The Mechanism Of Interleukin-13Induced Mucus Hypersecretion And The Treatment Study In The Human Bronchial Epithelial Cells

Goblet cell hyperplasia and mucus hypersecretion in airwayepithelium are hallmarks of chronic inflammatory lung diseases, includingchronic obstructive pulmonary disease (COPD) and asthma.Overproduction of mucin in pathological conditions leads to exacerbationof these diseases by impairing mucociliary clearance, causing severeairway narrow. Mucus hypersecretion and obstruction in the airway lumencould be life threatening in chronic inflammatory lung disease patients.During the pahtogenesis of chronic inflammatory lung disease, humanneutrophil elastase (HNE), reactive oxygen species (ROS) and manycytokines may injure the airway and activate mucin gene regulation. Morethan20human mucin genes have been deposited in GenBank. Amongthese, mucin (MUC)5AC are major mucin in the human respiratory tractand are closely linked to goblet cell hyperplasia. Up-regulated MUC5AC expression is the central event in goblet cell metaplasia.Mucous cell hyperplasia can also be observed in the airways ofpatients with mild asthma. There appears to be an important link betweenoverproduction of mucus and the pathogenesis of asthma. In asthma, Thelper type2cytokines, such as IL-4, IL-9, and IL-13have been shown toplay pivotal roles in directly causing MUC5AC expression in airway cells.Among these cytokines interleukin (IL)-13is considered to be theprincipal stimulatory factors involved in the pathophysiology of asthmaand the most widely studied cause relevant to chronic inflammation in theairway.The imbalance of oxidant burden and antioxidant capacity has beenimplicated as an important contributing factor in the formation of mucusproduction. Exposure to IL-13results in the generation of ROS in theairway epithelial cells, which may damage cellular macromolecules suchas DNAs, lipids, and proteins, leading to oxidative stress-induced tissueinjury. Since oxidative stress is involved in the pathogenesis andexacerbation of chronic inflammation lung diseases, antioxidants shouldbe effective in decreasing mucin production and airway obstruction inasthma.Objective:This study is intended to investigate the effects of IL-13on mucussecretion and the associated intracellular signaling pathways. We also investigated the effects of AR inhibition on mucus secretion induced byIL-13and the related mechanism.Methods:The effects of IL-13on the expression of MUC5AC in the HBE16cellsThe HBE16cells were starved for24h in serum free medium. Afterstarvation, cells were divided into four groups:the control group,10nMIL-13group,25nM IL-13group,50nM IL-13group. The cell activity wasassessed by MTT method. The expression of MUC5AC mRNA wasanalyzed by RT-PCR. The expression of MUC5AC protein was detectedby ELISA.The mechanism of mucus hypersecretion induced IL-13in theHBE16cellsTo investigate the the potential signal transduction pathways involvedin the effect of IL-13on mucus secretion, The cells were stimulated withIL-13for24h after starved for24h to maintain a low basal level ofMUC5AC mucin production. A771726(10μmol/l) was used as aninhibitor against STAT6. The expression of STAT4, STAT6, p38andERK1/2mRNA was examined by RT-PCR.The phosphorylation ofSTAT6, p38and ERK1/2were examined by western blotting. Electrophoretic mobility shift assays (EMSA) were performed to examinethe DNA-binding activities of Forkhead box a2(FOXA2) and Activatorprotein-1(AP1). The expression of MARCKS and TMEM16A wereobserved by immunofluorescence and confocal laser technology.The Inhibition of Aldose Reductase on Mucus Production induced byInterleukin-13in the HBE16cellsThe HBE16cells were cultured with AR inhibitors (zopolrestat) orwere transfected with an AR small interfering (si) RNA. Subsequently, thecells were stimulated with10mM IL-13for2h. The levels of mucin(MUC)5AC mRNA and protein were measured by using RT-PCR orELISA. Intracellular ROS were measured fluorimetrically with theCM-H2DCFDA probe. Western blotting was performed to determine thelevels of AR, phosphorylated signal transducer and activator oftranscription6(p-STAT6) and phosphorylated janus kinase2(p-JAK2).Results:The effect of IL-13on mucus secreton in the HBE16cellsTo investigate the effects of IL-13on mucus secretion in the HBE16cells, we evaluated the MUC5AC mRNA and protein after the addition ofIL-13to the cells. We found that IL-13increased MUC5AC mRNA andprotein expression in the HBE16cells, when compared with the control group(P <0.05). IL-13depressed the cell activity in a dose-dependentmanner(P <0.05).The mechanism of mucus hypersecretion induced IL-13in theHBE16cellsRT-PCR and Western blotting analysis were performed to investigatethe signaling pathway in mucus hypersecretion induced by IL-13in theHBE16cells. The levels of STAT6was higher in IL-13group than thatin negative control group(P <0.05), whereas the expression of ERK1/2was not significantly affected. The expression of MUC5AC wasattenuated after treatment with A771726(a specific STAT6inhibitor).IL-13induced delayed phosphorylation of p3824-48h after stimulation(P<0.05). The generation of endogenous ROS induced by IL-13wassignificantly elevated within2h, and ROS production increased steadilyfor the following48h(P <0.05). There was a sharp decrease in FOXA2DNA-binding activity in the HBE16cells after stimulation with IL-13(P <0.05). Immunofluorescence demonstrated the expression of TMEM16Aand MARCKS increased after simulation with IL-13for24h(P <0.05).The Inhibition of Aldose Reductase on Mucus Productioninduced by Interleukin-13in the HBE16cellsThe results show that treatment with zopolrestat or transfection withAR-siRNA significantly suppressed IL-13-stimulated MUC5AC mRNA and protein in the HBE16cells (P <0.05). AR inhibition could suppressIL-13-induced ROS generation and, the phosphorylation of JAK2/STAT6pathway, thereby decreases mucus production in vitro (all P <0.05).Conclusions:1. IL-13can effectively stimulate the expression of MUC5AC mRNAand protein. IL-13induces MUC5AC expression in a dose-dependentmanner, while the cell activity decreases.2. The expression of STAT6mRNA and protein increased afterstimulation with IL-13, whereas the expression of ERK1/2was notsignificantly affected. There was a sharp decreasease in FOXA2DNA-binding activity in the HBE16cells after stimulation with IL-13. Itwas suggested that IL-13could up-regulates mucus secretion viaJAK-STAT6-FOXA2pathway in vitro.3. The p38MARPK pathway is involved in IL-13induced mucushypersecretion and MUC5AC expression in the HBE16cells. In addition,p38MAPK phosphorylation may require STAT6dependent proteinsynthesis induced by IL-13.4. It was demonstrated in the study that TMEM16A and MARCKSis responsible for mucus hypersecretion induced by IL-13in the HBE16cells, which gives a new insight into therapeutic development for mucushypersecretion in COPD and bronchial asthma. 5. Oxidative stress is implicated in the mechanism of mucushypersecretion induced by IL-13. IL-13induced oxidative stress could beameliorated by zopolrestat or AR siRNA.6. It was demonstrated in the study that AR plays an important role inthe formation of mucus production in chronic inflammation lung disease.Inhibition of AR could ameliorate the ROS-induced signaling pathwayand thereby decrease mucus production in vitro. Treatment withzopolrestat or transfection with AR siRNA could suppressIL-13-stimulated ROS generation and the phosphorylation ofJAK2/STAT6pathway, thereby decreases mucus production in theHBE16cells.