The Effects Of Soluble Advanced Glycosylated Receptors On Airway Inflammation In Neutrophil Asthma

Part I Comparison of the Efficacy of sRAGE Gene Transfection in a Mouse Model with Neutrophil Asthma Via Tail Vein and Nasal DripObjective To investigate the efficacy of caudal vein injection and nasal drip in treating mice with neutrophil asthma by transfection of adenovirussRAGE into the lungsof mice.Methods1.Establishment of neutrophil asthma mouse model: 16 female C57BL/6 mice were randomly divided into neutrophil asthma group(NA group)and normal control group(NC group).NA group wasdrip from nasopharyngeal airway 100 ug OVA(Grade Ⅴ)and 1 ug LPS sensitization at 1st day,7th day and14 th days.From day 21 st,the mice were atomized 1 hoursper day with l % OVA(Grade Ⅱ)solution for 7 days.NC group wasstimulated with equal amountsof PBS.The symptomsof the mice during atomization and airway resistance wasobserved.Hematoxylin-eosin and the proportion of BALF neutrophilswere used to evaluate the inflammatory cell infiltration,and to evaluate whether the NA model wassuccessfully established.2.Comparison of effectivenessof mice transfected with tail vein and nasalgland virusmediated sRAGE: 32 healthy female C57BL/6 mice were selected and randomly divided into NA group,sRAGE tail vein group,sRAGE nasal group and NC group,with 8 mice in each group.At day 1st,the sRAGE tail vein group wastransfected with AAV 9-sRAGE 100 ul.In the sRAGE nasal group,AAV 9-sRAGE 100 ul were given by nasal drip.The NA group and NC group wasgiven PBS100 ul in two waysat the same time.At the 7th day,OVA+LPS combined with nasal drop sensitization and atomization were applied to the NA group,sRAGE tail vein injection group and sRAGE nasal drop group according to the NA modeling method.The NC group wasreplaced by the same amount of PBS.The effectivenessof the two methodswascompared by detecting the level of sRAGE in BALF and observing the expression of GREEN fluorescent protein in lung frozen sections.Results1.Compared with the NC group,the NA group frequently showed such stimulating symptomsassneezing,shortnessof breath,scratching of head and face,etc.Airway resistance in the NA group washigher than that in the NC group after acetylcholine stimulation at 12.5mg/ml,25mg/ml and 50mg/ml,(P<0.05);The total number of BALF cellsand the proportion of neutrophils(54.88±10.96)% in the NA group were significantly higher than that in the NC group(0.7±0.63)%,(P<0.0001);Compared with the NC group,the mice in the NA group had narrowed trachea lumen,thickened tube wall,widened alveolar septum,a large number of inflammatory cellswere infiltrated around the bronchi,and the blood vesselswere dilated and congested.The score of inflammation in NA group(3.75±0.65)washigher than that in NC group(0.63±0.43),(P<0.05).2.sRAGE level in BALF of mice wasdetermined by ELISA,and the sRAGE level in the NA group(93.29±11.04)pg/ml wassignificantly decreased,while the sRAGE level in the tail vein injection group(229.05±11.4)pg/ml and the nasal drop group(227.67±22.44)pg/ml wasincreased.The level of sRAGE in the sRAGE tail vein group and sRAGE nasal group washigher than that in the NA group(P<0.05).there wasn’t a statistical difference between the tail vein injection group and the nasal drop group,(p>0.05)3.The expression of green fluorescent protein wasobserved in the lungsafter transfection with AAV9-sRAGR by tail vein injection and nasal drip.Conclusion Adeno-associated viruseswere effectively transfected into NA mice by both tail vein injection and nasal drip.Part Ⅱ The Effect of sRAGE Regulating Airway Inflammation in a Mouse Model with Neutrophil AsthmaObjective To investigate the inhibitory effect of sRAGE on airway inflammation in a mouse model of neutrophil asthma.Methods32 healthy female C57BL/6 mice were randomly divided into NA group,NA+sRAGE group,NA+sRAGE negative control group and NC group,with 8 mice in each group.On the 1st day,the NA+sRAGE group wastransfected with AAV-sRAGE 100 ul injected into the tail vein.NA+sRAGE negative control group had AAV9-CON336 100 ul by tail vein injection.The same dose of PBS were injected into the tail vein of NA group and NC group.On the 7th day,the mice in NA group,NA+sRAGE group,NA+sRAGE negative control group were treated with NA modeling,and NC group wasgiven PBS at the same time.The symptomsof mice were observed and recorded during atomization.After the atomization,the lung function indexesof mice in each group were measured and analyzed.And then HE staining,Masson trichromatic staining and PAS staining were performed to observe the microscopic pathological changesin each group,and semi-quantitative scoring wasperformed.sRAGE and IL-17 expression in BALF were detected by ELISA.Results1.The NA group and the NA+sRAGE negative control group showed obviousexcitation symptomsduring atomization,while NC group had no obvioussymptoms,and the NA+sRAGE group had similar mild manifestations.2.The airway resistance of NA group and NA+sRAGE negative control group washigher than that of NC group and NA+sRAGE group,after 12.5mg/ml and25mg/ml acetylcholine stimulation,(P<0.05);After 50mg/m L acetylcholine atomization,airway resistance in NA group and NA +sRAGE negative control group washigher than that in NC group(P<0.05),airway resistance in NA group and NA+sRAGE negative control group washigher than that in NA+sRAGE group,but there wasno significant difference(P>0.05).3.Compared with NA group and NA+sRAGE negative control group,the total number of BALF cellsand the proportion of neutrophilsin NA+sRAGE group were significantly reduced.4.Compared with the NA group and the NA+sRAGE negative control group,the infiltration of inflammatory cellsin the NA+sRAGE group wassignificantly improved,the collagen deposition wasreduced,and the mucussecretion of goblet cellswasreduced.5.The expression of sRAGE of NA group and NA+sRAGE negative control group waslower than that of NC group and NA+sRAGE group(P<0.05);The level of IL-17 in BALF of NA group and NA+sRAGE negative control group washigher than that of NC group and NA+sRAGE group(P<0.05);6.The expression of sRAGE wasnegatively correlated with IL-17(R=-0.526,P<0.014),and negatively correlated with the proportion of neutrophils(R=-0.80,P<0.0001),the level of IL-17 in BALF waspositively correlated with the proportion of neutrophils(R=0.480,P<0.018)Conclusion 1.The decreased expression level of sRAGE in BALF of NA mice wasnegatively correlated with the level of IL-17 and the percentage of neutrophils.2.Adenovirusoverexpression of sRAGE improved airway neutrophil inflammation in NA mice.