仲景治疗肺痿的辩证规律及相关实验研究

【作者】 牛潞芳；

【导师】 李宇航；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2007， 博士

【摘要】 特发性肺纤维化(idiopathic pulmonary fibrosis IPF)是指原因不明并以普通间质性肺炎为特征性病理改变的一种慢性炎症性间质性肺疾病。主要表现为弥漫性肺泡炎、肺泡单位结构紊乱和肺纤维化。病情一般进行性发展,最终因呼吸衰竭导致死亡。目前治疗特发性肺纤维化临床常用的药物为糖皮质激素、免疫抑制剂/细胞毒药物和抗纤维化制剂,长期应用这些药物有严重的副作用,使机体免疫力下降,引起继发感染。因此,寻找副作用小,疗效好的药物是目前研究的热点。特发性肺纤维化在祖国传统医学中没有完全相对应的病名,但多数医家根据其发病过程及临床表现将其归属于“肺痹”、“喘证”、“肺痿”等疾病范畴。笔者认为,根据其病理形态及临床特点,特发性肺纤维化属于祖国传统医学中“肺痿”的证候范畴。历代医家对于肺痿的认识和治疗方法对于肺痿的辨证论治具有重要意义,但是都散见于各代医家著作之中,未经系统总结归纳,本课题的理论研究将对历代医家和张仲景对肺痿的认识和治疗方法予以系统总结,旨在遵从前贤所论的基础上,结合现代医学发展,对某些混淆的问题予以阐述,探微索隐,为能在临床上更好的应用祖国传统医学治疗肺痿这种疑难重症提供理论依据。本课题的实验研究追踪该领域国际最新动态,与耶鲁大学医学院合作,采用中药复方加味桔梗汤,即桔梗汤加银花、连翘、贯众等药物治疗特发性肺纤维化模型小鼠,观察加味桔梗汤对特发性肺纤维化模型小鼠的疗效及对小鼠肺中骨形态发生蛋白-4(Bone Morphogenetic protein 4 BMP-4)的和骨形态发生蛋白-7(Bone Morphogenetic protein 7 BMP-7)影响,探索特发性肺纤维化过程中加味桔梗汤对IPF小鼠的治疗作用及其对BMP家族的调节作用,以及BMP-4、BMP-7在特发性肺纤维化模型小鼠肺中的含量变化,为在临床上应用经方治疗特发性肺纤维化提供实验依据,并进一步探索中药治疗特发性肺纤维化的作用靶点。1理论研究1.1系统总结历代医家对肺痿的认识及治疗法则查阅自秦以降内科、方剂、医案医话、杂著等古代中医文献,对其进行全面的分析、归纳和总结,探讨古代著名医家辨治肺痿的经验,揭示古代中医学在肺痿诊治方面的规律和巨大成就。1.2归纳总结仲景治疗肺痿的辨证规律“肺痿”病名首见于仲景之《金匱要略·肺痿肺痈咳嗽上气病脉证治第七》,仲景对于肺痿的认识及辨证方法,大多后人认为止于《金匱要略》之条文,而其实在《伤寒杂病论》中,仲景对于喘证、咳嗽、短气、虚劳的辨证方法,都可以运用于肺痿的辨证治疗中。研究仲景治疗肺痿的辨证规律,是为了拓宽《伤寒杂病论》中对肺痿的认识及治疗范围,为灵活的应用经方治疗肺系疑难重症提供理论依据。2实验研究2.1加味桔梗汤对IPF模型小鼠的治疗作用生存率实验取30只C57BL/6小鼠,将小鼠随机分为中药生存曲线组、平阳霉素生存曲线组,全部小鼠气管内滴注盐酸平阳霉素造特发性肺纤维化小鼠模型,造模后,中药生存曲线组小鼠每日予以加味桔梗汤灌胃,平阳霉素生存曲线组小鼠予以生理盐水灌胃,观察两组小鼠的生存状态及死亡情况,描绘生存曲线图,第29天将剩余小鼠全部处死。结果表明,中药生存曲线组小鼠28天生存率为46%,平阳霉素生存曲线组小鼠28天生存率为13%,这说明加味桔梗汤对特发性肺纤维化模型小鼠有明显的治疗作用,为我们应用具有清热解毒的中药复方治疗特发性肺纤维化提供了一个整体性实验依据。2.2IPF模型小鼠肺组织标本病理观察实验小鼠造模同支气管灌洗实验,在造模后第0天、1天、3天、5天、9天、14天、28天分别在平阳霉素组、加味桔梗汤组、生理盐水组取8只小鼠的部分右肺,将小鼠肺组织标本(右肺叶)用10%福尔马林固定,石蜡包埋,切片,HE染色,观察小鼠肺部病理变化。本实验结果表明,加味桔梗汤对IPF模型小鼠有显著的治疗作用,加味桔梗汤组小鼠肺部的炎症明显较平阳霉素组小鼠轻,炎性细胞减少,在造模后第28天,加味桔梗汤组小鼠没有出现肺部早期纤维化的趋势。2.3IPF模型小鼠支气管灌洗实验取200只C57BL/6小鼠,随机分为三组,即生理盐水组、平阳霉素组、加味桔梗汤组。生理盐水组取C57BL/6小鼠60只,平阳霉素组、加味桔梗汤组分别取C57BL/6小鼠70只。生理盐水组小鼠不造模,平阳霉素组、加味桔梗汤组小鼠气管滴注博莱霉素造特发性肺纤维化小鼠模型,在造模后第0天、1天、3天、5天、9天、14天、28天分别在平阳霉素组、加味桔梗汤组、生理盐水组取8只小鼠,进行肺部病理变化肉眼观察,并进行支气管灌洗,于10倍显微镜下进行总细胞记数,Jamsa染色后进行中性粒细胞、巨噬细胞、淋巴细胞百分比记数。中性粒细胞是在早期急性肺损伤/肺胞炎阶段的主要炎性细胞。在造模后前五天,平阳霉素组、加味桔梗汤组的中性粒细胞占细胞总数的百分比都出现增高,与生理盐水组比较都有非常显著性差异,这说明,加味桔梗汤组和平阳霉素组在造模后早期都存在肺泡炎和肺部的损伤,从造模后第九天开始,平阳霉素组、加味桔梗汤组的中性粒细胞占细胞总数的百分比都趋于下降,加味桔梗汤组的中性粒细胞占细胞总数的百分比下降幅度较平阳霉素组占细胞总数的百分比下降幅度大,到第二十八天,平阳霉素组与生理盐水组比较仍有显著性差异,而加味桔梗汤组与生理盐水组比较没有显著性差异。说明加味桔梗汤促进了IPF小鼠肺部炎症的吸收、感染的痊愈以及肺组织的修复。IPF病变早期,肺泡巨噬细胞能合成和释放活性氧介质、肿瘤坏死因子α等对组织起损伤作用,还可分泌各种细胞因子如血小板源性生长因子(PDGF)、转化生长因子β等调控成纤维细胞在损伤区聚集、活化和增殖,促进细胞外基质的合成。在造模后,平阳霉素组、加味桔梗汤组的巨噬细胞占细胞总数的百分比较生理盐水组都出现下降,但没有表现出一定的趋势。平阳霉素组巨噬细胞占细胞总数的百分比虽然出现下降,但是平阳霉素组小鼠自造模后巨噬细胞绝对数依然高于生理盐水组,加味桔梗汤组巨噬细胞绝对数低于平阳霉素组,说明中药一定程度上抑制了巨噬细胞的增殖。在造模后,平阳霉素组、加味桔梗汤组的淋巴细胞数占细胞总数的百分比都出现升高,平阳霉素组淋巴细胞数占细胞总数的百分比从造模后第三天起呈逐渐增高的趋势,到第九天时达到高峰,到第十四天后有所下降,但是与生理盐水组比较仍有显著性差异。加味桔梗汤组的淋巴细胞数占细胞总数的百分比也出现增高,但从造模后第五天,加味桔梗汤组的淋巴细胞占细胞总数的百分比又逐渐下降,到造模后第二十八天,与生理盐水组相比较已经没有显著性差异。淋巴细胞则是继巨噬细胞和中性粒细胞之后出现的炎性细胞,但淋巴细胞在较长时间内保持高水平状态。本研究发现肺内巨噬细胞和中性粒细胞的增多先于淋巴细胞的浸润也支持这一观点。在本研究中也发现,当第七天肺内淋巴细胞浸润达高峰时,成纤维细胞也明显增多,而造模十四天后,随着成纤维细胞的增多和胶原沉积的增加,肺内炎性细胞的浸润也以淋巴细胞为主,表明淋巴细胞与成纤维细胞的增殖和胶原合成的增多有关。2.4 ELISA法测IPF模型小鼠左肺组织BMP蛋白水平实验在造模后第0天、1天、3天、5天、9天、14天、28天分别取平阳霉素组、加味桔梗汤组、生理盐水组8只小鼠的左肺,用酶联接免疫吸附剂测定( Enzyme-Linked Immunosorbnent Assay ELISA)方法测小鼠左肺组织BMP-4和BMP-7蛋白水平。实验结果表明,平阳霉素组及加味桔梗汤组小鼠的BMP-7蛋白含量在造模后五天内与生理盐水组小鼠相比较均呈下降趋势,但加味桔梗汤组小鼠逐渐抑制住了BMP-7蛋白含量的下降趋势,在第九天BMP-7蛋白含量开始回升,在第十四天基本恢复正常。而平阳霉素组的BMP-7蛋白含量一直趋于下降趋势,到第二十八天与生理盐水组比较仍有显著性差异。但中药治疗没有抑制住加味桔梗汤组小鼠BMP-4蛋白含量的下降趋势,与生理盐水组比较有显著性差异。2.5 RT-PCR法测IPF小鼠右肺BMPmRNA基因表达实验在IPF小鼠造模后第0天、1天、3天、5天、9天、14天、28天分别取平阳霉素组、加味桔梗汤组、生理盐水组8只小鼠的右肺,用RT-PCR方法测小鼠右肺组织BMP-4和BMP-7mRNA基因表达水平。实验结果表明,加味桔梗汤组和平阳霉素组小鼠肺部BMP-4mRNA的基因表达都较生理盐水组降低,从造模后开始呈持续性下降,说明中药加味桔梗汤对特发性肺纤维化模型小鼠肺部BMP-4mRNA的基因表达没有明显的调节作用。加味桔梗汤组和平阳霉素组小鼠肺部的BMP-7mRNA的基因表达在造模后都较生理盐水组出现降低,平阳霉素组小鼠肺部的BMP-7mRNA的基因表达从造模后开始呈持续性下降,加味桔梗汤组小鼠肺部BMP-7mRNA的基因表达在造模后也出现下降,但是从第五天开始出现缓慢的回升,这说明,中药加味桔梗汤可以提高特发性肺纤维化模型小鼠肺部BMP-7mRNA的基因表达,BMP-7有可能是中药治疗特发性肺纤维化模型小鼠的作用靶点。本课题的实验研究从特发性肺纤维化模型小鼠的生存状态、肺部病理变化动态观察、肺部BMP-4、BMP-7含量动态监测几个方面来研究加味桔梗汤对特发性肺纤维化模型小鼠的治疗作用。通过以上实验研究,可以得出以下结论1中药生存曲线组小鼠28天生存率远高于平阳霉素生存曲线组小鼠,说明加味桔梗汤对特发性肺纤维化模型小鼠有显著的治疗作用。2加味桔梗汤组小鼠肺部的炎症和肺损伤明显较平阳霉素组小鼠轻,淋巴细胞、中性粒细胞等炎症细胞渗出减少,在造模后28天,加味桔梗汤组小鼠没有出现肺部的纤维化。3 IPF模型小鼠肺中BMP-4、BMP-7蛋白含量和mRNA基因表达均出现下降,中药加味桔梗汤对IPF模型小鼠肺中的BMP-4的蛋白含量和mRNA基因表达没有调节作用,但加味桔梗汤提高了IPF模型小鼠肺中BMP-7的蛋白含量和mRNA基因表达。说明中药加味桔梗汤治疗特发性肺纤维化的作用靶点有可能是BMP-7。以上实验结果都表明,中药加味桔梗汤对IPF模型小鼠有治疗作用,其作用机制是提高IPF模型小鼠肺部的BMP-7蛋白含量和mRNA基因表达,减轻IPF模型小鼠肺部的炎症渗出,修复IPF模型小鼠受损伤的肺组织,BMP-7有可能是加味桔梗汤治疗IPF的作用靶点,但是还需要进行进一步的作用机制探索。更多还原

【Abstract】 Idiopathic pulmonary fibrosis(IPF) is an chronic inflammation Interstitial lung disease ,it’s marked pathologic change is interstitial pneumonitis.The main clinical manifestation of IPF is diffuse airsacculitis, alveoli of lung disorder,pulmonary fibrosis.The pathogenetic condition of IPF patient deteriorate,many IPF patients result in death because of respiratory failure.Now ,the drugs to treat IPF include glucocorticosteroid, immunodepressant /cytotoxic drug et al.These drugs have grave adverse reaction If patients long-term take these medicines,will cause body immunity to descend and secondary infection.So,it is investigation hot spot to search eutherapeutic, little side effect drugs.IPF don’t have corresponding syndrome name in traditional Chinese medition (TCM),it is relegated to“pulmonary arthralgia”“pulmonary paralysis”“the syndrome of dyspnea”by many traditional Chinese physicians.I think, IPF is belong to“pulmonary paralysis”of TCM.Traditional Chinese physicians of past dynasties have a great deal therapeutic methods to pulmonary paralysis,but all scatter in writings of traditional Chinese physicians of past, there is no system summary and induction. The theory research of my topic will systematic summarize therapeutic methods of traditional Chinese physicians to pulmonary paralysis, and bind to modern medicine, to explain some promiscuous questions , supply theory foundation for treat pulmonary paralysis by traditional Chinese medition.The empirical study of my topic will tracing latest development of international, to cooperate with Yale university, apply Jia Wei Jie Geng Decoction to cure IPF model mice, observe Jia Wei Jie Geng Decoction’s curative effect to IPF model mice , explore action mechanism of Jia Wei Jie Geng Decoction cure IPF , survey content change of bone morphogenetic protein-4 (BMP-4) and bone morphogenetic protein-7 (BMP-7), supply experiment foundation for treat pulmonary paralysis by classical prescription..1.Theory study1.1 Systematic summarize therapeutic methods of Traditional Chinese physicians to pulmonary paralysis.To look up TCM documents such as internal medicine, prescription, medical record, medical talks et.al, analysis and summary these TCM documents, probe experience and method of antiquity great physician treat pulmonary paralysis, reveal rule and achievement of TCM in diagnosis and treat pulmonary paralysis.1.2 Systematic summarize Biangzheng pattern of Zhongjng treat pulmonary paralysis.2. Empirical study2.1 survival rate experimentTake thirty C57BL/6 mice, random divided to the Chinese medicine survive group and the Bleomycin A5 survive group,all mice instillation Bleomycin A5 in airtube to make IPF model , the mice of the traditional Chinese medicine survive group intragastric administrate Jia Wei Jie Jeng Decoction,the mice of the Bleomycin A5 survive group intragastric administrate isotonic Na chloride, observe the survival condition of two group mice, depict survive curve, put to death all mice in twenty nine day after make IPF model .Experiment result indicate the survival rate of Chinese medicine survive group is 46%,survival rate of the Bleomycin A5 survive group is13%,this result indicate that the Jia Wei Jie Jeng Decoction has noticeable therapeutical effect to IPF model mice. 2.2 The pathological observation experiment of IPF mice model pulmonary tissue sampleTake eight mice part right lung in every group after make IPF model 0 day、1 day、3 day、5 day、9 day、14 day、28 day, fixed mice pulmonary tissue by 10%formalin, paraffin imbedding and slice, HE dyeing, to observe the pulmonary tissue pathological change of IPF mice model. The experiment result indicate that the Jia Wei Jie Geng Decoction group has marked therapeutic action to IPF mice model. Mice pneumonitis symptom of the Jia Wei Jie Geng Decoction group lessen than the Bleomycin A5 group, inflammatory cell grow downwards,the Jia Wei Jie Geng Decoction group mice have not appear pulmonary fibrosis.2.3 Bronchial lavage experimentTake two hundred C57BL/6 mice, random divided to three group: the Jia Wei Jie Geng Decoction group ,the Bleomycin A5 group and the isotonic Na chloride group.The isotonic Na chloride group has 60 mice, the Jia Wei Jie Geng Decoction group has 70 mice, the Bleomycin A5 group has 70 mice, Mice of the Isotonic Na chloride group don’t make model, the Jia Wei Jie Geng Decoction and the Bleomycin A5 group make IPF model by instillation Bleomycin A5 in mice airtube.Take eight mice in every group after make IPF model 0 day、1 day、3 day、5 day、9 day、14 day、28 day, macroscopic observation pathological change of mice pulmonary and make bronchial lavage, calculate cell amount by decuple micro,then make Jamsa dyeing, calculate cell percentage of neutrophil, macrophage,lymphocyte.Neutrophil is main inflammatory corpusclecute of lung injury morning stage, the neutrophil percentage of the Bleomycin A5 group and the Jia Wei Jie Geng Decoction group raise up after make the IPF mice model five days, have highly significant difference with the isotonic Na chloride group, this experiment result demonstrate that mice of the Bleomycin A5 group and the Jia Wei Jie Geng Decoction group have injury of lungs and alveolitis in the morning stage after make the IPF mice model. the neutrophil percentage of the Bleomycin A5 group and the Jia Wei Jie Geng Decoction group to descend after make the IPF mice model nine days,but the descend extent of the Jia Wei Jie Geng Decoction group super than the Bleomycin A5 group, after make the IPF mice model twenty eight days, Bleomycin A5 group have significant difference with the isotonic Na chloride group,but the Jia Wei Jie Geng Decoction group have no difference with the isotonic Na chloride group. This experiment result demonstrate that the Chinese medicine promote mouse pneumonitis to assimilate and tissue repair.In the morning stage of IPF, lung alveolus macrophage could synthesis and release active oxygen mediator and tumor necrosis factorαto damage pulmonary tissue, lung alveolus macrophage still secrete various kinds cell factor such as platelet-derived growth factor, transforming growth factorβ, these cell factors control fibroblast to gather, activation, proliferate. After make the IPF mice model, Macrophage percentage of the Bleomycin A5 group and the Jia Wei Jie Geng Decoction group lower than the isotonic Na chloride group,but have no determinate tendency. Macrophage absolute number of the Bleomycin A5 group still super than the isotonic Na chloride group, Macrophage absolute number of the the Jia Wei Jie Geng Decoction group lower than the Bleomycin A5 group, demonstrate the Chinese medicine restraint the macrophagocyte multiplication in certain extent.After make IPF mice model , Lympholeukocyte percentage of the Jia Wei Jie Geng Decoction group and the Bleomycin A5 group all raise up, lympholeukocyte percentage of the Bleomycin A5 group gradually ascend, reach peak after make IPF mice model nine day, gradually descend after make IPF mice model fourteenth day,but lympholeukocyte percentage of the Bleomycin A5 group still have significant difference with the isotonic Na chloride group after make IPF mice model twenty eight day. Lympholeukocyte percentage of the Jia Wei Jie Geng Decoction group also raise up, but gradually descend after make IPF mice model five day,lympholeukocyte percentage of the Jia Wei Jie Geng Decoction group have no significant difference with the isotonic Na chloride group after make IPF mice model twenty eight day.Lymphocyte is inflammatory cell which appear after macrophage and neutrophil,but lymphocyte maintain high level in long time.our research discovery that macrophage and neutrophil grow in number precede lymphocytic infiltration. .our research also discovery lymphocyte obviously grow in number after make IPF mice model nine day,at the same time fibroblast also increase, after make IPF mice model fourteen day, fibroblast obviously increase and collogen deposition, at the same time lymphocyte is main inflammatory cell in the lung,this experiment result indicated lymphocyte relevant with fibroblast generation and collogen deposition.2.4 Survey mice left lung BMP protein level by enzyme-linked immunosorbentassayTake eight mice left lung in every group after make IPF model 0 day、1 day、3 day、5 day、9 day、14 day、28 day, Survey mice left lung BMP-4 and BMP-7 protein level by enzyme-linked immunosorbentassay, the experiment result indicate,the BMP-7 protein level of the Bleomycin A5 group mice and the Jia Wei Jie Geng Decoction group mice not so many as the isotonic Na chloride group mice,but this downtrend had been hold back by Chinese medition,so the BMP-7 protein level of the Jia Wei Jie Geng Decoction group mice begin to advance in ninth day, to achieve normal after make IPF mice model twenty eight day. The BMP-7 protein level of the Bleomycin A5 group mice constantly descend, have significant difference to compare with the isotonic Na chloride group mice. the BMP-4 protein level of the the Jia Wei Jie Geng Decoction group mice not so many as the isotonic Na chloride group mice,this downtrend had not been hold back by Chinese medition, the BMP-4 protein level of the the Jia Wei Jie Geng Decoction group have significant difference to compare with the isotonic Na chloride group after make IPF mice model twenty eight day. 2.5 Survey mice right lung BMP mRNA level by reverse transcription-polymerase chain raction.Take eight mice right lung in every group after make IPF model 0 day、1 day、3 day、5 day、9 day、14 day、28 day, Survey mice right lung BMP-4 and BMP-7 mRNA level by reverse transcription-polymerase chain raction (Rt-PCR). the experiment result indicate,the BMP-4 mRNA level of the Bleomycin A5 group mice and the Jia Wei Jie Geng Decoction group mice continuity to descend after make IPF mice model, have significant difference to compare with the isotonic Na chloride group mice.This result indicate the Chinese medition has not regulation effect to BMP-4 mRNA level of IPF model mice. The BMP-7 mRNA level of the Bleomycin A5 group mice continuity descend after make IPF mice model, have significant difference to compare with the isotonic Na chloride group mice. The BMP-7 mRNA level of the Jia Wei Jie Geng Decoction group mice descend after make IPF mice model, then slowly to ascend after make IPF mice model five day, this result indicate the Chinese medition can increase BMP-7 mRNA level of IPF model mice.The BMP-7 may the effect target of the Chinese medition cure the IPF mice model.The empirical study of our topic investigate the therapeutic action of Jia Wei Jie Geng Decoction to IPF mice model from these aspect: living state of IPF mice model; observe pathological change of IPF mice model; kinesis monitor BMP-4、BMP-7 level of IPF mice model.We can make this conclusion by these empirical study:1. The 28 day survival rate of Chinese medicine survive group super than the Bleomycin A5 survive group,this result indicate that the Jia Wei Jie Jeng Decoction has noticeable therapeutical effect to IPF model mice.2. Mice pneumonitis symptom and the injury of pulmonary of the Jia Wei Jie Geng Decoction group lessen than the Bleomycin A5 group, inflammatory cell grow downwards,the Jia Wei Jie Geng Decoction group mice have not appear pulmonary fibrosis after make IPF mice model. 3.The protein level and the mRNA level of BMP-4 all descend,the Jia Wei Jie Geng Decoction group have no effect to improve BMP-4 level of IPF mice model,but the Jia Wei Jie Geng Decoction group increase the BMP-7 level of IPF mice model, this result indicate that the BMP-7 may be the effect target of the Chinese medition to cure IPF mice model,the concrete effect mechanism need further explore.更多还原