The Effects Of Heme Oxygenase-1 On Mucous Hypersecretion In The Airway

ObjectiveInvestigate the effects of heme oxygenase-1 on airway mucous hypersecretion under inflammation and its signal transduction mechanism, detect the level of MUC5AC, to further enrich and improve the theory adout the mechanism of mucus hypersecretion in the chronic airway inflammation, to provide a new theoretical basis for the treatment of airway mucus hypersecretion.This study is divided into four parts:1. Establish the experimental cell model of airway mucus hypersecretion.2. Study on the effects of heme oxygenase-1 on MUC5AC expression.3.Investigate the signal transduction mechanism that heme oxygenase-1 downregulates the level of MUC5AC.Methods1. Cultured human bronchial epithelial cell line HBE16 cells with different concentrations of human neutrophil elastae(HNE), detect the protein expression changes of mucin(MUC)5AC with enzyme-linked immunosorbent assay(ELISA), assess the cell activity with methyl thiazolyl tetrazolium method(MTT).2. MTT determine the effects of Hemin and ZnPPIX of different concentrations on HBE16 cells activity. The cells were divided into 4 groups: (1) control group: serum-free culture medium cultured HBE16 cells; (2) HNE group: adding HNE to HBE16 cells; (3) Hemin group: adding Hemin pretreat HBE16 cells for one hour, then join the HNE; (4) ZnPPIX group: adding ZnPPIX pretreat HBE16 cells for one hour, then join the HNE. Culture cells in each group for 24 hours, then extract the intracellular total protein in each group, detect the protein expression changes of MUC5AC with ELISA. Observe the protein morphological changes of MUC5AC by immunofluorescence and confocal laser technology.3. Experimental group: Same as Part II. Culture cells in each group for 24 hours, then extract the total ribonucleic acid (RNA) and intracellular protein. Determine the changes of HO-1 mRNA, Duox1 mRNA, EGFR mRNA and MUC5AC mRNA by the use of reverse transcriptase-polymerase chain reaction(RT-PCR). Detect the protein expression changes of EGFR, p-EGFR, Duox1 and HO-1 by Western blot. Results1.When concentrations of HNE were 0, 10, 25, 50 and 100 nmol / L, the level of MUC5AC in HBE16 cells were (106.71+8.39)μg/mg, (129.52+9.24)μg/mg, (157.50+9.15)μg/mg, (196.36+7.60)μg/mg and (235.84+5.33)μg/mg, and the cell survival rates in different concentrations were 100%, (92.9+3.1)%, (89.2+4.1)%, (78.6 +7.2)%, and (63.5 +5.0)%.2. When the concentrations of Hemin below 100μmol / L and the concentrations of ZnPPIX below 50μmol / L, the cells had no significant damage, the cell survival rate decreased significantly with the increasing concentration. The intracellular MUC5AC protein content in HNE group was (192.68 +4.71)μg / mg, which increased significantly (P<0.01) as compared to the control group (104.95 +6.82)μg / mg. After the cells were pretreated with Hemin, the level of MUC5AC (155.02 +9.87)μg / mg decreased significantly as compared to the HNE group(P<0.01). While in ZnPPIX group the intracellular MUC5AC protein content was (193.01 +3.70)μg / mg , which increased significantly (P<0.01) as compared to the control group. Immunocytochemistry confocal laser imaging of HBE16 cells showed that MUC5AC protein mainly located in the cytoplasm under normal circumstances. The level of MUC5AC increased significantly in HNE group as compared with the control group; while in Hemin group MUC5AC expression decreased significantly as compared with the HNE group; after the cells were pretreated with ZnPPIX, the level of MUC5AC was markedly enhanced as compared with the control group.3. The mRNA level of MUC5AC, EGFR, HO-1 and Duox1, the protein level of p-EGFR, EGFR, HO-1 and Duox1 in HNE group increased significantly (all P<0.01)than those in the control group.The HO-1 mRNA and protein increased significantly in Hemin group as compared with the HNE group(both P <0.01), while the mRNA level of Duox1, EGFR and MUC5AC, the protein level of p-EGFR, EGFR and Duox1 decreased significantly as compared with the HNE group(all P<0.01). After the cells were pretreated with ZnPPIX, the increase of HO-1 mRNA and protein were not significant as compared to the control group(P>0.05), while the Duox1, EGFR, and MUC5AC in mRNA levels and p-EGFR, EGFR, Duox1 protein increased significantly (all P<0.01).Conclusion1.The expression of MUC5AC protein in HBE16 cells in the HNE group increased significantly as compared with the control group, and HNE may enhance MUC5AC expression by a concentration-dependent manner. When the concentrations of HNE below 50nmol/L, the cells had no significant damage, the cell survival rate was significantly reduced with increasing concentration, so the follow-up experiment used this concentration to process.2. HO-1 inhibited the HNE-induced airway MUC5AC overexpression. Once the protective effect of HO-1 was canceled by its inhibitor ZnPPIX, the overexpression of MUC5AC presentated again.3.HNE can activated EGFR to start MUC5AC transcription by Duox1-ROS-TACE-TGF-αligand-dependent pathway.4. HNE induced MUC5AC overexpression as the same time it can induce HO-1 overexpression to play a certain amount of anti-oxidant effect. As enhanced this anti-oxidant effect by its inducer Hemin, the level of HO-1 was furtherly increased. Thereby HO-1 blocked Duox1-ROS-TACE-TGF-α-EGFR signal transduction pathway by downregulate the expression level of Duox1, inhibited the overexpression of MUC5AC.