阿奇霉素通过基质金属蛋白酶9抑制气道黏液高分泌的实验研究．

Inhibition of airway mucous hypersecretion by azithromycin through matrix metailoproteinase 9

目的探讨阿奇霉素（AZT）通过基质金属蛋白酶9（MMP9）抑制气道黏液高分泌的作用机制。方法培养人支气管上皮细胞株HBE16细胞，以中性粒细胞弹性蛋白酶（NE）为刺激物。AZT和表皮生长因子受体（EGFR）拮抗剂BIBX1522为干扰因素，分别对NE刺激的HBE16细胞进行预处理。培养细胞分为对照组、NE组、AZT＋NE组和BIBX1522＋NE组。用RT-PCR检测各组细胞黏蛋白（MUC）5ACmRNA和MMP9mRNA；ELISA检测培养上清液中MUC5AC蛋白含量；明胶酶谱法测定培养细胞内MMP9活性；Western印迹检测细胞中MMP9、前酶原MMP9（pro—MMP9）和金属蛋白酶类组织抑制剂（TIMP-1）蛋白含量及磷酸化的EGFR（p-EGFR）和磷酸化的信号末端调节激酶（P-ERK）。结果与对照组相比，NE组中MUCSAC与MMP9的基因转录（0．83±0．17，0．79±0．16）和蛋白表达水平（0．84±0．15，0．88±0．16）明显高（均P〈0．01），并且MMP9的活性指标高（392．33±18．33，P〈0．01），而pro—MMP9蛋白含量低（0．17±0．10，P〈0．01），TIMP-1含量未见明显改变，P—EGFR（0．86±0．23）和P—ERK（0．85±0．22）蛋白水平明显高（均P〈0．01）；与NE组相比，AZT＋NE组MUC5AC与MMP9的基因转录（0．36±0．15，0．41±0．09）和蛋白表达水平（0．30±0．08，0．37±0．14）明显低（均P〈0．01），MMP9活性也较低（295．33±14．54，P〈0．01），pro—MMP9（0．46±0．14）和TIMP-1（0．67±0．17）蛋白含量高（均P〈0．05），p-EGFR的蛋白水平未见明显改变，而p-ERK蛋白水平低（0．40±0．19，P〈0．01）；与NE组相比，BIBX1522＋NE组中除MIJC5ACmRNA（0．37±0．14）、MMP9mRNA（0．37±0．13）、p-EGFR（0．36±0．13）和p-ERK（0．37±0．18）明显低外（均P〈0．01），其余指标无明显改变，差异无统计学意义。结论AZT能抑制气道上皮HBE16细胞内MMP9的活性及其产生，由此发挥抑制气道黏液高分泌的作用。

Objective To investigate the mechanism of azithromycin （AZT） inhibiting the airway mucous hypersecretion through matrix metalloproteinase 9 （MMPg）. Methods After culturing, the airway epithelial cells of line HBE16 were stimulated with neutrophil elastase （NE） and pretreated with AZT and epidermal growth factor receptor （EGFR） antagonist BIBX1522. Then the cells were divided into 4 groups： control group, NE-stimulated group, AZT-pretreated ＆ NE-stimulated group and BIBX1522-pretreated ＆ NE-stimulated group. The mucin （MUC）SAC mRNA and MMP9 mRNA levels were analyzed by RT-PCR （reverse transcription-polymerase chain reaction ）. And the MUCSAC protein content in supernatant was detected by ELISA （ enzyme-linked immunosorbent assay ）. Gelatin zymogrphy was employed to assay the MMP9 activity. Western blot was used to detect the protein levels of MMPg, prozymogen MMP9 （pro- MMPg）, tissue inhibitors of metalloproteinases 1 （ TIMP-1 ）, phosphorylated EGFR （p-EGFR） and phosphorylated external-signal regulated kinase （p-ERK）. Results As compared with the control group, the levels of MUC5AC and MMP9 gene transcription （0. 83 ±0. 17, 0.79 ± 0. 16 ） and protein expression （0. 84 ±0. 15, 0. 88 ±0. 16） in the NE stimulated group were obviously higher than those of the control group （ all P 〈 0. 01 ）. And the activity of MMP9 increased （392. 33 ± 18. 33, P 〈 0. 01 ） while the protein level of pro-MMP9 decreased （0. 17 ±0. 10, P 〈0. 01 ）. But the expression of TIMP-1 showed no significant change. The protein expressions of p-EGFR and p-ERK increased （0. 86 ± 0. 23, 0. 85 ± 0. 22, both P 〈 0. 01）; as compared with the NE-stimulated group, there was a reduction of MUC5AC and MMP9 gene transcription （0. 36 ± 0. 15, 0.41 ± 0. 09, both P 〈 0. 01 ） and protein expression levels （0. 30 ±0. 08, 0. 37 ±0. 14, both P 〈 0.01 ） in the AZT-pretreated and NE-stimulated group. The MMP9 activity decreased （295.33 ±14. 54, P 〈 0. 01 ）, the protein levels of pro-MMP9 and TIMP-1 increased （0. 46 ±0. 14, 0. 67 ± 0. 17, both P 〈 0. 05 ） , p-ERK protein level decreased （ 0. 40 ± 0. 19, P 〈 0. 01 ） while the expression of p-EGFR showed no significant decline. As compared with the NE-stimulated group, except for the expressions of MUC5AC mRNA （0. 37 ±0. 14）, MMP9 mRNA （0. 37 ± 0. 13）, p-EGFR （0. 36 ± 0. 13） and p-ERK （0. 37 ± 0. 18） decreasing （all P 〈 0. 01 ） in BIBX1522-pretreated and NE-stimulated group, the other results had no obvious change. Conclusion AZT may suppress the activity and production of MMP9 in HBE16 cells so as to inhibit the airway mucous hyperseeretion.