喘咳宁方对发作期哮喘的临床和实验研究
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摘要
目的
支气管哮喘(简称哮喘)是一种由嗜酸性粒细胞、肥大细胞和淋巴细胞等多种炎症细胞参与的慢性非特异性气道炎症，产生多种炎症介质及细胞因子，作用于炎性细胞本身及气道上皮细胞、平滑肌细胞等气道结构细胞，一方面促进炎症的发展，同时也导致气道重塑，气道反应性增加，气道壁增厚，通气受阻，肺功能下降；并直接或间接导致哮喘气道内血管生成。气道重塑和气道血管生成已经成为哮喘重要的病理特征。
哮喘气道重塑是一个集合性术语，特指哮喘患者气道壁结构的改变不同于正常气道组织学改变，在哮喘患者气道中除有炎性细胞浸润外主要表现为上皮脱落、气道平滑肌层的增生和肥大及基底的增厚及其玻璃样变。1992年哮喘时“呼吸道重塑(airway remoding)”概念被明确提出。气道重塑可导致气流不可逆阻塞和持续性的气道高反应性，并能成为顽固性哮喘的病理学基础，研究它有利于防止哮喘病情的恶化；气道重塑不仅发生在哮喘的晚期，而且出现在哮喘的早期，随着哮喘的发展，气道重塑呈进行性加重，即使哮喘症状得到缓解，气道重塑仍呈持续性发展，治疗哮喘若不从气道重塑上下手，哮喘的治疗就不能从根本上得到缓解。转化生长因子β1(transforming growth factor-β1，TGF-β1)在气道重塑中发挥了重要作用。
气道血管生成是指从原有血管长出新生血管，表现为血管数目增多。血管在气道固有层中的数量和所占面积比例增加。即血管密度增加。哮喘气道的血管变化可引起气道功能的改变。炎症反应与血管生成密切相关，作为炎性细胞进入气道的通路，气道血管结构和功能的异常也有助于哮喘气道炎症反应的持续，可引起以下病理生理和临床异常：气道壁水肿，加重气道狭窄，增加气道高反应性，增加炎性细胞的渗出，加重气道炎症等；哮喘发作时释放的炎症介质亦有部分促血管形成的作用，血管内皮生长因子(Vascular endothelial growth factor，VEGF)在其中起主要作用。哮喘急性发作期气道的平滑肌和小血管痉挛，气道组织缺氧严重，在缺氧的环境中VEGF显著增加，促进血管的构建及生长，并诱导血管的扩张。多种炎症介质的促血管形成作用都是由VEGF的作用介导的。
本课题研究的目的就是通过对发作期哮喘的临床和实验研究观察气道重塑及气道内血管生成的改变，初步探讨其机制及意义。研究中药在抑制气道血管生成，延缓气道重塑方面的作用，提高中药治疗哮喘的临床效果。探讨中药治疗哮喘的可能机制，为中医药治疗哮喘提供可靠的实验和临床依据
一、临床研究内容与方法
方法
1．一般资料
自2004年12月至2007年3月共治疗发作期哮喘56例，病例来源于中国中医科学院望京医院和北京市协和医院及河南中医药大学附属医院，为住院或门诊病人，见下表将所得数据利用Excel软件进行统计分析。
2．病例选择标准
观察病例纳入标准：
(1)符合支气管哮喘诊断标准和中医哮病证类诊断标准者；
(2)符合支气管哮喘严重度分级标准之轻、中度者；
(3)年龄≥14周岁。
观察病例排除标准：
(1)符合纳入标准，但伴有已(或未)明确诊断的、可造成呼吸急促的其他疾病；
(2)符合纳入条件，但伴有心、肝、肾等一种以上脏器功能不全或(和)肿瘤、血液以及严重传染性疾病，影响疗效观察和安全性判定；
(3)符合纳入条件，但观察期间出现停药或加用与治疗哮喘有关的药物，而影响疗效判定；
(4)符合纳入条件，但对观察药物中之任意一成分有过敏史，以及于观察过程中出现过敏反应且不能排除观察药物之因素者。
剔除病例标准：
(1)因不良反应终止治疗者；
(2)不能按时复诊或失访而无法判断疗效或资料不全者；
(3)过敏体质或对本药物过敏者；
(4)治疗期间合并各种感染者。
实验过程中患者满足上述条件中任一条即可剔除。
3．临床观测指标
症状、体征
(1)症状(喘息、咳嗽、咯痰)
(2)体征(哮鸣音、舌象、脉象)
疗效性指标
(1)疗程前后测肺功能(FEV_1、PEF)
(2)疗程前后测血气分析、嗜酸性粒细胞计数(EOS)；
安全性指标
(1)血、尿、便常规
(2)心电图，肝、肾功能
4．统计学处理
全部数据运用大型统计分析软件SPSS 11.0进行计算机处理，其中
(1)等级资料采用Ridit分析(Ridit analydid)；
(2)计数资料采用卡方检验(Chi-squared test)；
(3)计量资料采用t检验(t-test)。
结果
1．喘咳宁方是治疗发作期哮喘的有效方剂，其总有效率与氨茶碱相近。
喘咳宁组的总有效率为94％，略低于氨茶碱组，氨茶碱组总有效率为95％，两组相比无统计学差异，说明二者的临床疗效相近。
2．喘咳宁方对中医证类疗效的比较。
喘咳宁组对热哮、寒哮的控显率(88％、50％)及总有效率(100％、83％)比较，统计学均有显著性差异(P＜0.01)。表明喘咳宁方对热哮的治疗作用最佳。
喘咳宁方对寒哮、虚哮的控显率(50％、50％)及总有效率(83％、88％)比较，无统计学差异(P＞0.05)，表明喘咳宁方对这两型的治疗作用相近。
3．喘咳宁方对西医证类疗效比较。
喘咳宁组对内源性、外源性、混合性哮喘的控显率分别为70％、71.42％、66.67％；总有效率分别90％、92.86％、91.67％，喘咳宁方对三者的疗效无统计学差异(均P＞0.05)，说明喘咳宁方对外源性、内源性及混合性哮喘的疗效相近。
4．喘咳宁方具有良好的止咳化痰、平喘作用，作用优于氨茶碱组。
喘咳宁组对喘息、哮鸣音症状改善的控显率(75％、72.22％)及总有效率(94.44％、94.44％)与氨茶碱组的控显率(80％、75％)及总有效率(95％、95％)比较，两者无统计学差异(均P＞0.05)，说明在缓解喘息及哮鸣音方面喘咳宁组与氨茶碱组作用相当；喘咳宁组对咳嗽及咯痰症状改善的控显率(80.56％、83.34％)及总有效率(97.22％、97.22％)与氨茶碱组的控显率(55％、45％)及总有效率(80％、70％)比较，两者存在统计学上的差异(分别为P＜0.05及P＜0.01)，喘咳宁方优于氨茶碱组，说明喘咳宁方止咳化痰作用优于氨茶碱。
5．喘咳宁方能显著改善肺功能。
疗程后喘咳宁组及氨茶碱组第1秒用力呼气量及最大呼气流速较治疗前都有所提高(P＜0.05)，说明两组都能缓解或解除支气管平滑肌痉挛，缓解气道狭窄，改善通气功能；喘咳宁组与氨茶碱组治疗效果无统计学差异(P＞0.05)，说明两组在改善肺功能方面疗效相当。
6．喘咳宁方可显著降低哮喘时外周血中异常升高的嗜酸性粒细胞(eosinophilic catonic protein，EOS)数。
疗程后喘咳宁组及氨茶碱组均能显著降低哮喘时外周血中异常升高的EOS数，减轻气道炎症，降低气道高反应性，消除管壁肿胀，使支气管分泌物减少；统计学有显著性差异(P＜0.01)，两组疗效相比无统计学差异(P＞0.05)。说明两组治疗作用相近。
7．喘咳宁方能明显降低哮喘患者血清IgE水平。
疗程后喘咳宁组及氨茶碱组均能明显降低哮喘患者血清IgE水平抑制变应性炎症发展，有效改善临床症状，统计学上有极显著性差异(P＜0.01)；两组疗效相比有统计学差异(P＜0.05)。说明喘咳宁组在抑制变应性炎症方面作用优于氨茶碱组。
8．喘咳宁方安全性评价
喘咳宁方可使异常增高的白细胞降低，统计学有显著性差异(P＜0.01)，说明喘咳宁方有较好的抗炎作用。
临床实验期间，未见患者血、尿、便常规、心电图及肝肾功能有异常改变，未见患者有其他不良反应，并能使血常规中异常增高的白细胞降低；表明喘咳宁方临床安全性良好。
结论
喘咳宁可通过降低哮喘时外周血中异常升高的EOS数、降低哮喘患者血清IgE水平、改善肺功能，而减轻气道炎症及气道高反应性，抑制变应性炎症发展，缓解或解除支气管平滑肌痉挛，缓解气道狭窄，改善通气功能，达到治疗哮喘的目的。
二、实验研究内容与方法
方法
1．一般资料
清洁级BALB／c小鼠60只，雌雄各半，体质量(20.0±2.0)g。
2．模型制备
参照《中药药理实验方法学》实验性哮喘致敏小鼠模型的建立及有关文献制备。具体操作步骤：
(1)小鼠在清洁级实验动物房适应性饲养1周后，除A组外，其余各组小鼠均经腹腔注射0.01％卵蛋白生理盐水0.2ml／20g，第7天、14天用同样的方法再各注射1次；
(2)第3次注射后于当天上午并此后每日上午8～10时给予超声雾化吸入质量分数为5％的卵蛋白生理盐水30min以激发哮喘，每日1次，连续用14天。激发时间以目的小鼠出现中度哮喘症状为度。
3．动物分组
取6-8周龄雄性BALB／c小鼠60只，体质量(20.0±2.0)g，随机分为正常对照组(A组)、模型组(B组)、喘咳宁小剂量组(C组)、喘咳宁中剂量组(D组)、喘咳宁大剂量组(E组)、地塞米松组(F组)，每组10只。
4．用药方法
每次激发哮喘前1h：
A组用生理盐水代替抗原液进行腹腔注射、雾化吸入和灌胃，用量和时间与其他组完全相同；
B组每只小鼠给予生理盐水，按0.2ml／10g灌胃。
5．标本采集
各组小鼠分别于最后一次激发后24h脱颈椎处死，眼科剪剖开胸腔，暴露气管、双肺；靠近左肺门处结扎左主支气管。于气管靠近头端剪一斜行切口，钝头18#注射器针头行气管插管，0.5ml生理盐水灌洗左肺，回收灌洗液，反复3次，收集BALF1.0-1.2ml，1500r／min室温下离心5min，取上清液分装，-20℃保存。
取右肺门区组织(约0.5cm×0.5cm)迅速置入体积分数为4％的多聚甲醛溶液中固定24h，随后置入TSJ-IA型自动组织脱水机中处理，60℃石蜡包埋，QPJ-IC生理病理切片机上切取5μm薄片4张，进行免疫组化染色。
6．统计学处理
数据以x±s标准差表示，方差齐性时，组间均数比较采用单因素方差分析，两两比较采用LSD法；否则采用秩和检验，相关分析采用Pearson法。采用SPSS11.0统计软件进行分析。
结果
1．各组引喘潜伏期延长时间动态观察比较
喘咳宁各剂量组与地塞米松组均能明显延长引喘潜伏期(P＜0.01)，其中以喘咳宁中剂量效果最好，与地塞米松组疗效相近，两者相比无统计学差异(P＞0.05)。喘咳宁大剂量组与小剂量组相比疗效相近，统计学无显著性差异(P＞0.05)。
用药第一周，地塞米松组疗效优于喘咳宁组，很快达到药效高峰；第二周，喘咳宁中剂量组效果稍优于地塞米松组，达到了中药的最佳疗效。
2．各组引咳潜伏期时间比较
喘咳宁各剂量组与地塞米松组均能明显延长引咳潜伏期(P＜0.01)，其中以喘咳宁中剂量效果最佳，从第一周开始疗效就稍优于地塞米松组，第二周止咳作用也稍优于地塞米松组，但两组相比无统计学差异(P＞0.05)，说明两者疗效相近。喘咳宁大剂量组与小剂量组相比疗效相近，统计学无显著性差异(P＞0.05)。
3．各组哮喘严重度比较
喘咳宁各剂量组均能显著降低哮喘严重度，其中以喘咳宁中剂量组效果最佳；
喘咳宁中剂量组与地塞米松组相比无统计学差异(P＞0.05)，说明两组降低哮喘严重度方面作用相近；
喘咳宁小剂量组与喘咳宁中剂量组相比无统计学差异(P＞0.05)，说明两组疗效相当；
在动态观察实验过程中，喘咳宁小剂量组和中剂量组及地塞米松组各有一例动物因喘促休克而死亡，大剂量组有三例，可能与用药量过大，小鼠耐受力下降有关。
4．喘咳宁方对发作期哮喘气道重塑的实验研究
各组BALF中TGF-β1浓度水平变化显示：
发作期哮喘小鼠BALF中TGF-β1水平显著高于正常组并且在接受变应原刺激后明显升高，统计学有极显著性差异(P＜0.01))。
喘咳宁方能显著抑制TGF-β1的表达，其中又以中剂量组效果最好。大剂量组与小剂量组治疗效果相近，统计学无显著性差异(P＞0.05)。
喘咳宁组与地塞米松组比较，两组治疗效果相近，无统计学差异(P＞0.05)。
免疫组化染色显示：
附图1正常对照组支气管肺组织结构大致正常，各级支气管上皮细胞完整，无明显炎性浸润，无明显TGF-β1合成及表达。
附图2模型组可见大量TGF-β1合成及分泌增多。证明哮喘发作期小鼠气道中TGF-β1合成及分泌增多。
附图4中剂量组及附图6地塞米松组TGF-β1合成及分泌显著减少，说明喘咳宁中剂量组及地塞米松组对发作期哮喘治疗效果最佳，两者治疗作用相近；
附图3小剂量组及附图5大剂量组TGF-β1合成及分泌显著减少，说明喘咳宁大剂量组及小剂量组对发作期哮喘有较好治疗作用。
5．喘咳宁方对发作期哮喘气道血管生成及其机制的研究
各组BALF中VEGF浓度水平变化显示：
发作期哮喘小鼠BALF中VEGF水平显著高于正常组并且在接受变应原刺激后明显升高，统计学有极显著性差异(P＜0.01))。
喘咳宁方能显著抑制VEGF的表达，其中又以中剂量组效果最好。大剂量组与小剂量组治疗效果相近，统计学无显著性差异(P＞0.05)。
喘咳宁组与地塞米松组比较，两组治疗效果相近，无统计学差异(P＞0.05)。
免疫组化染色显示：
附图7正常对照组支气管肺组织结构大致正常，各级支气管上皮细胞完整，无明显炎性浸润，无明显VEGF合成及表达。
附图8模型组可见大量VEGF表达。证明哮喘发作期小鼠气道VEGF合成及分泌增多。
附图10中剂量组及附图12地塞米松组VEGF合成及分泌显著减少，说明喘咳宁中剂量组及地塞米松组对发作期哮喘治疗效果最佳，两者治疗作用相近。
附图9小剂量组及附图11大剂量组VEGF合成及分泌显著减少，说明喘咳宁大剂量组及小剂量组对发作期哮喘有较好治疗作用。
结论
哮喘气道中TGF-β1表达明显增高，提示TGF-β1过度表达是导致气道重塑的重要因素，在哮喘气道炎症和气道重塑中发挥重要作用。
哮喘气道中VEGF表达明显增高，提示VEGF可能是哮喘气道血管生成的主要诱导因子。
喘咳宁方能显著降低VEGF表达，抑制TGF-β1。是治疗发作期哮喘的有效方药，中剂量组疗效最佳，与地塞米松组相比疗效相近，均优于喘咳宁大剂量组及喘咳宁小剂量组。喘咳宁大剂量与小剂量组相比，疗效相当。
喘咳宁方可以抑制哮喘气道血管生成和气道重塑，且提示喘咳宁方可能通过降低VEGF，抑制TGF-β1表达，从而抑制气道血管生成，延缓气道重塑。
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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 reasch
Materials and Methods
1．Patients
Fifty-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 FEV_1>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 Tests
FEV 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). FEV_1 was measured 2min after each inhalation. Airway hyperresponsiveness was expressed as provocative concentration of inhaled histamine causing a 20% fall in FEV_1 (PC20) . PC20 was determined by linear interpolation from the log10 concentration response curve. Bronchial challenge was performed in 28 asthmatic patients with FEV_1>70% predicted.
3．IgE Measurement
The 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 Measurement
The 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．Statistics
Student'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.
Results
1．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.Conclusion
In 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 Methods
1．Animals
Male 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 challenge
Mice 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 treatment
Mice 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 histology
The 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 levels
The 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 analysis
Results 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.Results
1．The CKN can prolong the latent period of cough and phlegm obviously
The 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.Conclusion
In 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 experiment
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 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.
支气管哮喘(简称哮喘)是一种由嗜酸性粒细胞、肥大细胞和淋巴细胞等多种炎症细胞参与的慢性非特异性气道炎症，产生多种炎症介质及细胞因子，作用于炎性细胞本身及气道上皮细胞、平滑肌细胞等气道结构细胞，一方面促进炎症的发展，同时也导致气道重塑，气道反应性增加，气道壁增厚，通气受阻，肺功能下降；并直接或间接导致哮喘气道内血管生成。气道重塑和气道血管生成已经成为哮喘重要的病理特征。
哮喘气道重塑是一个集合性术语，特指哮喘患者气道壁结构的改变不同于正常气道组织学改变，在哮喘患者气道中除有炎性细胞浸润外主要表现为上皮脱落、气道平滑肌层的增生和肥大及基底的增厚及其玻璃样变。1992年哮喘时“呼吸道重塑(airway remoding)”概念被明确提出。气道重塑可导致气流不可逆阻塞和持续性的气道高反应性，并能成为顽固性哮喘的病理学基础，研究它有利于防止哮喘病情的恶化；气道重塑不仅发生在哮喘的晚期，而且出现在哮喘的早期，随着哮喘的发展，气道重塑呈进行性加重，即使哮喘症状得到缓解，气道重塑仍呈持续性发展，治疗哮喘若不从气道重塑上下手，哮喘的治疗就不能从根本上得到缓解。转化生长因子β1(transforming growth factor-β1，TGF-β1)在气道重塑中发挥了重要作用。
气道血管生成是指从原有血管长出新生血管，表现为血管数目增多。血管在气道固有层中的数量和所占面积比例增加。即血管密度增加。哮喘气道的血管变化可引起气道功能的改变。炎症反应与血管生成密切相关，作为炎性细胞进入气道的通路，气道血管结构和功能的异常也有助于哮喘气道炎症反应的持续，可引起以下病理生理和临床异常：气道壁水肿，加重气道狭窄，增加气道高反应性，增加炎性细胞的渗出，加重气道炎症等；哮喘发作时释放的炎症介质亦有部分促血管形成的作用，血管内皮生长因子(Vascular endothelial growth factor，VEGF)在其中起主要作用。哮喘急性发作期气道的平滑肌和小血管痉挛，气道组织缺氧严重，在缺氧的环境中VEGF显著增加，促进血管的构建及生长，并诱导血管的扩张。多种炎症介质的促血管形成作用都是由VEGF的作用介导的。
本课题研究的目的就是通过对发作期哮喘的临床和实验研究观察气道重塑及气道内血管生成的改变，初步探讨其机制及意义。研究中药在抑制气道血管生成，延缓气道重塑方面的作用，提高中药治疗哮喘的临床效果。探讨中药治疗哮喘的可能机制，为中医药治疗哮喘提供可靠的实验和临床依据
一、临床研究内容与方法
方法
1．一般资料
自2004年12月至2007年3月共治疗发作期哮喘56例，病例来源于中国中医科学院望京医院和北京市协和医院及河南中医药大学附属医院，为住院或门诊病人，见下表将所得数据利用Excel软件进行统计分析。
2．病例选择标准
观察病例纳入标准：
(1)符合支气管哮喘诊断标准和中医哮病证类诊断标准者；
(2)符合支气管哮喘严重度分级标准之轻、中度者；
(3)年龄≥14周岁。
观察病例排除标准：
(1)符合纳入标准，但伴有已(或未)明确诊断的、可造成呼吸急促的其他疾病；
(2)符合纳入条件，但伴有心、肝、肾等一种以上脏器功能不全或(和)肿瘤、血液以及严重传染性疾病，影响疗效观察和安全性判定；
(3)符合纳入条件，但观察期间出现停药或加用与治疗哮喘有关的药物，而影响疗效判定；
(4)符合纳入条件，但对观察药物中之任意一成分有过敏史，以及于观察过程中出现过敏反应且不能排除观察药物之因素者。
剔除病例标准：
(1)因不良反应终止治疗者；
(2)不能按时复诊或失访而无法判断疗效或资料不全者；
(3)过敏体质或对本药物过敏者；
(4)治疗期间合并各种感染者。
实验过程中患者满足上述条件中任一条即可剔除。
3．临床观测指标
症状、体征
(1)症状(喘息、咳嗽、咯痰)
(2)体征(哮鸣音、舌象、脉象)
疗效性指标
(1)疗程前后测肺功能(FEV_1、PEF)
(2)疗程前后测血气分析、嗜酸性粒细胞计数(EOS)；
安全性指标
(1)血、尿、便常规
(2)心电图，肝、肾功能
4．统计学处理
全部数据运用大型统计分析软件SPSS 11.0进行计算机处理，其中
(1)等级资料采用Ridit分析(Ridit analydid)；
(2)计数资料采用卡方检验(Chi-squared test)；
(3)计量资料采用t检验(t-test)。
结果
1．喘咳宁方是治疗发作期哮喘的有效方剂，其总有效率与氨茶碱相近。
喘咳宁组的总有效率为94％，略低于氨茶碱组，氨茶碱组总有效率为95％，两组相比无统计学差异，说明二者的临床疗效相近。
2．喘咳宁方对中医证类疗效的比较。
喘咳宁组对热哮、寒哮的控显率(88％、50％)及总有效率(100％、83％)比较，统计学均有显著性差异(P＜0.01)。表明喘咳宁方对热哮的治疗作用最佳。
喘咳宁方对寒哮、虚哮的控显率(50％、50％)及总有效率(83％、88％)比较，无统计学差异(P＞0.05)，表明喘咳宁方对这两型的治疗作用相近。
3．喘咳宁方对西医证类疗效比较。
喘咳宁组对内源性、外源性、混合性哮喘的控显率分别为70％、71.42％、66.67％；总有效率分别90％、92.86％、91.67％，喘咳宁方对三者的疗效无统计学差异(均P＞0.05)，说明喘咳宁方对外源性、内源性及混合性哮喘的疗效相近。
4．喘咳宁方具有良好的止咳化痰、平喘作用，作用优于氨茶碱组。
喘咳宁组对喘息、哮鸣音症状改善的控显率(75％、72.22％)及总有效率(94.44％、94.44％)与氨茶碱组的控显率(80％、75％)及总有效率(95％、95％)比较，两者无统计学差异(均P＞0.05)，说明在缓解喘息及哮鸣音方面喘咳宁组与氨茶碱组作用相当；喘咳宁组对咳嗽及咯痰症状改善的控显率(80.56％、83.34％)及总有效率(97.22％、97.22％)与氨茶碱组的控显率(55％、45％)及总有效率(80％、70％)比较，两者存在统计学上的差异(分别为P＜0.05及P＜0.01)，喘咳宁方优于氨茶碱组，说明喘咳宁方止咳化痰作用优于氨茶碱。
5．喘咳宁方能显著改善肺功能。
疗程后喘咳宁组及氨茶碱组第1秒用力呼气量及最大呼气流速较治疗前都有所提高(P＜0.05)，说明两组都能缓解或解除支气管平滑肌痉挛，缓解气道狭窄，改善通气功能；喘咳宁组与氨茶碱组治疗效果无统计学差异(P＞0.05)，说明两组在改善肺功能方面疗效相当。
6．喘咳宁方可显著降低哮喘时外周血中异常升高的嗜酸性粒细胞(eosinophilic catonic protein，EOS)数。
疗程后喘咳宁组及氨茶碱组均能显著降低哮喘时外周血中异常升高的EOS数，减轻气道炎症，降低气道高反应性，消除管壁肿胀，使支气管分泌物减少；统计学有显著性差异(P＜0.01)，两组疗效相比无统计学差异(P＞0.05)。说明两组治疗作用相近。
7．喘咳宁方能明显降低哮喘患者血清IgE水平。
疗程后喘咳宁组及氨茶碱组均能明显降低哮喘患者血清IgE水平抑制变应性炎症发展，有效改善临床症状，统计学上有极显著性差异(P＜0.01)；两组疗效相比有统计学差异(P＜0.05)。说明喘咳宁组在抑制变应性炎症方面作用优于氨茶碱组。
8．喘咳宁方安全性评价
喘咳宁方可使异常增高的白细胞降低，统计学有显著性差异(P＜0.01)，说明喘咳宁方有较好的抗炎作用。
临床实验期间，未见患者血、尿、便常规、心电图及肝肾功能有异常改变，未见患者有其他不良反应，并能使血常规中异常增高的白细胞降低；表明喘咳宁方临床安全性良好。
结论
喘咳宁可通过降低哮喘时外周血中异常升高的EOS数、降低哮喘患者血清IgE水平、改善肺功能，而减轻气道炎症及气道高反应性，抑制变应性炎症发展，缓解或解除支气管平滑肌痉挛，缓解气道狭窄，改善通气功能，达到治疗哮喘的目的。
二、实验研究内容与方法
方法
1．一般资料
清洁级BALB／c小鼠60只，雌雄各半，体质量(20.0±2.0)g。
2．模型制备
参照《中药药理实验方法学》实验性哮喘致敏小鼠模型的建立及有关文献制备。具体操作步骤：
(1)小鼠在清洁级实验动物房适应性饲养1周后，除A组外，其余各组小鼠均经腹腔注射0.01％卵蛋白生理盐水0.2ml／20g，第7天、14天用同样的方法再各注射1次；
(2)第3次注射后于当天上午并此后每日上午8～10时给予超声雾化吸入质量分数为5％的卵蛋白生理盐水30min以激发哮喘，每日1次，连续用14天。激发时间以目的小鼠出现中度哮喘症状为度。
3．动物分组
取6-8周龄雄性BALB／c小鼠60只，体质量(20.0±2.0)g，随机分为正常对照组(A组)、模型组(B组)、喘咳宁小剂量组(C组)、喘咳宁中剂量组(D组)、喘咳宁大剂量组(E组)、地塞米松组(F组)，每组10只。
4．用药方法
每次激发哮喘前1h：
A组用生理盐水代替抗原液进行腹腔注射、雾化吸入和灌胃，用量和时间与其他组完全相同；
B组每只小鼠给予生理盐水，按0.2ml／10g灌胃。
5．标本采集
各组小鼠分别于最后一次激发后24h脱颈椎处死，眼科剪剖开胸腔，暴露气管、双肺；靠近左肺门处结扎左主支气管。于气管靠近头端剪一斜行切口，钝头18#注射器针头行气管插管，0.5ml生理盐水灌洗左肺，回收灌洗液，反复3次，收集BALF1.0-1.2ml，1500r／min室温下离心5min，取上清液分装，-20℃保存。
取右肺门区组织(约0.5cm×0.5cm)迅速置入体积分数为4％的多聚甲醛溶液中固定24h，随后置入TSJ-IA型自动组织脱水机中处理，60℃石蜡包埋，QPJ-IC生理病理切片机上切取5μm薄片4张，进行免疫组化染色。
6．统计学处理
数据以x±s标准差表示，方差齐性时，组间均数比较采用单因素方差分析，两两比较采用LSD法；否则采用秩和检验，相关分析采用Pearson法。采用SPSS11.0统计软件进行分析。
结果
1．各组引喘潜伏期延长时间动态观察比较
喘咳宁各剂量组与地塞米松组均能明显延长引喘潜伏期(P＜0.01)，其中以喘咳宁中剂量效果最好，与地塞米松组疗效相近，两者相比无统计学差异(P＞0.05)。喘咳宁大剂量组与小剂量组相比疗效相近，统计学无显著性差异(P＞0.05)。
用药第一周，地塞米松组疗效优于喘咳宁组，很快达到药效高峰；第二周，喘咳宁中剂量组效果稍优于地塞米松组，达到了中药的最佳疗效。
2．各组引咳潜伏期时间比较
喘咳宁各剂量组与地塞米松组均能明显延长引咳潜伏期(P＜0.01)，其中以喘咳宁中剂量效果最佳，从第一周开始疗效就稍优于地塞米松组，第二周止咳作用也稍优于地塞米松组，但两组相比无统计学差异(P＞0.05)，说明两者疗效相近。喘咳宁大剂量组与小剂量组相比疗效相近，统计学无显著性差异(P＞0.05)。
3．各组哮喘严重度比较
喘咳宁各剂量组均能显著降低哮喘严重度，其中以喘咳宁中剂量组效果最佳；
喘咳宁中剂量组与地塞米松组相比无统计学差异(P＞0.05)，说明两组降低哮喘严重度方面作用相近；
喘咳宁小剂量组与喘咳宁中剂量组相比无统计学差异(P＞0.05)，说明两组疗效相当；
在动态观察实验过程中，喘咳宁小剂量组和中剂量组及地塞米松组各有一例动物因喘促休克而死亡，大剂量组有三例，可能与用药量过大，小鼠耐受力下降有关。
4．喘咳宁方对发作期哮喘气道重塑的实验研究
各组BALF中TGF-β1浓度水平变化显示：
发作期哮喘小鼠BALF中TGF-β1水平显著高于正常组并且在接受变应原刺激后明显升高，统计学有极显著性差异(P＜0.01))。
喘咳宁方能显著抑制TGF-β1的表达，其中又以中剂量组效果最好。大剂量组与小剂量组治疗效果相近，统计学无显著性差异(P＞0.05)。
喘咳宁组与地塞米松组比较，两组治疗效果相近，无统计学差异(P＞0.05)。
免疫组化染色显示：
附图1正常对照组支气管肺组织结构大致正常，各级支气管上皮细胞完整，无明显炎性浸润，无明显TGF-β1合成及表达。
附图2模型组可见大量TGF-β1合成及分泌增多。证明哮喘发作期小鼠气道中TGF-β1合成及分泌增多。
附图4中剂量组及附图6地塞米松组TGF-β1合成及分泌显著减少，说明喘咳宁中剂量组及地塞米松组对发作期哮喘治疗效果最佳，两者治疗作用相近；
附图3小剂量组及附图5大剂量组TGF-β1合成及分泌显著减少，说明喘咳宁大剂量组及小剂量组对发作期哮喘有较好治疗作用。
5．喘咳宁方对发作期哮喘气道血管生成及其机制的研究
各组BALF中VEGF浓度水平变化显示：
发作期哮喘小鼠BALF中VEGF水平显著高于正常组并且在接受变应原刺激后明显升高，统计学有极显著性差异(P＜0.01))。
喘咳宁方能显著抑制VEGF的表达，其中又以中剂量组效果最好。大剂量组与小剂量组治疗效果相近，统计学无显著性差异(P＞0.05)。
喘咳宁组与地塞米松组比较，两组治疗效果相近，无统计学差异(P＞0.05)。
免疫组化染色显示：
附图7正常对照组支气管肺组织结构大致正常，各级支气管上皮细胞完整，无明显炎性浸润，无明显VEGF合成及表达。
附图8模型组可见大量VEGF表达。证明哮喘发作期小鼠气道VEGF合成及分泌增多。
附图10中剂量组及附图12地塞米松组VEGF合成及分泌显著减少，说明喘咳宁中剂量组及地塞米松组对发作期哮喘治疗效果最佳，两者治疗作用相近。
附图9小剂量组及附图11大剂量组VEGF合成及分泌显著减少，说明喘咳宁大剂量组及小剂量组对发作期哮喘有较好治疗作用。
结论
哮喘气道中TGF-β1表达明显增高，提示TGF-β1过度表达是导致气道重塑的重要因素，在哮喘气道炎症和气道重塑中发挥重要作用。
哮喘气道中VEGF表达明显增高，提示VEGF可能是哮喘气道血管生成的主要诱导因子。
喘咳宁方能显著降低VEGF表达，抑制TGF-β1。是治疗发作期哮喘的有效方药，中剂量组疗效最佳，与地塞米松组相比疗效相近，均优于喘咳宁大剂量组及喘咳宁小剂量组。喘咳宁大剂量与小剂量组相比，疗效相当。
喘咳宁方可以抑制哮喘气道血管生成和气道重塑，且提示喘咳宁方可能通过降低VEGF，抑制TGF-β1表达，从而抑制气道血管生成，延缓气道重塑。
?
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 reasch
Materials and Methods
1．Patients
Fifty-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 FEV_1>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 Tests
FEV 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). FEV_1 was measured 2min after each inhalation. Airway hyperresponsiveness was expressed as provocative concentration of inhaled histamine causing a 20% fall in FEV_1 (PC20) . PC20 was determined by linear interpolation from the log10 concentration response curve. Bronchial challenge was performed in 28 asthmatic patients with FEV_1>70% predicted.
3．IgE Measurement
The 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 Measurement
The 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．Statistics
Student'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.
Results
1．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.Conclusion
In 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 Methods
1．Animals
Male 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 challenge
Mice 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 treatment
Mice 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 histology
The 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 levels
The 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 analysis
Results 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.Results
1．The CKN can prolong the latent period of cough and phlegm obviously
The 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.Conclusion
In 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 experiment
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 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.
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