LL-37 Restored Glucocorticoid Sensitivity Impaired By Virus DsRNA In Lung

Background:Glucocorticoids play an important role in the treatment of chronic lung diseases such as chronic airway disease and interstitial lung diseases.They are the cornerstones in the treatment of chronic airway diseases such as bronchial asthma.Respiratory RNA virus infections including respiratory syncytial virus(HSV)and human rhinovirus(HRV)are the main causes of acute exacerbation or onset of chronic lung disease.What's more,it can cause insensitivity to glucocorticoid treatment,which called glucocorticoid resistance.It is reported that about 5%to 25%of refractory asthma,most chronic obstructive pulmonary disease,and most interstitial lung disease are not sensitive to glucocorticoids.Glucocorticoid resistance caused by RNA virus infection causes a huge medical burden.The antibacterial peptide LL-37 is the only member of the human cathelicidin family.It has a wide range of antimicrobial activities,including antiviral activity.Previous study of our group showed that LL-37 can promote anti-inflammatory effect,but it is unclear whether LL-37 can alleviate glucocorticoid resistance caused by RNA virus and its mechanism.Objective:In this study,we focus on whether LL-37 relieves dsRNA-induced glucocorticoid resistance and its possible mechanism through in vitro and in vivo experiments.Methods:In vitro:In this part,type ?(BEAS-2B)and type ?(A549)lung epithelial cell lines were used to evaluate the effect of LL-37 on dsRNA-induced glucocorticoid resistance and explore its possible mechanism.The study used Poly I:C to mimic RNA virus infection.1.Immunofluorescence and Western Blot were used to verify whether exogenous LL-37 can enter lung epithelial cells.2.Immunofluorescence co-localization and gel electrophoresis were used to verify that LL-37 can bind to dsRNA.3.Using Western Blot,it is clear that LL-37 can inhibit the expression of glucocorticoid-induced anti-inflammatory protein PLZF and Glucocorticoid Receptor(GR)phosphorylation.4.Western Blot and nuclear separation extraction were used to show the influence of LL-37 on GR transfer from cytoplasm to nucleus.5.Use luciferase reporter gene detection method to study the effect of LL-37 on the activation of glucocorticoid responsive elements(GRE)induced by glucocorticoids.6.Detect the phosphorylation of Erk and Akt pathways by Western Blot and evaluate the effect of LL-37 on the above pathways.In vivo:This part was verified in vivo by using LL-37 mouse homolog mCRAMP in asthma model mice.1.The study first tested the airway resistance of the lung function challenge test in mice to confirm the success of the asthma model.2.Hematoxylin-Eosin Staining(HE)staining of lung tissue was used to evaluate the inflammation around the airways and alveoli in asthmatic mice after glucocorticoid treatment.3.The expression of proinflammatory cytokine KC(Keratinocyte-derived Cytokine)and antiviral cytokine IFN-?(Interferon-?)in alveolar lavage fluid were evaluated using Enzyme Linked Immunosorbent Assay(Elisa)method.4.Western Blot was used to verify the effect of LL-37 on Erk and Akt pathways in animal models.Conclusions:1.LL-37 can alleviate glucocorticoid resistance caused by dsRNA by promoting PLZF expression,GRE activation,GR phosphorylation and GR transfer to the nucleus.2.The mechanism by which LL-37 alleviates dsRNA-induced glucocorticoid resistance may be through down-regulation of Erk and Akt pathways by combining with dsRNA.3.After administration of Poly I:C,glucocorticoids could not inhibit the airway inflammatory cell infiltration and secretion of inflammatory cytokines in asthma model mice,and LL-37 mouse homolog mCRAMP can relieve inflammation in asthma model mice.4.Asthma animal models confirmed that the mechanism by which LL-37 alleviates dsRNA-induced glucocorticoid resistance may be through down-regulation of Erk and Akt pathways.Indicate that LL-37 may play an important role in the treatment of chronic inflammatory lung disease virus infection.