【作者】 王谦；

【导师】 史利卿；

【作者基本信息】 北京中医药大学 ， 中医内科学（专业学位）， 2017， 博士

【摘要】 研究背景咳嗽作为生理上可以有效清除呼吸道分泌物及阻挡有害物质的保护性动作有着非常重要的意义。但由于个体差异或者疾病严重导致这种动作频繁、剧烈、无法控制,影响人们说话、睡眠,尤其是长时间的咳嗽,对于患者身体、心理、日常生活质量及经济都有着严重的影响。有研究表明,医院呼吸科门诊中,10%-38%的患者是以不明原因的慢性咳嗽来就诊,不明原因慢性咳嗽患病率高于3.3%。上世纪80年代美国的Irwin从神经解剖学角度系统论述咳嗽的病因,让人们认识到慢性咳嗽在病因、病理学上的独特之处,区别于传统的感染后咳嗽。咳嗽变异性哮喘(cough variant asthma,CVA)在慢性咳嗽病因中占33.3%。气道的反应性增高是各种原因导致的咳嗽,尤其是慢性咳嗽,的中心环节。气道高反应性的产生的主要病理机制是气道炎症,并且认为,气道炎症的持续和加重与存在于气道的神经末梢及其所联系的中枢神经系统和周围神经系统之间存在复杂的相互作用。已有实验明确的表明,其感受器存在于气道的感觉神经及其分泌的神经因子可能直接参与气道炎症的形成,并提出气道神经源性炎症的概念。典型的气道高反应性疾病是哮喘,众多实验证明哮喘状态下神经生长因子(nerve growthfactor,NGF)及其受体表达升高。气道神经源性炎症的存在是咳嗽敏感性增高、咳嗽经久不愈的重要原因。近来NGF通过膜受体酪氨酸蛋白激酶信号传导途径(Ras/MAPK通路)来对下游因子如P物质(subs tance P,SP)的调控作用,及其所发挥生物学效应越来越受到关注。中医对咳嗽的认识与治疗源远流长,发现其的独特性,并且将咳嗽列为单独疾病之一。中医在改善咳嗽症状,尤其是对于顽固性咳嗽的气道高反应有着较好的疗效和较多的经验。CVA以干咳为主,无痰或少痰,咽痒即咳,多为阵发性,有时表现为难以抑制的刺激性咳嗽,讲话、灰尘、冷热空气、异味等容易诱发,夜间咳嗽为主,病程较长,属于中医"风咳"范畴,中医对于"风咳"的认识与治疗有着长久的历史,随着现代研究的不断完善,对于"风咳"有了新的认识。导师史利卿教授提出"风咳"为内伤咳嗽,临床研究表明"风邪伏肺"是慢性咳嗽的首要病机。CVA患者中"风邪伏肺"证为主要证型,自拟"祛风宣肺方"为主方根据兼夹证型加减用药治疗CVA疗效显著。本课题组既往临床研究表明祛风宣肺方治疗感染后咳嗽疗效优于阿斯美,实验研究表明"祛风宣肺方"对于咳嗽豚鼠模型有降低气道高反应性、减轻气道炎症的作用,并且通过调节Ras-Raf-MAPK通路相关因子降低豚鼠气道神经源性炎症。但对于其作用于人气道上皮降低气道神经源性炎症的作用物质基础仍有待于进一步研究。临床研究(一)研究目的通过观察CVA "风邪伏肺"证患者应用导师自拟祛风宣肺方治疗效果,旨在科学、客观的阐明祛风宣肺方治疗CVA的临床效果,通过中药组与安慰剂组的疗效对比,研究中医药治疗该病的疗效,并且对于"风邪伏肺"理论治疗CVA临床应用进行评价。(二)研究方法纳入符合CVA "风邪伏肺"证诊断患者,共72例,采用随机对照的研究方法,分成治疗组与对照组,服用中药配方颗粒祛风宣肺方治疗的40例患者为治疗组,服用安慰剂治疗的32例患者为对照组。观察两组患者用药前、14天后主观咳嗽严重程度、咳嗽次数、咽痒、气急、咳痰程度积分变化情况,比较两组患者的积分差异,并运用统计学方法进行分析。(三)研究结果1.一般资料:1)入组情况:入组80例,祛风宣肺方组因未能按规律服药脱落1名;安慰剂组因不能坚持服药脱落7名。2)年龄:青年31人,占43.1%;中年20人,占27.8%;轻老年17人,占23.6%;老年4人,占5.6%。3)性别比例,女51人,男21人。2.疗效评价1)"风邪伏肺"证候评分:治疗前,两组患者证候评分无明显差异(P>0.05),治疗后中药组证候评分明显低于治疗前(P<0.01),疗后中药组证候评分下降明显高于安慰剂组(P<0.01)。2)咳嗽视觉评分:治疗前,两组患者咳嗽视觉评分无明显差别(P>0.05),治疗后,中药组咳嗽视觉评分明显低于治疗前(P<0.01),疗后中药组咳嗽视觉评分下降明显高于安慰剂组(P<0.01)。3)昼夜咳嗽症状积分:治疗前,两组咳嗽昼夜总积分无明显差别(P>0.05),治疗后,中药组咳嗽昼夜总积分明显低于治疗前(P<0.01),疗后中药组咳嗽昼夜总积分下降明显高于安慰剂组(P<0.01)。4)基于咳嗽症状积分的有效率判断:祛风宣肺方有效率:62.5%,安慰剂有效率21.9%,祛风宣肺方有效率明显高于安慰剂组(P<0.01)。5)咳嗽缓解时间判定:对于祛风宣肺方有效患者(包括有效、显效、痊愈)为25人,平均咳嗽缓解时间为3.24±1.01天。(四)结论1.祛风宣肺方对于CVA "风邪伏肺"证候、患者咳嗽自主感觉、咳嗽昼夜程度都有明显改善,明显优于安慰剂;2.安慰剂对于"风邪伏肺"证候略有改善,有些患者咳嗽自主感觉加重,对咳嗽昼夜症状无明显改善;3.祛风宣肺方有效率为62.5%高于安慰剂;4.祛风宣肺方治疗CVA的起效时间为3.24±1.01天。实验研究(―)研究目的通过正常SPF级Hartley豚鼠进行生理盐水、中药、西药灌胃获取含药血清进行人气道上皮细胞培养,观察中药祛风宣肺方对神经源性炎症信号转导通路的影响,探讨Ras-Raf-MAPK通路在气道神经源性炎症及咳嗽敏感增高中的作用及祛风宣肺方治疗气道神经源性炎症的物质基础。(二)研究方法把15只SPF级Hartley豚鼠随机分成5组,进行生理盐水,西药阿斯美,中药祛风宣肺方(高、中、低剂量)分组灌胃一周,取动脉含药血清。通过体外实验,NGF作用于人气道上皮细胞造模,分别加入空白血清作为模型组,加入中、西药血清作为治疗组观察其对人气道上皮细胞Ras/Raf/MAPKK/MAPK/c-fos通路相关因子的调控作用。(三)研究结果1.细胞增殖、凋亡及细胞周期细胞增殖结果显示PD98059和西药血清对NGF的作用明显抑制,中药高剂量血清具一定的抑制,同时呈剂量依赖性;单独PD98059作用对细胞增殖无明显影响。细胞凋亡结果显示,仅PD98059组细胞凋亡大于10%,其他各组凋亡均小于等于10%;细胞周期检测,显示各组细胞周期大致相同。2.Western-Blot 结果(1)NK1R蛋白电泳:中药中剂量组、中药小剂量组NK1R蛋白表达均低于模型组,且中药中剂量组抑制NK1R蛋白表达效果最佳。(2)Pan-Ras蛋白电泳:中药中剂量组Pan-Ras蛋白表达较模型组低。(3)P-Erk1/2蛋白电泳:西药组、中药大、中、小剂量组P-Erk1/2蛋白表达比模型组低,且西药组、中药大剂量两组降低比较明显。(4)C-fos蛋白表达:中药中剂量组、中药小剂量组C-fos蛋白表达均低于模型组,且中药中剂量组对C-fos蛋白抑制较中药小剂量组明显。3.Rt-PCR 结果(1)NK1R基因RT-PCR:西药、中药大、中、小剂量组均能明显抑制NK1R基因表达(P<0.01)中药大、中剂量组效果明显优于西药组(P<0.01),中药中剂量组效果明显优于中药大剂量组和中药小剂量组(P<0.01)。(2)Pan-Ras基因RT-PCR:中药大、中、小剂量均能减少Pan-Ras基因表达(P<0.01),且中、小剂量更明显(P<0.01),西药不能减少Pan-Ras基因表达(P＞0.05)。(3)C-fos基因RT-PCR:西药、中药都能显著降低C-fos基因的表达(P<0.01),且中药效果优于西药(P<0.01),中剂量中药效果明显(P<0.05);(四)结论1.NGF作用于人气道上皮,气道上皮细胞Ras/MAPK通路激活,相关因子表达,提示模型制备成功。2.中药祛风宣肺、阿斯美均能不同程度的下调Ras/MA PK通路相关因子的蛋白和基因层面的表达,且中药中剂量较大、小剂量效果更明显。更多还原

【Abstract】 Back GroundCough is a protective action that can effectively clear the respiratory secre tions and the harmful substances that are blocked in the airway.However,du e to individual differences or the impaction of serious illness,the cough becom es frequent,intense,uncontrollable,affecting people to talk or sleep.Especiall y,the long-time cough do seriously bad to the patients in physical,mental and economic aspect.In the outpatient service,there is no clear diagnosis on the patients,given a large number of empirical use of antibiotics and antitussive drugs.This phenomenon leads to the heavy medical and health expenditures of family and even the country.In the department of respiration clinic,10%-38%of patients is diagnosed with unexplained chronic cough,and prevalence rate of chronic cough is higher than 3.3%.In recent years,due to environmental pollution,the incidence and visiting rate of cough in China is increasing.Chro nic cough has been paid more and more attention,requiring the classification a nd targeted treatment.From neuroanatomy perspective,Irwin systematically dis cussed cough,making people aware of that chronic cough is unique in etiolog y and pathology from traditional cough after infection,in the 1980s.The stud y of this type of cough has become a hot topic.Cough variant asthma in the chronic cough accounts for 33.3%,which is firstly discovered by Glauser in 1972.This disease has relations with allergic sensitization,and its pathophysio logyical characteristics is similar with typical asthma,such as,airway inflamma tion,airway hyperresponsiveness.It is believed that the increased of airway high reactivity is the core of c ough caused by any reason,especially chronic cough.Airway abnormally incr easing response to normal stimulation results in frequent cough and prolonged healing.Modern medichine is generally believed that the airway inflammation is the main mechanism of airway hypperresponsiveness,and there are complex interactions between airway inflamation and the nerve endings in airway which conact the central and peripheral nervous system.It has been proved that the sensory nerve in the airway and its secreted nerve factors may be directly invo lved in the formation of airway inflammation.Which,we called airway neuro genic inflammation.The typical airway hyperresponsiveness disease is asthma,and,NGF and its receptor are high expressed in asthma.The airway neuroge nic inflammation is an important cause of increased cough sensitivity and prolo nged cough.Recently,tyrosine protein kinase membraine receptor signal transd uction pathway(Ras/MAPK pathway)which NGF acts on and its downstream factors,such as,substance P,which play biological effect have attracted more and more attention.Traditional Chinese Medicine has a long history of knowledge and treatme nt of cough,and has a unique understanding of cough,recognized cough as a single diagnosis.The Inner Canon of Huangdi says "all the five internal orga ns make people cough";Valuable Prescriptions for Emergencies proposed ten types cough classification method;Zhang Jingyue of Ming Dynasty divided cou gh into two major categories of exogenous and internal injuries.Which impro ved clinical practice.Exogenous fator refers to the six evils invaded the lung s,such as,wind,cold,heat,dampness,dryness,fire.Internal injury refers to weakness of the viscera,disfunction,and forming syndrome,such as,phlegmy wetness aggregating lung,liver-fire invading lung and kidney deficiency.TCM has a better curative effect and more experience in improving symptoms,espe cially for airway hyperresponsiveness of intractable cough.CVA has a lot of symptoms,such as,dry cough,throat itching,the paroxysmal and irritating cou hg,induction by speech,dust,hot and cold air,cough at night and long cours e.All the symptoms above belong to the TCM "wind cough".TCM has a 1 ong history of recognition and treatment of "wind cough".With the increasing of clinical research on the treatment of CVA in recent years,"wind cough" pl ay the key role.Professor Shi Liqing recognized that "incubative wind in lun g" is the critical pattern of syndrome in many,and get good curative results o f treating CVA with "expelling wind and releasing lung fomula".Clinical Research(1)Research PurposeThrough the observation and comparison of 72 cases of patients with CVA"incubative wind in long" pattern of syndrome,respectively,clarifying the e ffect of the treatments of "expelling wind and releasing lung fomula" and p lacebo.Inorder to clarify the effect of the fomula in the treatment of CV A scientifically and objectively.Comparing with placebo,studying the curati ve result of TCM,and evaluating the clinical application of "incubative win d in lung" theory in the treatment of CVA.(2)Research MethodFinding 70 patients of CVA with "incubative wind in lung",choosing rando mized controlled trial method.The 40 patients take "expelling wind and re leasing lung fomula" as treatment group,and 32 patients takes placebo as c ontrol group.Observe the change of two groups of patients’ subjective ord er of cough,times of cough,degree of cough,pharyngeal itching,short of breath and expectoration on the day before treating and 14th day after the t reatment.Compare the differences in cough scores between two groups,an d analyzed them using the statistical method.(3)Research Results1.General situation:1)Number:80 cases in group,treatment group losed 1 case caused by irregula r medication;Placebo group losed 7 cases caused by stoping taking medicin e.2)Age:31 young people,20 middle-aged people.17 light old people.4 o ld people;3)Gender:51 female,21 male;4)The patients diagnosed of aller gic rhinitis:20 cases,accounting for 27.8%.2."Incubative wind in lung" syndrome score:Before treatment,there is no diffe rence between two groups(P>0.05);After treatment,the score of treatment g oup is significantly lower than before treatment(P<0.01);After treatment,the score of treatment group is significantly lower than placebo group(P<0.01).3.Cough visual score:Before treatment,there is no difference between two gro ups(P>0.05);After treatment,the score of treatment goup is significantly lo wer than before treatment(P<0.01);After treatment,the score of treatment gr oup is significantly lower than placebo group(P<0.01).4.Cough symptom score:Before treatment,there is no difference between two groups(P>0.05);After treatment,the score of treatment goup is significantly lower than before treatment(P<0.01);After treatment,the score of treatment group is significantly lower than placebo group(P<0.01).5.Effective rate based on cough symptom score:Effective rate of "expelling wi nd and dispersing lung fomula" is 62.5%,and effective rate of placebo is 21.9%.The effective rate of "Expelling wind and dispersing lung fomula" is significantly higher than placebo group(P<0.01).6.Cough relief time:The patients who responded to "expelling wind and disper sing lung fomula" is 25,and the average cough reduce time is 3.24± 1.01 day s.(4)Conclusion1.It has been obviously improved by "expelling wind and dispersing lung fomula" that "Incubative wind in lung" patern of syndrome,felling of cough a nd symptoms of cough both day and night,better than placebo.2.The placeb o plays a little role in feeling of cough,and it makes the patients feel worse.Placebo does no good to symptoms of cough both day nand night.3.The e ffective rate of fomula is 62.5%,far higher than placebo.5.The onset time of fomula treating CVA is 3.24±1.01 days.Experimental Study(1)Research PurposeGive the SPF Hartley guinea pigs by gavage with normal saline,Chinese h erbal decoction,and extract the serum containing drug.Culture human bronchia 1 epithelial cells with the serum containing drug,to observe the influence of"expelling wind and dispersing lung fomula" on signal transduction of airway neurogenic inflammation.Investigate the role of Ras-Raf-MAPK signal pathwa y in airway eurogenic inflammation and cough hypersensitivity,and substance basis for the treatment of neurogenic inflammation of the airways by "expellin g wind and dispersing lung fomula".(2)Research MethodGive the SPF Hartley guinea pigs by gavage with normal saline,Asmeton or three doses of "expelling wing and dispersing lung fomula"(high dose,midd le dose,low dose)for one week,and take the drug containing serum from art ery.Trough in-vitro experiment,observe the influence of different kinds of ser ums on related factors in Ras/Raf/MAPKK/MAPK/C-fos signal passway.(3)Research Results1.Cell proliferation,apoptosis and cell cycle:The effect of PD98059 and Asmeton serum on the inhibition of NGF is signifi cant.The high dose of "expelling wind and dispersing lung fomula"has a cert ain inhibition function,and the inhibition is in a dose dependent manner.PD98059 alone has no effect on cell proliferation.The results of apoptosis of cell s shows:only the apoptosis rate of group of PD98059 is more than 10%,and other groups are all less than 10%.Cell cycle test shows that the cell cycle of eache group is approximately the same.2.Western-BlotNK1R:The expression of NK1R protein in the medium dose and low dose of Chinese medicine serum group is lower than the model group.And the inhibiti on of NK1R caused by medium dose group is the best.Pan-Ras:The expression of Pan-Ras protein in the medium dose group is lowe r than that in the model group.C-fos:The expression of C-fos protein in the medium dose group and low dos e group is lower than that in the model group.And,the inhibition of C-fos protein in the medium dose group is significantly lower than that in the low d ose group.P-Erk1/2:The expressions of P-Erkl/2 protein in Asmeton group,three doses o f Chinese medicine are lower than the model group.And,P-Erkl/2 protein in the Asmeton group and high dose of Chinese medicine decrease significantly.3.RT-PCRPan-Ras:Large,medium and low doses of Chinese medicine can reduce the ge netic expression of Pan-Ras(P<0.01),and medium and low doses are more obv ious(P<0.01),Asmeton cannot reduce the genetic expression of Pan-Ras(P>0.05).C-fos:Asmeton and Chinese medicine can significantly reduce the genetic expr ession of C-fos(P<0.01).And,the effect of Chinese medicine is superior to As meton(P<0.01).The inhibitive effect of medium dose of Chinese medicine is t he most(P<0.05).NK1R:Asmeton and three does of Chinese medicine can significantly inhibit t he genetic expression of NK1R(P<0.01).The effect of high dose and medium dose of Chinese medicine are better than Asmeton(P<0.01).And,medium dos e is obviously better than the other two doses(P<0.01).(4)Conclusion1.NGF plays an important role in human airway epithelial cells through the a ctivation of Ras/Raf/MAPK pathway and the expression of related factors.All these show that the model preparation is successful.Chinese medicine and Asmeton can promote the expression of protein and genetic expression of Ras/Raf/MAPK pathway related factors in different degrees.And,the m edium dose of Chinese medicine is better than the other two doses.更多还原