Objective: To investigate the mechanism of azithromycin (AZT) inhibiting the airway mucous hypersecretion through matrix metalloproteinase 9 (MMP9).
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)5AC mRNA and MMP9 mRNA levels were analyzed by RT-PCR (reverse transcription-polymerase chain reaction). And the MUC5AC 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 MMP9, prozymogen MMP9 (pro-MMP9), 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 hypersecretion.