Potential Contribution Of Soluble Advanced Glycosylation Receptor To Neutrophilic Asthma

Neutrophilic asthma(NA)is a subtype of asthma that was characterized by neutrophil inflammation of the airway.It has various clinical phenotypes and has a poor reaction to glucocorticoid treatment.Airway neutrophil inflammation was related to irreversible airflow obstruction of asthma.Impaired lung function in children with asthma played a crucial role in some adult chronic respiratory diseases.Recent studies have shown that adverse conditions in early life(eg.asthma)are associated with the occurrence of some adult chronic obstructive pulmonary disease(COPD).Long-term cohort studies abroad have shown that childhood asthma is a risk factor for COPD and COPD may originate from early life.Studies even considered that COPD might be a childhood disease,it just was obvious in adulthood with COPD phenotype.NA is dominated by airway neutrophil inflammation and is related to airflow restriction.COPD is also characterized by airway neutrophil inflammation and fixed airflow obstruction.Whether children with NA may be the phenotype of COPD in childhood? To explore the potential association between NA and COPD,we tried to find the common pathogenesis of NA and COPD.Soluble advanced glycosylation receptor(s RAGE)as a predictive indicator of airway neutrophil inflammation and a biomarker of COPD,was decreased in the blood and bronchoalveolar lavage fluid of patients with COPD.Moreover,it was negatively correlated with COPD grade and lung function.Whether s RAGE is involved in the pathogenesis of NA and the common pathological process of NA and COPD?Airway remodeling is a common pathological process of asthma and COPD and is also an important cause of airflow limitation.Studies showed that children with asthma developed airway remodeling in early childhood.Whether s RAGE participate in airway remodeling of NA and play a role in subsequent COPD phenotypes? To study the function of s RAGE in NA,we established a mouse model with NA to explore the relationship between s RAGE and airway remodeling.Airway neutrophil inflammation,cytokines,and mucus hypersecretion may result in airway remodeling in which epithelial-mesenchymal transition(EMT)is considered to be one of the mechanisms.We further investigated the relationship between s RAGE and EMT in NA mice and explored the mechanism of s RAGE participating in EMT in vitro and in vivo.Part I Effect of s RAGE on Airway Remodeling in Mice with Neutrophil AsthmaObjective To investigate the level of sRAGE and the effect of s RAGE on neutrophil airway inflammation and airway remodeling in a mouse model with neutrophil asthma(NA).Methods The NA mouse model was established,and the s RAGE level in BALF was detected by ELISA.The correlation between s RAGE and airway neutrophils,airway resistance was observed.Mice were transinfected with overexpressed s RAGE using an adeno-associated virus 9(AAV-9)vector,and the effects of s RAGE intervention on neutrophil airway inflammation and total inflammatory cells in airways were assessed by Diff-quick staining.Hematoxylin-eosin(HE),Periodic Acid-Schiff(PAS),and Masson trichrome staining were used to evaluate the effects of s RAGE on inflammatory cell infiltration,goblet cell proliferation,and collagen deposition in the lung.Real-time PCR and Western Blotting were applied to detect airway remodeling markers.Results 1.The sRAGE level decreased in BALF,while BALF neutrophil count and airway resistance increased in NA mice.2.The expression of green fluorescent protein in the lung tissue was significantly increased in mice transinfected with AAV-9 overexpressing s RAGE by tail vein.3.s RAGE decreased the airway resistance induced by 25mg/m L acetylcholine in the NA+s RAGE group.4.s RAGE reduced airway neutrophil inflammation,lung tissue inflammatory cell infiltration,airway goblet cell metaplasia,and collagen deposition in the NA+s RAGE group.5.TGF-β1 elevated in the NA group,while s RAGE administration inhibited the expression of TGF-β1.VEGF,MMP-9,and α-SMA exhibited no difference between the NC group and the NA group.Conclusion 1.s RAGE level decreases in NA mice BALF and is negatively correlated with airway neutrophil inflammation.2.AAV9-s RAGE can effectively transfect mouse models by tail-vein injection and partially reduce the airway resistance of NA mice.4.AAV9-s RAGE inhibits airway neutrophil inflammation,goblet cell metaplasia,and collagen deposition in NA mice.5.AAV9-s RAGE inhibits the expression of airway remodeling marker-TGF-β1.Part II Possible Regulatory Mechanism of s RAGE on Airway Remodeling in Neutrophil Asthma MiceObjective To investigate the effect of s RAGE on inflammatory mediators,mucins,airway EMT markers,proliferation,apoptosis,and autophagy phenotypes in NA mice,and to explore the possible mechanism of s RAGE regulating NA mice phenotype.Methods A mouse model of NA was established and treated with AAV9-s RAGE by tail-vein injection.Inflammatory mediators and mucins were detected by ELISA.Real-time PCR was undergone to determine transcription levels of mucins,markers of proliferation,apoptosis,autophagy,and EMT markers,as well as RAGE ligands,RAGE,PI3 K,and other signaling pathway molecules.HMGB1 inhibitor,RAGE inhibitor,and PI3 K inhibitor were used to intervene in the mouse model with NA,respectively.EMT markers were detected by Real-time PCR and Western Blotting.Results 1.The levels of IL-17 and IL-6 increased in NA mice BALF,while decreased after s RAGE intervention.2.The transcription and protein expression of MUC5 AC and MUC5 B elevated in lung tissue and BALF of NA mice,whereas declined in the NA+s RAGE group.3.The expression of E-cadherin decreased and Vimentin increased in the lung tissue of NA mice.After administration of s RAGE,E-cadherin expression was up-regulated and Vimentin expression was down-regulated.4.Ki67 level was increased and the Bcl-2 level was decreased in the NA group,while s RAGE administration reversed the expression of Ki67 and Bcl-2.P62 was down-regulated in NA mouse lung tissue,however,there was no difference after s RAGE intervention.Moreover,there was no significant difference in Caspase3 and LC3 expressions in the lung tissue between the NC group and the NA group.5.The expression of RAGE ligands,HMGB1,and SPARC,was up-regulated in the NA group,whereas the expressions were reduced by s RAGE intervention.6.RAGE,PI3 K,and p-ERK levels were increased in the NA group and decreased in the NA+s RAGE group.However,the expression of β-catenin and JNK was declined in the NA group and elevated in the NA+s RAGE group.7.HMGB1 inhibitor,RAGE inhibitor,and PI3 K inhibitor intervention improved the E-cadherin expression,however,had no significant effect on Vimentin expression.Conclusion 1.s RAGE inhibits the expression of IL-17 and IL-6 in NA mice BALF.2.s RAGE down-regulates MUC5 AC and MUC5 B expressions in the lung tissue and BALF.3.s RAGE reverses the decrease of E-cadherin and the increase of Vimentin in NA mice.4.s RAGE suppresses the high expression of Ki-67 and the low expression of anti-apoptotic protein Bcl-2 in NA mice.5.s RAGE reduces the expression of HMGB1 and SPARC.6.s RAGE regulates the expression of pathway molecules RAGE,PI3 K,and p-ERK.Part III Mechanism of s RAGE Regulating Epithelial-mesenchymal Transition in Human Airway Epithelial CellsObjective To explore the possible mechanism of s RAGE regulating epithelial-mesenchymal transition(EMT)in human airway epithelial cells.Methods HMGB1 was used to induce EMT phenotype in 16 HBE cells.The expression of epithelial and mesenchymal markers was detected using real-time PCR.s RAGE was administrated to 16 HBE cells induced by HMGB1 followed by observing the wound healing and detecting the expression of EMT markers,signaling molecules,and related inflammatory mediators.A rescue assay was performed by pathway inhibitors to explore the signal pathways and the mechanism of s RAGE on EMT induced by HMGB1 in 16 HBE cells.Results 1.The expression of E-cadherin decreased while Vimentin increased in 16 HBE cells induced by HMGB1.2.The migration ability of 16 HBE cells intervened by HMGB1 was enhanced,whereas it was reduced in the HMGB1+s RAGE group.3.Compared with the HMGB1 group,the expression of E-cadherin was elevated while Vimentin declined in the HMGB1+s RAGE group.4.The transcript expression of RAGE and PI3 K increased in 16 HBE cells treated with HMGB1,and the expression decreased after s RAGE intervention.5.Compared with the HMGB1 group,E-cadherin levels were improved while Vimentin was reduced in the HMGB1+RAGE inhibitor and HMGB1+PI3K inhibitor group.6.MUC5 AC expression increased in 16 HBE cells intervened by HMGB1,while decreased in HMGB1+s RAGE group,HMGB1+RAGE inhibitor group,and HMGB1+PI3K inhibitor group.No change of MUC5 B expression in HMGB1-induced 16 HBE cells was observed.7.Compared with the control group,there was no significant difference in the transcription levels of TGF-β1,VEGF,MMP-9,and α-SMA in the HMGB1 intervention group.Conclusion 1.s RAGE can inhibit the migration ability of 16 HBE cells induced by HMGB1.2.s RAGE reverses the decrease of E-cadherin and the increase of Vimentin in 16 HBE cells caused by HMGB1.3.s RAGE inhibits RAGE and PI3 K transcription expression.4.HMGB1 may regulate the expression of E-cadherin and Vimentin expression in 16 HBE cells through RAGE and PI3 K.5.s RAGE,RAGE,and PI3 K inhibit MUC5 AC expression in HMGB1-induced 16 HBE cells.6.The induction effect of HMGB1 on TGF-β1,VEGF,MMP-9,and α-SMA in 16 HBE cells is not obvious.