| Objective:To investigate the effects of Naringenin on airway mucous hypersecretion under inflammation and the relative molecular mechanism.Methods: (1) The airway epithelial cells of the line HBE16 were cultured, and then they were divided into 7 groups: 0, 20, 50, 80, 100, 200, 400μmol/L of naringenin treatment, respectively. The cell activity after Naringenin treatment was assessed by methylthiazolyl tetrazolium method, and the dose of naringenin was determined; (2) The cells were divided into 5 groups: a negative control group, HNE treatment groups were 5nmol/L, 25nmol/L, 50nmol/L respectively, and 100μmol/L naringenin group, the cells had been treatment for 24h, MUC5AC mRNA was examined by the use of reverse transcriptase-polymerase chain reaction (RT-PCR); The protein expression changes of MUC5AC were detected by enzyme-linked immunosorbent assay; The relative content of reactive oxygen species (ROS) were assayed by special kit; (3) The cells were divided into 5 groups: ①the control group;②an HNE treatment group (50nmol/L) ;③a naringenin treatment group (100μmol/L);④ROS scavenger DMTU treatment group (20μmol/L);⑤a Naringenin + DMTU group. The changes of MUC5AC mRNA and EGFR mRNA were examined by the use of reverse transcriptase-polymerase chain reaction (RT-PCR); The protein expression changes of EGFR, p-EGFR, PI3K, PKB, p-PKB and NF-κB were measured by Western blot; The protein expression changes of MUC5AC were detected by enzyme-linked immunosorbent assay; while the protein morphological changes of MUC5AC were observed by immunofluorescence and confocal laser technology.Results:(1) MTT results showed that naringenin concentrations of less than 100μmol/L when the cells had no significant damage, with increasing concentration, ability of cell adhesion reduced and death cells increased while concentration of naringenin were 200μmol/L and 400μmol/L, the survival rate of cell was decreased significantly, which indicated that the best role of the dose of naringenin was 100μmol/L. (2) HNE significantly increased MUC5AC gene expression and protein synthesis in dose-dependent manners, the content of ROS increased as also a dose-dependent manner, after pretreatment with Naringenin, the relative content of ROS decreased significantly compared with the 50nmol/L HNE group (P<0.01). (3) The expression levels of MUC5AC mRNA and its protein in the HNE group increased significantly as compared to those in the control group (P<0.01), The protein expression changes of P-EGFR, PI3K, PKB, P-PKB and NF-κB were increased significantly as compared with the normal control group, too. The difference was statistically significant (P<0.01); The expression levels of MUC5AC mRNA and EGFR mRNA were significantly decreased in the Naringenin group as compared with the HNE group, The protein expression changes of EGFR, p-EGFR, PI3K, PKB, p-PKB and NF-κB were significantly decreased, too (P<0.05); The above measurements were decreased more significantly in Naringenin + DMTU group, as compared with Naringenin pretreatment alone or DMTU pretreatment alone (P<0.05); Enzyme-linked immunosorbent assay results showed that MUC5AC protein expression reduced in the pretreatment naringenin group; We also observed that MUC5AC protein expression decreased in the cytoplasm of HBE16 in the naringenin group by laser confocal microscopy.Conclusions:(1) HNE induced MUC5AC gene expression and mucous hypersecretion in the airway epithelial cells HBE16,with a high concentration of ROS, while naringenin can reduce the level of ROS; (2) HNE by increasing ROS content, increase the levels of EGFR gene and protein expression, and also increase level of phosphor-EGFR, which caused to increase MUC5AC mucin generation. (3) Naringenin inhibited ROS generation, reduced the level of EGFR expression by decreasing relative content of ROS, and blocked EGFR phosphorylation partially, and involved in PI3K/PKB signaling pathway was blocked partially, therefore attenuated airway mucous hypersecretion under inflammation. |
