CLKC1治疗小儿上呼吸道感染后咳嗽药效学研究及机理探讨

【作者】 温丽娜；

【导师】 孙建宁；

【作者基本信息】 北京中医药大学 ， 中医学， 2010， 硕士

【摘要】 小儿上呼吸道感染后咳嗽是指患儿感冒好转后遗留咳嗽久而不愈,又称感冒后咳嗽,简称CPI。上呼吸道感染是儿科常见病,多发于冬季,常常导致小儿上感后咳嗽。部分患儿在上呼吸道感染恢复后出现咳嗽的呼吸道症状,可持续数周至数月,经常被误诊为支气管炎、喘息性支气管炎或肺炎,反复使用抗生素、止咳药等无效,给患儿带来了一定的痛苦,也给父母造成了经济负担。由于小儿上呼吸道感染后咳嗽的发病率较高,近年来其药物治疗已经倍受关注。目前,西医学治疗小儿上感后咳嗽的药物主要有组胺受体拮抗剂、中枢性镇咳药、麻醉剂、激素、肾上腺素能神经受体拮抗剂等。西药治疗小儿上感后咳嗽疗效欠佳。中医学治疗咳嗽历史悠久,经验丰富,与我国得天独厚的中药资源相结合,为中医药治疗小儿上呼吸道感染后咳嗽提供了广阔的空间。中成药携带方便,易于服用,因此将疗效显著的中药复方开发成治疗小儿上感后咳嗽的中成药,将具有广泛的经济效益和社会效益。CLKC1是以临床疗效为基础,由中医经验方开发而成的颗粒剂。该方由黄芪、金荞麦等中药组成,具有清肺化痰,止咳利咽的功能。主治小儿痰热阻肺,肺失宣肃所致咳嗽不已,咳声重浊,痰稠色黄或黄白相兼,咽干不利,或大便秘结；小儿感冒或感冒后咳嗽见上述证候者。本校中药药剂教研室将其开发为颗粒剂,拟用于小儿上呼吸道感染后咳嗽的治疗。目前研究认为上呼吸道感染后咳嗽的致病机理为气道高反应性。本课题以CLKC1的功能主治为依据,结合小儿上呼吸道感染后咳嗽的西医学发病机制,借助常规药理学动物模型对CLKC1的药效及其作用机制进行了研究。主要包括两部分研究内容：CLKC1治疗小儿上呼吸道感染后咳嗽的药效学研究；CLKC1治疗小儿上呼吸道感染后咳嗽的作用机制研究。在进行药效学研究的同时探讨CLKC1治疗小儿上呼吸道感染后咳嗽的作用机制,以期阐明其发挥药效的作用机理,为CLKC1的临床应用和新药的申报提供实验依据。1CLKC1治疗小儿上呼吸道感染后咳嗽的药效学研究1.1CLKC1镇咳作用的试验研究目的：观察CLKC1对咳嗽动物模型咳嗽潜伏时和咳嗽次数的影响,以确定其是否具有镇咳作用。方法：分别采用二氧化硫引咳小鼠模型和枸橼酸引咳豚鼠模型,预防治疗给药后观察二氧化硫引咳小鼠和枸橼酸引咳豚鼠的咳嗽潜伏时和咳嗽次数。结果：CLKC1能够减少二氧化硫、枸橼酸引发的动物咳嗽的反应次数,并且大、中、小剂量组具有良好的量效关系(p<0.05)。但对咳嗽潜伏时的影响不明显。结论：CLKC1具有明显的镇咳作用。1.2祛痰作用的试验研究目的：观察CLKC1对小鼠气管段酚红排泌量的影响,确定其是否具有祛痰作用。方法：采用气管段酚红试验法,给药后观察气管段酚红的排泌量。结果：同对照组小鼠的气管段酚红排泌量相比较,CLKC1大、中、小三个剂量组小鼠的气管段酚红排泌量均增加,大剂量增加的更为明显(p<0.05)。结论：CLKC1可以促进气管酚红的排泌,具有较明显的祛痰作用。1.3 CLKC1平喘作用的试验研究目的：通过观察CLKC1对引喘动物喘息潜伏期和倒仆时间的影响评价其平喘作用。方法：采用Ach、组胺喷雾引喘法,观察给药后Ach、组胺引喘豚鼠的喘息潜伏期和倒仆时间的。结果：CLKC1各剂量组可明显减少豚鼠的咳嗽次数且具有良好的量效关系,但对咳嗽潜伏时无明显延长作用(p<0.05)。结论：CLKC1具有平喘作用。1.4 CLKC1肠推进作用的试验研究目的：通过观察CLKC1对小肠推进运动的影响,评价其通便的药理作用.方法：以伊文思蓝灌胃,分离从幽门端至回盲部的肠管,测量其在小鼠肠内的推进距离,计算伊文思蓝推进的百分率。结果：CLKC1各剂量组可明显促进小肠蠕动,提高小肠推进率(p<0.01)结论：CLKC1可促进肠推进运动,具有通便作用。1.5 CLKC1抗炎作用的试验研究目的：通过观察CLKC1对急慢性炎症动物模型的影响,确定其是否具有抗炎作用。方法：采用大鼠足跖皮下注射角叉菜胶造成的急性炎症模型,观察CLKC1对致炎后大鼠足肿胀的影响；采用大鼠腹股沟皮下埋置棉球造成的慢性炎症模型,观察CLKC1对致炎后大鼠棉球肉芽肿的影响。结果：CLKC1中、小剂量组在多个时间点可明显减轻大鼠的足肿胀度(p<0.01),但对大鼠棉球肉芽肿的干、湿重无明显影响。结论：CLKC1在炎症早期具有抗炎作用,而在炎症晚期无明显效果。2 CLKC1治疗小儿上呼吸道感染后咳嗽的作用机制研究2.1 CLKC1对哮喘大鼠气道高反应性的影响目的：通过研究CLKC1对哮喘大鼠气道阻力,HMGB-1、IgE、IL-4、IL-13表达的影响,考察其对气道高反应性的抑制作用.方法：以卵蛋白和灭活结核杆菌菌苗激发大鼠哮喘模型,予CLKC1干预后测定其气道阻力、血清HMGB-1、肺匀浆中IgE、IL-4、IL-13的含量.结果：CLKC1可以抑制大鼠气道阻力的增加以及血清HMGB-1.肺匀浆中IgE、IL-4、IL-13含量的增高.结论：CLKC1可以通过抑制炎症介质、细胞因子含量的增高降低哮喘大鼠的气道高反应性。2.2 CLKC1对哮喘小鼠气道高反应性的影响目的：通过研究CLKC1对哮喘小鼠辣椒素咳嗽敏感性试验,NO、ET-1、IL-4、IL-13表达的影响,考察其对气道高反应性的抑制作用。方法：以卵蛋白激发Ba1b/c小鼠哮喘模型,予CLKC1干预后测定咳嗽敏感性试验辣椒素的阈值以及肺匀浆中NO、ET-1、IL-4、IL-13的含量。结果：CLKC1可以增加辣椒素咳嗽敏感性试验的阈值,抑制肺匀浆中NO、ET-1、IL-4、IL-13含量的增高。结论：CLKC1可能从减轻气道炎症、促进气道重建角度降低哮喘小鼠的气道高反应性。2.3 CLKC1对晚期炎症介质HMGB-1含量的影响目的：观察CLKC1对晚期炎症介质高迁移率族蛋白B-1(HMGB-1)含量的影响。方法：测定LPS尾静脉注射致急性肺损伤小鼠不同时段血清中HMGB-1的含量,取含量最高的时间点,CLKC1预防治疗给药,测定在含量最高时间点时急性肺损伤小鼠血清中HMGB-1的含量。结果：CLKC1能够抑制HMGB-1含量的增高(P<0.05)。结论：CLKC1对晚期炎症介质HMGB-1含量的增高具有一定的抑制作用。结论1.药效学研究表明CLKC1具有止咳、祛痰、平喘、通便、抗炎的药理作用。2.作用机制研究表明CLKC1治疗小儿上呼吸道感染后咳嗽的机理可能为从抑制细胞因子及炎性介质含量的增高,促进气道重建等角度减小气道阻力,降低气道高反应性。创新点1.监测了急性肺损伤小鼠模型血清中HMGB-1的含量随时间的变化情况,研究了中药颗粒剂对晚期炎症介质HMGB-1含量的影响。2.首次将辣椒素咳嗽敏感性试验与气道高反应动物模型相结合,以辣椒素的阈值检测气道高反应性,并研究了中药颗粒剂对气道高反应动物模型辣椒素咳嗽敏感性试验阈值的影响。更多还原

【Abstract】 Pediatric coughing after upper respiratory tract infection refers to the children’s coughing for a long time without healing while the cold restores, which is also called cough post infection, referred to as CPI. The upper respiratory tract infection is a common pediatric disease, frequently happens in winter, often leads to the post infection cough. For parts of the patients, respiratory symptoms of cough appear after the recovery of upper respiratory tract infection, lasting for several weeks to mohths, often misdiagnosed as bronchitis or pneumonia, and the repeated use of antibiotics, cough killer etc is of no effect. It not only brings the children of pain, but also causes economic burden for parents. Because of the high incidence of coughing after upper respiratory tract infection, the drug therapy has been more concerned in recent years.At present, the therapic drugs of CPI are mainly histamine receptor antagonist, central antitussive, anaesthetic, hormone, epinephrine never receptor antagonist, etc. Western medicine is not of great good effects in treating pediatric CPI. In treating cough, TCM has a long history and rich experience, our national unique traditional Chinese medicine resourse has provided a vast space for the application of TCM in treating CPI. Chinese patent medicines is convenient to carry and easy to use, so making Chinese prescription develope into Chinese patent medicines will have broad economic and social benefit based on the remarkable curative effect in treating CPI.CLKC1 is developed from experienced prescription. The function is lung-clearing and expectorant, antitussive and throat-clearing. It has been made into granules by pharmaceutical department of Beijing University of Chinese Medicine, using for the treatment of CPI.Generally speaking the main pathogenesis of CPI is AHR.With the function basis of CLKC1, combining the western medicine pathogenesis of CPI,we study pharmacological effect and function mechanism by conventional animal models. Two parts are mainly included:first a study on pharmacological effect; second the study on function mechanism. We try to clarify the function mechanism of CLKC1’s pharmacological effect, in order to provide sufficient experimental data for the clinical using and new drug’s application.1 Study on pharmacodynamics of CLKC1 in CPI treatment1.1 Study on antitussive effects of CLKC1Objective:To observe the antitussive effects of CLKC1. Methods:Antitussive effects were observed through mice cough test induced by SO2steaming and through guinea pigs cough test induced by citric acid. Results:CLKC1 decreased the frequency of mice cough induced by ammonia and guinea pigs cough induced by citric acid, prolonged the cough latent period,dose groups have good concentration response relations (p<0.05). Conclusion: CLKC1 shows certain Antitussive effects.1.2 Study on expectorant effects of CLKC1Objective:To observe expectorant effect of CLKC1. Methods:Expectorant effectswere evaluated by measuring the excretion of phenol red in mice trachea. Results: Increased the excretion of phenol red from mice trachea, promoted the movement of prepared Chinese ink in the mice trachea (p<0.05). Conclusion:CLKC1 shows certain expectorant effects.1.3 Study on Antiasthmatic effects of CLKC1Objective:To observe antiasthmatic effect of CLKC1. Methods:Antiasthmatic actions in guinea pigs were observed by histamine and acetylcholine ultrasonic atomization test. Results:CLKC1 can inhibited the guinea pigs asthma introduced by histamine and acetylcholine solution (p<0.05). Conclusion:CLKC1 shows certain antiasthmatic effects. 1.4 Study on bowel propulsion effects of CLKC1Objective:To observe the effect of CLKC1 on bowel propulsion in mice. Methods: Comput and compare the EB propulsion percentages in bowel in CLKC1 group, model group (treated with bowel propulsion supp ressants) and blank group, and the data were analyzed statistically 1. Results:CLKC1 dose groups could significantly increase EB propulsion percentages in mice(p<0.01).Conclusion:CLKC1 can promote intestine propulsive movement.1.5 Study on anti-inflammatory effects of CLKC1Objective:To observe anti-inflammatory effects of CLKC1. Methods:anti-inflammatory effects were observed by the swelling of hind paw in rats induced by carrageenin and granuloma induced by cotton pellets in rats. Results:CLKC1 medium and small doses groups can inhibite the paw edema induced by carrageenin (p<0.01), but not the granuloma induced by cotton pellet in SD rats. Conclusion:CLKC1 can inhibite early inflammation but without anti-inflammatory effects on later inflammation.2 Study on Function mechanism of CLKC1 in CPI treatment2.1 Study on effect of CLKC1 on AHR in allergic asthma rats Objective:To investigate the effects of CLKC1 on the inhibition of airway hyperresponsiveness(AHR) in allergen induced asthma in rats. Methods:Model group and CLKC1 group were challenged with ovalbumin (OVA) to reproduce asthma., the blank group only receied an injection of same amount of Al(OH)3. On the 22nd day determine airway resistance, the content of HMGB-1、IgE、IL-4、IL-13 in lung homogenate. Results: CLKC1 can reduce the rat airway resistance, and inhibit the content’s increase of HMGB-1、IgE、IL-13、IL-4 in lung homogenate(P<0.05). Conclusion:CLKC1 can reduce airway resistance and inhibit AHR.2.2 Study on effect of CLKC1 on AHR of chronic asthma murineObjective:To investigate the effects of CLKC1 on the inhibition of airway hyperresponsiveness(AHR) in allergen induced asthma in Balb/c rats. Methods:model group and CLKC1 group were challenged with ovalbumin (OVA) to reproduce asthma.,the blank group only receied an injection of same amount of Al(OH)3 On the 22nd day determine the threshold of capsaicin cough sensitivity test, the content of NO、ET-1、IL-4、IL-13 in lung homogenate and their physiological and pathological changes. Results:CLKC1 can increase the threshold of capsaicin cough sensitivity test, inhibit the content’s increase of lung homogenate’s NO、ET-1、IL-4、IL-13(P<0.05). Conclusion:CLKC1 can inhibit AHR.2.3 Study on effect of CLKC1 on serum HMGB-1 level of sepsis micObjective:To observe the effects of CLKC1 on HMGB-1 protein expression in lung tissues of mice with endotoxin shock. Methods:Except the blank group, all other groups are injected LPS (5mg/kg)by the tails vein to establish the lung tissue injury models, The blank group only receied an injection of same amount of saline. Testing the serum HMGB-1 level at 24h,36h,48h,72h,96h. Study the effect of CLKC1 on serum HMGB-1 level of sepsis mic at the point of 72h. Results:72h after tail vail injection of LPS, serum HMGB-1 gets to the highest level; CLKC1 can inhibit the content’s increase of HMGB-1 (P<0.05). Conclusion: CLKC1 can decrease serum HMGB-1 level.Conclusion1 The pharmacodynamics study shows that CLKC1 has antitussive, expectorant, antiasthmatic, bowel propulsion, anti-inflammatory effect pharmacological activity.2 Study on function mechanism of CLKC1 in treating coughing after pediatric upper respiratory tract infection indicates that CLKC1 can reduce airway resistance, inhibit the content’s increase of inflammatory mediators and cytokines, promote airway remodeling so as to decrease AHR. Innovation1 First studied the effect of Chinese medicine granule on the expression of. terminal inflammatory mediators HMGB-1.2 Combine capsaicin cough sensitivity test with AHR animal models for the first time, detecting airway resistance by the threshold of capsaicin, and also study the effect of Chinese medicine granule on the threshold of capsaicin.更多还原