蕃茄红素对气道上皮细胞黏液高分泌的作用

Roles of lycopene in inflammatory airway mucus hypersecretion

目的探讨蕃茄红素(lycopene)对人中性粒细胞弹性蛋白酶(human neutrophil elastae,HNE)诱导的气道上皮细胞黏液高分泌的影响。方法分别用0、25、50、75、100、200、400μmol/L的番茄红素处理人支气管上皮细胞株HBE16,采用MTT法检测细胞活性,确定番茄红素试验浓度;用HNE刺激HBE16细胞构建气道黏液高分泌模型,用番茄红素及AG1478进行干预,实验共分5组:对照组(无血清培养基培养)、HNE组(加入终浓度为50 nm/L的HNE)、HNE+蕃茄红素组(加入终浓度为100μmol/L的蕃茄红素,作用24 h后,给予终浓度为50 nm/L的HNE刺激)、HNE+AG1478组(加入5μmol/L AG1478预处理30 min后,给予终浓度为50 nmol/L的HNE刺激)、HNE+蕃茄红素+AG1478组(先加入100μmol/L的蕃茄红素作用24 h,再给予5μmol/L的AGl478处理30 min后,给予终浓度为50 nmoL/L的HNE刺激)。各组均于处理后24 h取样,采用RT-PCR法检测各组黏蛋白5ac(mucin,MUC5ac)基因、表皮生长因子受体(epidermal growth factor receptor,EGFR)基因mRNA的转录水平;Western blot法检测各组EGFR和磷酸化表皮生长因子受体(phosphorylated EGFR,P-EGFR)蛋白的表达;ELISA法检测各组MUC5ac蛋白的表达。结果番茄红素浓度低于100μmol/L时对细胞无明显损伤,以100μmol/L番茄红素作为实验浓度。与对照组比较,HNE组MUC5ac和EGFR基因mRNA转录水平均明显升高,HNE、HNE+番茄红素、HNE+AG1478组EGFR和P-EGFR蛋白的表达均明显升高(P均<0.01);与HNE组比较,HNE+番茄红素、HNE+AGl478和HNE+番茄红素+AG1478组MUC5ac和EGFR基因mRNA转录水平均明显降低,EGFR和P-EGFR蛋白的表达水平均明显降低(P均<0.01)。与对照组比较,HNE组MUC5ac蛋白含量明显升高(P<0.01);与HNE组比较,HNE+番茄红素、HNE+AG1478和HNE+番茄红素+AG1478组MUC5ac蛋白含量均明显降低(P均<0.01)。与HNE+AG1478组比较,HNE+番茄红素+AG1478组中MUC5ac、EGFR mRNA及蛋白表达水平、P-EGFR蛋白表达水平均明显降低(P均<0.01)。结论蕃茄红素通过下调EGFR水平对炎性气道上皮细胞黏液高分泌有抑制作用。

Objective To investigate the effect of lycopene on airway mucus hypersecretion induced by human neutrophil elastae（HNE）. Methods Human bronchial epithelial HBE16 cells were stimulated with 0,25,50,75,100,200 and 400 μmol / L lycopene,and determined for activity by MTT method,based on which the lycopene concentration was optimized. The model of airway mucus hypersecretion was established by stimulation with HNE,and treated with lycopene and AG1478. HBE16 cells were divided into five groups. The cells in control group were cultured in serum-free medium, while those in HNE group were treated with HNE at a final concentration of 50 nmol / L,those in HNE ＋ lycopene group were treated with lycopene at a final concentration of 100 μmol / L for 24 h then stimulated with HNE at a final concentration of 50 nmol / L,those in HNE ＋ AG1478 group were pre-treated with 5 μmol / L AG1478 for 30 min then stimulated with 50 nmol / L HNE at a final concentration of 50 nmol / L,and those in HNE ＋ lycopene ＋ AG1478 group were treated with 100 μmol / L lycopene for 24 h,5 μmol / L AG1478 for 30 min,than stimulated with HNE at a final concentration of 50 nmol / L. Samples were taken from various groups 24 h later,and determined for the transcription levels of mucin（MUC5ac）,epidermal growth factor receptor（EGFR）mRNAs by RT-PCR,and for expression of EGFR and phosphorylated EGFR（P-EGFR）by Western blot. The expressions of MUC5ac in various groups were determined by ELISA. Results The lycopene at concentrations of less than 100 μmol / L showed no significant damage to cells,so the lycopene concentration for test was served as 100 μmol / L. As compared with those in control group,the transcription levels of MUC5ac and EGFR mRNAs in HNE group as well as expression levels of EGFR and P-EGFR in HNE,HNE ＋ lycopene and HNE ＋ AG1478 groups increased significantly（each P〈0. 01）. However,as compared with those in HNE group,the transcription levels of MUC5ac and EGFR mRNAs,as well as expression levels of EGFR and P-GEFR in HNE ＋ lycopene,HNE ＋ AG1478 and HNE ＋ lycopene ＋ AG1478 groups decreased significantly（each P〈0. 01）. The MUC5ac protein content was significantly higher in HNE group than in control group（P〈0. 01）,and in HNE ＋ lycopene, HNE ＋ AG1478 and HNE ＋ lycopene ＋ AG1478 groups than in HNE group（each P〈0. 01）. However,as compared with those in HNE ＋ AG1478 group,the expression levels of MUC5ac and EGFR mRNAs and proteins as well as P-EGFR protein in HNE ＋ lycopene ＋ AG1478 group decreased significantly（each P〈0. 01）. Conclusion Lycopene inhibited the inflammatory airway mucus hypersecretion by down-regulating eGFR level.