The Establishment Of Eosinophilic Bronchitis Mouse Model And Proteome Study Of Eosinophilic Bronchitis Mechanisms

Background:Eosinophilic bronchitis (EB) patients have chronic cough, sputum evidence of EB, but normal spirometry, no evidence of airway hyperresponsiveness (AHR), normal Peak Expiratory Flow (PEF) variability. Researches of EB are mainly focused on clinical patients. At present there is no animal model uesed on systematically study of EB. Since there are obvious ethical and experimental limitations on people, it is urgent to establish an animal model of EB. The mouse is most commonly used for genetic manipulation, and its use in physiological studies is rapidly increasing. Therefore,we aimed to establish a mouse model of EB.Chronic cough is the mainly symptom of EB, which should be possesed by EB mouse model. But mouse cough detection is not generally accepted internationally. The mouse cough sound examination becomes the barriar to model establishment.Recent developments of proteomics techniques provide powerful tools for studying molecular mechanisms and functions of key proteins involving in many diseases. The goals of proteomics are to improve molecular classification of diseases and to discover sensitive biomarkers that are useful for diagnosing, treating, and predicting the prognosis.Objective:1. To develop a mouse cough detection method based on mouse sound monitoring.2. To set up an EB mouse model.3. To study EB mechanisms with with proteomic technique.Contents:Part I: Establishment of mouse cough detection methodMethods: Mouse respiratory waveforms were detected by Finepointe software, mouse cough sound were heard through microphone attached with the Buxco whole body plethysmography chamber. Sound wave shapes were recorded by cooledit software. Mouse body movements were observed by operator. Codeine, capasazepine and capsaicin were pretreated to observe the changes of mouse cough counts. Manual verses automated counts were also compared in mice exposed to capsaicin.Results: There were two kinds of respiratory shapes indicate mouse cough. Observation of mice movements and analysis of sound shapes solelly were of useless meaning. Mouse cough numbers decreased significantly after drugs pretreated. Mean manual and automatic counts of cough were very similar for each mouse. The correlation and coincidence between FP software automatic and manual counts is 0.964 and 0.956. Conclusion: Mouse cough detection method based on mouse cough sound had been established. The FP mouse cough detection software had good correlation with manual counts.Part II: Establishment of eosinophilic bronchitis mouse modelSegment 1: Research of the intranasal OVA dosage that could not introduce mouse airway hyperresponsivenessMethods: Mice were divided into asthma (AS), normal saline (NS) and three model groups. AS mice were intranasally challenged with 200μg ovalbumin (OVA). The model groups were challenged with one of three OVA doses (10μg, 20μg, or 100μg). NS group mice were treated with normal saline. Changes in lung resistance (RL) were determined after exposure to increasing doses of methacholine (MCh). Differential inflammatory cell counts in bronchoalveolar lavage fluid (BALF) were performed. Lung histological sections were observed to evaluate inflammatory infiltration.Results: RL in the 10-μg OVA-challenged model group was not significantly different compared with NS group at any MCh concentration while it had significant difference compared with AS group (P < 0.01). RL in the other two model groups were intermediate between AS and NS groups. Eosinophils in BALF significantly increased in all model and AS groups compared with NS group, but no significant differences were observed among model and AS groups. Inflammatory cells were observed around bronchioles and capillaries in model and AS groups but not in the NS group.Conclusion: A mouse model of eosinophilic airway inflammation without AHR could be established by 10μg OVA sensitization followed by 10μg OVA intranasal challenges. Segment 2: Study of the effect of cough detection on airway resistance examinationMethods: 24 female BALB/c mice were divided into NS, model-1 and AS groups. Invasive lung resistance was examined to ensure the model were successfully constructed. Mouse cough detection was executed in NS group mice just before airway resistance examination to make clear about the influence of capsaisin on airway resistance. 24 mice were randomly divided into M-2, M-3 and M-4 groups according to the time phase of mouse cough detection(6h,12h and 24h). Invasive lung resistance was examined 24h after the last OVA intranasal challenge.Results: Cough numbers in NS, M-2, M-3 and M-4 group mice were (16±5), (24±6), (22±6) and (25±6)/6min. Cough numbers among 3 model groups had no significant difference, but higher than NS group mice. AS mice had higher RL compared with NS and M-1 group mice. M-1 group mice had no significant difference compared with NS group ones. Airway resistance of M-2, M-3 and M-4 group mice had no difference compared with NS group mice and M-1 group mice which did not suffer capsaisin stimulation, but had significant difference compared with AS group mice.Conclusion: Mouce cough detection executed at 6h,12h and 24h could not affect AHR results. A suitable time (6h after the last OVA intranasal challenge) was chosen for mouse cough detection before airway resistance examination by experimental arrangement. Segment 3: The effect of dexmethasone on cough number and airway resistance of model miceMethods: 32 female BALB/c mice were divided into NS,model,AS and dexamethasone (DEX) groups. DEX group mice were treated as model ones and DEX was used total 4 times 1h before OVA intranasal challenges and capsaisin stimulation. Mouse cough detection was detected 6h and airway resistance was examined 24h after the last OVA challenge. BALF differential inflammatory cell counts were performed. Lung histological sections were observed to evaluate inflammatory infiltration.Results: DEX could diminish the cough number of model mice to NS group level. Except higher than NS group mice at 12.5mg/ml MCh concentration, DEX group mice had no significant difference compared with NS group mice. DEX could lower the proportion of eosinophils in BALF and eosinophils infiltration in lung tissue but not returned to NS group level.Conclusion: DEX could decrease model mice cough numbers, diminish airway EOS inflammation, whereas could not affect airway resistance.Summary of Part II: Compared with NS group mice, model group mice had more cough counts, airway eosinophilic inflammation, no airway hyperresponsiveness and effective response to DEX. These features are in accordance with EB pathological process. It is suggested that an eosinophilic bronchitis mouse model can be established by 10μg OVA intraperitoneal sensitization followed by 10μg OVA intranasal challenges.Part III: Proteomic study of eosinophilic bronchitis mechanismsSegment 1: Proteomic analysis between EB, asthma patients and healthy volunteers Methods: 7 EB, 9 asthma (including cough variant asthma) and 5 healthy people were enrolled. BAL was performed in the right middle lobe. Total proteins extracted from BALF cells were separated using two-dimensional electrophoresis (2-DE). After silver nitrate staining, the gel image analysis was carried out using ImageMaster 2D Elite 5.0 analysis software to identify the proteins differentially expressed in EB, asthma and healthy groups. The differential expression proteins were identified by peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) .Results: There were 5 among total 8 differential proteins were up-regulated and 3 proteins were down-regulated in EB group compared with asthma group. 2 proteins were up- regulated in healthy group compared with EB group.Conclusion: LASP1, Actin-like protein 2 and PDLIM1 may be relatted with no AHR in EB mechanisms, and HSPB1 and CLPP may relatted with EB airway inflammation.Segment 2: Initial proteomic analysis between EB, asthma and normal control miceMethods: 12 female BALB/c mice were divided into NS, EB and AS groups. Mice were immunized as before. Total proteins extracted from the lung tissue were separated using 2-DE. After coomassie brilliant blue staining, the gel image analysis was carried out using Image Master 2D Elite 5.0 analysis software to identify the proteins differentially expressed. The differential expression proteins were identified by PMF using MALDI-TOF-MS.Results: 11 among total 22 differential proteins were up-regulated and 11 were down- regulated in EB group compared with AS group. 4 proteins were up-regulated in EB group compared with NS group. Transgelin-2 (TAGL2) has close relationship with airway smooth muscle cells.Conclusion: Cytoskeletal proteins may be related with airway responsiveness. TAGL2, which is tightly related with smooth muscle cells, shows to be a good biomarker to indicate why EB has no AHR.Summary of the thesis:1. Mouse cough examination method was successfully set up. Criteria of mouse cough detection was set up based on mouse sound monitor and respiratory waves changes. rIt is the first time to record and analyze mouse cough sound internationally. The method was gradually simplified and developed in the process of advancement. It is a good mouse cough model for further cough mechanisms study and antitussive research.2. Eosinophilic bronchitis mouse model was successfully established. 10μg OVA intraperitoneal sensitization followed by 10μg OVA intranasal challenges could successfully set up a mouse model with EB features: enhanced cough numbers, eosinophilic airway inflammation, no airway hyperresponsiveness and good response to corticosteroids. It is the first time internationally to set up an EB animal model. This model not only suuplies for EB mechanisms research, but also supplies a better control model for AHR study compared with asthma model.3. New differential proteins related with AHR were identified in clinical and experimental studies. Cytoskeletal proteins are also important with EB mechanisms in animal experiments. Interestingly, a protein tightly related with smooth muscle cells, TAGL2, was up-regulated in asthma group mice, suggests that it is perhaps to be a molecular biomarker used to explain why EB has no AHR.