The Affect Of Kv1.3 Channel On The Airway Inflammation In Neutrophilic Asthma Through ERK1/2/NF-κB Signal Pathway

Objecti:Allergic asthma is a chronic inflammatory disease of the airways.Of the different lower airway-infiltrating immune cells participate in asthma,T lymphocytes play important roles in pathogenesis.The voltage-gated Kv1.3 potassium channel may be a target for preferential inhibition of thefunction of T lymphocytes.Here,we investigated the effects of PAP-1,a selective Kv1.3 channel blocker,for the treatment of asthma.The ordinary alum-mediated model is not useful for studying the function of neutrophils in asthma.We sensitized the mouse model with Ovalbumin(OVA)and lipopolysaccharide(LPS)to reflect the impact of the unique lung environment on developing immune responses which mainly induced by neutrophlils,related with Th17 function.This research aim to establish a Th17-mediated allergic asthma mouse model with increased neutrophils around airways,to detect the function of Kv1.3 channel in the neutrophlial asthma model and the mecchanism.Methods:1.Establish of the Th17-mediated allergic asthma mouse model with increased neutrophils1.1 Experimental group:24 female BALB/c mice according to different interference factors,randomly divided into 3groups,the control group(control);and neutrophilic asthma group(NA):Ovalbumin(OVA)and lipopolysaccharide(LPS)were applied for sensitization;eosinophilic asthma group(EA):Ovalbumin(OVA)and aluminum hydroxide were applied for sensitization.1.2 Animals noninvasive lung function examination to test airway resistance in mice1.3 the amount of total BALF cells and the number of eosinophil cells,neutrophil cells to test validation of asthma model1.4 HE staining and Masson staining evaluated the severity of airway inflommation2.The effect of Kv1.3 channel on the airway inflammation in neutrophilic asthma model2.1 Experimental group:32 female BALB/c mice according to different interference factors,randomly divided into 4groups,the control group(control);and neutrophilic asthma group(NA):Ovalbumin(OVA)and lipopolysaccharide(LPS)were applied for sensitization;PAP-1 group treated with 5-(4-phenoxybutoxy)psoralen 30min before aerosol inhalation;DEX group:treated with Dexamethasone 30min before aerosol inhalation2.2 Animals noninvasive lung function examination to test airway resistance in mice2.3 the amount of total BALF cells and the number of eosinophil cells,neutrophil cells to test validation of asthma model2.4 HE staining and Masson staining evaluated the severity of airway inflommation2.5 Test the Kv1.3 channel proein level in lung and the change of current density2.5.1 Detection of Kv1.3 channel in lung tissue by immunohistofluorescence2.5.2 Test the Kv1.3 channel proein level in lung by western blot2.6 Detection of IL-10、IL-17、IFN-γ、IL-4 level in both BALF and serum by enzyme-linked immuno sorbent assay3.The effort of Kv1.3 channel on Foxp3+Treg、Th17 cells in neutrophilic asthma model and the correlation with ERK1/2/NF-κB signal pathway3.1 Experimental group:24female BALB/c mice according to different interference factors,randomly divided into 4groups,the control group(control);and neutrophilic asthma group(NA):Ovalbumin(OVA)and lipopolysaccharide(LPS)were applied for sensitization;PAP-1 group treated with 5-(4-phenoxybutoxy)psoralen 30min before aerosol inhalation;3.2 Test the Erk1/2、p-Erk1/2、IκBα、p-IκBα、NF-κB level in lung by western blot3.3 Culture of splenic monocytes and group:(1)Control group:splenic monocytes of normal control(2)NA group:splenic monocytes of neutrophilic asthma model(3)PAP-1 group:splenic monocytes of neutrophilic asthma model PAP-1 group treated with PAP-1 30min before aerosol inhalation(4)PAP-1+PD98059 group:splenic monocytes of neutrophilic asthma model PAP-1 group treated with PAP-1 30min before aerosol inhalation.Cultured with PD98059(5)NA+PD98059 group:splenic monocytes of neutrophilic asthma model1Cultured with PD98059(6)NA+EGF group:splenic monocytes of neutrophilic asthma model1Cultured with Epidermal Growth Factor3.4 Detect the change of Kv1.3 channel current density by whole-cell patch clamp technique3.5 Detect the shift of the Foxp3~+Treg and Th17 cells Result:1.We successfully sensitized the mouse model with Ovalbumin(OVA)and lipopolysaccharide(LPS)to reflect the impact of the unique lung environment on developing immune responses which mainly induced by neutrophlils,related with Th17 function.2.Kv1.3 protein level increased in both NA and EA model.Kv1.3 channel Current Intensity increased in both NA and EA model.3.PAP-1 could decrease the airway hyperreactivity in both NA and EA model decreasd the IL-17 and IL-4 level.4.PAP-1could inhibit the activation of ERK1/2/NF-κB signal pathway.5.inhibit the activation of ERK1/2/NF-κB signal pathway could decrease Kv1.3channel current Intensity.6.There might be other signal pathway participated in the mechanism in which Kv1.3channel regulated theshift of the Foxp3+Treg and Th17 cells.conclusion:kv1.3 take part in the inflammation of the neutrophilic asthma induced by Ovalbumin(OVA)and lipopolysaccharide(LPS).ERK1/2/NF-κB signal pathway also take part in the inflammation of the neutrophilic asthma.The antagonist of kv1.3channel may regulate the inflammation partly through ERK1/2/NF-κB signal pathway.