多次盐酸灌注豚鼠食管气道高反应动物模型呼吸道及内脏传入部位神经源性炎症的探讨
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摘要
近年来,胃食管反流性疾病(gestroesophageal reflux disease,GERD)与支气管哮喘的关系一直是共同关注的焦点,随着有效的抑酸药物和便携式pH测试仪的出现,逐渐意识到胃食管反流(gestroesophageal reflux,GER)GER可能是哮喘的一个激发因素。为探讨GERD与哮喘的关系,合适的动物模型的建立是不可缺少的手段之一。目前认为GER性呼吸系统疾病的主要原因是食管-支气管反射和神经源性炎症,即由于盐酸刺激食管内的酸敏感受体促发迷走神经活动产生的,GER通过该反射引起气道高反应。在酸性胃食管反流状态下,由于食管-支气管反射,气道感觉神经受到刺激,一方面,通过轴突反射释放神经肽,引起神经源性炎症;另一方面,冲动传至中枢,与孤束核及相应节段脊髓背角的神经元形成突触,引起呼吸反射。研究发现该反射至少部分通过迷走神经介导,反射弧的感受器分布在气道上皮的感觉神经末梢,它们的胞体位于结状神经节和上段脊神经节(C_7-T_5)。P物质(substance P,SP)作为神经肽家族中最重要的成员,其发挥多种生物学效应是通过与其高亲和力受体NK-1的结合来完成的。研究显示,SP参与了哮喘和酸灌注食管的病理过程,但SP是否参与了胃食管反流性气道高反应的发病过程以及食管灌注盐酸豚鼠模型的C_7-T_5脊神经节和相应节段脊髓背角内的SP是否有变化目前尚不清楚。神经生长因子(nerve growth factor,NGF)是哮喘发病过程中的一个重要介质,支气管哮喘动物模型的研究结果显示NGF能引起气道高反应,但NGF是否参与GER性哮喘的发病机制目前尚不清楚。故以多次盐酸灌注豚鼠食管动物模型为研究对象,探讨SP在呼吸道和内脏传入部位的表达和NGF在肺组织中的表达。进一步研究食管-支气管反射及其相关的神经源性炎症的作用,试图为食管一支气管反射找出明确的解剖学证据。
1、多次盐酸灌注豚鼠食管气道高反应动物模型的建立
普通级健康雄性白化豚鼠30只,体重350g—450g,随机分为3组:A组:PBS对照组;B组:HCL模型组;C组:盐酸灌注+SR140333干预组。食管酸灌注法:盐酸氯胺酮注射液50g/L腹腔注射(1mL/kg)轻度麻醉豚鼠,仰卧固定于手术台,经口插5F胃管入食管的中、下段,以8滴/min速率缓慢灌注0.1mmol/L HCl(含0.5%胃蛋白酶)溶液,每次20min,每天一次,连续14d。对照组:用PBS代替盐酸灌注食管,方法同上。拮抗剂用法:将SR140333溶于1%DMSO的蒸馏水中,配成0.5mg/ml,按1mg/kg在每次灌酸前30分钟进行腹腔注射。
2、气道反应性测定
所有豚鼠于末次灌注后24h行气道反应性测定,戊巴比妥腹腔注射麻醉,将三组豚鼠应用AniRes2005实验动物肺功能检测分析系统(北京贝兰博科技有限公司)检测气道阻力。
3、取支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALA),BALF细胞成分计数;血管灌注,豚鼠处死,取豚鼠食管,气管,肺组织,C_7-T_5段脊髓和相应节段脊神经节。
4、HE染色观察食管,气管和肺组织病理变化
将豚鼠食管、气管和肺组织OCT包埋、切片后,进行HE染色,光镜下观察并照相。
5、免疫组织化学染色检测SP和NGF的表达
取对照组、实验组、干预组的气管、肺组织、C_7-T_5段脊髓和相应节段脊神经节切片,参照二步法免疫组织化学试剂盒步骤测定各部位SP和NGF的表达情况。
6、RT-PCR检测肺组织、C_7-T_5段脊髓SP的表达,图像分析。
7、免疫印迹检测肺组织中NGF的表达
肺组织中蛋白提取和定量后,取等量样品进行聚丙烯凝胶电泳,经一抗和二抗孵育后显色,图像分析。
8、统计学分析
所有原始数据采用SPSS11.5统计软件进行数据分析。所有结果均表示为(?)±s,样本均数比较采用单因素方差分析方法,正态分布及方差齐时,两两比较采用LSD检验;非正态分布采用轶和检验,p＜0.05为差异有统计学意义
实验结果
1、三组豚鼠食管、气管和肺组织病理检测
食管病理学HE染色显示,可见正常对照组豚鼠食管结构基本正常;模型组豚鼠食管上皮层不同程度增厚,以基底层及刺层细胞增生为主,上皮角化过度,乳头延长;固有层中性粒细胞、嗜酸性粒细胞、巨噬细胞和淋巴细胞浸润;粘膜下层内血管扩张,充血,符合轻度食管炎改变。支气管肺组织病理学HE染色显示,对照组豚鼠支气管肺组织结构基本正常;模型组气管可见纤毛柱状上皮明显增生,部分上皮脱落,粘膜和粘膜下层血管充血、扩张,腺体增生、肥大,杯状细胞增多,炎症细胞如巨噬细胞、中性粒细胞、嗜酸性粒细胞和淋巴细胞浸润,肺组织、细支气管管壁及管腔内可见大量淋巴细胞及嗜酸性粒细胞浸润,细支气管管壁增厚,管腔狭窄,部分可见粘液栓。SR140333干预组模型的病理变化均较实验组有所减轻。
2、气道反应性测定
随着乙酰胆碱浓度的成倍递增,模型组与对照组的呼气阻力均有增加,当浓度到达25μg/kg体重以上时,模型组与对照组比较有显著差异(P＜0.01)。给予SR140333干预后,呼气阻力较实验组明显改善。
3、BALF中白细胞计数及分类
模型组BALF中细胞总数及嗜酸细胞百分比均高于对照组(P＜0.01)。给予SR140333干预后,与模型组比较明显减少。
4、免疫组织化学染色及分析
正常对照组、模型组和SR140333干预组支气管、肺组织、C_7-T_5段脊神经节和相应节段脊髓背角中均有SP的表达,但强弱不同,模型组明显高于其他两组(P＜0.01)。模型组下呼吸道NGF的表达明显高于正常对照组(P＜0.01)。
5、RT-PCR检测SP的表达
各组肺组织和C_7-T_5段脊髓背角中SP含量不同,模型组高于正常对照组、SR140333抗体组(P＜0.01)。
6、免疫印迹检测NGF的表达
模型组和对照组NGF蛋白含量不同,模型组高于对照组。
结论
1、成功建立了多次盐酸灌注豚鼠食管气道高反应动物模型,为进一步探讨GERD与哮喘的关系提供了帮助。
2、SP在豚鼠下呼吸道、C_7-T_5段脊模型较对照组明显增加。提示胃食管反流性气道高反应存在气道神经源性炎症,并且神经源性炎症参与了胃食管反流性气道高反应的中枢神经的发病机制。
3、NK-1受体拮抗剂可以降低多次盐酸灌注豚鼠食管气道高反应动物模型的气道阻力。NK-1受体拮抗剂可以下调多次盐酸灌注豚鼠食管气道高反应动物模型下呼吸道和内脏传入部位SP的表达,即阻断了其气道神经源性炎症。
4、NGF在豚鼠下呼吸道有表达,NGF在GER性气道高反应豚鼠下呼吸道中表达明显,提示NGF可能参与了GER性哮喘的发病。
AIM
Gastro-oesophageal reflux disease(GERD) is believed to lead to extra-oesophageal symptoms and complications,primarily in the respiratory tract,such as asthma.With appearance of the drug of effective acid-inhibitor and the 24h PH-monitoring,that GER might be an excitation factor of asthma is recognized gradually.To discuss the relationship between the GER and asthma,appropriate animal model is necessary.The main reason of GER in respiratory tract is the oesophageal-bronchial reflux,that is the acid sensitive receptors in the oesophagus are stimulated by acid which prime the vagus nerve,the airway hyperresponsiveness is caused by this reflux.Sensory nerve in the airway is stimulated because of the oesophageal-bronchial reflux,releases neuropeptide through axon-reflux,causes neurogenic inflammation.On the other hand,impulse conducts to the CNS,forms synaptic with nTS and correspondent spinal dorsal horn,causes respiratory reflux which induces by vagus nerve partly.The receptors of the reflex arc innervate in the airway epithelium and its bady locates in the vagal nodose ganglia and the vagal jugular ganglia.Substance P acts as an important role of neuropeptide family,the diverse actions of SP are often fulfilled by binding to its high-affinity specific receptor,the NK-1.SP is confirmed to participate in the pathogenesis of asthma and acid perfusion of the esophagus,but whether SP also takes part in the process of GER inducing hyperresponsiveness and whether it expresses in the spinal ganglia and corresponding spinal dorsal horn remain open questions.Nerve growth factor,(NGF) is an important medium in pathogenesis of asthma,but whether it take part in the pathogenesis of GER inducing hyperresponsiveness is unknowed.In this study,we detected the expression of SP and NGF in the airway and viscerosensory afferent sites in the external repetitive acid-perfusion oesphageal guinea pig model to test the oesophagus - branchus reflex and neuroginic inflammation.
Material and methods
1、Animals
Specific pathogen-free albino guinea pigs weighing 350-450 g of both sexes were provided by the Laboratory Animal Center,College of Basic Medicine,China Medical University.Thirty guinea pigs were randomized into 3 groups:A group:namely the phosphate buffer solution(PBS)control(n=10);B group:experimental group(n=10);C group:SR140333 group(n=10).In the experimental group the oesophagi were challenged with a solution of hydrochloric acid(HCI) containing 1 g/L pepsin(HCl-P; pH 1.0).In the control group phosphate buffered saline(PBS)(pH 7.0) was used to treat the oesophagi.HCl was used in a 0.1 N solution,the same concentration found in the human stomach.Pepsin was added to simulate the gastric contents during the digestive process.In the HCl-P group,on the day of experimentation,guinea pigs were maintained under ketamine anesthesia delivered intraperitoneally at 50 mg/kg,with additional injections given as necessary.Animals were placed in supine position,and a 5-Fr catheter was inserted through the mouth and into the lumen of the middle and lower oesophagus.The oesophagus of each animal was perfused with HCl-P for 20 min/d for 14 d at a flow rate of 0.3 ml/min via a recirculating system.In the control group,animals received an oesophageal perfusion of PBS.During perfusion,all animals were in a 30°anti-Trendelenburg position.The guinea pigs in C group were injected pertioneally with ST 140333 30min before acid perfusion.
2、Determination of airway hypersensitivity
The determination of airway responsiveness to acetylcholine(ACh) was measured 24 hr after the last perfusion,pentobarbital anesthesia.Measure the airway hypersensitivity with AniRes 2005 animal lungs function analysis(Beijing Beilanbo Technology Co.Ltd.)
3、Vascular perfusion,sacrifice of guinea pigs,sample preparation
Cell numbers in BALF were detected;pathological changes of the lower oesophagus,bronchial and lung tissues of different groups were detected by HE staining.
4、Hematoxylin-Eosin(HE)staining of the lower oesophagus, bronchial and lung tissues pathology
The lower oesophagus,right main bronchus,and right lung tissues were removed and fixed in 4%polyformaldehyde for subsequent frozen section HE staining to observe the pathological changes using light microscopy(Olympus BX51,Japan).
5、Detecting the expression of SP by means of immunohistochemistry technique in lower respiratory tract and viscerosensory afferent sites of the guinea pigs among different groups.
SP protein was observed in groups of A B C,referencing to two-step immunohistochemistrical kits.
6、The mRNA levels of SP in the lung and spinal dorsal horn were analyzed by RT-PCR,withβ-actin as an internal control..
7、Western blot method to detect protein level of NGF in the lung.
After extraction and quantification of protein from the lung,the samples were electrophoresed with SDS-PAGE.The samples were culture with primary and secondary antibody and detected.
8、Statistical Analysis
The data were analyzed with SPSS 13.0 software.The results are expressed as mean values±standard deviation.Differences between the groups were analyzed by t-test and one-way analysis of variance(ANOVA) followed by Fisher's LSD test.A P value of＜0.05 was considered statistically significant.
Results
1.Pathological changes of the lower oesophagus,bronchial and lung tissues
No pathological changes of the oesophagus were observed in the PBS control animals.In the oesophagi of HCl-P-perfused animals,we observed infiltration of the mucosa by inflammatory cells,basal cell and spinous cell hyperproliferation,papillae hypertrophy,epithelia hyperkeratinization,squamous cell expansion,and increases in the number of fibrin cells.These results were consistent with the typical histological findings associated with low-grade reflux oesophagitis.Guinea pigs in the PBS control group presented with normal tracheal structures.In the HCl-P-treated model group, obvious hyperplasia,duplicated layer array,and nucleolus darkening of the ciliated columnar epithelium were observed.We observed large numbers of inflammatory cells, namely lymphocytes and eosinophils,infiltrating the submucosa.Airway epithelial desquamation was also evident in experimental animals.We also observed goblet cell metaplasia,ciliated columnar epithelium necrosis and degeneration,alveolus epithelium hyperproliferation,as well as in capillary vessels.Mucus plugs were seen in some of the bronchioles.Constitution of SR140333 group is releaser than that of the HCl-P-perfused group.
2.The measurement of airway hyperresponsiveness
In response to increasing doses of intravenously administered ACh,all experimental guinea pigs showed dose-dependent increases in Re.However,when the dose of ACh was increased to 25 g/kg,the airway responsiveness increased significantly in HCl-P model animals when compared with PBS control animals. Airway resistance is more lessened in SR140333 group than the HCl-P-perfused.
3.Cell numbers and types observed in guinea pig BALF
The total cell number,as well as lymphocyte and eosinophil counts were significantly greater in the BALF of HCl-P model animals than that of the PBS control animals(P＜0.01).The total cell number and eosinophil is more lessened in SR140333 group than the HCl-P-perfused.
4.Immunohistochemistry analysis for SP
SP in the lung tissues,C_7-T_5 spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups(P＜0.01).
5.The mRNA expression of SP
The ratios of mRNA/β-actin mRNA in HCL-P model animals were increased significantly compared with control group and SR140333 group.
6.Detectiong the expression of NGF in lung with Western blot experiment
Western blotting revealed significantly more intense expression of NGF in the lung from model guinea pigs than in the control(P＜0.01).
Conclusion
1.A novel external oesophageal perfusion model to induce oesophageal,tracheal and pneumonic histological injury similar to that associated with gastroesophageal reflux(GER) was evaluated successfully.
2.SP in the lung tissues,C_7-T_5 spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups,which suggested that the neurognic inflammation of airway participated in the pathogenesis in the centre nerve of GER.
3.NK-1 receptor antagonist may reduce the airway resistance in external oesophageal perfusion guinea pigs for reflux-associated respiratory symptoms.NK-1 receptor antagonist may also reduce the SP expression in the in lower respiratory tract and viscerosensory afferent sites of HCl-P-perfused group,which suggested that it restrained the neurognic inflammation of airway.
4.NGF expression in the lung of control and model groups,and found increased NGF expression in the lung of model,suggest that NGF participates in the pathogenesis of GER inducing asthma.
1、多次盐酸灌注豚鼠食管气道高反应动物模型的建立
普通级健康雄性白化豚鼠30只,体重350g—450g,随机分为3组:A组:PBS对照组;B组:HCL模型组;C组:盐酸灌注+SR140333干预组。食管酸灌注法:盐酸氯胺酮注射液50g/L腹腔注射(1mL/kg)轻度麻醉豚鼠,仰卧固定于手术台,经口插5F胃管入食管的中、下段,以8滴/min速率缓慢灌注0.1mmol/L HCl(含0.5%胃蛋白酶)溶液,每次20min,每天一次,连续14d。对照组:用PBS代替盐酸灌注食管,方法同上。拮抗剂用法:将SR140333溶于1%DMSO的蒸馏水中,配成0.5mg/ml,按1mg/kg在每次灌酸前30分钟进行腹腔注射。
2、气道反应性测定
所有豚鼠于末次灌注后24h行气道反应性测定,戊巴比妥腹腔注射麻醉,将三组豚鼠应用AniRes2005实验动物肺功能检测分析系统(北京贝兰博科技有限公司)检测气道阻力。
3、取支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALA),BALF细胞成分计数;血管灌注,豚鼠处死,取豚鼠食管,气管,肺组织,C_7-T_5段脊髓和相应节段脊神经节。
4、HE染色观察食管,气管和肺组织病理变化
将豚鼠食管、气管和肺组织OCT包埋、切片后,进行HE染色,光镜下观察并照相。
5、免疫组织化学染色检测SP和NGF的表达
取对照组、实验组、干预组的气管、肺组织、C_7-T_5段脊髓和相应节段脊神经节切片,参照二步法免疫组织化学试剂盒步骤测定各部位SP和NGF的表达情况。
6、RT-PCR检测肺组织、C_7-T_5段脊髓SP的表达,图像分析。
7、免疫印迹检测肺组织中NGF的表达
肺组织中蛋白提取和定量后,取等量样品进行聚丙烯凝胶电泳,经一抗和二抗孵育后显色,图像分析。
8、统计学分析
所有原始数据采用SPSS11.5统计软件进行数据分析。所有结果均表示为(?)±s,样本均数比较采用单因素方差分析方法,正态分布及方差齐时,两两比较采用LSD检验;非正态分布采用轶和检验,p＜0.05为差异有统计学意义
实验结果
1、三组豚鼠食管、气管和肺组织病理检测
食管病理学HE染色显示,可见正常对照组豚鼠食管结构基本正常;模型组豚鼠食管上皮层不同程度增厚,以基底层及刺层细胞增生为主,上皮角化过度,乳头延长;固有层中性粒细胞、嗜酸性粒细胞、巨噬细胞和淋巴细胞浸润;粘膜下层内血管扩张,充血,符合轻度食管炎改变。支气管肺组织病理学HE染色显示,对照组豚鼠支气管肺组织结构基本正常;模型组气管可见纤毛柱状上皮明显增生,部分上皮脱落,粘膜和粘膜下层血管充血、扩张,腺体增生、肥大,杯状细胞增多,炎症细胞如巨噬细胞、中性粒细胞、嗜酸性粒细胞和淋巴细胞浸润,肺组织、细支气管管壁及管腔内可见大量淋巴细胞及嗜酸性粒细胞浸润,细支气管管壁增厚,管腔狭窄,部分可见粘液栓。SR140333干预组模型的病理变化均较实验组有所减轻。
2、气道反应性测定
随着乙酰胆碱浓度的成倍递增,模型组与对照组的呼气阻力均有增加,当浓度到达25μg/kg体重以上时,模型组与对照组比较有显著差异(P＜0.01)。给予SR140333干预后,呼气阻力较实验组明显改善。
3、BALF中白细胞计数及分类
模型组BALF中细胞总数及嗜酸细胞百分比均高于对照组(P＜0.01)。给予SR140333干预后,与模型组比较明显减少。
4、免疫组织化学染色及分析
正常对照组、模型组和SR140333干预组支气管、肺组织、C_7-T_5段脊神经节和相应节段脊髓背角中均有SP的表达,但强弱不同,模型组明显高于其他两组(P＜0.01)。模型组下呼吸道NGF的表达明显高于正常对照组(P＜0.01)。
5、RT-PCR检测SP的表达
各组肺组织和C_7-T_5段脊髓背角中SP含量不同,模型组高于正常对照组、SR140333抗体组(P＜0.01)。
6、免疫印迹检测NGF的表达
模型组和对照组NGF蛋白含量不同,模型组高于对照组。
结论
1、成功建立了多次盐酸灌注豚鼠食管气道高反应动物模型,为进一步探讨GERD与哮喘的关系提供了帮助。
2、SP在豚鼠下呼吸道、C_7-T_5段脊模型较对照组明显增加。提示胃食管反流性气道高反应存在气道神经源性炎症,并且神经源性炎症参与了胃食管反流性气道高反应的中枢神经的发病机制。
3、NK-1受体拮抗剂可以降低多次盐酸灌注豚鼠食管气道高反应动物模型的气道阻力。NK-1受体拮抗剂可以下调多次盐酸灌注豚鼠食管气道高反应动物模型下呼吸道和内脏传入部位SP的表达,即阻断了其气道神经源性炎症。
4、NGF在豚鼠下呼吸道有表达,NGF在GER性气道高反应豚鼠下呼吸道中表达明显,提示NGF可能参与了GER性哮喘的发病。
AIM
Gastro-oesophageal reflux disease(GERD) is believed to lead to extra-oesophageal symptoms and complications,primarily in the respiratory tract,such as asthma.With appearance of the drug of effective acid-inhibitor and the 24h PH-monitoring,that GER might be an excitation factor of asthma is recognized gradually.To discuss the relationship between the GER and asthma,appropriate animal model is necessary.The main reason of GER in respiratory tract is the oesophageal-bronchial reflux,that is the acid sensitive receptors in the oesophagus are stimulated by acid which prime the vagus nerve,the airway hyperresponsiveness is caused by this reflux.Sensory nerve in the airway is stimulated because of the oesophageal-bronchial reflux,releases neuropeptide through axon-reflux,causes neurogenic inflammation.On the other hand,impulse conducts to the CNS,forms synaptic with nTS and correspondent spinal dorsal horn,causes respiratory reflux which induces by vagus nerve partly.The receptors of the reflex arc innervate in the airway epithelium and its bady locates in the vagal nodose ganglia and the vagal jugular ganglia.Substance P acts as an important role of neuropeptide family,the diverse actions of SP are often fulfilled by binding to its high-affinity specific receptor,the NK-1.SP is confirmed to participate in the pathogenesis of asthma and acid perfusion of the esophagus,but whether SP also takes part in the process of GER inducing hyperresponsiveness and whether it expresses in the spinal ganglia and corresponding spinal dorsal horn remain open questions.Nerve growth factor,(NGF) is an important medium in pathogenesis of asthma,but whether it take part in the pathogenesis of GER inducing hyperresponsiveness is unknowed.In this study,we detected the expression of SP and NGF in the airway and viscerosensory afferent sites in the external repetitive acid-perfusion oesphageal guinea pig model to test the oesophagus - branchus reflex and neuroginic inflammation.
Material and methods
1、Animals
Specific pathogen-free albino guinea pigs weighing 350-450 g of both sexes were provided by the Laboratory Animal Center,College of Basic Medicine,China Medical University.Thirty guinea pigs were randomized into 3 groups:A group:namely the phosphate buffer solution(PBS)control(n=10);B group:experimental group(n=10);C group:SR140333 group(n=10).In the experimental group the oesophagi were challenged with a solution of hydrochloric acid(HCI) containing 1 g/L pepsin(HCl-P; pH 1.0).In the control group phosphate buffered saline(PBS)(pH 7.0) was used to treat the oesophagi.HCl was used in a 0.1 N solution,the same concentration found in the human stomach.Pepsin was added to simulate the gastric contents during the digestive process.In the HCl-P group,on the day of experimentation,guinea pigs were maintained under ketamine anesthesia delivered intraperitoneally at 50 mg/kg,with additional injections given as necessary.Animals were placed in supine position,and a 5-Fr catheter was inserted through the mouth and into the lumen of the middle and lower oesophagus.The oesophagus of each animal was perfused with HCl-P for 20 min/d for 14 d at a flow rate of 0.3 ml/min via a recirculating system.In the control group,animals received an oesophageal perfusion of PBS.During perfusion,all animals were in a 30°anti-Trendelenburg position.The guinea pigs in C group were injected pertioneally with ST 140333 30min before acid perfusion.
2、Determination of airway hypersensitivity
The determination of airway responsiveness to acetylcholine(ACh) was measured 24 hr after the last perfusion,pentobarbital anesthesia.Measure the airway hypersensitivity with AniRes 2005 animal lungs function analysis(Beijing Beilanbo Technology Co.Ltd.)
3、Vascular perfusion,sacrifice of guinea pigs,sample preparation
Cell numbers in BALF were detected;pathological changes of the lower oesophagus,bronchial and lung tissues of different groups were detected by HE staining.
4、Hematoxylin-Eosin(HE)staining of the lower oesophagus, bronchial and lung tissues pathology
The lower oesophagus,right main bronchus,and right lung tissues were removed and fixed in 4%polyformaldehyde for subsequent frozen section HE staining to observe the pathological changes using light microscopy(Olympus BX51,Japan).
5、Detecting the expression of SP by means of immunohistochemistry technique in lower respiratory tract and viscerosensory afferent sites of the guinea pigs among different groups.
SP protein was observed in groups of A B C,referencing to two-step immunohistochemistrical kits.
6、The mRNA levels of SP in the lung and spinal dorsal horn were analyzed by RT-PCR,withβ-actin as an internal control..
7、Western blot method to detect protein level of NGF in the lung.
After extraction and quantification of protein from the lung,the samples were electrophoresed with SDS-PAGE.The samples were culture with primary and secondary antibody and detected.
8、Statistical Analysis
The data were analyzed with SPSS 13.0 software.The results are expressed as mean values±standard deviation.Differences between the groups were analyzed by t-test and one-way analysis of variance(ANOVA) followed by Fisher's LSD test.A P value of＜0.05 was considered statistically significant.
Results
1.Pathological changes of the lower oesophagus,bronchial and lung tissues
No pathological changes of the oesophagus were observed in the PBS control animals.In the oesophagi of HCl-P-perfused animals,we observed infiltration of the mucosa by inflammatory cells,basal cell and spinous cell hyperproliferation,papillae hypertrophy,epithelia hyperkeratinization,squamous cell expansion,and increases in the number of fibrin cells.These results were consistent with the typical histological findings associated with low-grade reflux oesophagitis.Guinea pigs in the PBS control group presented with normal tracheal structures.In the HCl-P-treated model group, obvious hyperplasia,duplicated layer array,and nucleolus darkening of the ciliated columnar epithelium were observed.We observed large numbers of inflammatory cells, namely lymphocytes and eosinophils,infiltrating the submucosa.Airway epithelial desquamation was also evident in experimental animals.We also observed goblet cell metaplasia,ciliated columnar epithelium necrosis and degeneration,alveolus epithelium hyperproliferation,as well as in capillary vessels.Mucus plugs were seen in some of the bronchioles.Constitution of SR140333 group is releaser than that of the HCl-P-perfused group.
2.The measurement of airway hyperresponsiveness
In response to increasing doses of intravenously administered ACh,all experimental guinea pigs showed dose-dependent increases in Re.However,when the dose of ACh was increased to 25 g/kg,the airway responsiveness increased significantly in HCl-P model animals when compared with PBS control animals. Airway resistance is more lessened in SR140333 group than the HCl-P-perfused.
3.Cell numbers and types observed in guinea pig BALF
The total cell number,as well as lymphocyte and eosinophil counts were significantly greater in the BALF of HCl-P model animals than that of the PBS control animals(P＜0.01).The total cell number and eosinophil is more lessened in SR140333 group than the HCl-P-perfused.
4.Immunohistochemistry analysis for SP
SP in the lung tissues,C_7-T_5 spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups(P＜0.01).
5.The mRNA expression of SP
The ratios of mRNA/β-actin mRNA in HCL-P model animals were increased significantly compared with control group and SR140333 group.
6.Detectiong the expression of NGF in lung with Western blot experiment
Western blotting revealed significantly more intense expression of NGF in the lung from model guinea pigs than in the control(P＜0.01).
Conclusion
1.A novel external oesophageal perfusion model to induce oesophageal,tracheal and pneumonic histological injury similar to that associated with gastroesophageal reflux(GER) was evaluated successfully.
2.SP in the lung tissues,C_7-T_5 spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups,which suggested that the neurognic inflammation of airway participated in the pathogenesis in the centre nerve of GER.
3.NK-1 receptor antagonist may reduce the airway resistance in external oesophageal perfusion guinea pigs for reflux-associated respiratory symptoms.NK-1 receptor antagonist may also reduce the SP expression in the in lower respiratory tract and viscerosensory afferent sites of HCl-P-perfused group,which suggested that it restrained the neurognic inflammation of airway.
4.NGF expression in the lung of control and model groups,and found increased NGF expression in the lung of model,suggest that NGF participates in the pathogenesis of GER inducing asthma.
引文
1 Alexander JA,Hunt LW,Patel AM.Revalence,pathophysiology,and treatment of patients with asthma and gastroesophageal reflux disease[J].Mayo Clin Prco,2000,75(10):1055-1063.
2 Harding SM.Gastroesophageal reflux and asthma:insight into the association[J].J Allergy Clin Immuno,1999,104(2 Pt 1):251-259.
3 Richter J E.Beyond heartburn:extraesophageal manifestations of gastroesophageal reflux disease[J].Am J Manag Care,2001,7(suppl):S6-S9.
4 刘春丽,赖克方,陈如冲,等.盐酸灌注豚鼠食管反流性疾病的模型的建立[J].中国病理生理杂志,2006,22(3):620-1,624.
5 Havemann BD,Henderson CA,El-serag HB.The association between gastro-oesophageal reflux disease and asthma:a systematic review[J].Gut.2007 Dec;56(12):1654-1664.
6 Daoui S,D'Agostino B,Gallelli L,et al.Tachykinins and airway microvascular leakage induced by HCL intra-oesophageal instillation[J].Eur Respirr J.2002.20(2):268-273
7 Harding SM,Guzzo MR.Richter JE.24-h esophageal pH testing in asthmatics:respiratory symptom correlation with esophageal acid events[J].Chest,1999,115(3):654-659
8 Wu DN,Tanifuji Y,Kobayashi H,et al.Effects of esophageal acid perfusion on airway hyperresponsiveness in patients with bronchial asthma[J].Chest,2000,118(6):1553-1556.
9 Bagnato GF,Gulli S,Giacobbe O,et al.Bronchial hyperresponsiveness in subjects with gastroesophageal reflux[J].Respiration,2000,67(5):507-509.
10 Vincent D,Cohen-Jonathan AM,Leport J,et al.Gastro-oesophageal reflux prevalence and relationship with bronchial reactivity in asthma.Eur Respir J,1997;10(10):2255-2259
11 Irwin RS,French CL,Curley FJ,et al.Chronic cough due to gastroesophageal reflux.Clinical,diagnostic,and pathogenetic aspects[J].Chest,1993,104:1511-1517.
12 Irwin RS,Madison JM,Fraire AE.The cough reflex and its relation to gastroesophageal reflux[J].Am J Med,2000,108(Suppl 4a):73s-78s.
13 Patterson RN,Johnston BT,Ardill JE,et al.Increased tachykinin levels in induced sputum from asthmatic and cough patients with acid reflux[J].Thorax,2007,62:491-495
14 刘春丽,赖克方,陈如冲等。迷走神经在盐酸灌注食管诱导的气道神经源性炎症中的作用[J]。广东医学,2007年7月第28卷第7期1027-1029
15 Wunderlich AW,Murray JA.Temporal correlation between chronic cough and gastroesophagesl reflux disease[J].Dig Dis Sci,2003,48:1050-1056.
16 Kohroqi H,Hamamoto J,Kawano O,et al.The role of substance P release in the lung with esophageal acid[J].Am J Med,2001,111(8A):25s-30s
17 Daoui S,D'Agostino B,Gallelli L,et aL Tachykinins and airway microvascular leakage induced by HCL intra-oesophageal instillation[J].Eur Respirr J,2002,20(2):268-273
18 Lundberg JM,Hokfelt T,Martlmg CR,et aL Substance P immunoreactive sensory nerves in the lower respiratory tract of various mammals including man[J]. Cell Tissue Res, 1984,235;251-256
19 Uchida Y, Nomurra A, Ohtsuka M,et al. Neurokinin A as a potent bronchoconstrictor[J]. Am Rev Respir Dis, 1987, 136:718-721
20 Magge CA,Patacchini R. Tachykinin receptors and tachykinin receptor antagonists[J].J Auton Pharmacol,1993,13;23-93
21 F Lembeck. Zur Frage der zentralen Ubertragung afferenter Impulse. Mitteilung. Das Vorkommen und die bedeutung der Substanz P. in den dorsalen Wurzein des Ruckenmarks[J].Arch Exp Pathol Pharmakol,1953,219:197-213
22 Maggi CA.Tachykinin receptors and airway pathophysiology.Eur Respir J.1993, 6:735-42
23 Zhang XL, Mak JC, Barnes PJ . Character2 ization and autoradiographic mapping of[~3H] CP96,345, a nonpeptide selective NK21 receptor antagonist in guinea pig lung. Peptides, 1995, 16 :867-72.
24 Walsh DA ,Sakmon M ,Featherstone R,et al.Differences in the distribution and characteristics of tachykinin NK21 binding sites between human and guinea pig lung. Br J Pharmacol, 1994,113:1407-15.
25 Ichikawa S, Sreedharan SP, Owen RL, et al. Immunochemical localization of type I VIP receptor and NK-1-type substance P receptor in rat lung. Am J Physiol, 1995 ,268 :584-8.
26 Bai TR, Zhou DY, Weir T ,et al.Substance P (NK1) and neurokinin A (NK2)receptor gene expression in inflammatory airway disease. Am J Physiol, 1995, 269 :309-17.
27 Khawaja AW, Rogers DF. Tachykinins : receptor to effector. Int J Biochem Cell Biol, 1996, 28:721-38
28 Hirayama Y, Yu HL,Barnes PJ ,et al. Effects of two noval tachykinin antagonists ,FK224 and FK888, on neurogenic airway plasma exudation, broncho constriction and systemic hypotension in guinea-pig in vivo.Br J Pharmacol, 1993, 108 :844-51.
29 Kaltreider HB, Ichikawa S, Byrol PK, et al. Up regulation of neuropeptides and neuropeptide receptors in a murine model of immune inflammation in lung parenchyma. Am J Respir Cell Mol Biol, 1997, 16:133-44.
30 Adcock IM, Peters M, Gelder C, et al. Increased tachykinin receptor gene expression in asthmatic lung and its modilation by steroids. Joural of Molecular Endocrinology, 1993, 11 :127-31.
31 Ichinose M, Nakajima N, Takahashi T, et al. Protection against bradykinin2 induced bronchoconst riction in asthmatic patients by neurokinin receptor antagonist . Lancet ,1992, 340:1248-51.
32 Ichinose M, Miura M, Yamauchi H, et al .A neurokinin 1-receptor antagonist improves exercise induced airway narrowing in asthmatic patients. Am J Respir Crit CareMed, 1996, 153 :93-9
33 Emonds-Alt, X., Doutremepuich, J. D., Heaulme, M., et al. In vitro and in vivo biological activities of SR 140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol. 1993,250,403-13
34 Di Sebastiano P, Grossi L, Di Mola F,et al. SR140333, a substance P receptor antagonist, influences morphological changes in rat experimental colitis. Dig Dis Sci. 1999 ,44,439-44
35 Amann R, Schuligoi R, Holzer P, Donnerer J. The non-peptide NK.1 receptor antagonist SR140333 produces long-lasting inhibition of neurogenic inflammation, but does not influence acute chemo- or thermonociception in rats. Naunyn Schmiedebergs Arch Pharmacol. 1995, 352,201-5
36 Boichot E, Germain N, Emonds-Alt X, Advenier C, Lagente V. Effects of SR 140333 and SR 48968 on antigen and substance P-induced activation of guinea-pig alveolar macrophages. Clin Exp Allergy, 1998, 28, 1299-1305
37 Veron M, Guenon I, Nenan S, et al. Interactions of tachykinin receptor antagonists with lipopolysaccharide-induced airway inflammation in mice. Clin. Exp PharmacolPhysiol. 2004, 31,634-640
38 Zijlstra FK, Hynna-Liepert TT, Dinda PK, Beck IT, Paterson WG. Microvascular permeability increases early in the course of acid-induced esophageal injury. Gastroenterology. 1991; 101:295-302
39 Underwood S, Foster M, Raeburn D, Bottoms S, Karlsson JA. Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness. Eur RespirJ, 1995,8:2104-2113
40 Friess H, Zhu Z, Liard V, et al. Neurokinin-1 receptor expression and its potential effects on tumor growth in human pancreatic cancer. Lab Invest.2003 May;83:731-42
1 Menes T, Lelcuk S, Spivak H. Pathogenesis and current management of gastroesophageal reflux-related asthma [ J ] .J Surg, 2000, 166 (8) :596-601
2 Harding SM. Pulmonary complications of gastroesophageal reflux. In: Castell DO, Richter JE, eds. In:The esophagus, 4th, edn. Philadelphia: Lippincott Williams and Wilkins, 2004:530-545.
3 Kiljander TO, Laitinen JO. The prevalence of gastroesophageal reflux disease in adult asthmatics [ J ]. Chest, 2004, 126 ( 5) :1490-1494.
4 Debley J S, Carter ER, Redding GJ . Prevalence and impact of gast roesophageal reflux in adolescent s wit h asthma : apopulation2based study [ J ] . Pediat r Pulmonol, 2006, 41 (5) :475-481
5 Diette GB, Krishnan JA, Dominici F, et al. Asthma in older patients .Factors associated with hospitalization. Arch Intern Med, 2002, 162(10);1123-1132
6 Gislason T, Janson C, Vermeire P, et al. Respiratory symptoms and nocturnal gastroesophageal reflux. A population-based study of young adults in three European countries.Chest, 2002, 121(1):158-163
7 Tomonaga T, Awad ZTT, Filipi CJ, et al. Symptom predictability of reflux-inducedrespiratory disease.Dig Dis Sci, 2002, 47(1);9-14
8 Clinquette M, Miceli S, Voltolina C, et al. The pattern of gastroesophageal reflux in asthmatic children.J Asthma, 2002, 39(2); 135-142
9 el-Serag H, Gilger M, Kuebeler M, et al.Extrsesophageal associations of gastroesophageal reflux disease in children without neruologic defects. Gastroenterology, 2001, 11(6); 1294-1299
10 Avidan B, Sonnenberg A, Schnell TG, et al.Temporal associations between coughing or wheezing and acid reflux in asthmatics.Gut, 2001, 49(6);767-772
11 Field SK. A critical review of the studies of the effects of simulated or real gastroesophageal reflux on pulmonary function in asthmatic adults. Chest. 1999; 115: 848-856.
12 Sontag SJ. Why do the published data fail to clarify the relationship between gastroesophageal reflux and asthma? Am J Med. 2000;108(suppl 4A):159S-169S.
13 Field SK. Gastroesophageal reflux and asthma: are they related? J Asthma. 1999;36:631-644
14 Bocskei C, Viczian M, Bocskei R, et al. The influence of gastroesophageal reflux disease and its treatment on asthmatic Cough [J ] . Lung, 2005, 183 (1) :53-62.
15 Kiljander TO. The role of proton pump inhibitors in the management of gast roesophageal reflux disease-related asthma and chronic cough[J ] . Am J Med, 2003, 115 (3) :65S-71S
16 Tuchman DN, Boyle JT, Pack Al et al. Comparison of airway responses following tracheal or esophageal acidification in the cat.Gastroenterology, 1984; 87(4):872-881
17 Jack CI, Calverley PM, Donnelly RJ, et al. Simultaneous tracheal and oesophageal pH measurements in asthmatic patients with gastro-oesophageal reflux. Thorax, 1995; 50(2) : 201-204
18 Ricciardolo FLM. Mechanisms of citric acid-induced bronchoconstriction [J ] . Am J Med, 2001, 111 (8): 18-24.
19 Irwin RS, Madison JM, Fraire AE. The cough reflex and its relation to gastroesophageal reflux.Am J Med, 2000, 108(Suppl 4a):73s-78s.
20 Canning BJ, Mazzone SB. Reflex Mechanisms in Gastroesophageal Reflux Disease and Asthma [J ] . Am J Med, 2003, 115 (3) :45S-48S
21 Field SK. A critical review of the studies of the effects of simulated or real gastroesophageal reflux on pulmonary function in function in asthmatic asults. Chest, 1999; 115(3);848-856
22 Schan CA, Harding SM, Haile JM, et al. Gastroesophageal reflux-induced bronchoconstriction: an intraesophageal acidinfusion study using state-of-the-art technology. Chest 1994;106:731-737
23 Harding SM, Schan CA, Guzzo MR, et al. Gastroesophageal reflux-induced bronchoconstriction: is microaspiration a factor? Chest 1995; 108:1220-1227
24 Harding SM, Guzo MR, Maples RV, et al. Gastroesophageal reflux induced bronchoconstriction: vagolytic doses of atro-pine diminish airway responses to esophageal acid infusion [abstract]. Am J Respir Crit Care Med 1995; 156-159
25 Baluk P .Neurogenic inflammationin skin and airways. J In vestig Dermatol Symp Proc, 1997, 2(1):76-78.
26 Hunter, DD, Undem, BJ. Identification and substance P content of vagal afferent neurons innervating the epithelium of the guinea pig trachea. Am J Respir Crit Care Med 1999; 159, 1943-1948.
27 Undem, BJ, Chuaychoo, B, Lee, MG, et al .Subtypes of vagal afferent C-fibres in guinea-pig lungs. J Physiol 2004; 556, 905-917.
28 Lee, LY, Pisarri, TE. Afferent properties and reflex functions of bronchopulmonary C-fibers. Respir Physiol 2001; 125, 47-65.
29 Richardson JD, Vasko MR.Cellular mechanisms of neurogenic inflammation. Pharmacol Exp Therapeut, 2002, 302(3);839-845
30 Lee LY, Pisarri TE. Afferent properties and reflex functions of bronchopulmonary C-fibers. Respir Physiol, 2001, 125:47-65.
31 Sant'Ambrogio G, Widdicombe J. Reflexes from airway rapidly adapting receptors. Respir Physiol, 2001, 125,33-45.
32 Widdicombe JG. Afferent receptors in the airways and cough. Respir Physio, 1998, 114:5-15.
33 Springer J, Geppetti P, Fischer A, et al. Calcitonin gene-related peptide as inflammatory mediator. Pulm Pharmacol Ther, 2003, 16:121-130.
34 Ricciardolo FL. Mechanisms of citric acid-induced bronchoconstriction. Am J Med, 2001, 111(Suppl 8A):18S-24S.
35 Sekizawa K,Jia YX,Ebihara T,et al.Role of substance P in cough.Pulm Pharmacol,1996,9:323-328.
36 Levi-Montalcini R,Dal Toso R,della Valle F,et al.Update of the NGFsaga.J Neurol Sci.1995;130(2):119-127
37 Aloe L,Bracci-Laudiero L,Bonini S,et al.The wxpanding role of nerve growth factor:from neurotrophic activity to immunologic diseases.Allergy.1997;52(9):883-894
38 周敏,徐永健,熊盛道,等。神经生长因子在哮喘患者诱导痰炎性细胞的表达。中华内科杂志,2003:42(11):764-767
39 Otten U,Baumann JB,Girard J.Nerve growth factor induces plasma extravasation in rat skin.Eur J Pharmacol,1984,106(1):199-201
40 Aloe L,Levi Montalcine R,Mast cells increase in tissue of neonatal rats injected with the nerve growth factor.Brain Res,1977,133:358-366
41 Horigome K,Bullock ED,Johnson EM.Effect of nerve growth factor on rat peritoneal mast cells,survival promotion and immediate-early gene induction.J Biol Chem,1994,269:1695-1702
42 Susaki Y,Shimizu S,Katakura K,et al.Functional properties of murine macrophages promoted by nerve growth factor.Blood,1996,88(12):4630-4637
43 Hunter DD,Myers AC,Undem BJ.Nerve growth factor-induced phenotypic switch in guinea pig airway sensory neurons.Am J Respir Crit Care Med.2000;161:1985-1991
44 Joos GF,De Swert KO,Pauwels RA.Airway inflammation and tachykinins:prospects for the development oftachykinin receptor antagonists.Eur J Pharmacol,2001,429(1-3);239-250
45 Hirotsugu K.The role of substance P release in the lung with esophageal acid.Am J Med,2001,111(8A);25S-30S
46 Daoui S,Agostino L.Tachykinins and airway microvascular leakage induced by HCL intra-oesophageal instillation.Eur Respir J,2002,20(2);268-273
47 47.WuD N,T anifujiY,K obayashiH,et al.Effects of esophageal acid perfusion on airway hyperresponsiveness in patients with bronchial asthma.Chest,2000;118(6):1553-6
48 Bagnato GF,Gulli S,Giaoobbe,et al.Bronchial hyperresponsiveness in subjects with gastroesophageal reflux.Respiration,2000;67(5):507-509
49 Vincent D,Cohen-Jonathan AM,Leport J,et al.Gastro-oesophageal reflux prevalence and relationship with bronchial reactivety in asthma.Eur Respir J,1997;10(10):2255-2259
50 Field SK.A critical review of the studies of the effects of simulated or real gastroesophageal reflux on pulmonary function in asthmatic asults.Chest,1999;115(3);848-856
51 Scarupa MD,Mori N,Canning BJ.Gast roesophageal reflux disease in children with asthma [J].Pediat r Drugs,2005,7(3):177-186.
52 Inhaled P22adrenergic agoniston lower esophageal function, a dose response study [J] . Chest, 2001, 120 (4) :1184-1189.
53 Lazenby J P, Guzzo MR, Harding SM, et al . Oral corticosteroids increase esomonitoringphageal acid contact times in patient s wit h stable asthma [J] . Chest, 2002, 121 (2) :625-634.
2 Harding SM.Gastroesophageal reflux and asthma:insight into the association[J].J Allergy Clin Immuno,1999,104(2 Pt 1):251-259.
3 Richter J E.Beyond heartburn:extraesophageal manifestations of gastroesophageal reflux disease[J].Am J Manag Care,2001,7(suppl):S6-S9.
4 刘春丽,赖克方,陈如冲,等.盐酸灌注豚鼠食管反流性疾病的模型的建立[J].中国病理生理杂志,2006,22(3):620-1,624.
5 Havemann BD,Henderson CA,El-serag HB.The association between gastro-oesophageal reflux disease and asthma:a systematic review[J].Gut.2007 Dec;56(12):1654-1664.
6 Daoui S,D'Agostino B,Gallelli L,et al.Tachykinins and airway microvascular leakage induced by HCL intra-oesophageal instillation[J].Eur Respirr J.2002.20(2):268-273
7 Harding SM,Guzzo MR.Richter JE.24-h esophageal pH testing in asthmatics:respiratory symptom correlation with esophageal acid events[J].Chest,1999,115(3):654-659
8 Wu DN,Tanifuji Y,Kobayashi H,et al.Effects of esophageal acid perfusion on airway hyperresponsiveness in patients with bronchial asthma[J].Chest,2000,118(6):1553-1556.
9 Bagnato GF,Gulli S,Giacobbe O,et al.Bronchial hyperresponsiveness in subjects with gastroesophageal reflux[J].Respiration,2000,67(5):507-509.
10 Vincent D,Cohen-Jonathan AM,Leport J,et al.Gastro-oesophageal reflux prevalence and relationship with bronchial reactivity in asthma.Eur Respir J,1997;10(10):2255-2259
11 Irwin RS,French CL,Curley FJ,et al.Chronic cough due to gastroesophageal reflux.Clinical,diagnostic,and pathogenetic aspects[J].Chest,1993,104:1511-1517.
12 Irwin RS,Madison JM,Fraire AE.The cough reflex and its relation to gastroesophageal reflux[J].Am J Med,2000,108(Suppl 4a):73s-78s.
13 Patterson RN,Johnston BT,Ardill JE,et al.Increased tachykinin levels in induced sputum from asthmatic and cough patients with acid reflux[J].Thorax,2007,62:491-495
14 刘春丽,赖克方,陈如冲等。迷走神经在盐酸灌注食管诱导的气道神经源性炎症中的作用[J]。广东医学,2007年7月第28卷第7期1027-1029
15 Wunderlich AW,Murray JA.Temporal correlation between chronic cough and gastroesophagesl reflux disease[J].Dig Dis Sci,2003,48:1050-1056.
16 Kohroqi H,Hamamoto J,Kawano O,et al.The role of substance P release in the lung with esophageal acid[J].Am J Med,2001,111(8A):25s-30s
17 Daoui S,D'Agostino B,Gallelli L,et aL Tachykinins and airway microvascular leakage induced by HCL intra-oesophageal instillation[J].Eur Respirr J,2002,20(2):268-273
18 Lundberg JM,Hokfelt T,Martlmg CR,et aL Substance P immunoreactive sensory nerves in the lower respiratory tract of various mammals including man[J]. Cell Tissue Res, 1984,235;251-256
19 Uchida Y, Nomurra A, Ohtsuka M,et al. Neurokinin A as a potent bronchoconstrictor[J]. Am Rev Respir Dis, 1987, 136:718-721
20 Magge CA,Patacchini R. Tachykinin receptors and tachykinin receptor antagonists[J].J Auton Pharmacol,1993,13;23-93
21 F Lembeck. Zur Frage der zentralen Ubertragung afferenter Impulse. Mitteilung. Das Vorkommen und die bedeutung der Substanz P. in den dorsalen Wurzein des Ruckenmarks[J].Arch Exp Pathol Pharmakol,1953,219:197-213
22 Maggi CA.Tachykinin receptors and airway pathophysiology.Eur Respir J.1993, 6:735-42
23 Zhang XL, Mak JC, Barnes PJ . Character2 ization and autoradiographic mapping of[~3H] CP96,345, a nonpeptide selective NK21 receptor antagonist in guinea pig lung. Peptides, 1995, 16 :867-72.
24 Walsh DA ,Sakmon M ,Featherstone R,et al.Differences in the distribution and characteristics of tachykinin NK21 binding sites between human and guinea pig lung. Br J Pharmacol, 1994,113:1407-15.
25 Ichikawa S, Sreedharan SP, Owen RL, et al. Immunochemical localization of type I VIP receptor and NK-1-type substance P receptor in rat lung. Am J Physiol, 1995 ,268 :584-8.
26 Bai TR, Zhou DY, Weir T ,et al.Substance P (NK1) and neurokinin A (NK2)receptor gene expression in inflammatory airway disease. Am J Physiol, 1995, 269 :309-17.
27 Khawaja AW, Rogers DF. Tachykinins : receptor to effector. Int J Biochem Cell Biol, 1996, 28:721-38
28 Hirayama Y, Yu HL,Barnes PJ ,et al. Effects of two noval tachykinin antagonists ,FK224 and FK888, on neurogenic airway plasma exudation, broncho constriction and systemic hypotension in guinea-pig in vivo.Br J Pharmacol, 1993, 108 :844-51.
29 Kaltreider HB, Ichikawa S, Byrol PK, et al. Up regulation of neuropeptides and neuropeptide receptors in a murine model of immune inflammation in lung parenchyma. Am J Respir Cell Mol Biol, 1997, 16:133-44.
30 Adcock IM, Peters M, Gelder C, et al. Increased tachykinin receptor gene expression in asthmatic lung and its modilation by steroids. Joural of Molecular Endocrinology, 1993, 11 :127-31.
31 Ichinose M, Nakajima N, Takahashi T, et al. Protection against bradykinin2 induced bronchoconst riction in asthmatic patients by neurokinin receptor antagonist . Lancet ,1992, 340:1248-51.
32 Ichinose M, Miura M, Yamauchi H, et al .A neurokinin 1-receptor antagonist improves exercise induced airway narrowing in asthmatic patients. Am J Respir Crit CareMed, 1996, 153 :93-9
33 Emonds-Alt, X., Doutremepuich, J. D., Heaulme, M., et al. In vitro and in vivo biological activities of SR 140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol. 1993,250,403-13
34 Di Sebastiano P, Grossi L, Di Mola F,et al. SR140333, a substance P receptor antagonist, influences morphological changes in rat experimental colitis. Dig Dis Sci. 1999 ,44,439-44
35 Amann R, Schuligoi R, Holzer P, Donnerer J. The non-peptide NK.1 receptor antagonist SR140333 produces long-lasting inhibition of neurogenic inflammation, but does not influence acute chemo- or thermonociception in rats. Naunyn Schmiedebergs Arch Pharmacol. 1995, 352,201-5
36 Boichot E, Germain N, Emonds-Alt X, Advenier C, Lagente V. Effects of SR 140333 and SR 48968 on antigen and substance P-induced activation of guinea-pig alveolar macrophages. Clin Exp Allergy, 1998, 28, 1299-1305
37 Veron M, Guenon I, Nenan S, et al. Interactions of tachykinin receptor antagonists with lipopolysaccharide-induced airway inflammation in mice. Clin. Exp PharmacolPhysiol. 2004, 31,634-640
38 Zijlstra FK, Hynna-Liepert TT, Dinda PK, Beck IT, Paterson WG. Microvascular permeability increases early in the course of acid-induced esophageal injury. Gastroenterology. 1991; 101:295-302
39 Underwood S, Foster M, Raeburn D, Bottoms S, Karlsson JA. Time-course of antigen-induced airway inflammation in the guinea-pig and its relationship to airway hyperresponsiveness. Eur RespirJ, 1995,8:2104-2113
40 Friess H, Zhu Z, Liard V, et al. Neurokinin-1 receptor expression and its potential effects on tumor growth in human pancreatic cancer. Lab Invest.2003 May;83:731-42
1 Menes T, Lelcuk S, Spivak H. Pathogenesis and current management of gastroesophageal reflux-related asthma [ J ] .J Surg, 2000, 166 (8) :596-601
2 Harding SM. Pulmonary complications of gastroesophageal reflux. In: Castell DO, Richter JE, eds. In:The esophagus, 4th, edn. Philadelphia: Lippincott Williams and Wilkins, 2004:530-545.
3 Kiljander TO, Laitinen JO. The prevalence of gastroesophageal reflux disease in adult asthmatics [ J ]. Chest, 2004, 126 ( 5) :1490-1494.
4 Debley J S, Carter ER, Redding GJ . Prevalence and impact of gast roesophageal reflux in adolescent s wit h asthma : apopulation2based study [ J ] . Pediat r Pulmonol, 2006, 41 (5) :475-481
5 Diette GB, Krishnan JA, Dominici F, et al. Asthma in older patients .Factors associated with hospitalization. Arch Intern Med, 2002, 162(10);1123-1132
6 Gislason T, Janson C, Vermeire P, et al. Respiratory symptoms and nocturnal gastroesophageal reflux. A population-based study of young adults in three European countries.Chest, 2002, 121(1):158-163
7 Tomonaga T, Awad ZTT, Filipi CJ, et al. Symptom predictability of reflux-inducedrespiratory disease.Dig Dis Sci, 2002, 47(1);9-14
8 Clinquette M, Miceli S, Voltolina C, et al. The pattern of gastroesophageal reflux in asthmatic children.J Asthma, 2002, 39(2); 135-142
9 el-Serag H, Gilger M, Kuebeler M, et al.Extrsesophageal associations of gastroesophageal reflux disease in children without neruologic defects. Gastroenterology, 2001, 11(6); 1294-1299
10 Avidan B, Sonnenberg A, Schnell TG, et al.Temporal associations between coughing or wheezing and acid reflux in asthmatics.Gut, 2001, 49(6);767-772
11 Field SK. A critical review of the studies of the effects of simulated or real gastroesophageal reflux on pulmonary function in asthmatic adults. Chest. 1999; 115: 848-856.
12 Sontag SJ. Why do the published data fail to clarify the relationship between gastroesophageal reflux and asthma? Am J Med. 2000;108(suppl 4A):159S-169S.
13 Field SK. Gastroesophageal reflux and asthma: are they related? J Asthma. 1999;36:631-644
14 Bocskei C, Viczian M, Bocskei R, et al. The influence of gastroesophageal reflux disease and its treatment on asthmatic Cough [J ] . Lung, 2005, 183 (1) :53-62.
15 Kiljander TO. The role of proton pump inhibitors in the management of gast roesophageal reflux disease-related asthma and chronic cough[J ] . Am J Med, 2003, 115 (3) :65S-71S
16 Tuchman DN, Boyle JT, Pack Al et al. Comparison of airway responses following tracheal or esophageal acidification in the cat.Gastroenterology, 1984; 87(4):872-881
17 Jack CI, Calverley PM, Donnelly RJ, et al. Simultaneous tracheal and oesophageal pH measurements in asthmatic patients with gastro-oesophageal reflux. Thorax, 1995; 50(2) : 201-204
18 Ricciardolo FLM. Mechanisms of citric acid-induced bronchoconstriction [J ] . Am J Med, 2001, 111 (8): 18-24.
19 Irwin RS, Madison JM, Fraire AE. The cough reflex and its relation to gastroesophageal reflux.Am J Med, 2000, 108(Suppl 4a):73s-78s.
20 Canning BJ, Mazzone SB. Reflex Mechanisms in Gastroesophageal Reflux Disease and Asthma [J ] . Am J Med, 2003, 115 (3) :45S-48S
21 Field SK. A critical review of the studies of the effects of simulated or real gastroesophageal reflux on pulmonary function in function in asthmatic asults. Chest, 1999; 115(3);848-856
22 Schan CA, Harding SM, Haile JM, et al. Gastroesophageal reflux-induced bronchoconstriction: an intraesophageal acidinfusion study using state-of-the-art technology. Chest 1994;106:731-737
23 Harding SM, Schan CA, Guzzo MR, et al. Gastroesophageal reflux-induced bronchoconstriction: is microaspiration a factor? Chest 1995; 108:1220-1227
24 Harding SM, Guzo MR, Maples RV, et al. Gastroesophageal reflux induced bronchoconstriction: vagolytic doses of atro-pine diminish airway responses to esophageal acid infusion [abstract]. Am J Respir Crit Care Med 1995; 156-159
25 Baluk P .Neurogenic inflammationin skin and airways. J In vestig Dermatol Symp Proc, 1997, 2(1):76-78.
26 Hunter, DD, Undem, BJ. Identification and substance P content of vagal afferent neurons innervating the epithelium of the guinea pig trachea. Am J Respir Crit Care Med 1999; 159, 1943-1948.
27 Undem, BJ, Chuaychoo, B, Lee, MG, et al .Subtypes of vagal afferent C-fibres in guinea-pig lungs. J Physiol 2004; 556, 905-917.
28 Lee, LY, Pisarri, TE. Afferent properties and reflex functions of bronchopulmonary C-fibers. Respir Physiol 2001; 125, 47-65.
29 Richardson JD, Vasko MR.Cellular mechanisms of neurogenic inflammation. Pharmacol Exp Therapeut, 2002, 302(3);839-845
30 Lee LY, Pisarri TE. Afferent properties and reflex functions of bronchopulmonary C-fibers. Respir Physiol, 2001, 125:47-65.
31 Sant'Ambrogio G, Widdicombe J. Reflexes from airway rapidly adapting receptors. Respir Physiol, 2001, 125,33-45.
32 Widdicombe JG. Afferent receptors in the airways and cough. Respir Physio, 1998, 114:5-15.
33 Springer J, Geppetti P, Fischer A, et al. Calcitonin gene-related peptide as inflammatory mediator. Pulm Pharmacol Ther, 2003, 16:121-130.
34 Ricciardolo FL. Mechanisms of citric acid-induced bronchoconstriction. Am J Med, 2001, 111(Suppl 8A):18S-24S.
35 Sekizawa K,Jia YX,Ebihara T,et al.Role of substance P in cough.Pulm Pharmacol,1996,9:323-328.
36 Levi-Montalcini R,Dal Toso R,della Valle F,et al.Update of the NGFsaga.J Neurol Sci.1995;130(2):119-127
37 Aloe L,Bracci-Laudiero L,Bonini S,et al.The wxpanding role of nerve growth factor:from neurotrophic activity to immunologic diseases.Allergy.1997;52(9):883-894
38 周敏,徐永健,熊盛道,等。神经生长因子在哮喘患者诱导痰炎性细胞的表达。中华内科杂志,2003:42(11):764-767
39 Otten U,Baumann JB,Girard J.Nerve growth factor induces plasma extravasation in rat skin.Eur J Pharmacol,1984,106(1):199-201
40 Aloe L,Levi Montalcine R,Mast cells increase in tissue of neonatal rats injected with the nerve growth factor.Brain Res,1977,133:358-366
41 Horigome K,Bullock ED,Johnson EM.Effect of nerve growth factor on rat peritoneal mast cells,survival promotion and immediate-early gene induction.J Biol Chem,1994,269:1695-1702
42 Susaki Y,Shimizu S,Katakura K,et al.Functional properties of murine macrophages promoted by nerve growth factor.Blood,1996,88(12):4630-4637
43 Hunter DD,Myers AC,Undem BJ.Nerve growth factor-induced phenotypic switch in guinea pig airway sensory neurons.Am J Respir Crit Care Med.2000;161:1985-1991
44 Joos GF,De Swert KO,Pauwels RA.Airway inflammation and tachykinins:prospects for the development oftachykinin receptor antagonists.Eur J Pharmacol,2001,429(1-3);239-250
45 Hirotsugu K.The role of substance P release in the lung with esophageal acid.Am J Med,2001,111(8A);25S-30S
46 Daoui S,Agostino L.Tachykinins and airway microvascular leakage induced by HCL intra-oesophageal instillation.Eur Respir J,2002,20(2);268-273
47 47.WuD N,T anifujiY,K obayashiH,et al.Effects of esophageal acid perfusion on airway hyperresponsiveness in patients with bronchial asthma.Chest,2000;118(6):1553-6
48 Bagnato GF,Gulli S,Giaoobbe,et al.Bronchial hyperresponsiveness in subjects with gastroesophageal reflux.Respiration,2000;67(5):507-509
49 Vincent D,Cohen-Jonathan AM,Leport J,et al.Gastro-oesophageal reflux prevalence and relationship with bronchial reactivety in asthma.Eur Respir J,1997;10(10):2255-2259
50 Field SK.A critical review of the studies of the effects of simulated or real gastroesophageal reflux on pulmonary function in asthmatic asults.Chest,1999;115(3);848-856
51 Scarupa MD,Mori N,Canning BJ.Gast roesophageal reflux disease in children with asthma [J].Pediat r Drugs,2005,7(3):177-186.
52 Inhaled P22adrenergic agoniston lower esophageal function, a dose response study [J] . Chest, 2001, 120 (4) :1184-1189.
53 Lazenby J P, Guzzo MR, Harding SM, et al . Oral corticosteroids increase esomonitoringphageal acid contact times in patient s wit h stable asthma [J] . Chest, 2002, 121 (2) :625-634.