The Inhibitory Action Of Earthworm To Airway Remodeling In Asthma

Objective Epithelial cells play an important role in chronic inflammation and airway remodeling of asthma due to the release of cytokines,chemokines.Chronic inflammation and airway remodeling are significant characteristics.At present,glucocorticoid andβ2-receptor agonist are the first-line medicines for treatment,but they cannot inhibit the progress of airway remodeling,prolonged exposure may have adverse effects and be insensitivity.Earthworm is an effective antasthmatic,its pharmacological action is correlated with airway remodeling.To observe the influence of Earthworm parenteral solution to the bronchial epithelial cells(HBE) sensitized by house dust mite allergen Dermatophagoides pteronyssinus and human embryonic lung fibroblast(HLF),and investigate the value and explore the possible underlining mechanisms,on the treatment of asthma,then provide original thinking to clinical treatment for asthma.Methods Using different intervention and treatment,HBE cells were divided into 6 groups,control group(group A),Derp1 allergen(10μg/ml)group(group B), Derp1 allergen(10μg/ml)+Dexamethasone(DEX)(1×10^-6mol/l)group(group C), Derp1 allergen(10μg/ml)+Earthworm parenteral solution(0.9mg/ml)group(group D),Derp1 allergen(10μg/ml)+Earthworm parenteral solution(9mg/ml)group (group E),Derp1 allergen(10μg/ml)+Earthworm parenteral solution(18mg/ml) group(group F),cultured for 72 hours.Then detecting the expression of IL-13 mRNA, TGF-β₁ mRNA,Smad2 mRNA by RT-PCR,meantime collecting the supernatants of each group,culturing the HLF for 72 hours,and detecting the expression of Smooth muscle actin-α(α-SMA),Fibronectin(FN)by immunocytochemical method.All data are expressed as mean±SD.Statistical significance between groups was determined by one-way analysis of variance(ANOVA),multiple comparisons among groups was used.The dependability between two groups was rank correlation.Data analysis was performed using the SPSS 13.0 statistical package programme.p＜0.05 was considered as statistically significant.Results1.Morphologic changes of HBE influencend by Derp1:Observed by inverted phase contrast microscope,the cells were completely tile of cell monolayer in control group and were almost spherical or oval-shap(arrow of figure 1 A),and spindle cells were less.All cells were large and their arrangement were tight;In Derp1 allergen(10μg/ml)group cultured for 72 hours,the cells became lanky(arrow of figure 1 B),the gap between cells became large(triangle of figure 1 B).2.The expression of IL-13 mRNA,TGF-β₁ mRNA,Smad2 mRNA in HBE:Compared to each other,The concentration of the IL-13 mRNA,TGF-β₁ mRNA, Smad2 mRNA in HBE had a significant difference;In group B,IL-13 mRNA,TGF-β₁ mRNA,Smad2 mRNA were higher than group A,the difference was significant(p＜0.05);Group D,group E,group F and group C,theirs concentration had a little differences,but there was no statistically significant(p＞0.05);Compared with group B, the concentration of the IL-13 mRNA,TGF-β₁ mRNA,Smad2 mRNA in HBE of group D,group E,group F and group C were inhibited and had a significant difference(p＜0.05).Between group D,group E and group F,although there were some differences,there were no statistically significant(p＞0.05),that was mean,dose-effect relationship was not evident.3.The expression of protein:α-SMA,FN in HLF:Correspond to supernatants of group A,B,C,D,E,F,the HLF was divided into group A₁,B₁,C₁,D₁,E₁,F₁,the concentration ofα-SMA and FN in group B₁ was larger than that in group A₁ and had significant differences(p＜0.01);Group E₁,group F₁ and group C₁,theirs concentration had a little differences,but there was no statistically significant(P＞0.05);Compared with group B₁,The concentration ofα-SMA and FN in group D₁,group E₁,group F₁ and group C₁ was inhibited and had a significant differences(P＜0.01).Between group D₁,group E₁ and group F₁,although there were some differences,there were no statistically significant(P＞0.05).4.Analysis of dependability of some cell factors.The concentrations between TGF-β₁mRNA and Smad2 mRNA were positive correlation(r_s=0.9429,P＜0.05)The concentrations between TGF-β₁mRNA and FN were positive correlation (r_s=0.9429,P＜0.05).Conclusion1.Dermatophagoides pteronyssinus could result in the change of bronchial epithelial cells which were just like asthmatic,and were represented by the increasing concentration of the cytokines IL-13,TGF-β₁ and Smad2.2.The bronchial epithelial cells play important role in inducing airway remodeling,which was demonstrated by the increasing concentration of the cytokinesα-SMA and FN in HLF.HLF was cultured by the supernatants collected from HBE.α-SMA and FN could reflect to the degree of airway remodeling indirectly.3.The earthworm had an inhibitory effect on airway remodeling.It significantly depressed the signaling pathway of IL-13/TGF-β₁ and TGF-β₁/Smad2 which may be key in modulating mechanism of asthma;and supplied a new thinking to treat asthma.