House Dust Mite-induced Endoplasmic Reticulum Stress Mediates Mucus MUC5AC Hypersecretion Via TBK1 In Airway Epithelium

Objective: Bronchial asthma,referred to as asthma,is a chronic inflammatory disease characterized by reversible airway obstruction and airway hyperresponsiveness,which is primarily characterized by episodes of wheezing,cough and dyspnea due to chronic inflammation and airway hyperresponsiveness.Airway remodeling,airflow limitation and mucus hypersecretion are known symptoms of asthma pathology.Excessive mucus production leads to morbidity and mortality in many patients,especially those with more severe disease,and airway mucus plugging has been identified as a major cause of death in asthma patients.Therefore,inhibition of mucus hypersecretion plays a key role in the treatment of asthma.Endoplasmic reticulum(ER)stress is a major responder to organelle stress;loss of ER homeostasis triggers ER stress,and there is substantial evidence that ER stress is associated with the pathogenesis of a variety of diseases,such as neurodegenerative diseases,metabolic disorders,cardiovascular diseases,malignancies,and respiratory diseases.Endoplasmic reticulum(ER)stress is thought to be part of the regulation of mucus hypersecretion,which may promote gene expression by activating downstream factors through the TBK1 signaling pathway,a multifunctional serine/threonine protein kinase involved in multiple cellular signaling pathways and a central kinase in inflammatory IFN signaling and selective autophagy.In innate immune signaling,TBK1 acts as a receptor that mediates pathogen-detected integrator signaling leading to type I IFN-mediated T-cell responses.TBK1 is closely associated with inflammatory responses,and there is evidence that the TBK1 signaling pathway is involved in allergic inflammatory responses in the lung.However,the exact mechanism of whether ER stress promotes airway mucus secretion through the TBK1 pathway is not known.We therefore aimed to explore whether endoplasmic reticulum stress in house dust mite(HDM)-induced allergic asthma promotes MUC5 AC mucus hypersecretion through activation of the non-classical TBK1 signaling pathway.Methods: This experiment was divided into cellular and animal experimental parts.Firstly,the experiments were performed using HDM to construct asthma model mice,and the expression levels of P-TBK1,STAT6,P-NF-κB and MUC5 AC proteins were detected by immunoblotting,and the nuclear translocation of STAT6 and P-NF-κB were analyzed by immunofluorescence.Hematoxylin and eosin(HE)and periodate Schiff(PAS)staining were used to analyze airway inflammation and mucus secretion in mice;followed by the use of human bronchial epithelial BEAS-2B cells and transfection with si-NC,si-TBK1 lentiviral vectors to knock down the expression of TBK1 gene.According to the specific experiments were divided into normal BEAS-2B cells group,normal BEAS-2B cells + HDM group,lentiviral transfection si-NC group,si-TBK1 group,and si-NC+HDM,si-TBK1+HDM group,where HDM concentration was(200 μg/ml),and subsequent experiments were performed after treating the transfected cells for24 hours;then TBK1 phosphorylation was performed with TBK1 inhibitor Amlexanox(25 μM)or endoplasmic reticulum stress inhibitor 4-PBA(10 m M)was used to treat the cells.The experiments were divided into control group(PBS),experimental group(HDM),administration group alone(Amlexanox or4-PBA),and asthma treatment group(HDM+Amlexanox or HDM+4-PBA),and protein blotting was used to detect Bip,Chop,p-NF-κB,NF-κB,p-STAT6,STAT6,p-TBK1,and TBK1 expression levels,and immunofluorescence for nuclear translocation.Finally,24 healthy female C57BL/6J mice of SPF grade were selected and randomly divided into 4 groups of 6 mice each,which were labeled as control group(group A)(PBS),asthma group(group B)(HDM),drug administration alone group(group C)(4-PBA),and asthma administration group(group D)(HDM+Amlexanox or HDM+4-PBA).During sensitization of asthmatic mice,groups B and D immunized mice with a mixture of HDM(20μg)and aluminum hydroxide(1 mg)intraperitoneally on d 1,8 d,while groups A and C were given equal amounts of PBS buffer for intraperitoneal injection.Then,from d 15 to d 21,groups B and D were injected intranasally using HDM(20 μg)daily to induce airway allergic reactions,while mice in groups A and C were given equal amounts of PBS buffer for intranasal injection.amlexanox or4-PBA treatment groups were injected intraperitoneally(100 mg/kg).After successful modeling,the mice were cervically dissected and executed,and the desired tissue or cell samples were collected.The mouse lung tissue samples were stained with standard hematoxylin and eosin(HE)or periodic acid Schiff(PAS),and after successful staining,the pathological sections were placed under light microscope to observe and analyze the airway inflammation and mucus secretion in each group of mice and counted;Western blotting and immunofluorescence were used to analyze the cellular endoplasmic reticulum stress-related proteins Bip,Chop,P-TBK1,STAT6,P-NF-κB,MUC5 AC and their translocations were analyzed by western blotting and immunofluorescence.Results: Cellular assay WB and immunofluorescence results showed that the expression levels of MUC5 AC as well as P-TBK1,STAT6,P-NF-κB proteins and STAT6,P-NF-κB nuclear translocation were significantly increased in cells stimulated with HDM compared with the PBS control group(P<0.01);HDM group treated with si-TBK1 lentivirus transfection or inhibitor of TBK1 The expression of STAT6,P-NF-κB and MUC5 AC proteins decreased and STAT6,P-NF-κB nuclear translocation decreased after treatment in the HDM group compared with the HDM group(P<0.01);after treatment with4-PBA in the HDM group compared with the HDM group,the protein expression levels of Bip,Chop,MUC5 AC,P-TBK1,STAT6,P-NF-κB decreased and nuclear translocation of STAT6 and P-NF-κB decreased(P<0.01).Histopathological sections of the lungs of mice in animal experiments showed a large infiltration of inflammatory cells around the airways of mice in the HDM group compared with those in the PBS control group,increased airway cupped cell chemosis and increased airway mucus secretion(P<0.01);after treatment with 4-PBA,the airways of mice in the HDM+4-PBA group had reduced inflammatory cell infiltration,decreased airway cupped cell chemosis and mucus secretion compared with those in the HDM group(P<0.01).was reduced(P<0.01).WB and immunofluorescence results of mouse lung tissue showed that the expression levels of MUC5 AC as well as P-TBK1,STAT6,P-NF-κB protein and nuclear translocation of STAT6 and P-NF-κB were significantly increased in the HDM group compared with the PBS control group(P<0.01);after treatment with 4-PBA in the HDM group compared with the HDM group,Bip,Chop MUC5 AC,P-TBK1,STAT6,and P-NF-κB protein expression levels decreased and STAT6 and P-NF-κB nuclear translocation decreased after treatment with 4-PBA in the HDM group compared with the HDM group(P<0.01).The results suggest that mice exposed to HDM are characterized by ER stress and excessive secretion of mucus Muc5 ac in airway epithelial cells(p<0.001).Both in vivo and in vitro studies suggest that HDM-induced endoplasmic reticulum stress leads to MUC5 AC protein overexpression through TBK1 signaling.Conclusion : Our study shows that HDM stimulates BEAS-2B cells leading to increased Muc5 ac mucus secretion by a mechanism that is achieved through endoplasmic reticulum stress;TBK1 plays a key role in HDM-induced ER stress leading to MUC5 AC overproduction in the asthmatic airway epithelium through activation of the NF-κB/STAT6 signaling pathway;by inhibiting the TBK1 pathway or The use of ER stress inhibitor 4-PBA could alleviate airway inflammation and significantly downregulate the expression of NF-κB/STAT6 signaling pathway to some extent,significantly reducing the overproduction of MUC5 AC mucus.This suggests that our endoplasmic reticulum stress-induced TBK1-NF-κB/STAT6 signaling pathway may become a potential therapeutic target for patients with chronic allergic asthma and be used to develop new therapeutic agents.