三小汤治疗慢性阻塞性肺病的药效学及临床观察
摘要
研究背景
慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是一种常见的多发的具有高致残率和高致死率的慢性呼吸系统疾病,以不完全可逆的气流受限为特点。据研究,该病已排在当前死亡原因的第4位,预计至2020年将成为世界疾病经济负担的第5位。感染、吸烟、大气污染、职业性粉尘和有害气体吸入等外界致病因素在易患个体导致气道、肺实质和肺血管的慢性炎症。中性粒细胞、肺泡巨噬细胞、淋巴细胞等多种炎症细胞通过释放多种生物活性物质而参与该慢性炎症的发生,通过不同环节促进气道慢性炎症的发生和发展,如IL-1、IL-4、IL-8、TNF-a等。其中气道粘液高分泌状态和炎症反应被认为是COPD的重要原因之一,最终导致气道阻塞和气流受限。
COPD可归属于中医学的“咳嗽”、“喘证”、“痰饮”、“肺胀”等病证范畴,乃本虚标实之证,肺气虚是其根本,痰湿水饮内阻贯穿始终。张仲景《伤寒论》指出:“伤寒表不解,心下有水气,干呕,发热而咳,或渴,或利,或噎,或小便不利,少腹满,或喘者,小青龙汤主之”。“伤寒心下有水气,咳而微喘,发热不渴。服汤已,渴者,此寒去欲解也,小青龙汤主之。”《金匮要略·痰饮咳嗽病脉证并治》中指出:“病溢饮者,当发其汗,大青龙汤主之,小青龙汤亦主之。”“咳逆倚息不得卧,小青龙汤主之。”因此,小青龙汤是治疗外寒内饮证的重要方剂。小青龙汤组成为麻黄、桂枝、细辛、半夏、干姜、五味子、白芍、甘草。麻黄发汗、平喘、利水,配桂枝则增强通阳宜散之力；芍药与桂枝配伍,调和营卫；干姜大辛、大热合细辛散寒温肺化饮；五味子味酸性温敛肺止咳,防诸辛散药发散太过,耗伤正气；半夏味辛性温,降逆止呕,燥湿祛痰；炙甘草调和诸药。诸药配合共奏温里化饮,止咳平喘之功效。本方临床多用于呼吸系统疾病,为后世治疗咳喘的圣方。导师陈宝田教授延续古人经验,通过辨证施治,自拟三小汤加味,在临床中治疗慢阻肺取得良好疗效。三小汤是小青龙汤、小柴胡汤、小陷胸汤三方合并创立的新方。其中,小青龙汤是治疗外寒内饮的常用方剂。陈老认为,慢性咳喘症多有血弱气尽,腠理开,邪气因入,与正气相搏,结于胁下,故见小柴胡汤症,尤以“胸胁苦满,不欲饮食”多见。将三方合用治疗慢性阻塞性肺病,切中病机,无论寒热都可应用,适应症广。加用鱼腥草清热解毒。具体应用时,因病位深浅、病情轻重、兼夹症异同、并发症多少而灵活加减用药。
本课题组构建了慢阻肺的动物模型,实验观察的基础上证明三小汤对大鼠慢性阻塞性肺病模型具有止咳、平喘、化痰、抗过敏等疗效。因为气道粘液高分泌状态和炎症反应被认为是COPD发病的重要原因之一。所以我们在药效学的基础上,根据引起慢阻肺的重要发病原因,进一步研究三小汤治疗慢阻肺的相关药理机制。
白细胞介素-4(白介素-4,Interleukin-4,IL-4)是Ⅱ型辅助T细胞(Th2细胞)分泌的细胞因子。白细胞介素-4的生物作用,包括刺激活化B细胞和T细胞增殖、CD4+T细胞分化成Ⅱ型辅助T细胞。IL-4可能通过调节嗜酸粒细胞性炎症反应、促进气道纤维化、诱导气道黏液高分泌和气道高反应性(AHR)等途径参与COPD的发病。
白细胞介素-8(白介素-8,Interleukin-8,IL-8)是细胞趋化因子家族中的主要组分,是许多炎性疾病的重要介质,与COPD的发生、发展关系密切,是COPD病情监测和疗效判断的较好指标。
TNF-a是一种具有广泛生物学活性的前炎症细胞因子,吸烟等多种COPD的危险因素导致支气管上皮细胞受损,TNF-a分泌增加,促进炎症细胞趋化和活化,导致气道结构破坏。有研究提示多种因素导致COPD患者血清TNF-a水平升高。
水通道蛋白(aquaporins waterchannel protein, AQPs)是一种膜通道蛋白家族,负责生物体内多种组织和细胞的水的快速跨膜转运。AQP1主要在气道周围毛细血管、淋巴管及肺泡毛细血管内皮细胞和脏层胸膜间皮细胞上表达。本研究中通过检测水通道蛋白1(AQP1)在肺中的表达情况了解三小汤对改善肺水肿的作用。
我们试图通过观察影响慢性阻塞性肺病发病的3个重要的指标：白细胞介素-4、白细胞介素-8、肿瘤坏死因子观察三小汤可能的作用机制。同时观察三小汤对慢阻肺大鼠的咳嗽和呼吸频率、气管直径及观察肺部病理图片,从而推断三小汤对慢阻肺大鼠模型的止咳、平喘、抗过敏等药效学作用,具备创新性。我们希望通过以上实验研究能为该药开发提供科学依据。同时观察三小汤治疗慢性阻塞性肺病患者临床症状的改善程度及安全性评价。
第一部分大鼠慢性阻塞性肺病模型的建立及三小汤的干预作用
研究目的
研究三小汤治疗慢性阻塞性肺病的药效学。构建Wistar大鼠慢性阻塞性肺病的模型；观察三小汤不同剂量对慢性阻塞性肺病大鼠咳嗽、呼吸、肺泡灌洗液有核细胞百分率、血清细胞因子及肺内支气管扩张、水通道蛋白表达情况的影响。
研究方法
1清洁级健康Wistar大鼠,适应性饲养7d后,随机分为正常组、模型组、低剂量组、中剂量组、高剂量组和阳性对照组(沐舒坦组),每组8只。
2采用气管注脂多糖加熏烟方法构建慢性阻塞性肺病模型,4周后造模完成。各组分别给予不同处理,三小汤高剂量组(10g/kg),三小汤中剂量组(5g/kg),三小汤低剂量组(2.5g/kg),沐舒坦片西药对照组(10.4mg/kg),模型对照组(生理盐水)和正常的对照组(生理盐水),自由饮食。
3药物治疗过程中及治疗后1周观察实验动物单位时间内咳嗽和呼吸频率。
4处死大鼠,抽取肺泡灌洗液检测各种有核细胞百分率,进一步分析抗过敏效应。
5常规肺组织切片,检查支气管扩张水平。
6采用大鼠IL-4、IL-8、TNF-aELISA试剂盒检测大鼠血清内IL-4、IL-8、 TNF-a的含量。
7采用Envision二步法免疫组织化学技术检测水通道蛋白1(AQP1)在肺组织中的表达。
8统计方法
(1)计量资料结果以均数±标准差表示；
(2)不同分组用药前及用药后各组间比较应用SPSS13.0统计软件包采用one-way ANOVA方法分析及组间比较采用LSD法,如方差不齐则选择近似F检验Welch法,及Dunnett's T3组间比较。各组大鼠给药前后咳嗽频率、呼吸频率比较采用协方差分析；以P<0.05为差异有显著性。
实验结果
1造模1周后各组大鼠出现明显的咳嗽、呼吸急促症状。
2给药前三小汤低剂量组、中剂量组、高剂量组、阳性对照组、模型组咳嗽频率(F=0.220,P=0.925)、呼吸频率(F=6.879,P=0.000)差异均无统计学意义,但与正常组比较差异具有统计学意义(P<0.05),提示造模后大鼠的症状显著,造模成功。给药后整体组间咳嗽频率(F=49.040,P=0.000)、呼吸频率(F=12.990,P=0.000)比较差异具有统计学意义,三小汤高剂量组、中剂量组、低剂量组、阳性对照组与模型组比较差异均有统计学意义,提示三小汤可改善慢性阻塞性肺病大鼠的咳嗽和呼吸急促。
3治疗后1周各组大鼠肺泡灌洗液中淋巴细胞百分率(F=15.091,P=0.000)、中性粒细胞百分率(F=11.534,P=0.000)、嗜酸性粒细胞百分率(F=3.045,P=0.020)、嗜碱性粒细胞百分率(F=2.479,P=0.047)整体组间比较差异具有统计学意义,提示三小汤可有效降低肺内升高的有核细胞百分率,减轻过敏反应。
4肺组织支气管扩张整体组间比较差异具有统计学意义(F=18.557,P=0.000),三小汤低剂量组、中剂量组、高剂量组、阳性对照组与模型组比较差异均具有统计学意义(P=0.000,P=0.000,P=0.000,P=0.000),提示三小汤可有效减轻慢性阻塞性肺病大鼠肺内支气管扩张水平。
5给药后各组大鼠血清中IL-4含量(F=15.078,P=0.000)、IL-8含量(F=29.442,P=0.000)、TNF-a含量(F=18.648,P=0.000)整体组间比较差异具有统计学意义,三小汤低剂量组、中剂量组、高剂量组、阳性对照组、正常组与模型组比较差异均具有统计学意义,提示三小汤可有效降低血清IL-4、IL-8、 TNF-a的含量,减轻炎症反应。
6水通道蛋白1(AQP1)在肺中的阳性表达情况整体组间比较差异具有统计学意义(F=27.382,P=0.000),三小汤低剂量组、中剂量组、高剂量组、阳性对照组与模型组比较均具有统计学意义,提示三小汤有效促进水通道蛋白1(AQP1)在肺中的表达上调,从而增加清除和转运肺水,改善水液代谢,减轻肺水肿状态,有效保护急性肺损伤。
讨论
三小汤中药可以有效改善慢性阻塞性肺病的相关临床症状,降低肺内有核细胞的比例,减轻过敏反应,有效改善体内IL-4、IL-8、TNF-a细胞因子含量,降低炎症反应,可以减轻肺组织中支气管扩张状况并促进水通道蛋白1的表达上调,从而增加清除和转运肺水,改善水液代谢,减轻肺水肿状态以减轻肺组织损伤。
第二部分三小汤的临床疗效观察
研究目的
观察三小汤治疗慢性阻塞性肺病患者临床症状的改善程度及安全性评价。
研究方法
所有病例符合中华医学会呼吸系病学会制定的《慢性阻塞性肺疾病诊治指南(2007年修订版)》诊断标准；病例来源于南方医院中医科、呼吸内科的门诊和病房,性别不限,年龄在48岁—70岁,共60例,随机分为对照组和观察组,每组30例。对照组给予卧床休息、抗感染、扩张支气管、降低气道阻力、糖皮质激素、化痰等常规西医治疗,观察组在对照组治疗的基础上加用三小汤加味。
三小汤：细辛l0g,半夏l0g,甘草10g,五味子10g,干姜10g,桂枝10g,麻黄10g,白芍l0g,柴胡20g,黄芩15g,党参10g,生姜l0g,大枣10g,黄连10g,瓜萎30g,鱼腥草30g。
治疗前后两组均做三大常规、肝功能、肾功能检查。根据《中药新药临床研究指导原则》对临床症状进行评定。
所有数据均采用SPSS13.0统计软件进行统计学分析,计量资料用均数±标准差(x±s)表示。两组治疗疗效比较用两独立样本非参数检验,治疗后血清细胞因子含量比较应用协方差分析,治疗前后各组均数比较应用单因素方差分析(One-way ANOVA)进行统计分析,方差齐时多重比较采用LSD-t法检验；方差不齐时,用近似方差分析的Welch法,P<0.05为有统计学意义。
实验结果
1用药14天后,观察组和对照组的总有效率分别为93.33%和73.33%,观察组疗效明显优于对照组,两组比较差异有统计学意义(P=0.039),提示在常规西医治疗的基础上加用三小汤加味,可以更加有效的改善慢性阻塞性肺病患者的临床症状,提高生活质量。
2三小汤+常规西医治疗慢性阻塞性肺病与单纯常规西医治疗慢性阻塞性肺病治疗前两组血清IL-4、IL-8、TNF--a含量比较差异均无统计学意义,治疗后IL-4(F=10.874,P=0.002)、IL-8(F=378.696,P=0.000)、TNF-a(F=404.358, P=0.000)含量差异有统计学意义,提示三小汤可改善慢性阻塞性肺病患者血清内IL-4、IL-8、TNF-a细胞因子含量,减轻炎症反应。
讨论
三小汤联合常规西药治疗慢性阻塞性肺病的疗效优于单纯常规西医治疗,且安全可靠。因此,三小汤具有重要的临床意义及潜在的临床开发价值。
Background
Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease with high rate of disability and mortality, with a characteristic of Incomplete airflow limitation. As reported, this disease has ranked to the fourth cause of death, and is expected to be the world's fifth economic burden of disease in2020. Some pathogenic factors, such as infections, smoking, air pollution, occupational dust harmful gas and so on, can lead to chronic inflammation of airway, lung parenchyma, and pulmonary vasculature. But many kinds of inflammatory cells,such as, neutrophils, alveolar macrophages, lymphocytes,can release various bioactive substances(e.g. IL-1、IL-4、IL-8、TNF-α) to promote the occurrence and development of chronic airway inflammation. The airway mucus hypersecretion and inflammatory reaction is considered to be one of the important causes of COPD, which eventually can lead to airway obstruction and airflow limitation.
COPD belongs to lung disease category in Chinese traditional medicine of cough, asthma", phlegm, whose root is deficiency of air. Zhang Zhongjing in Treatise pointed out that typhoid table puzzled, heart gas, retching, fever and cough, or thirsty, or profit, or choking, or dysuria, less abdominal fullness, or asthma, which all can be treated by Xiaoqinglong Decoction. Golden Eagle and treatment of phlegm and cough pointed that Xiaoqinglong Decoction can treat the disease of anasarca, when the person is sweating. Xiaoinglong Decoction is an important prescription for treating drinking outside in cold syndrome. The composition of Xiaoqinglong Decoction is Ephedra sinica, cinnamon twig, Herba Asari, Rhizoma Pinelliae, Rhizoma Zingiberis, Fructus Schisandrae Chinensis, radices paeoniae alba, Radix liquiritiae. Ephedra sinica can deal with sweating, asthma, and diuresis, and cinnamon twig can improve the force of dispersion. The combination of cinnamon twig and peony can recuperate the body. This method is often used for the treatment of diseases of the respiratory system. Professor Chen Baotian continues the ancient experience, and has achieved good effect on the clinical treatment of COPD with sanxiaotang. Sanxiaotang includes Koshiba Koyu, Xiao Xianxiong Decoction and Xiaoqinglong decoction, which is the commonly used formula in the treatment with the cold drink. Professor chen thought that Sanxiaotang can be widely used to treat the COPD. Our research group plan to establish animal model of COPD, and explore the effect of sanxiaotang on chronic obstructive pulmonary disease in the aspects of preventing cough, phlegm, asthma, allergy. On the basis of pharmacodynamics, we further study the related pharmacological mechanism of sanxiaotang in the treatment of COPD. Interleukin-4(IL-4) secreted by II T helper cell (Th2cell) has many kinds of Biological effects, such as, the activation of B cells and T cells proliferation, differentiation of CD4+T cells into II T helper cell and so on. IL-4can participate in COPD pathogenesis by regulating the eosinophilic inflammation, promoting airway fibrosis, inducing airway mucus hypersecretion and airway hyperresponsiveness (AHR). Interleukin-8(IL-8) is the main group of chemotactic factor's family, is important mediator of many inflammatory diseases, has close relationship with the occurrence and development of COPD, and is a better indicator to monitor and assess the therapeutic effect. TNF-a is a proinflammatory cytokine with widely biological activity of. Smoking and other risk factors of COPD can lead to bronchial epithelial cell injury, increase TNF-a secretion, promote the inflammatory cell chemotaxis and activation, leading to airway structure damage. Studies suggest that many factors lead to the increase of serum level of TNF-a in COPD.
Aquaporins waterchannel protein(AQPs) is a membrane channel protein family, responsible for water rapid transmembrane transportation in various tissues and cells. AQP1is mainly expressed in airway peripheral vascular, lymphatic and alveolar capillary endothelial cells and visceral pleural mesothelial cells. In this study, we detect the expression of AQP1in lung in order to explore the effect of sanxiaotang on pulmonary edema.
We tried to explore the possible effect mechanism of sanxiaotang on COPD through three important indexes:interleukin-4, interleukin-8, tumor necrosis factor. At the same time, we observed cough and breathing frequency, pipe diameter and observation of pulmonary pathology images in the COPD rat in order to explore the effect of sanxiaotang on cough, asthma, allergy. The aim of the research is to provide the scientific basis for sanxiaotang development. Degree of improvement of clinical symptoms and safety evaluation were also observed in the patients with chronic obstructive pulmonary disease.
Part One The construction of COPD rat model and intervention effect of sanxiaotang
Objective
Explore the pharmacodynamics of sanxiaotang on COPD. To construct COPD model in Wistar rats; to observe the influence of sanxiaotang with three different doses on cough and respiratory frequency, the percentage of nuclear cells in bronchoalveolar lavage fluid, cytokines in serum, lung bronchiectasis and water channel protein expression in COPD rat.
Methods
1. Healthy Wistar rats of clean grade, adaptive feeding for7d, were randomly divided into normal group, model group, low dose group, middle dose group, high dose group and positive control group (ambroxol group),8rats in each group.
2. Using injection of LPS and smoke in trachea method to construct the model of chronic obstructive pulmonary disease for4weeks. Each group were given different treatment, sanxiaotang high dose group (10g/kg), middle dose group (5g/kg), low dose group (2.5g/kg), ambroxol tablets control group (10.4mg/kg), model control group (saline) and normal control group (physiological saline), free diet.
3. Observe cough and breathing frequency of the experimental animal per unit time in the process of drug treatment and1weeks after the treatment.
4. Sacrifice the rats, extract the bronchoalveolar lavage fluid to detect various percentage of mononuclear cells, in order to further analysis of anti-allergic effect.
5. Normal lung tissue section, check the bronchiectasis level.
6. IL-4, IL-8, TNF-a ELISA kits are used to detect IL-4, IL-8, TNF-a of serum in rat.
7. The two step immunohistochemical technique of Envision is used to detect water channel protein1(AQP1) expression in the lung tissue.
8Statistic methods
(1) Measurement results can be represented by mean±SD (2) different groups before and after treatment was compared by SPSS13statistical software package using the analysis and one-way ANOVA method, comparison between groups apply the LSD method; if the variance not neat, approximate F test Welch method will be applied, and Dunnett's T3is used to comparison between groups. The rats cough frequency, respiratory frequency before and after treatment are compared using analysis of covariance; P<0.05for the difference was significant.
Results
1. One weeks after modeling, rats in each group showed the symptoms of cough, shortness of breath.
2. Prior to the administration of three low dose group, middle dose group, high dose group, western medicine group, model group, cough frequency (F=0.220, P=0.925), respiratory rate (F=6.879, P=0.000) had no significant difference, but the difference compared with the normal group had statistical significance (P<0.05), suggested that symptoms in rats after building the model is significantly different from before. After administration, the cough frequency (F=49.040, P=0.000), respiratory rate (F=12.990, P=0.000) overall group were significant different. Sanxiaotang high dose group, middle dose group, low dose group, western medicine group compared with the model group all were statistically significant, suggesting sanxiaotang can improve the cough and shortness of breath in chronic obstructive pulmonary disease rats.
3. One week after treatment, lymphocyte percentage solution (F=15.091, P=0.000), percentage of neutrophil (F=11.534, P=0.000), percentage of eosinophils (F=3.045, P=0.020), basophil percentage (F=2.479, P=0.047) in rats bronchoalveolar were statistically significant among the groups, suggested that sanxiaotang can effectively reduce the increased lung cells percentage, reduce the allergic reaction.
4. Lung bronchiectasis was statistically significant (F=18.557, P=0.000) among groups. Sanxiaotang low dose group, middle dose group, high dose group, western medicine group compared with the model group, the difference was statistically significant (P=0.000, P=0.000, P=0.000, P=0.000), suggested that the sanxiaotang can effectively alleviate intrapulmonary bronchial dilatation level in chronic obstructive pulmonary disease rat.
5. After administration of the content of IL-4(F=15.078, P=0.000),the content of IL-8(F=29.442, P=0.000), the content of TNF-a(F=18.648, P=0.000) in serum of rats in each group were statistically significant among groups.Sanxiaotang low dose group, middle dose group, high dose group, western medicine group, normal group compared with the model group the differences were statistically significant, suggested that the sanxiaotang can effectively reduce the content of serum IL-4, IL-8, TNF-a and the inflammatory reaction.
6. AQP1expression in the lung of the overall group is statistically significant (F=34.297, P=0.000). Sanxiaotang low dose group, middle dose group, high dose group, western medicine group and model group were statistically different, suggested that the sanxiaotang can effectively promote the water channel protein1(AQP1) expression in lung, thereby increase the removal and transport of lung water, improve water metabolism, reduce the state of pulmonary edema, and effectively protect acute lung injury.
Discussion
Traditional Chinese medicine sanxiaotang can effectively improve clinical symptoms of the chronic obstructive pulmonary disease, reduce the proportion of lung cells, reduce the allergic reaction, effectively improve IL-4, IL-8, TNF-a content, reduce inflammation, can alleviate the situation of bronchiectasis pulmonary in lung tissue and promote the up-regulation of expression of aquaporins1, thereby increase the clearance and transport of lung water, improve water metabolism, reduce the state in order to reduce the loss of lung tissue of pulmonary edema.
Part Two Clinical observation of sanxiaotang
Objective
Observe the effect of sanxiaotang on patients with chronic obstructive pulmonary disease in the aspects of improvement degree of clinical symptoms and safety evaluation.
Methods
All cases conform to the society of respiratory system formulation of the chronic obstructive pulmonary disease diagnosis and treatment guideline of Chinese Medical Association (2007Edition). Diagnostic criteria:patients from respiratory department of Internal Medicine Department of traditional Chinese medicine, Nanfang hospital outpatient and ward, sex not limited, at the age of48-70years old, a total of60cases, were randomly divided into control group and the observation group,30cases each group. The control group was treated with bed rest, antibiotics, bronchodilators, reduce airway resistance, glucocorticoid, phlegm and other conventional treatment of Western medicine. The observation group were treated with sanxiaotang on the basis of the treatment of the control group.
Sanxiaotang:Asarum10g, Pinellia tuber10g, Licorice10g, Schisandra10g, dried ginger10g, Cassia twig10g, Ephedra10g, Radix Paeoniae Alba10g, Bupleurum20g, Scutellaria15g, Codonopsis10g, Ginger10g, Jujube10g, Coptis chinensis10g, Trichosanthes30g, Houttuynia cordata30g.
The patients in the two groups were under three conventional tests, liver function, renal function tests before and after treatment. We assess the clinical symptom according to the guiding principle of research on new drugs.
All data were analyzed with statistical software SPSS13.0.(1) Measurement results can be represented by mean±SD.(2)The treatment efficacy of the two groups was compared with two independent samples nonparametric tests. The comparison of cytokine content in the serum after treatment applied analysis of covariance. Each group before and after treatment was compared using analysis of variance (One-way ANOVA) for statistical analysis. When the variance is neat using LSD-t multiple comparison test; variance not neat, with approximate Welch method of analysis of variance, P<0.05was statistically significant.
Results
1Fourteen days after treatment, the total efficiency of the observation group and the control group was93.33%and73.33%respectively. The observation group was better than control group, there was significant difference between two groups (P=0.039). This suggested that on the basis of conventional treatment, sanxiaotang can more effectively improve the clinical symptoms of chronic obstructive pulmonary disease of the patients, improve the quality of life.
2The comparison of IL-4, IL-8, TNF between routine western medicine treatment group and sangxiaotang+western medicine treatment group before treatment had no statistically significant different. IL-4(F=10.874, P=0.002), IL-8(F=378.696, P=0.000), TNF a (F=404.358, P=0.000) after treatment have statistical differences. This suggested that the sanxiaotang can improve the content of IL-4, IL-8, TNF in the serum of patients with chronic obstructive pulmonary disease, and can reduce the inflammatory reaction.
Discussion
Sanxiaotang combined with conventional western medicine in the treatment of chronic obstructive pulmonary disease has more curative effect than the simple routine treatment of Western medicine, while it's safe and reliable. Therefore, sanxiaotang has important clinical significance and potential clinical value.
慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是一种常见的多发的具有高致残率和高致死率的慢性呼吸系统疾病,以不完全可逆的气流受限为特点。据研究,该病已排在当前死亡原因的第4位,预计至2020年将成为世界疾病经济负担的第5位。感染、吸烟、大气污染、职业性粉尘和有害气体吸入等外界致病因素在易患个体导致气道、肺实质和肺血管的慢性炎症。中性粒细胞、肺泡巨噬细胞、淋巴细胞等多种炎症细胞通过释放多种生物活性物质而参与该慢性炎症的发生,通过不同环节促进气道慢性炎症的发生和发展,如IL-1、IL-4、IL-8、TNF-a等。其中气道粘液高分泌状态和炎症反应被认为是COPD的重要原因之一,最终导致气道阻塞和气流受限。
COPD可归属于中医学的“咳嗽”、“喘证”、“痰饮”、“肺胀”等病证范畴,乃本虚标实之证,肺气虚是其根本,痰湿水饮内阻贯穿始终。张仲景《伤寒论》指出:“伤寒表不解,心下有水气,干呕,发热而咳,或渴,或利,或噎,或小便不利,少腹满,或喘者,小青龙汤主之”。“伤寒心下有水气,咳而微喘,发热不渴。服汤已,渴者,此寒去欲解也,小青龙汤主之。”《金匮要略·痰饮咳嗽病脉证并治》中指出:“病溢饮者,当发其汗,大青龙汤主之,小青龙汤亦主之。”“咳逆倚息不得卧,小青龙汤主之。”因此,小青龙汤是治疗外寒内饮证的重要方剂。小青龙汤组成为麻黄、桂枝、细辛、半夏、干姜、五味子、白芍、甘草。麻黄发汗、平喘、利水,配桂枝则增强通阳宜散之力；芍药与桂枝配伍,调和营卫；干姜大辛、大热合细辛散寒温肺化饮；五味子味酸性温敛肺止咳,防诸辛散药发散太过,耗伤正气；半夏味辛性温,降逆止呕,燥湿祛痰；炙甘草调和诸药。诸药配合共奏温里化饮,止咳平喘之功效。本方临床多用于呼吸系统疾病,为后世治疗咳喘的圣方。导师陈宝田教授延续古人经验,通过辨证施治,自拟三小汤加味,在临床中治疗慢阻肺取得良好疗效。三小汤是小青龙汤、小柴胡汤、小陷胸汤三方合并创立的新方。其中,小青龙汤是治疗外寒内饮的常用方剂。陈老认为,慢性咳喘症多有血弱气尽,腠理开,邪气因入,与正气相搏,结于胁下,故见小柴胡汤症,尤以“胸胁苦满,不欲饮食”多见。将三方合用治疗慢性阻塞性肺病,切中病机,无论寒热都可应用,适应症广。加用鱼腥草清热解毒。具体应用时,因病位深浅、病情轻重、兼夹症异同、并发症多少而灵活加减用药。
本课题组构建了慢阻肺的动物模型,实验观察的基础上证明三小汤对大鼠慢性阻塞性肺病模型具有止咳、平喘、化痰、抗过敏等疗效。因为气道粘液高分泌状态和炎症反应被认为是COPD发病的重要原因之一。所以我们在药效学的基础上,根据引起慢阻肺的重要发病原因,进一步研究三小汤治疗慢阻肺的相关药理机制。
白细胞介素-4(白介素-4,Interleukin-4,IL-4)是Ⅱ型辅助T细胞(Th2细胞)分泌的细胞因子。白细胞介素-4的生物作用,包括刺激活化B细胞和T细胞增殖、CD4+T细胞分化成Ⅱ型辅助T细胞。IL-4可能通过调节嗜酸粒细胞性炎症反应、促进气道纤维化、诱导气道黏液高分泌和气道高反应性(AHR)等途径参与COPD的发病。
白细胞介素-8(白介素-8,Interleukin-8,IL-8)是细胞趋化因子家族中的主要组分,是许多炎性疾病的重要介质,与COPD的发生、发展关系密切,是COPD病情监测和疗效判断的较好指标。
TNF-a是一种具有广泛生物学活性的前炎症细胞因子,吸烟等多种COPD的危险因素导致支气管上皮细胞受损,TNF-a分泌增加,促进炎症细胞趋化和活化,导致气道结构破坏。有研究提示多种因素导致COPD患者血清TNF-a水平升高。
水通道蛋白(aquaporins waterchannel protein, AQPs)是一种膜通道蛋白家族,负责生物体内多种组织和细胞的水的快速跨膜转运。AQP1主要在气道周围毛细血管、淋巴管及肺泡毛细血管内皮细胞和脏层胸膜间皮细胞上表达。本研究中通过检测水通道蛋白1(AQP1)在肺中的表达情况了解三小汤对改善肺水肿的作用。
我们试图通过观察影响慢性阻塞性肺病发病的3个重要的指标：白细胞介素-4、白细胞介素-8、肿瘤坏死因子观察三小汤可能的作用机制。同时观察三小汤对慢阻肺大鼠的咳嗽和呼吸频率、气管直径及观察肺部病理图片,从而推断三小汤对慢阻肺大鼠模型的止咳、平喘、抗过敏等药效学作用,具备创新性。我们希望通过以上实验研究能为该药开发提供科学依据。同时观察三小汤治疗慢性阻塞性肺病患者临床症状的改善程度及安全性评价。
第一部分大鼠慢性阻塞性肺病模型的建立及三小汤的干预作用
研究目的
研究三小汤治疗慢性阻塞性肺病的药效学。构建Wistar大鼠慢性阻塞性肺病的模型；观察三小汤不同剂量对慢性阻塞性肺病大鼠咳嗽、呼吸、肺泡灌洗液有核细胞百分率、血清细胞因子及肺内支气管扩张、水通道蛋白表达情况的影响。
研究方法
1清洁级健康Wistar大鼠,适应性饲养7d后,随机分为正常组、模型组、低剂量组、中剂量组、高剂量组和阳性对照组(沐舒坦组),每组8只。
2采用气管注脂多糖加熏烟方法构建慢性阻塞性肺病模型,4周后造模完成。各组分别给予不同处理,三小汤高剂量组(10g/kg),三小汤中剂量组(5g/kg),三小汤低剂量组(2.5g/kg),沐舒坦片西药对照组(10.4mg/kg),模型对照组(生理盐水)和正常的对照组(生理盐水),自由饮食。
3药物治疗过程中及治疗后1周观察实验动物单位时间内咳嗽和呼吸频率。
4处死大鼠,抽取肺泡灌洗液检测各种有核细胞百分率,进一步分析抗过敏效应。
5常规肺组织切片,检查支气管扩张水平。
6采用大鼠IL-4、IL-8、TNF-aELISA试剂盒检测大鼠血清内IL-4、IL-8、 TNF-a的含量。
7采用Envision二步法免疫组织化学技术检测水通道蛋白1(AQP1)在肺组织中的表达。
8统计方法
(1)计量资料结果以均数±标准差表示；
(2)不同分组用药前及用药后各组间比较应用SPSS13.0统计软件包采用one-way ANOVA方法分析及组间比较采用LSD法,如方差不齐则选择近似F检验Welch法,及Dunnett's T3组间比较。各组大鼠给药前后咳嗽频率、呼吸频率比较采用协方差分析；以P<0.05为差异有显著性。
实验结果
1造模1周后各组大鼠出现明显的咳嗽、呼吸急促症状。
2给药前三小汤低剂量组、中剂量组、高剂量组、阳性对照组、模型组咳嗽频率(F=0.220,P=0.925)、呼吸频率(F=6.879,P=0.000)差异均无统计学意义,但与正常组比较差异具有统计学意义(P<0.05),提示造模后大鼠的症状显著,造模成功。给药后整体组间咳嗽频率(F=49.040,P=0.000)、呼吸频率(F=12.990,P=0.000)比较差异具有统计学意义,三小汤高剂量组、中剂量组、低剂量组、阳性对照组与模型组比较差异均有统计学意义,提示三小汤可改善慢性阻塞性肺病大鼠的咳嗽和呼吸急促。
3治疗后1周各组大鼠肺泡灌洗液中淋巴细胞百分率(F=15.091,P=0.000)、中性粒细胞百分率(F=11.534,P=0.000)、嗜酸性粒细胞百分率(F=3.045,P=0.020)、嗜碱性粒细胞百分率(F=2.479,P=0.047)整体组间比较差异具有统计学意义,提示三小汤可有效降低肺内升高的有核细胞百分率,减轻过敏反应。
4肺组织支气管扩张整体组间比较差异具有统计学意义(F=18.557,P=0.000),三小汤低剂量组、中剂量组、高剂量组、阳性对照组与模型组比较差异均具有统计学意义(P=0.000,P=0.000,P=0.000,P=0.000),提示三小汤可有效减轻慢性阻塞性肺病大鼠肺内支气管扩张水平。
5给药后各组大鼠血清中IL-4含量(F=15.078,P=0.000)、IL-8含量(F=29.442,P=0.000)、TNF-a含量(F=18.648,P=0.000)整体组间比较差异具有统计学意义,三小汤低剂量组、中剂量组、高剂量组、阳性对照组、正常组与模型组比较差异均具有统计学意义,提示三小汤可有效降低血清IL-4、IL-8、 TNF-a的含量,减轻炎症反应。
6水通道蛋白1(AQP1)在肺中的阳性表达情况整体组间比较差异具有统计学意义(F=27.382,P=0.000),三小汤低剂量组、中剂量组、高剂量组、阳性对照组与模型组比较均具有统计学意义,提示三小汤有效促进水通道蛋白1(AQP1)在肺中的表达上调,从而增加清除和转运肺水,改善水液代谢,减轻肺水肿状态,有效保护急性肺损伤。
讨论
三小汤中药可以有效改善慢性阻塞性肺病的相关临床症状,降低肺内有核细胞的比例,减轻过敏反应,有效改善体内IL-4、IL-8、TNF-a细胞因子含量,降低炎症反应,可以减轻肺组织中支气管扩张状况并促进水通道蛋白1的表达上调,从而增加清除和转运肺水,改善水液代谢,减轻肺水肿状态以减轻肺组织损伤。
第二部分三小汤的临床疗效观察
研究目的
观察三小汤治疗慢性阻塞性肺病患者临床症状的改善程度及安全性评价。
研究方法
所有病例符合中华医学会呼吸系病学会制定的《慢性阻塞性肺疾病诊治指南(2007年修订版)》诊断标准；病例来源于南方医院中医科、呼吸内科的门诊和病房,性别不限,年龄在48岁—70岁,共60例,随机分为对照组和观察组,每组30例。对照组给予卧床休息、抗感染、扩张支气管、降低气道阻力、糖皮质激素、化痰等常规西医治疗,观察组在对照组治疗的基础上加用三小汤加味。
三小汤：细辛l0g,半夏l0g,甘草10g,五味子10g,干姜10g,桂枝10g,麻黄10g,白芍l0g,柴胡20g,黄芩15g,党参10g,生姜l0g,大枣10g,黄连10g,瓜萎30g,鱼腥草30g。
治疗前后两组均做三大常规、肝功能、肾功能检查。根据《中药新药临床研究指导原则》对临床症状进行评定。
所有数据均采用SPSS13.0统计软件进行统计学分析,计量资料用均数±标准差(x±s)表示。两组治疗疗效比较用两独立样本非参数检验,治疗后血清细胞因子含量比较应用协方差分析,治疗前后各组均数比较应用单因素方差分析(One-way ANOVA)进行统计分析,方差齐时多重比较采用LSD-t法检验；方差不齐时,用近似方差分析的Welch法,P<0.05为有统计学意义。
实验结果
1用药14天后,观察组和对照组的总有效率分别为93.33%和73.33%,观察组疗效明显优于对照组,两组比较差异有统计学意义(P=0.039),提示在常规西医治疗的基础上加用三小汤加味,可以更加有效的改善慢性阻塞性肺病患者的临床症状,提高生活质量。
2三小汤+常规西医治疗慢性阻塞性肺病与单纯常规西医治疗慢性阻塞性肺病治疗前两组血清IL-4、IL-8、TNF--a含量比较差异均无统计学意义,治疗后IL-4(F=10.874,P=0.002)、IL-8(F=378.696,P=0.000)、TNF-a(F=404.358, P=0.000)含量差异有统计学意义,提示三小汤可改善慢性阻塞性肺病患者血清内IL-4、IL-8、TNF-a细胞因子含量,减轻炎症反应。
讨论
三小汤联合常规西药治疗慢性阻塞性肺病的疗效优于单纯常规西医治疗,且安全可靠。因此,三小汤具有重要的临床意义及潜在的临床开发价值。
Background
Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease with high rate of disability and mortality, with a characteristic of Incomplete airflow limitation. As reported, this disease has ranked to the fourth cause of death, and is expected to be the world's fifth economic burden of disease in2020. Some pathogenic factors, such as infections, smoking, air pollution, occupational dust harmful gas and so on, can lead to chronic inflammation of airway, lung parenchyma, and pulmonary vasculature. But many kinds of inflammatory cells,such as, neutrophils, alveolar macrophages, lymphocytes,can release various bioactive substances(e.g. IL-1、IL-4、IL-8、TNF-α) to promote the occurrence and development of chronic airway inflammation. The airway mucus hypersecretion and inflammatory reaction is considered to be one of the important causes of COPD, which eventually can lead to airway obstruction and airflow limitation.
COPD belongs to lung disease category in Chinese traditional medicine of cough, asthma", phlegm, whose root is deficiency of air. Zhang Zhongjing in Treatise pointed out that typhoid table puzzled, heart gas, retching, fever and cough, or thirsty, or profit, or choking, or dysuria, less abdominal fullness, or asthma, which all can be treated by Xiaoqinglong Decoction. Golden Eagle and treatment of phlegm and cough pointed that Xiaoqinglong Decoction can treat the disease of anasarca, when the person is sweating. Xiaoinglong Decoction is an important prescription for treating drinking outside in cold syndrome. The composition of Xiaoqinglong Decoction is Ephedra sinica, cinnamon twig, Herba Asari, Rhizoma Pinelliae, Rhizoma Zingiberis, Fructus Schisandrae Chinensis, radices paeoniae alba, Radix liquiritiae. Ephedra sinica can deal with sweating, asthma, and diuresis, and cinnamon twig can improve the force of dispersion. The combination of cinnamon twig and peony can recuperate the body. This method is often used for the treatment of diseases of the respiratory system. Professor Chen Baotian continues the ancient experience, and has achieved good effect on the clinical treatment of COPD with sanxiaotang. Sanxiaotang includes Koshiba Koyu, Xiao Xianxiong Decoction and Xiaoqinglong decoction, which is the commonly used formula in the treatment with the cold drink. Professor chen thought that Sanxiaotang can be widely used to treat the COPD. Our research group plan to establish animal model of COPD, and explore the effect of sanxiaotang on chronic obstructive pulmonary disease in the aspects of preventing cough, phlegm, asthma, allergy. On the basis of pharmacodynamics, we further study the related pharmacological mechanism of sanxiaotang in the treatment of COPD. Interleukin-4(IL-4) secreted by II T helper cell (Th2cell) has many kinds of Biological effects, such as, the activation of B cells and T cells proliferation, differentiation of CD4+T cells into II T helper cell and so on. IL-4can participate in COPD pathogenesis by regulating the eosinophilic inflammation, promoting airway fibrosis, inducing airway mucus hypersecretion and airway hyperresponsiveness (AHR). Interleukin-8(IL-8) is the main group of chemotactic factor's family, is important mediator of many inflammatory diseases, has close relationship with the occurrence and development of COPD, and is a better indicator to monitor and assess the therapeutic effect. TNF-a is a proinflammatory cytokine with widely biological activity of. Smoking and other risk factors of COPD can lead to bronchial epithelial cell injury, increase TNF-a secretion, promote the inflammatory cell chemotaxis and activation, leading to airway structure damage. Studies suggest that many factors lead to the increase of serum level of TNF-a in COPD.
Aquaporins waterchannel protein(AQPs) is a membrane channel protein family, responsible for water rapid transmembrane transportation in various tissues and cells. AQP1is mainly expressed in airway peripheral vascular, lymphatic and alveolar capillary endothelial cells and visceral pleural mesothelial cells. In this study, we detect the expression of AQP1in lung in order to explore the effect of sanxiaotang on pulmonary edema.
We tried to explore the possible effect mechanism of sanxiaotang on COPD through three important indexes:interleukin-4, interleukin-8, tumor necrosis factor. At the same time, we observed cough and breathing frequency, pipe diameter and observation of pulmonary pathology images in the COPD rat in order to explore the effect of sanxiaotang on cough, asthma, allergy. The aim of the research is to provide the scientific basis for sanxiaotang development. Degree of improvement of clinical symptoms and safety evaluation were also observed in the patients with chronic obstructive pulmonary disease.
Part One The construction of COPD rat model and intervention effect of sanxiaotang
Objective
Explore the pharmacodynamics of sanxiaotang on COPD. To construct COPD model in Wistar rats; to observe the influence of sanxiaotang with three different doses on cough and respiratory frequency, the percentage of nuclear cells in bronchoalveolar lavage fluid, cytokines in serum, lung bronchiectasis and water channel protein expression in COPD rat.
Methods
1. Healthy Wistar rats of clean grade, adaptive feeding for7d, were randomly divided into normal group, model group, low dose group, middle dose group, high dose group and positive control group (ambroxol group),8rats in each group.
2. Using injection of LPS and smoke in trachea method to construct the model of chronic obstructive pulmonary disease for4weeks. Each group were given different treatment, sanxiaotang high dose group (10g/kg), middle dose group (5g/kg), low dose group (2.5g/kg), ambroxol tablets control group (10.4mg/kg), model control group (saline) and normal control group (physiological saline), free diet.
3. Observe cough and breathing frequency of the experimental animal per unit time in the process of drug treatment and1weeks after the treatment.
4. Sacrifice the rats, extract the bronchoalveolar lavage fluid to detect various percentage of mononuclear cells, in order to further analysis of anti-allergic effect.
5. Normal lung tissue section, check the bronchiectasis level.
6. IL-4, IL-8, TNF-a ELISA kits are used to detect IL-4, IL-8, TNF-a of serum in rat.
7. The two step immunohistochemical technique of Envision is used to detect water channel protein1(AQP1) expression in the lung tissue.
8Statistic methods
(1) Measurement results can be represented by mean±SD (2) different groups before and after treatment was compared by SPSS13statistical software package using the analysis and one-way ANOVA method, comparison between groups apply the LSD method; if the variance not neat, approximate F test Welch method will be applied, and Dunnett's T3is used to comparison between groups. The rats cough frequency, respiratory frequency before and after treatment are compared using analysis of covariance; P<0.05for the difference was significant.
Results
1. One weeks after modeling, rats in each group showed the symptoms of cough, shortness of breath.
2. Prior to the administration of three low dose group, middle dose group, high dose group, western medicine group, model group, cough frequency (F=0.220, P=0.925), respiratory rate (F=6.879, P=0.000) had no significant difference, but the difference compared with the normal group had statistical significance (P<0.05), suggested that symptoms in rats after building the model is significantly different from before. After administration, the cough frequency (F=49.040, P=0.000), respiratory rate (F=12.990, P=0.000) overall group were significant different. Sanxiaotang high dose group, middle dose group, low dose group, western medicine group compared with the model group all were statistically significant, suggesting sanxiaotang can improve the cough and shortness of breath in chronic obstructive pulmonary disease rats.
3. One week after treatment, lymphocyte percentage solution (F=15.091, P=0.000), percentage of neutrophil (F=11.534, P=0.000), percentage of eosinophils (F=3.045, P=0.020), basophil percentage (F=2.479, P=0.047) in rats bronchoalveolar were statistically significant among the groups, suggested that sanxiaotang can effectively reduce the increased lung cells percentage, reduce the allergic reaction.
4. Lung bronchiectasis was statistically significant (F=18.557, P=0.000) among groups. Sanxiaotang low dose group, middle dose group, high dose group, western medicine group compared with the model group, the difference was statistically significant (P=0.000, P=0.000, P=0.000, P=0.000), suggested that the sanxiaotang can effectively alleviate intrapulmonary bronchial dilatation level in chronic obstructive pulmonary disease rat.
5. After administration of the content of IL-4(F=15.078, P=0.000),the content of IL-8(F=29.442, P=0.000), the content of TNF-a(F=18.648, P=0.000) in serum of rats in each group were statistically significant among groups.Sanxiaotang low dose group, middle dose group, high dose group, western medicine group, normal group compared with the model group the differences were statistically significant, suggested that the sanxiaotang can effectively reduce the content of serum IL-4, IL-8, TNF-a and the inflammatory reaction.
6. AQP1expression in the lung of the overall group is statistically significant (F=34.297, P=0.000). Sanxiaotang low dose group, middle dose group, high dose group, western medicine group and model group were statistically different, suggested that the sanxiaotang can effectively promote the water channel protein1(AQP1) expression in lung, thereby increase the removal and transport of lung water, improve water metabolism, reduce the state of pulmonary edema, and effectively protect acute lung injury.
Discussion
Traditional Chinese medicine sanxiaotang can effectively improve clinical symptoms of the chronic obstructive pulmonary disease, reduce the proportion of lung cells, reduce the allergic reaction, effectively improve IL-4, IL-8, TNF-a content, reduce inflammation, can alleviate the situation of bronchiectasis pulmonary in lung tissue and promote the up-regulation of expression of aquaporins1, thereby increase the clearance and transport of lung water, improve water metabolism, reduce the state in order to reduce the loss of lung tissue of pulmonary edema.
Part Two Clinical observation of sanxiaotang
Objective
Observe the effect of sanxiaotang on patients with chronic obstructive pulmonary disease in the aspects of improvement degree of clinical symptoms and safety evaluation.
Methods
All cases conform to the society of respiratory system formulation of the chronic obstructive pulmonary disease diagnosis and treatment guideline of Chinese Medical Association (2007Edition). Diagnostic criteria:patients from respiratory department of Internal Medicine Department of traditional Chinese medicine, Nanfang hospital outpatient and ward, sex not limited, at the age of48-70years old, a total of60cases, were randomly divided into control group and the observation group,30cases each group. The control group was treated with bed rest, antibiotics, bronchodilators, reduce airway resistance, glucocorticoid, phlegm and other conventional treatment of Western medicine. The observation group were treated with sanxiaotang on the basis of the treatment of the control group.
Sanxiaotang:Asarum10g, Pinellia tuber10g, Licorice10g, Schisandra10g, dried ginger10g, Cassia twig10g, Ephedra10g, Radix Paeoniae Alba10g, Bupleurum20g, Scutellaria15g, Codonopsis10g, Ginger10g, Jujube10g, Coptis chinensis10g, Trichosanthes30g, Houttuynia cordata30g.
The patients in the two groups were under three conventional tests, liver function, renal function tests before and after treatment. We assess the clinical symptom according to the guiding principle of research on new drugs.
All data were analyzed with statistical software SPSS13.0.(1) Measurement results can be represented by mean±SD.(2)The treatment efficacy of the two groups was compared with two independent samples nonparametric tests. The comparison of cytokine content in the serum after treatment applied analysis of covariance. Each group before and after treatment was compared using analysis of variance (One-way ANOVA) for statistical analysis. When the variance is neat using LSD-t multiple comparison test; variance not neat, with approximate Welch method of analysis of variance, P<0.05was statistically significant.
Results
1Fourteen days after treatment, the total efficiency of the observation group and the control group was93.33%and73.33%respectively. The observation group was better than control group, there was significant difference between two groups (P=0.039). This suggested that on the basis of conventional treatment, sanxiaotang can more effectively improve the clinical symptoms of chronic obstructive pulmonary disease of the patients, improve the quality of life.
2The comparison of IL-4, IL-8, TNF between routine western medicine treatment group and sangxiaotang+western medicine treatment group before treatment had no statistically significant different. IL-4(F=10.874, P=0.002), IL-8(F=378.696, P=0.000), TNF a (F=404.358, P=0.000) after treatment have statistical differences. This suggested that the sanxiaotang can improve the content of IL-4, IL-8, TNF in the serum of patients with chronic obstructive pulmonary disease, and can reduce the inflammatory reaction.
Discussion
Sanxiaotang combined with conventional western medicine in the treatment of chronic obstructive pulmonary disease has more curative effect than the simple routine treatment of Western medicine, while it's safe and reliable. Therefore, sanxiaotang has important clinical significance and potential clinical value.
引文
[1]张红军.慢性阻塞性肺疾病发病机制及治疗进展[J].中国中医药现代远程教育.2010.8(8)：206.
[2]A. D. Lopez, K. Shibuya, C. Rao. Chronic obstructive pulmonary disease: current burden and future projections[J].Eur Resp ir J,2006,27:397-412.
[3]中华医学会呼吸病学分会慢性阻塞性肺疾病学组.慢性阻塞性肺疾病诊治指南(2007年修订版)[S].中华结核和呼吸杂志,2007,30(1)：8-17.
[4]Vogelmeier, C.;Worth, H.COPD-a historical review, current management and research perspectives[J].Pneumologie,2010,64(9):550-554.
[5]Wang, D.; Wang, Y; Liu, Y. N.Experimental pulmonary infection and colonization of Haemophilus influenzae in emphysematous hamsters[J]. Pulm Pharmacol Ther,2010,23(4):292-299.
[6]Kherad O, Bridevaux P O, Kaiser L, et al. [Viral infection in acute exacerbation of COPD][J]. Rev Med Suisse,2011,7(317):2222-2226.
[7]Buttafuoco F, Peche R, Vander E B, et al. Mycobacterium marinum infection causing extensive tissue destruction in a hypoxic COPD patient[J]. Acta Clin Belg,2011,66(4):315-317.
[8]Global Initiative for Chronic Obstructive Lung Disease.(2006) [R/OL].http://:www.goldcopd.org
[9]陈杭薇.慢性阻塞性肺疾病的治疗进展[J].中国全科医学,2002,5(6)：499.
[10]周小斌.慢性阻塞性肺疾病的护理体会[J].中国误诊学杂志,2007,(16)：52.
[11]郭慧.急性肺栓塞39例的心电图分析[J].临床和实验医学杂志,2008,7(4)：148.
[12]彭评志,罗飞.慢性肺源性心脏病并发急性心肌梗死20例临床分析[J].临床和实验医学杂志,2008,7(4)：145.
[13]陈宝田,谢炜.经方临床应用[M]广东科技出版社,2004.8.26.
[14]Kasaian MT, Miller DK. IL-3 as a therapeutic target f or respiratory dis ease[J]. Bioch em Pharmacol,2008,76(2):147-155.
[15]Reyes, E.; Prieto, A.; de la Hera, A.Treatment with AM3 restores defective T-cell function in COPD patients[J].Chest,2006,129(3):527-535.
[16]Shirai, T.; Suda, T.; Inui, N.Correlation between peripheral blood T-cell profiles and clinical and inflammatory parameters in stable COPD[J].Allergol Int,2010, 59(1):75-82.
[17]张明,金小玲.慢性阻塞性肺病患者血清白细胞介素—8的测定及临床意义[J].上海医学检验杂志,2003,18(2)：114-115.
[18]DI STEFANO A, CAPELLI A, DONNER C F. Role of interleukin-8 in the pathogenesis and treatment of COPD [J]. Chest,2004,126(3):926-934.
[19]Ouagued, M.; Martin-Chouly, C. A.; Brinchault, G.The novel phosphodiesterase 4 inhibitor, CI-1044, inhibits LPS-induced TNF-alpha production in whole blood from COPD patients[J].Pulm Pharmacol Ther,2005,18(1):49-54.
[20]Brogger, J.; Steen, V. M.; Eiken, H. GGenetic association between COPD and polymorphisms in TNF, ADRB2 and EPHX1[J]. Eur Respir J,2006,27(4): 682-688.
[21]Basyigit, I.; Yildiz, F.; Yildirim, E.Body mass index and serum and sputum TNF-alpha levels relation in asthma and COPD[J].Tuberk Toraks,2004,52(3): 256-261.
[22]GOLD Executive Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease(Revised 2011). www.goldcopd.com.
[23]王勃.多靶点抗炎治疗老年慢性阻塞性肺疾病急性加重期临床研究[J].中国全科医学,2010,13：1760-1762.
[24]Ryu JH, Colby TV, Hartman TE, et al. Smoking-related international lung disease:a concise review[J]. ERS J,2009,17:122-132.
[25]Spurzem JR, Rennard SI. Pathogenesis of COPD[J]. Semin Respir Crit Care Med,2009,26:142-153.
[26]戴莉莉,秦斌斌,陈晓红.慢性阻塞性肺疾病合并PIF的临床研究[J].上海第二医科大学学报,2007,24：764-768.
[27]Roche WR. Inflammatory and structural changes in the small airways in bronchial asthma[J]. Am J Respir Crit Care Med,2008,157:191-194.
[28]柳涛、蔡柏蔷慢性阻塞性肺疾病诊断、处理和预防全球策略(2011年修订版)介绍。中国呼吸与危重监护杂志,2011(1)：1-12.
[29]慢性阻塞性肺疾病诊治指南(2007年修订版).中华医学会呼吸病学分会慢性阻塞性肺疾病学组.中华结核和呼吸杂志,30(1)：8-17.
[30]张建全,钟小宁,蒋明,等.慢性支气管炎、肺气肿时肺动脉高压与肺血管壁改建与炎症的关系[J].中国病理生理杂志,2006,22(9)：1811-1815.
[31]陈国忠,陈宏斌,曹霞,等.慢性阻塞性肺疾病相关性肺动脉高压与炎症反应关系的研究[J].中国综合临床,2009,25(6)：614-617.
[32]石志红,全红艳*,李忠民,等.COPD患者血中性粒细胞基础代谢的变化及其意义[J].西安医科大学学报,2000,4(21)：317-319.
[33]FeskeS, GiltnaneJ, DolmetschR, etal. Gene regulation mediated by calcium signals in T lymphocytes [J]. Nat Immunol,2001,2(4):3162-324.
[34]Kell-Welch AE, Hanson EH, Boothby HR, et al. Interleukin-4 and interleukin-13 signaling connections maps[J]. Science,2003;300(5625):1527-1528.
[35]曹岩,黄颖,钟莉莉,等.老年支气管哮喘病人血清中白细胞介素及肿瘤坏死因子的检测及意义[J].中国实验诊断学,2009；13(2)：197-199.
[36]Soler Nm, Ewig S, Torres A, et al. Airway inflammation and bronchial microbial patterns in patients with stable chronic obstructive pulmonary disease[J]. Eur Respir J,1999; 14(5):1015-1022
[37]Alaina JA, Rebecca KH, Yassine A, et al. Tumor necrosis factor a induced secret ion of RANTES and interleuk in-6 from human airway smoot-muscle cells[J]. Am J Respir Cell Mol Biol.2000,23:794-802.
[38]Sugiya H, Matsuki M. AQPs and control of vesicle volume in secretory cells[J]. J Membr Biol,2006,210(2):155-159.
[39]Kotimaki J, Sorri M, Aantaa E, et al. Prevalence of Meniere disease in Finland[J]. Laryngoscope,1999,109(5):748-753.
[40]何迎春,陈海玲,张如富.补中益气汤加减改善慢性阻塞性肺病稳定期患者生活质量的临床疗效观察[J].中华中医药刊.2010,28(3)：506-507.
[41]徐婷贞,姚晓岚.冬病夏治方治疗COPD稳定期的临床疗效观察[J].中华中医药学刊.2012,30(3)：570-572.
[42]张进.自拟清肺汤治疗COPD急性加重期30例临床观察[J].中外医学研究.2011,9(18)：75-76.
[2]A. D. Lopez, K. Shibuya, C. Rao. Chronic obstructive pulmonary disease: current burden and future projections[J].Eur Resp ir J,2006,27:397-412.
[3]中华医学会呼吸病学分会慢性阻塞性肺疾病学组.慢性阻塞性肺疾病诊治指南(2007年修订版)[S].中华结核和呼吸杂志,2007,30(1)：8-17.
[4]Vogelmeier, C.;Worth, H.COPD-a historical review, current management and research perspectives[J].Pneumologie,2010,64(9):550-554.
[5]Wang, D.; Wang, Y; Liu, Y. N.Experimental pulmonary infection and colonization of Haemophilus influenzae in emphysematous hamsters[J]. Pulm Pharmacol Ther,2010,23(4):292-299.
[6]Kherad O, Bridevaux P O, Kaiser L, et al. [Viral infection in acute exacerbation of COPD][J]. Rev Med Suisse,2011,7(317):2222-2226.
[7]Buttafuoco F, Peche R, Vander E B, et al. Mycobacterium marinum infection causing extensive tissue destruction in a hypoxic COPD patient[J]. Acta Clin Belg,2011,66(4):315-317.
[8]Global Initiative for Chronic Obstructive Lung Disease.(2006) [R/OL].http://:www.goldcopd.org
[9]陈杭薇.慢性阻塞性肺疾病的治疗进展[J].中国全科医学,2002,5(6)：499.
[10]周小斌.慢性阻塞性肺疾病的护理体会[J].中国误诊学杂志,2007,(16)：52.
[11]郭慧.急性肺栓塞39例的心电图分析[J].临床和实验医学杂志,2008,7(4)：148.
[12]彭评志,罗飞.慢性肺源性心脏病并发急性心肌梗死20例临床分析[J].临床和实验医学杂志,2008,7(4)：145.
[13]陈宝田,谢炜.经方临床应用[M]广东科技出版社,2004.8.26.
[14]Kasaian MT, Miller DK. IL-3 as a therapeutic target f or respiratory dis ease[J]. Bioch em Pharmacol,2008,76(2):147-155.
[15]Reyes, E.; Prieto, A.; de la Hera, A.Treatment with AM3 restores defective T-cell function in COPD patients[J].Chest,2006,129(3):527-535.
[16]Shirai, T.; Suda, T.; Inui, N.Correlation between peripheral blood T-cell profiles and clinical and inflammatory parameters in stable COPD[J].Allergol Int,2010, 59(1):75-82.
[17]张明,金小玲.慢性阻塞性肺病患者血清白细胞介素—8的测定及临床意义[J].上海医学检验杂志,2003,18(2)：114-115.
[18]DI STEFANO A, CAPELLI A, DONNER C F. Role of interleukin-8 in the pathogenesis and treatment of COPD [J]. Chest,2004,126(3):926-934.
[19]Ouagued, M.; Martin-Chouly, C. A.; Brinchault, G.The novel phosphodiesterase 4 inhibitor, CI-1044, inhibits LPS-induced TNF-alpha production in whole blood from COPD patients[J].Pulm Pharmacol Ther,2005,18(1):49-54.
[20]Brogger, J.; Steen, V. M.; Eiken, H. GGenetic association between COPD and polymorphisms in TNF, ADRB2 and EPHX1[J]. Eur Respir J,2006,27(4): 682-688.
[21]Basyigit, I.; Yildiz, F.; Yildirim, E.Body mass index and serum and sputum TNF-alpha levels relation in asthma and COPD[J].Tuberk Toraks,2004,52(3): 256-261.
[22]GOLD Executive Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease(Revised 2011). www.goldcopd.com.
[23]王勃.多靶点抗炎治疗老年慢性阻塞性肺疾病急性加重期临床研究[J].中国全科医学,2010,13：1760-1762.
[24]Ryu JH, Colby TV, Hartman TE, et al. Smoking-related international lung disease:a concise review[J]. ERS J,2009,17:122-132.
[25]Spurzem JR, Rennard SI. Pathogenesis of COPD[J]. Semin Respir Crit Care Med,2009,26:142-153.
[26]戴莉莉,秦斌斌,陈晓红.慢性阻塞性肺疾病合并PIF的临床研究[J].上海第二医科大学学报,2007,24：764-768.
[27]Roche WR. Inflammatory and structural changes in the small airways in bronchial asthma[J]. Am J Respir Crit Care Med,2008,157:191-194.
[28]柳涛、蔡柏蔷慢性阻塞性肺疾病诊断、处理和预防全球策略(2011年修订版)介绍。中国呼吸与危重监护杂志,2011(1)：1-12.
[29]慢性阻塞性肺疾病诊治指南(2007年修订版).中华医学会呼吸病学分会慢性阻塞性肺疾病学组.中华结核和呼吸杂志,30(1)：8-17.
[30]张建全,钟小宁,蒋明,等.慢性支气管炎、肺气肿时肺动脉高压与肺血管壁改建与炎症的关系[J].中国病理生理杂志,2006,22(9)：1811-1815.
[31]陈国忠,陈宏斌,曹霞,等.慢性阻塞性肺疾病相关性肺动脉高压与炎症反应关系的研究[J].中国综合临床,2009,25(6)：614-617.
[32]石志红,全红艳*,李忠民,等.COPD患者血中性粒细胞基础代谢的变化及其意义[J].西安医科大学学报,2000,4(21)：317-319.
[33]FeskeS, GiltnaneJ, DolmetschR, etal. Gene regulation mediated by calcium signals in T lymphocytes [J]. Nat Immunol,2001,2(4):3162-324.
[34]Kell-Welch AE, Hanson EH, Boothby HR, et al. Interleukin-4 and interleukin-13 signaling connections maps[J]. Science,2003;300(5625):1527-1528.
[35]曹岩,黄颖,钟莉莉,等.老年支气管哮喘病人血清中白细胞介素及肿瘤坏死因子的检测及意义[J].中国实验诊断学,2009；13(2)：197-199.
[36]Soler Nm, Ewig S, Torres A, et al. Airway inflammation and bronchial microbial patterns in patients with stable chronic obstructive pulmonary disease[J]. Eur Respir J,1999; 14(5):1015-1022
[37]Alaina JA, Rebecca KH, Yassine A, et al. Tumor necrosis factor a induced secret ion of RANTES and interleuk in-6 from human airway smoot-muscle cells[J]. Am J Respir Cell Mol Biol.2000,23:794-802.
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