| BACKGROUND AND AIM:Asthma,as a most common chronic respiratory disease,has caused lots of trouble for human being. Statistic suggests that there are300million people,10percent are Chinese,are suffering from asthma.The number is increasing.Thanks to the standardized therapeutic schedule given by GINA,lots of symptoms about asthmahas been controled to certain level. However, the therapy can’t inhibit the pot ential developping trend of the disease. So the pathogenesis of asthma is still indistinct, which need more researchs to study the pathogenesis of asthma and find new therapeutic targets for it.AECs,as the first gate to against outside stimuli, plays an essential role in keeping the balance and stability of airway internal environment. AECs plays a central role in the onset and development of asthma. Although asthma is a TH-2type-dominant chronic respiratory diseases.It is insufficient to explain the origins of the disease and why the airway epithelium of asthma patients were so susceptible to external stimuli with chronic airway inflammation alone.Researchs showed that there exits airway epithelia barrier dysfunction among asthma patients, and the expression of tight junction proteins and adherens junctions was less than that of non-asthma people, which was also related to infiltration of inflammatory cells especially eosinophil cells.The changed structure of the airway epithelium caused by the disruption of tight junction and adherens junctions disables its protection function, which makes a high way for outside stimuli to pass throuth airway epithelium easily and contact with immunological and inflammatory cells like DC cells which could express lots of proinflammatory cytokines、chemokines and growth factors. The growth factors can also interact with the underlying mesenchyme to promote airway remodeling and a more powerful and long-lasting inflammatory reaction.Occupational Asthma is,a common disease in industrial country, a Work-related lung disease. Its prevalence accounts for15%of asthma patients.According to collected data, Diisocyanates is the most common reason for Occupational Asthma by now. The often-seen Diisocyanates consists of toluene diisocyanate (TDI), diphenyl-methane diisocyanate(MDI), and hexamethylene diisocyanate (HDI),which are highly reactive chemicals used in industry.TDI was most widely used in home and architectural decoration and with widespread use in the manufacture of polyurethane foams, Polyurethane elastomers, paint and plastics because of its lower price,so the number of people exposed to TDI is very huge.The prevalence of asthma among diisocyanate-exposed workers is estimated to range from5%to15%, while the global incidence rate of asthma in the common population is0.1-8%, China0.11-2.03%. In recent years, for its high prevalence and high socio-economic cost of OA, OA caused by TDI, has draw great attention,but the pathogenesis responsible for the disease is still ambiguous,so it is immportant to develop experiments about the pathoqenic mechanism of TDI-induced asthma.Diisocyanates which was characterized by the N=C=O group, are extremely cross-linking agents, making them very valuable in the production of polyurethane foams, paints and elastomers. However, this properties also contributes to toxicity and diisocyanate-related asthma. As in other types of asthma, the interaction between diisocyanates and AECs plays central role in the pathogenesis of diisocyanate-induced occupational asthma.The barrier structure and protective function of the AECs can be disrupted when exposed to high concentrations of diisocyanates. Diisocyanate exposure at high concentrations can induce pyknosis of cells, DNA fragmentation, disorganization of tight and gap junctions leading to damage of transepithelial resistance, or even cell death.Our research team have conformed that TDI increased the permeability of HBE cell monolayers, partly through a VEGF-mediated pathway in vitro. However, it doesn’t show how TDI influence the expression and distribution of tight junctions (like E-cadherin and so on) in normal airway epithelial cells and under allergic conditions.The therapeutic role of glucocorticoids(GCs) in human allergic asthma is very immportant,which is mainly bacause of its powerful anti-inflammatory characteristic.The most inmmportant mechanism of the anti-inflammatory function of GCs is their ability to inhibit the infiltration of inflammaory cells into affected tissues,human airway epithelia cells also express functional GC receptors. A recent report demonstrated GCs enhance airway barrier intergrity through re-distribution of TJ proteins(ZO-1and OCLN) and that EGFR signaling could be involved in this process. However, the protective effects of Glucocorticoids on the distribution and expression of E-cadherin under allergic conditions have not been clarified.Accordingly, the purpose of the present study was further to assess the influence of TDI on the expression and distribution of E-cadherin in a TDI-induced asthma model and define the mechanism involved in vitro. An additional aim of this study was to evaluate the inhibitory effect of dexamethasone (DEX) during this process. METHODSAnimal experiments1. TDI-asthma model were prepared by a modification of Hoet’s method.60Specific-pathogen-free,6-week-old male BALB/c mice(Southern Medical University) were randomly divided into three groups(20mice/group):(1) The vehicle(AOO)-sensitized, AOO-challenged, and PBS-treated mice (AOO group);(2) TDI-sensitized, TDI-challenged, and PBS-treated (TD1group), and (3) TDI-sensitized, TDI-challenged, and DEX-treated mice (DEX group:DEX21-phosphate dissolved in endotoxin-free PBS (1mg/kg) was given to the TDI-induced asthma mice via intraperitoneal injection1day before the TDI challenge for8consecutive days.).2. Airway hyperresponsiveness to MeCh (Sigma;0,3.125,6.25,12.5,25mg/ml in isotonic saline) was assessed by whole body plethysmography (Buxco), Penh value of different groups were recorded and compared.3. Twenty-four hours after measuring the airway parameters,the mice were sacrificed with pentobarbital (100mg/kg, i.p. weight), Retro-auricular nodes were obtained from the mice and kept on ice in RPMI-1640,and cell suspensions were obtained by pressing the lymph nodes through a cell strainer (100μm) and rinsing with10ml tissue culture medium (RPMI-1640). Then Lymphocytes were washed three times and suspended (107cells/ml) in complete tissue culture medium (RPMI-1640supplemented with10%heat-inactivated fetal bovine serum).At last,Cells were seeded into48-well culture plates at a density of106cells/ml and incubated in complete RPMI-1640medium for48h with3.5mg/ml of concanavaline A (ConA). Cells were then centrifuged (3000×g,10min) and supernatants were stored at-80℃.4. BAL fluid was isolated, total cells recovered were counted, and the cellular composition of BALF was determined using Hematoxylin-eosin staining.5. Pulmonary histopathology:mice lungs were excised and fixed. These tissues were then embedded in paraffin, cut sections and stained with hematoxylin and eosin (H&E).6. The expression of E-cadherin was examined by western blotting, Quantity One software was used to analyze the comparative production of E-cadherin protein.Cells experiments1. The TDI-human serum albumin (TDI-HSA) conjugate was prepared by a modified Son’s method.2. Methyltetrazolium (MTT) assay was used to assess the16HBE cell viability under different concentrations of TDI-HSA and HSA, groups as follows:The normal control group, TDI-HSA20ug/ml group、TDI-HSA40ug/ml group、TDI-HSA60ug/ml group、TDI-HSA100ug/ml group, HSA20ug/ml group. HSA40ug/ml group、HSA60ug/ml group、HSA100ug/ml group, stimulated16HBE for24hours, then MTT colorimetric assay detected cell viability.3. Western blot and immunohistochemistry were used to detect the expression and distribution of E-cadherin of16HBE under different concentrations of TDI-HSA, groups as follows:The normal control group, HSA100ug/ml group,TDI-HSA40ug/ml group, TDI-HSA60ug/ml group, TDI-HSA100ug/ml group.After stimulated for24h, the cells were collected.4. The inhibitory effect of dexamethasone (DEX) and PDP8095on the expression and distribution of E-cadherin when cells were treated with TDI-HSA. Groups as follows:The normal control group, HSA100ug/ml group,TDI-HSA100ug/ml group, dexamethasone pretreated group (10-4M or10"6M), PD98095pretreated group(10"7M), dexamethasone treated group (10-4M or10-6M). After stimulated for24h, the cells were collected and Western blot and immunohistochemistry were used to detect the expression and distribution of E-cadherin.5. The influence of TDI-HSA on the levels of p-ERK and the inhibitory effect of dexamehasone.Groups as follows:The normal control group, HSA100ug/ml group,TDI-HSA100ug/ml group, dexamethasone pretreated group (10-6M),cells were collected after stimulated for10minutes and30minutes.Statistical analysisSPSS13.0analysis statistical software was used for date analysis. Data was expressed as mean±SD, One-way analysis of variance (one-way ANOVA) was used to compare the overall mean when the variance was Homogeneity, and LSD method was used for Multiple comparisons among the groups; when the variance was not Homogeneity, Welch method was used to compare the overall mean, Tamhane’s was used for Multiple comparisons among the groups. Significance was accepted when P<0.05.Results:Animal experiments1. We succeeded to set up the TDI-induced asthmatic mode of mouse. These asthmatic mice appeared dyspneic.Airway responsiveness to MeCh increased along with its concentration in different groups. Mice in the TDI group have the most high%baseline Penh value compared to mice in other groups. The differences of Penh values between TDI group and AOO group were significant when stimulated by Ach (12.5and25.0mg/ml)(p<0.001, p<0.001).,which was significantly reduced in mice treated with DEX when stimulated by Ach (12.5and25.0mg/ml)(p<0.001,p=0.02) in comparison with TDI group.2. The concentration of IL-4、IFN-γ in retro-auricular nodes supernatants and total IgE in serum were significantly increased in TDI group(p<0.001,p<0.001,p <0.001),which were significantly reduced by DEX treatment(p<0.001, p=0.001,p <0.001).3. DEX significantly inhibits TDI-induced increase in the number of eosinophils(P=0.031) but not the total cells lymphocytes, and macrophages recruitment in the BALF. And the number of neutrophils was increased in TDI group(P=0.017),and it was further increased in DEX group(P=0.026).On histological examination, TDI-sensitized/challenged mice had a heavy infiltration of inflammatory cells, epithelial cell proliferation and some epithelial shedding. DEX significantly reduced inflammatory infiltrate in the peribronchial and perivascular regions.4. In the AOO group, E-cadherin localized at the lateral side and apicolateral border. The immunoreactivity of E-cadherin significantly decreased at the epithelial cell-cell contact and diffused in the cytoplasm in the TDI group. And in the DEX group, the immunoreactivity of E-cadherin was partly recovered.Cells experiments1. The effect of different concentrations of TDI-HSA and HSA on cell viability of normal human bronchial epithelial cells(16HBE):TDI-HSA with the concentrations of20ug/ml,40ug/ml,60ug/ml,100ug/ml and HSA with the concentrations of20ug/ml,40ug/ml,60ug/ml,100ug/ml for16HBE did not significantly affect cell viability(.P=0.78).2. Changes in the expression and localization of E-cadherin Protein induced by TDI-HSA:Confocal microscopy showed that strong E-cadherin staining in16HBE cell line located at the plasma membrane level at sites of cell-cell contact in untreated cells and HSA treated cells. TDI-HSA (40to100ug/ml) exposure for24h induced a dose-dependent disassembly of E-cadherin. But no evidence was provided for a signifantly alteration in E-cadherin expression after treatment with TDI-HSA(P=0.99). 3. Protection role of DEX and PD98095on TDI-HSA-induced E-cadherin aberrant of16HBE:In comparison with TDI-HSA treated group, pretreatment with DEX for1h, the immunoreactivity of E-cadherin was almost recovered to control levels, as a similar extent as with PD98095treated group. These suggested that ERK activation might be involved in the aberrant distribution of E-cadherin induced by TDI-HSA. But no evidence was provided for a signifantly alteration in E-cadherin expression between groups(P=0.79).4. Pretreatment with DEX could inhibit ERK1/2activation.Western blot showed that TDI-HSA exposure increased threonine/tyrosine phosphorylation of ERK1/2after10min and30min stimulation (P<0.001,P=0.03), and it was stronger after10min of stimulation in comparison with30min of stimulation.and the activation of P-Erk was partly inhibited by DEX(p=0.034,p=0.025).Conclusion1. TDI can significantly alter the distribution of E-Cadherin in airway epithelial cells in a TDI-induced asthma model and in normal16HBE cells.2. TDI-HSA can increase the threonine/tyrosine phosphorylation of ERK1/2, inhibition of ERK can attenuate TDI-HSA-induced redistribution of E-cadherin.3. DEX attenuate TDI-HSA-induced redistribution of E-cadherin partly by inhibiting p-ERK level.4. TDI-sensitized/challenged mice had a heavy infiltration of inflammatory cells, epithelial cell proliferation and some epithelial shedding. DEX significantly reduced inflammatory infiltrate in the peribronchial and perivascular regions. |
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