治疗肺纤维化复方的组方原理及配伍优化研究

发布时间：2018-03-17 05:28

本文选题：肺纤维化　切入点：咳嗽　出处：《北京中医药大学》2014年硕士论文　论文类型：学位论文

【摘要】：特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)以进行性呼吸困难,随病情发展,气不相续发为喘,同时伴刺激性干咳为主要症状。系统整理发掘仲景辨治咳嗽与喘证的用药规律,并结合近年中医药治疗IPF的用药经验,制定出了用于治疗IPF配伍优化的复方。运用混料均匀设计的方法将复方设计成药物组成相同而剂量配比不同的多首复方,将各复方作用于以博莱霉素诱导的雄性ICR小鼠肺纤维化模型,以模型小鼠7d、28d死亡率及Hyp含量为药效指标来寻找该复方最优配伍模式,为中医临床治疗IPF合理用药提供实验依据。理论研究理论研究一：仲景关于咳嗽与喘证的辨治规律探析系统整理并研究《伤寒论》及《金匮要略》中出现的有关“咳”、“喘”及“短气”的条文,揭示仲景在治疗咳喘方面的用药规律。在辨证论治的基础上,基于药证相应、舍性取用理论,指出仲景治咳常用药物组合为干姜、细辛、五味子；对于喘证的治疗仲景擅用麻黄,因麻黄功擅宣肺平喘,为治喘之要药,且麻黄常与半夏为伍,共奏降逆平喘之功。理论研究二：用治IPF复方的选药依据基于中医对IPF病程一般发展规律的认识,以症状学为切入点,认为本病早期可归属中医“咳嗽”与“喘证”的范畴,其基本的病理改变为肺气阴两虚；随着病情的发展,症状又可见唇指发绀、舌质紫暗等瘀血证候,以及手足逆冷、皮肤发凉等阳虚证候；病情进一步发展,又会出现咳喘加剧、动则喘甚等肺肾两虚证候,并伴见乏力、厌食、消瘦等虚劳证候。基于对IPF中医证候演变发展规律的认识,从仲景辨治咳喘的规律、中医临床治疗IPF主要方药的文献报道寻找用于治疗IPF的中药复方,最终确定了用治IPF复方优化筛选的具体药物组成为：麦冬、法半夏、干姜、五味子、炙麻黄、炙黄芪、党参、炙甘草、当归、丹参、黄芩、桔梗。实验研究实验一：用治IPF复方不同配伍比例的药效实验研究实验方法：将195只雄性ICR小鼠随机分为13组,即正常组、模型组和11个中药复方组。小鼠麻醉后向气管内注射博莱霉素,造成肺纤维化模型。复方组小鼠每日给予混料均匀设计好的总剂量相等而配比不同的11种复方,正常组及模型组小鼠给予等量的生理盐水,连续灌胃28d。观察各组小鼠的生存状态、体重变化,统计死亡情况；碱水解法测定肺组织Hyp含量；HE染色和Masson染色,观察小鼠肺部病理变化。实验结果及讨论： 1小鼠一般状况变化：第7d时,复方第1、5组小鼠体重较模型组增加,复方第11组小鼠体重较模型组下降；第14d时,复方第1组小鼠体重较模型组增加；第28d时,复方第1组小鼠体重较模型组增加。 2各复方对小鼠死亡率的影响及配伍优化：(1)复方对小鼠死亡率的影响：第2、5组复方对于提高小鼠肺纤维化早期(炎症期)的生存率有帮助；第1、2、5、6组复方对于提高肺纤维化模型小鼠整个周期的生存率有帮助。(2)各组小鼠28d生存率与28d平均体重有直线相关关系,说明肺纤维化小鼠体重越高,生存优势越明显。(3)以7d死亡率为药效指标的配伍优化：麦冬、当归、五味子、桔梗、黄芩对于死亡率的降低起主导作用,干姜、炙黄芪对于死亡率的降低起辅助作用。(4)以28d死亡率为药效指标的配伍优化：麦冬、炙黄芪、当归、桔梗四味药物起主导作用。 3各复方对小鼠肺组织28dHyp含量的影响及配伍优化：(1)复方对小鼠肺组织28dHyp含量的影响：第1组复方能显著降低小鼠肺组织Hyp含量,第11组复方可升高小鼠肺组织Hyp含量。(2)各组小鼠28d死亡率与28dHyp含量有直线相关关系,说明有效抑制肺纤维化组织中胶原的沉积对于生存率的提高有积极意义。(3)以小鼠28d肺组织Hyp含量为药效指标的配伍优化：五味子桔梗、炙黄芪、炙甘草、黄芩起主导作用,麦门冬、丹参起一定的辅助作用。 4肺组织形态学观察：(1)HE染色观察炎症细胞浸润：复方1、2、5组在一定程度上有减轻小鼠肺泡炎,促进肺实质细胞修复的作用,并具有一定的抑制肺纤维化形成的作用。复方3、6组在一定程度上具有减轻小鼠肺泡炎和促进肺实质修复的作用。(2) Masson染色观察纤维化程度：复方第1、2、5、6组胶原含量较模型组明显减少,表明这些复方在减轻胶原纤维沉积和抗纤维化方面的作用显著。以Hyp含量为药效指标的优化出的药物基本包含了以7d死亡率和28d死亡率为药效指标优化出的药物。说明优化结果因药效指标的不同而有所差异,但无论以哪个指标进行优化,结果中的主要药物均在五味子、桔梗、炙黄芪、炙甘草、黄芩、麦门冬、当归之中,只是它们的配伍比例有所不同,提示这些药物配伍组成的复方对于肺纤维化的治疗有一定作用。实验二：药效学再验证实验实验方法：将60只雄性ICR小鼠随机分为6组,分别为正常组、模型组、复方第5组、7d死亡率优化组、28d死亡率优化组、序贯治疗组。检测各组小鼠肺组织Hyp含量及CTGF、IL-6、Foxp3mRNA含量。实验结果及讨论： 1序贯治疗组无论对于减少肺组织中胶原的沉积,还是下调某些细胞因子(如CTGF、IL-6)的基因表达水平都有显著疗效,这为IPF的分期治疗(早期在补肺滋阴的同时兼顾清热解毒,后期注重滋阴助阳、补气活血养血)提供了实验依据。 2序贯治疗组和28d死亡率优化组复方可升高Foxp3mRNA表达水平,28d死亡率优化组复方可降低CTGFmRNA和IL-6mRNA表达水平,序贯治疗组复方可降低CTGFmRNA表达水平。结果表明CTGF及IL-6基因表达过量、Foxp3基因表达不足可能是IPF发病的重要因素。
[Abstract]:Idiopathic pulmonary fibrosis (idiopathic pulmonary, fibrosis, IPF) with dyspnea, with the progression of the disease, not continue to breathe air, at the same time with the irritating cough as the main symptom. The system finishing excavation of Zhongjing cough and asthma and drug laws, combined with the clinical experience of Chinese medicine in treating IPF in recent years. Developed for treatment of compound optimization. Using IPF with uniform mixing design method will be designed into the same composition and compound drug dose of different proportion of the first compound, the compound effect on male ICR mice pulmonary fibrosis model induced by bleomycin in mice, with 7d, 28d and Hyp content for the efficacy of mortality to find the optimal parameters of compound compatibility mode, to provide the experimental basis for the clinical treatment of IPF medication.
theoretical research
A study on the theory of Zhongjing on cough and asthma treatment rules of
Collect and Study on typhoid fever and < < > > in the Golden Chamber "cough", "asthma" and "short" provisions, reveal Zhongjing medication rules in the treatment of cough and asthma aspects. In the basis of syndrome differentiation and treatment, based on the corresponding medicine, take care theory, pointed out that the secondary kageharu cough drugs commonly used combination of ginger, asarum, Schisandra chinensis; for asthma treatment in Zhongjing using ephedra, ephedra for work at Xuanfei asthma, for the treatment of asthma to medicine, and Ma Huang and Pinellia company, played a total of relieving asthma of power.
Theoretical study two: the basis for the treatment of IPF compound
Understanding the general law of development of IPF course based on traditional Chinese medicine, with symptoms as the starting point, that the disease attributable TCM "cough" and "asthma" category, the basic pathological changes of the lung qi and yin deficiency; with the development of disease symptoms, but also refers to the lip cyanosis, dark purple tongue blood stasis wait, and hand foot skin against the cold cold Yang deficiency syndrome; further development of the disease, will appear cough aggravating, while activing lung and kidney deficiency syndromes such as two, and accompanied by fatigue, anorexia, weight loss and other asthenia syndrome. Understanding of IPF TCM syndrome evolution law based on the Zhongjing treating cough of TCM clinical treatment of IPF mainly medicine reported in the literature for Chinese herbal compound in the treatment of IPF, and ultimately determine the composition of IPF treated with compound optimization specific drug screening: Ophiopogon japonicus, pinellia, ginger, ephedra, Schisandra, astragalus root, dangshen, roasted sweet Grass, angelica, Salvia miltiorrhiza, Scutellaria, Platycodon.
experimental study
Experiment 1: Experimental Study on the efficacy of different proportions of IPF compound
Experimental methods:
195 male ICR mice were randomly divided into 13 groups: normal group, model group and 11 compound Chinese medicine group. The mice were anesthetized after intratracheal injection of bleomycin, pulmonary fibrosis caused by compound model. Mice were given daily uniform mixing design total dose equal and different ratio of 11 kinds of compound, normal saline group and model group were given equal weight change, continuous intragastric 28d. mice were observed, the survival of the state, death statistics; Hyp content of lung tissue was measured by alkaline hydrolysis; HE staining and Masson staining to observe the pathologic changes in the lung.
Experimental results and discussions:
1, general condition changes of mice: at 7d, the weight of compound 1,5 group increased compared with the model group, and the weight of compound eleventh group mice decreased compared with the model group. At the time of 14d, the weight of compound first group mice increased compared with the model group. When 28d was added, the weight of first groups of compound mice increased compared with the model group.
2 the compound effect on the mortality of mice and the compatibility of Optimization: (1) the effect of compound on the mortality of mice: Group 2,5 compound for improving early pulmonary fibrosis in mice (inflammation) the survival rate of help; the group 1,2,5,6 compound to help improve the survival rate of the entire cycle of the pulmonary fibrosis model in mice. (2) a a linear correlation between the average weight of the mice survival rate of 28d and 28d, the higher the weight of mouse pulmonary fibrosis, a survival advantage is more obvious. (3) to 7d mortality formula optimization efficacy indicators: Radix, Angelica sinensis, Schisandra chinensis, Platycodon grandiflorum, Scutellaria for reducing the mortality play a leading role, ginger, astragalus root to reduce mortality play a supporting role. (4) to 28d mortality formula optimization efficacy indicators: Radix, astragalus root, angelica, Balloonflower four drugs play a leading role.
3 the compound effect on the 28dHyp content in the lung tissue of mice and the compatibility of Optimization: (1) effect on the content of 28dHyp in the lung tissue of mice in first groups: compound can significantly reduce the Hyp content in lung tissue of mice, eleventh groups of compound can increase the content of Hyp in lung tissue of mice. (2) the death rate of mice 28d and 28dHyp content line correlation between that inhibit collagen deposition in pulmonary fibrosis has a positive significance to improve the survival rate. (3) the content of Hyp in lung tissue of mice 28d compatibility optimization efficacy indicators: Schisandra astragalus root, licorice root, Platycodon root, Scutellaria plays a leading role, Ophiopogon japonicus, Salvia miltiorrhiza auxiliary function.
Observation of 4 lung tissue morphology: (1) to observe the infiltration of inflammatory cells in HE staining: 1,2,5 group compound to a certain extent alleviated alveolitis in mice, promote the repair of lung parenchyma cells, and inhibit the formation of pulmonary fibrosis has certain effect. The compound 3,6 group to a certain extent with ease and promote pulmonary alveolitis in mice the essence of repair. (2) to observe the degree of fibrosis Masson staining: group 1,2,5,6 compound collagen content was significantly reduced compared with model group showed that the compound in reducing collagen deposition and anti fibrosis effect.
The contents of Hyp was optimized by drug efficacy index basically contains 7d and 28d mortality for drug efficacy index optimization. The optimization results show that the pharmacodynamic indexes vary, but whichever index is optimized, the main results in drug Schisandra, astragalus root, licorice root, Platycodon grandiflorum. Baicalin, Ophiopogon, angelica, but they have different compatibility proportion, suggesting that compound composition of these drugs have certain effect for the treatment of pulmonary fibrosis.
Experiment two: revalidation experiment of pharmacodynamics
Experimental methods:
60 male ICR mice were randomly divided into 6 groups: normal group, model group, compound fifth group, 7d mortality optimization group, 28d mortality optimization group and sequential treatment group. The Hyp content and CTGF, IL-6 and Foxp3mRNA contents in lung tissue of each group were detected.
Experimental results and discussions:
1 sequential therapy group for reducing collagen deposition in the lung tissue, or down-regulation of some cytokines (CTGF, IL-6) gene expression levels have a significant effect, which is IPF (in the early stage treatment of Bufei Ziyin both detoxification, late on the Yin and Yang, Qi and nourishing blood) provides on the basis of experiment.
2 sequential therapy group and 28d group compound can increase the mortality to optimize the expression level of Foxp3mRNA, 28d group compound optimization can reduce CTGFmRNA mortality and IL-6mRNA expression level, sequential therapy group compound can reduce the expression level of CTGFmRNA. The results showed that overexpression of CTGF and IL-6, the expression of Foxp3 gene deficiency may be an important factor in the pathogenesis of IPF.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R289;R259

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