Azithromycin Inhibits Airway Mucous Hypersecretion Through Matrix Metalloproteinase 9

Objective To investigate the mechanism of azithromycin (AZT) inhibits airway mucous hypersecretion through matrix metalloproteinase 9 (MMP9).Methods (1) The airway epithelial cells of line HBE16 were cultured and stimulated with neutrophil elastase (NE), pretreated the cells with AZT and epidermal growth factor receptor (EGFR) antagonist BIBX1522. The cells were divided into four groups: control group, NE stimulated group, AZT pretreated and NE stimulated group, BIBX1522 pretreated and NE stimulated group. Using Western blot to detect MMP9,tissue inhibitors of metalloproteinases 1 (TIMP-1) and prozymogen MMP9 (pro-MMP9) proteins; gelatin zymography assayed MMP9 activity in cultured cells. (2) MMP9 mRNA level were analyzed by RT-PCR; Western blot detected the protein levels of phosphorylated EGFR (p-EGFR) and phosphorylated external-signal regulated kinase (p-ERK) in cells. (3) Mucin(MUC)5AC protein level in supernate of HBE16 cells was detected by ELISA; RT-PCR analyzed MUC5AC mRNA level. Results (1) Compared to the control group, MUC5AC protein expression in the NE stimulated group was obviously higher than control group (P<0.01), and the activity of MMP9 increased (P<0.01), the protein level of pro-MMP9 reduced (P<0.01), but the expression of TIMP-1 didn't significantly change; Compared to NE stimulated group, in AZT pretreated and NE stimulated group MMP9 protein expression and MMP9 activity decreased (all P<0.01), the protein levels of pro-MMP9 and TIMP-1 advanced (both P<0.05); Compared to NE stimulated group, in BIBX1522 pretreated and NE stimulated group, results of MUC5AC protein expression,MMP9 activity,pro-MMP9 and TIMP-1 didn't altered obviously. (2) Compared with control group, MMP9 gene transcription in the NE stimulated group were obviously higher (P<0.01), the expressions of p-EGFR and p-ERK increased (both P<0.01); Compared to NE stimulated group, in AZT pretreated and NE stimulated group MMP9 mRNA decreased (P<0.01), p-ERK protein level reduced (P<0.01) but the expression of p-EGFR didn't reduce significantly; Compared to the NE stimulated group, in BIBX1522 pretreated and NE stimulated group, the expressions of MMP9 mRNA,p-EGFR and p-ERK didn't alter obviously. (3) Compared to the control group, MUC5AC gene transcriptionin and protein expression in NE stimulated group increased (P<0.01); Compared with NE stimulated group, in AZT pretreated and NE stimulated group MUC5AC gene transcription and protein expression levels decreased (all P<0.01); Compared to NE stimulated group, in BIBX1522 pretreated and NE stimulated group, the expressions of MUC5AC mRNA decreased (P<0.01), MUC5AC protein expression t didn't change obviously.Conclusions (1) AZT can inhibit the activation of pro-MMP9 in airway epithelial cells that stimulated by NE, and increase the expression of MMP9 inhibitor TIMP-1, thereby inhibiting the activity of MMP9. (2) AZT can suppress EGFR/ERK1/2 signaling pathway, inhibit the generation of additional MMP9. (3) AZT can inhibit airway mucus hypersecretion.