Exacerbation Of Asthma. The Chuankening Side Of Clinical And Experimental Study,

?Asthma is a common disorder of the airways in which they contract too much and too easily. The burden of asthma appears to be increasing worldwide, especially in societies undergoing rapid urbanization, and both morbidity and mortality from asthma have increased in many parts of the world, making it a global health concern. The Global Strategy for Asthma Management and Prevention Report stated that the definition of asthma is based on the functional consequences of airway inflammation, i.e. "Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation causes an associated increase in airway responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and cough, particularly at night and/or early morning. These episodes are usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with treatment." The treatment of asthma is internationally agreed upon and guidelines have been developed for the management of it. The treatment of asthma is directed against airway obstruction and inflammation.Airway hyperreactivity has been attributed to chronic airway inflammation and the severity of this hyperreactivity correlates with the airway remodeling and bronchial angiogenesis. In summary, we report that allergen-induced airway inflammation in OVA-sensitized/challenged mice was reduced by the broad-spectrum caspase inhibitor, consistent with an important role of caspases in transforming growth factor-β1,(TGF-β1 )and Vascular endothelial growth factor, (VEGF).一. Clinical reaschMaterials and Methods1．PatientsFifty-fife patients (30 men and 25 women) with unstable,steroid-naive, atopic asthma (age range, 14 to 73 years; medianage, 45 years) were examined at the outpatient department of the Hospital Therapeutic Clinic of Wang Jing, China. They had nocturnal wheezing and daily asthma symptoms. Asthma was diagnosed according to Chinese Thoracic Society criteria. 12 Diagnosis was based on clinical history, reversibility of FEV₁>15%, and diurnal variability of peak expiratory flow rate>20%. The mean duration of asthma was 8．41±3．79 ?years (mean±SD). All patients were nonsmokers. The study was approved by the local ethics committee. The participants were informed in writing, and their written consent was obtained.2．Lung Function TestsFEV was measured by dry spirometry (Vitalograph; Buckingham, UK). Airway hyperresponsiveness was assessed by histamine challenge (MasterScope automatic spirometer; Jaeger GmbH; Wurzburg, Germany). After an initial 0．9% sodium chloride inhalation, patients were exposed to doubling concentrations of histamine delivered as five breaths from a dosimeter (Dosimeter APS pro; Jaeger GmbH). FEV₁ was measured 2min after each inhalation. Airway hyperresponsiveness was expressed as provocative concentration of inhaled histamine causing a 20% fall in FEV₁ (PC20) . PC20 was determined by linear interpolation from the log10 concentration response curve. Bronchial challenge was performed in 28 asthmatic patients with FEV₁>70% predicted.3．IgE MeasurementThe concentration of IgE in serum was measured. Blood samples were obtained at 9 am to 10 am after an overnight fast. After centrifugation, serum was frozen and stored at ₂0℃within 14 days until the assay.4．EOS MeasurementThe concentration of ECP in serum was measured by using radioimmunoassay in duplicate. Blood samples were obtained at 9 am to 10 am after an overnight fast. Clotting time was 60±10 min. After centrifugation, serum was frozen and stored at-20℃within 14 days until the assay.5．StatisticsStudent's unpaired two-tailed t test, Pearson correlation, Statistical significance was assumed at P< 0．05, and the data are expressed as mean±SD.Statistical analyses were performed using SPSS 11．0 software.Results1．The CKN has a satisfactory curative effect on attacking-phrase asthma.The observed rate of therapeutic effect is 94% for CKN and 95% for Aminophylline. CKN show the similar function with control group.2．The CKN can ameliorate the symptoms of cough and relieve phlegm extraordinary.The CKN demonstrate the better effect on ameliorate the symptoms of cough and phlegm at attacking-phrase asthma than Aminophylline.3．It has the same function for different types.The CKN didn't show significant distinguish on different types of asthma, the effect is alike.4．The CKN can improve the Lung Function obviously.The CKN can release the spasm of bronchus, perfect the function of ventilation by improving the Lung Function.5．The CKN can lower the abnormal serum level of EOS and IgE.Activation of inflammatory cells, and particularly eosinophils, is the prominent feature of airway inflammation in patients with asthma. Antigen challenge caused a significant increase in eosinophils recovered in the serum. The concentrations of abromal EOS in serum were significantly elevated in asthmatic patients compared to normal subjects. Most of the asthmatic patients had an increased level of EOS. This may indicate an enhanced production of oxidants and/or decreased antioxidant capacity of asthmatic airways. Therefore, elevated concentrations of EOS may result from an enhanced number and activity of inflammatory cells in the airways. Eosinophils releaseseveral mediators, including EOS, that may amplify the inflammatory process in the airways. EOS is suggested to be an indirect marker of airway inflammation. We found an increased level of EOS in serum in our patients. EOS may diffuse from inflammatory cells in the airways to the blood compartment. There was a correlation between enhanced activity of eosinophils in bronchial mucosa and serum levels of EOS. Suppression of eosinophilic inflammation by inhaled corticosteroids reduces the concentrations of EOS in serum. Another explanation may be that eosinophils are activated both locally in the lungs and in the blood. Perhaps, this may reflect activation of eosinophils in the asthmatic airways.The CKN can lower the increased level of EOS and IgE apparently by our experiment..6．We couldn't find his side effect by a series of examinations, so we feel relieved when we take it.ConclusionIn conclusion, our study has shown ongoing airway inflammation and airway remodeling in adolescents in clinical remission of attacking-phase asthma. We speculate that airway inflammation may well determine the risk of asthma relapse later in life. Furthermore, we believe that subjects with evidence of airway inflammation during remission could benefit from treatment of the CKN in the short-term /or long-term. We propose that it can monitoring of asthmatic airway inflammation and bronchial responsiveness by reducing the serum abnormal level of EOS and IgE,improving the lung function in asthma.二. Experimental reaschMaterials and Methods1．AnimalsMale Balb/C mice (20±2 g, 6 weeks old), were obtained from Center of animal Laboratories .and housed for 1 week before experiments were initiated. Food and water were supplied ad libitum. Experiments were performed in accordance with the research institutes of Chinese traditional medicine .All animal use procedures were approved by the Chinese Animal Care Committee.2．Allergen sensitization and challengeMice were sensitized and later challenged with OVA. Mice were immunized with OVA (10mg) complexed with aluminum potassium sulfate in a 0．2ml volume, administered by i.p. injection on days 1, 7, and 14, mice were anesthetized with 0．2 ml of ketamine (10 mg/ml) and xylazine (1 mg/ml) diluted in 0．9% saline. Mice received 20g of OVA by intratracheal (i.t.) administration. Intratracheal challenges were performed. Mice were anesthetized by i.p. injection of a 0．2 ml of amixture of ketamine and xylazine (10 and 1 mg/ml, respectively) in normal saline and were placed on board in the supine position. The control group received normal saline with aluminum potassium sulfate.3．Ab treatmentMice were given 3 types dose of CKN at 30 min before OVA challenge from days 14 to 28．For control mice, Dexamethasone Acetate was given.4．Bronchoalveolar lavage (BAL)On day 28, after measurement of airway hyperreactivity, the mice were sacrificed by exsanguination by cardiac puncture and the left lung was isolated by tying off the left main stem bronchus. The right lung was lavaged with one wash of 1 ml of saline and then additional lavage of 1 ml in and out five times for a total of 2 ml. The total number of leukocytes per 0．05-ml aliquot was determined after methylene blue nuclear staining. The remaining BAL fluid was centrifuged at 200×g for 10 min at 4℃and supernatants were stored at-80℃until assay of cytokine protein levels. The cell pellets were resuspended in saline containing 10% BSA and smears were made on glass slides. 5．Lung histologyThe left lung tissue was fixed in Carnoy's solution at 20℃for 15 h. The tissues were embedded in paraffin and cut into 5-um sections. A minimum of 10 fields was randomly examined by light microscopy by a blinded observer. The intensity of the cellular infiltration around pulmonary blood vessels and airways was assessed on a semiquantitative scale ranging from 0 to 4+. Airway mucus (i.e., mucin and sulfated mucosubstances) was identified after staining with methylene blue, H&E, and Alcian blue as previously described. Occlusion of the airway diameter by mucus was assessed on a semiquantitative scale ranging from 0 to 4．Each airway section was assigned a score for airway diameter occlusion by mucus based on the following criteria: 0, 0-10% occlusion; 1, 10-30% occlusion; 2, 30-60% occlusion; 3, 60-90% occlusion; and 4, 90-100% occlusion (17) . Airway edema was assessed on a 0-4 scale (16) . An investigator blinded to the protocol design performed the morphometric analysis.6．ELISA for cytokine levelsThe expression of TGF-β1 and VEGF levels in BAL fluid and stimulated splenocytes medium were assayed using BD PharMingen OptEIA assays according to the manufacturer's protocol. The OD were read on a microplate reader (EL340; Bio-Tek Instruments, Winooski, VT) at 510 nm. Cytokine levels were determined by comparison with standards.7．Statistical analysisResults are reported as the means±SE of the combined experiments. Differences were analyzed for significance ( p< 0．05) by Student's twotailed t test for independent means. Differences in pulmonary function data were analyzed by linear regression followed by the Fisher's protected least significant difference test. Statistical analyses were performed using SPSS11．0 software.Results1．The CKN can prolong the latent period of cough and phlegm obviouslyThe CKN can prolong the latent period of cough and phlegm over Aminophylline in terms of therapeutic effect. There is no difference between every group. But, in the second week, CKN show the better effct than DexamethasoneAcetateTa-blets.2．The CKN can reduce the gravity of asthma.The CKN can reduce the gravity of asthma. The effect of middle dose groud is the best. There were no obvious distinguish between every group. 3．The experiment research on airway remodeling.Twenty-eight days after the final OVA challenge, BAL fluid was performed on the right lung and left lung tissue was obtained to assess the effect of histology on airway inflammation histologically. TGF-β1 expression in BAL fluid were measured by ELISA. TGF-β1 expression in lavage fluid were elevated correlate with treatment group. After CKN treatment on days 28, showed a dramatic reduction of factor-β1 of the airway parenchyma and surrounding blood vessels and airway mucus product, and the effect of middle group is the best. But there were no apparent difference between middle group and DexamethasoneAcetateTablets group.4．The experiment research on bronchial angiogenesis.VEGF expression in BAL fluid were measured by ELISA .Compared with the treatment group the expression in lavage fluid were elevated. After CKN treatment on days 28, showed a dramatic reduction of Vascular endothelial growth factor of the airway parenchyma and surrounding blood vessels and airway mucus production,the effect of middle group is the best. But there were no apparent difference between the middle group and the DexamethasoneAcetateTablets group.ConclusionIn conclusion, our study has shown high expression of transforming growth factor-β1and Vascular endothelial growth factor on attacking-phase asthma. We infer that connection with airway remodeling and bronchial angiogenesis, and they can lead to airway inflammation and bronchial responsiveness, those are the reasons of bad effect on asthma, through our experimentWe believe that subjects with evidence of airway inflammation during remission could benefit from treatment of the CKN in the short-term /or long-term. We propose that it can inhibiting of asthmatic airway inflammation and bronchial responsiveness by reducing the high expression of TGF-β1 and VEGF. We anticipated that inhibition of asthmatic airway inflammation and bronchial responsiveness would exacerbate allergen-induced lung inflammation by reducing the expression of TGF-β1 and VEGF.