK-β and MUC5AC in the LAMPs+si-IL17 group(respectively 1.12±0.37,1.67±0.27,7.53±0.31,1.26±0.17)were significantly reduced,the differences were statistically significant(all P＜0.01).After LAMPs+overexpression of IL-17,the protein expression levels of NF-κB signaling molecules NF-κB p65,p-NF-κB p65,IKK-β and MUC5AC(respectively 1.87±0.19,3.07±0.33,3.94±0.22,2.93±0.15)were significantly increased compared with the control group,IL-17 group,and LAMPs group,the differences were statistically significant(F=6.72,12.84,21.19,11.31;all P＜0.05).After treatment with NF-κB inhibitor,the expression of MUC5AC(1.21±0.10)in LAMPs-induced cells decreased significantly compared with before treatment(1.59±0.12),the difference was statistically significant(t=2.5 1,P=0.012).Conclusion MP-LAMPs stimulated the expression of MUC5AC in HBEC cells by inducing the NF-κB signaling pathway with IL-17.
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