论文部分内容阅读
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 mucus hypersecretion of human airway epithelial cells induced by human neutrophil elastae (HNE). Methods Human bronchial epithelial cell line HBE16 was treated with 0, 25, 50, 75, 100, 400 and 400 μmol / L lycopene respectively. The cell viability was measured by MTT assay and the concentration of lycopene was determined. HBE16 cells were stimulated with HNE The model of mucus hypersecretion in the airway was treated with lycopene and AG1478. The experiment was divided into 5 groups: control group (serum-free medium), HNE group (HNE at a final concentration of 50 nm / L), HNE + tomato red HNE + AG1478 group (pretreated with 5μmol / L AG1478 for 30min, then treated with 50μmol / L HNE for 24 hours) HNE + lycopene + AG1478 group (with 100μmol / L lycopene for 24 h, then treated with 5μmol / L AGl478 for 30 min, the final concentration was 50 nM L / L HNE stimulation). The rats in each group were sampled 24 h after the treatment, and the transcriptional levels of mucin (MUC5ac) and epidermal growth factor receptor (EGFR) mRNA were detected by RT-PCR. Western blot The expressions of EGFR and phosphorylated EGFR (P-EGFR) in each group were detected. The expression of MUC5ac protein in each group was detected by ELISA. Results Lycopene concentration of less than 100μmol / L, no significant damage to the cells to 100μmol / L lycopene as the experimental concentration. Compared with the control group, mRNA transcription levels of MUC5ac and EGFR in HNE group were significantly increased. The expression of EGFR and P-EGFR protein in HNE, HNE + lycopene and HNE + AG1478 group were significantly increased (all P <0.01); Compared with HNE group, mRNA transcription levels of MUC5ac and EGFR in HNE + lycopene, HNE + AGl478 and HNE + lycopene + AG1478 group were significantly decreased (P all <0.01). The content of MUC5ac in HNE + lycopene, HNE + AG1478, HNE + lycopene + AG1478 group were significantly lower than those in control group (P <0.01) 0.01). Compared with HNE + AG1478 group, MUC5ac, EGFR mRNA and protein expression and P-EGFR protein expression in HNE + lycopene + AG1478 group were significantly decreased (all P <0.01). Conclusion Lycopene can inhibit mucus hypersecretion of inflammatory airway epithelial cells by downregulating EGFR level.