The Mechanism Of 1,25-dihydroxyvitamin D₃ In Alleviates House Dust Mite/cigarette Smoke Extracts-induced Airway Epithelial Barrier Dysfunction

IntroductionChronic airway disease most commonly manifest as asthma or chronic obstructive pulmonary disease（COPD）,and they are a leading cause of morbidity and mortality throughout the world.Recent studies have shown that airway epithelial disruption is related to persistent airway inflammation in the pathology process of chronic airway disease,the structural damage and immune dysfunction of airway epithelium is the key of asthma trigger,it involved in the inflammation,hyperresponsiveness and remodeling of airway in the pathological process.Therefore,the further understanding the mechanism of airway epithelium damage and repair in the chronic airway disease will help to find new therapeutic targets.The airway epithelium forms the first structural barrier against the external harmful substances.The physical barrier was composed of airway epithelium connection,mucus scour and ciliary movement,and the immunologic barrier be composed of a variety of immune cells.The damage and repair of airway epithelial barrier depends on the balance between airway internal protection factors and external damage factors.This epithelial barrier function is maintained by the formation of tight junctions（TJs）,composed of zonula occludens（ZO）1-3,occludin and claudins 1-5,as well as adherens junctions（AJs）,consisting of E-cadherin,P-catenin and α-catenin.A variety of stimulus including allergens（such as dust mite,the most common allergen in asthma）,irritants（such as cigarette smoke,the most common irritant in asthma）,and viruses disrupted the integrity of epithelial barrier by changing the structure and expression of junction proteins.The damage of airway epithelial barrier maked the allergens can arrival in submucosa to activate the dendritic cells（DC）,activate the Thl or Th2 immune response,increase the release of proinflammatory factor,induce the Inflammatory reaction and associated with susceptibility.How to avoid and restore the airway epithelial damage is the key of asthma prevention.Recent researches proved that vitamin D increased the membrane resistance and the junction protein expression,reduced the epithelial permeability and enhanced the epithelial barrier function in the human gut cells and corneal epithelial cells.In the intestinal epithelium.Moreover,our recent research showed that the active form of vitamin D（1,25（OH）₂D₃）could not only significantly increase the membrane resistance of airway epithelial cells,but also alleviate toluene diisocyanate（TDI）-induced airway epithelial barrier disruption and the expression and distribution abnormalities of E-cadherin.However,in the common risk factors of chronic airway diseases（such as dust mites,tobacco smoke）-induced airway injury model,the detailed mechanisms involved still remain poorly defined.House dust mites（HDM）is one of the most common allergens in asthma.Studies have shown:HDM makes the allergens are more likely to reach the submucosa,produce inflammation,and trigger asthma by destroying the airway epithelial integrity.The recent studies have found that:in the process of the pathology of asthma,bronchial epithelial cells,macrophages and Th2 cells could produce VEGF under the stimulation of HDM,viruses and pollutions in asthma,and the expression of VEGF induced allergic inflammation and promoted immune inflammatory responses.Moreover,the VEGF could induce airway and vascular remodeling in asthma pathology process.Clinical studies even showed that the levels of VEGF were positively correlated with the severity of asthma.Phosphoinositide 3-kinase（PI3K）/AKT pathway is a classical mediator of various immune response and inflammatory processes.Studies even have indicated that the PI3K/Akt pathway modulates airway high reactivity（AHR）,airway inflammation,and vascular permeability through the regulation of VEGF expression.Several experimental evidences have shown the activation of the PI3K/Akt signaling axis up-regulates the VEGF release and facilitate epithelial wound.Our previous studies have revealed that TDI could induce airway epithelial damage by increasing the VEGF expression,the inhibitor of VEGF could allivate these pathological changes.Meanwhile,our study also indentified the VEGF could directly induce airway epithelial barrier disruption,and the inhibitor of PI3K could allivate it.The above researches showed that the activation of PI3K/VEGF pathway is associated with the expression of in the pathological process of airway epithelial damage.Moreover,we have revealed that 1,25（OH）₂D₃ inhibited HDM-induced airway epithelial VEGF and VEGF mRNA expression.Based on the above results,we guess:PI3K/Akt pathway is involved in the regulation of VEGF expression and 1,25（OH）₂D₃ play the role of epithelial barrier protection by adjusting the VEGF expression via the PI3K/Akt pathway in the pathological process of HDM-induced airway epithelial damage.COPD is a kind of diseases,which is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases.The global incidence of COPD over 40 has been as high as 9%～10%.The pathogeny of COPD is not yet clear.Generally considered factors that related to chronic obstructive bronchitis and emphysema occurring factors are likely to be involved in the pathogenesis of COPD.Risk factors were divided into exogenous factors（environment）and internal（individual susceptibility factors）.External causes include smoking,inhalation of dust and chemicals,air pollution,respiratory infections,etc.Internal cause including hereditary factors,increased airway reactivity,etc.The cigarette smoke is one of the most common external stimulation factors of COPD.Current research is generally believed that cigarette smoke extracts（CSE）is related with COPD,asthma and other chronic respiratory diseases.CSE can activate calcium protease（calpain）,pyrolysis of intraepithelial adhesion proteins,destroy the connection between the airway epithenum,increase epithenai mucous memorane permeability,cause epithelial dysfunction,stimulate the submucosa pathological change,and eventually lead to the occurrence and progress of chronic airway disease.This shows that the activation of calcium protease is one of the ways for CSE induced the airway epithelial barrier damage.Calpain is calcium-activated cysteine protease protease,it almost expressed in all organizations,its activity is regulated by the MAPK pathway.Recent research suggests that vitamin D can inhibit biliary epithelial injury caused by E-cadherin cleaving after calpain activation.However,its specific mechanism is unclear now.Therefore,we conjecture that MAPK pathways involved in the regulation of calpain activation and 1,25（OH）₂D₃ can play the role of epithelial barrier protection by adjusting the calpain activation via MAPK pathway in the airway epithelial injury pathological process induced by CSE.In this study,we intended to identify:1.The relationship between the HDM induced airway epithelial barrier damage and VEGF,PI3K/Akt pathway.2.Whether 1,25（OH）₂D₃ preserve HDM-induced epithelial cells barrier dysfunction?whether the VEGF and PI3K/VEGF pathway inhibition are involved in this process?3.Further clarify whether the MAPK pathways is associated with the calpain activation and Whether 1,25（OH）₂D₃ preserve the epithelial cells barrier dysfunction in the pathological process of CSE-induced airway epithelial damage.Methods1.The human bronchial epithelial cell line 16HBE and BEAS-2B cultured in RPMI1640 medium with 10-15%fetal calf serum.The cells were then incubated at 37℃ in a 5%CO2 incubator.After cells reached complete confluence,they were passaged and seeded to proper culture plates at a density of 2.5×104 cells/cm2 for the next step of the experiment.All the experiments were using the bronchial epithelial cells within 3 to 10 generations in order to avoid the effect of cell ageing.2.Cell viability under different concentration of intervention factor was detected with a MTT colorimetric assay.3.Studying the effect of 1,25（OH）₂D₃ on HDM-induced airway epithelial barrier dysfunction.Cells were divided into four groups in the experiment:① control group（treated with culture medium）;②HDM group（treated with 400u/ml HDM）;③ 1,25（OH）₂D₃ control group（treated with 10-8mol/L1,25（OH）₂D₃）;④ 1,25（OH）₂D₃ treatment group（treated with 400u/ml HDM and 10-8mol/L 1,25（OH）₂D₃）。The evaluation indicators of barrier function were measured as following:1)Trans Epithellal Electric Resistance（TEER）was measured with a Millicell-Electrical Resistance System.Epithelial permeability was expressed as percent leakage of fluorescein isothiocyanate-dextran（FITC-dextran）from apical to basolateral compartments.2)Western Blot was used to analyze protein expression of adherent and tight junction proteins（E-cadherin,β-catenin,Occludin and ZO-1）.3)Immunofluorescence microscopy was measured to evaluate the distribution of junction proteins.4.To identify whether 1,25（OH）₂D₃ can inhibit HDM-induced VEGF release and decide the role of PI3K/Akt pathway during this process.Cells were divided into four groups in the experiment:①control group（treated with culture medium）;②HDM group（treated with 400u/ml HDM）;③1,25（OH）₂D₃ control group（treated with 10-8mol/L 1,25（OH）₂D₃）;④1,25（OH）₂D₃ treatment group（treated with 400u/ml HDM and 10-8mol/L 1,25（OH）₂D₃）.Western Blot was used to analyze protein expression of phosphorylated Akt（Thr308 and Ser473）and VEGF.Real-time PCR and ELISA were respectively used to investigate VEGF mRNA expression and VEGF-released.5.To further identify the role of PI3K/Akt pathway on VEGF release and epithelial barrier damage induced by the HDM,and to determine if 1,25（OH）₂D₃ decreases VEGF release and protects airway epithelial barrier by down-regulating the PI3K/Akt pathway in the airway epithelium.Cells were divided into six groups in the experiment:①control group（treated with culture medium）;②HDM group（treated with 400u/ml HDM）;③LY294002 control group（treated with 50μM LY294002）;④LY294002 treatment group（treated with 400u/ml HDM and 1 50μM LY294002）;⑤IGF-1 control group（treated with 100ng/ml IGF-1）;⑥IGF-1 treatment group（treated with 10-8mol/L 1,25（OH）₂D₃ and 100ng/ml IGF-1）.TEER and FITC-dextran permeability were used to evaluate airway barrier function.Real-time PCR and ELISA were used to investigate VEGF mRNA expression and VEGF-released.6.To indentify if 1,25（OH）₂D₃ inhibited VEGF-induced airway epithelial barrier disruption,16-HBE cells were divided into four groups in the experiment:①control group（treated with culture medium）;②VEGF group（treated with 50ng/ml VEGF）;③1,25（OH）₂D₃ control group（treated with 10-8mol/L 1,25（OH）₂D₃）;④1,25（OH）₂D₃ treatment group（treated with 50ng/ml VEGF and 10-8mol/L 1,25（OH）₂D₃）.And then the function of airway epithelial barrier was evaluated as the ’3’ descripted;Western Blot was used to analyze protein expression of phosphorylated Akt（Thr308 and Ser473）.7.To further indentify if 1,25（OH）₂D₃ inhibited CSE-induced airway epithelial barrier disruption,16-HBE cells were divided into four groups in the experiment:①control group（treated with culture medium）;②CSE group（treated with CSE）;③1,25（OH）₂D₃ control group（treated with 10-8mol/L 1,25（OH）₂D₃）;④1,25（OH）₂D₃ treatment group（treated with VEGF and 10-8mol/L 1,25（OH）₂D₃）.And then the function of airway epithelial barrier was evaluated as the ’3’ descripted;Western Blot was used to analyze protein expression of phosphorylated Akt（ERK,JNK,and p38）.8.Statistical analysis:SPSS 13.0 analysis statistical software was used for date analysis.Data was expressed as mean±SD（X±S）,One-way analysis of variance（one-way ANOVA）was used to compare the overall mean when the variance was Homogeneity,and Bonferroni method was used for Multiple comparisons among the groups;When the variance was not homogeneity,Welch method was used to compare the overall mean,Dunnett T3 was used for multiple comparisons among the groups.ANOVA of factorial design was used for two or more factors of multiple level measurement.Significance was accepted when P<0.05.Results1.The effect of 1,25（OH）₂D₃（100nM or below groups）and HDM（600u/ml or below groups）on 16HBE and BEAS-2B cell viability:All the experimental intervention did not affect cell viability（P>0.05,n=6）.2.1,25（OH）₂D₃ alleviated airway epithelial barrier dysfunction induced by the HDM:In 16HBE and BEAS-2B cells,the HDM resulted in the TEER significantly reduced（t=9.016,P=0.001 和 t=36.366,P=0.000）,the FITC-dextran permeability of epithelial layer increased（t=-4.022,P=0.016 and t=-4.657,P=0.010）,the expression of E-cadherin and ZO-1 decrease（P<0.01）,and the distribution of junction proteins（E-cadherin,β-catenin,Occludin and ZO-1）became discontinuous and diffuse,meanwhile,the 1,25（OH）₂D₃ restored the decreased TEER（t=11.750,P=0.020 and t=17.000,P=0.000）,the increased FITC-dextran permeability of epithelial layer（t=-3.355,P=0.028 and t=-3.615,P=0.022）,and the abnormal distribution and expression of junction proteins induced by HDM.It implied that the 1,25（OH）₂D₃ inhibited the HDM-induced epithelial cells barrier dysfunction in the 16HBE and BEAS-2B cells（P<0.05 vs.HDM group）.3.1,25（OH）₂D₃ inhibited HDM-induced airway epithelial VEGF expression and barrier disruption through PI3K/Akt signaling pathway.The western blot results showed,HDM increased the expression of VEGF and phosphorylated Akt（Thr308 and Ser473）（PT308=0.018,P s473=0.013 and PT308=0.012,P S473=0.011,vs.control group）,whereas,1,25（OH）₂D₃ inhibited the expression of VEGF（P=0.000,vs.HDM group）and phosphorylated Akt（PT308=0.012,PS473=0.016 and PT308=0.042,P s473=0.011,vs.control group）.Meanwhile,the PCR and ELISA also showed that VEGF mRNA expression（t=-11.078,P=0.000 and t＝-17.662,P=0.000,vs.control group）and VEGF release significantly increased（t＝-17.902,P=0.000 and t=-53.445,P=0.000,vs.control group）after HDM treatment,1,25（OH）₂D₃ could inhibit these pathological changes in bronchial epithelial cells(（t=-14.025,P=0.000 and t=-20.313,P=0.000 vs.HDM group）.4.1,25（OH）₂D₃ alleviates house dust mite（HDM）-induced airway epithelial barrier dysfunction via PI3K/VEGF pathway.As the HDM,IGF-1（100ng/ml）significantly decreased the TEER（P=0.000 and P=0.000,vs.control group）,increased FITC-dextran permeability of epithelial layer（P=0.000 and P=0.014,vs.control group）,and induced the expression of VEGF（P=0.003 and P=0.004,vs.control group）and VEGF mRNA(（P=0.000 and P=0.000,vs.control group）,1,25（OH）₂D₃ inhibit the decreased TEER（P=0.001 and P=0.000,vs.IGF-1 group）,the increased FITC-dextran permeability of epithelial layer（P=0.002 and P=0.019,vs.IGF-1 group）,the increased expression of VEGF（P=0.000 and P=0.000,vs.IGF-1 group）and VEGF mRNA（P=0.000 and P=0.000,vs.IGF-1 group）induced by IGF-1.Meanwhile,as the 1,25（OH）₂D₃,LY294002（50μM）also could inhibit these pathological changes（P=0.000 and P=0.000,vs.HDM group）.5.VEGF impair airway epithelial barrier disruption via PI3K/Akt signaling pathway.The VEGF significantly reduced the TEER（t=8.600,P=0.001,vs.control group）and increased the FITC-dextran permeability of epithelial layer（t=-5.361,P=0.006,vs.control group）in the 16-HBE cells.Western blot analysis revealed that the VEGF treatment did not affect the expression of junction proteins（E-cadherin,β-catenin,Occludin and ZO-1）in the 16-HBE cells.However,the results of immunofluorescence showed that the VEGF resulted in the distribution of junction proteins became discontinuous and diffuse in the 16-HBE cells.Western blot analysis revealed that VEGF165 could promote phosphorylated Akt（Thr308 and Ser473）,decrease TEER and increase permeability of FITC-dextran in 16-HBE cells（all P<0.01,vs.control group）.1,25（OH）₂D₃ pretreatment alleviated these pathological changes（all P<0.01 vs.VEGF group）.6.The effect of CSE on 16HBE cell viability:CSE（Concentration of 2.5%and below）did not affect cell viability（P>0.05,n=6）.7.1,25（OH）₂D₃ alleviated airway epithelial barrier dysfunction induced by the CSE:In 16HBE cells,the CSE resulted in the TEER significantly reduced（t=6.930,P=0.002 vs.control group）,the FITC-dextran permeability of epithelial layer increased（t=-12.954,P<0.001 vs.control group）,the expression of E-cadherin and ZO-1 decrease（all P<0.01,vs.control group）,and the distribution of junction proteins（E-cadherin,and P-catenin）became discontinuous and diffuse,meanwhile,the 1,25（OH）₂D₃ restored these pathological changes.It implied that the 1,25（OH）₂D₃ inhibited the CSE-induced epithelial cells barrier dysfunction in the 16HBE cells（all P<0.05 vs.CSE group）.8.1,25（OH）₂D₃ inhibited CSE-induced airway epithelial calpain-1 expression and barrier disruption through ERK signaling pathway.The western blot results showed,CSE increased the expression of calpain-1 and phosphorylated MAPK（ERK,P38 and JNK）（all P<0.05 vs.control group）,whereas,1,25（OH）₂D₃ inhibited the expression of calpain-1 and phosphorylated ERK（all P<0.05 vs.CSE group）.9.1,25（OH）₂D₃ alleviates CSE-induced airway epithelial barrier dysfunction via PI3K/VEGF pathway.As the U0126（inhibitor of ERK）and ALLM（inhibitor of calpain-1）significantly inecreased the TEER,decreased FITC-dextran permeability of epithelial layer,and reduced the expression of calpain-1（P<0.01 vs.CSE group）,Meanwhile,as the U0126 also could inhibit the phosphorylated ERK（P<0.01 vs.CSE group）.Conclusions:1.1,25（OH）₂D₃ inhibited the HDM-induced airway epithelial barrier dysfunction:the 1,25（OH）₂D₃ inhibited the HDM-induced TEER reduced,the FITC-dextran permeability of epithelial layer increased,the expression of E-cadherin and ZO-1 decrease,and the distribution of junction proteins（E-cadherin,β-catenin,Occludin and ZO-1）disruption.HDM induced airway epithelial barrier disruption through increasing the VEGF release via PI3K/Akt/VEGF signaling pathway.1,25（OH）₂D₃ protectd the epithelial barrier function by partially inhibited these changes.1,25（OH）₂D₃ alleviated VEGF-induced airway barrier disruption.2.1,25（OH）₂D₃ inhibited the CSE-induced airway epithelial barrier dysfunction:the 1,25（OH）₂D₃ inhibited the CSE-induced TEER reduced,the FITC-dextran permeability of epithelial layer increased,the expression of E-cadherin and β-catenin decrease,and the distribution of junction proteins（E-cadherin and P-catenin）disruption.CSE induced airway epithelial barrier disruption through increasing the calpain-1 release via MAPK signaling pathway.1,25（OH）₂D₃ protectd the epithelial barrier function by ERK/calpain-1 pathway.