清肺消炎丸及有效成分缓解哮喘的系统生物学研究
摘要
哮喘为众多细胞和细胞组分参与的气道炎症疾病。在体内外因素的作用下,可能导致支气管的高反应性、支气管上皮结构发生改变、气道和肺组织重构以及呼吸功能异常。全球哮喘疾病的发生发展都呈现上升趋势。目前哮喘治疗药物主要围绕两个方面,一是利用支气管扩张剂治疗急性哮喘发作症状,二是利用抗炎药物对于慢性炎症的长期治疗。临床使用哮喘治疗药物包括β2受体激动剂、M受体拮抗剂、糖皮质激素、白三烯调节剂、胰蛋白酶抑制剂、免疫抑制剂、抗气道重塑药物、基因治疗等。
清肺消炎丸是根据医圣张仲景所著《伤寒论》中的经典名方“麻杏石甘汤”为基础的复方丸剂,有清肺化痰、止咳平喘之功效。临床用于痰热阻肺、咳嗽气喘、胸胁胀痛、吐痰黄稠；以及上呼吸道感染、急性支气管炎、慢性支气管炎急性发作等证候者。我们前期研究中发现,清肺消炎丸对组胺致喘豚鼠的哮喘潜伏期有明显的延长作用,能有效减少豚鼠的咳嗽次数；对豚鼠离体气管收缩的抑制效果较好,但其平喘作用机理方面的研究尚不够深入。本课题旨在从系统生物学角度探讨清肺消炎丸缓解哮喘的作用机制。
在本研究中的清肺消炎丸的系统生物学研究部分,首先建立豚鼠哮喘模型,并给予清肺消炎丸干预治疗；发现清肺消炎丸给药组明显延缓哮喘发作开始时间,并且支气管和肺组织病理切片显示给药后气道和肺组织炎症、水肿、嗜酸性粒细胞浸润、气道重塑现象都明显减轻,表明清肺消炎丸缓解哮喘的药效良好。然后设计并定制Agilent4x44K双通道豚鼠全基因组表达谱芯片对模型组和给药组的肺组织进行基因表达差异的筛查,共筛选豚鼠差异基因55条。对差异表达基因的Cluster聚类分析结果显示,清肺消炎丸组的基因表达特征与正常对照组样本较为接近,提示给药后整体基因表达水平有恢复正常的趋势。经Blast比对获得相应的人源基因27条,参与了炎症信号传递、气道平滑肌细胞凋亡、平滑肌重构、基质蛋白分泌等生物学过程。其中MAPK3,RHO,VIM,CLU, GNB1,ENO1(3)基因的荧光定量PCR验证结果与芯片实验结果基本相符,表明了芯片实验结果可靠性。
而后,采用蛋白质二维电泳-基质辅助激光解析电离飞行时间质谱技术对模型组和给药组样本进行了蛋白质组学研究。通过二维电泳筛选差异蛋白,并对其中6条差异蛋白进行质谱鉴定,发现其与炎症、哮喘、机体对药物敏感性、肺组织重构等相关,并对其中Hsp90的基因和蛋白表达水平进行了验证。将上述差异基因及差异蛋白对应基因进行GO基因功能注释、KEGG信号通路分析,发现各差异分子分别定位于多种细胞通路,共同参与炎症反应、细胞骨架及胞外基质调控、平滑肌收缩等信号通路。将实验确定的总的差异基因,基于HPRD数据库蛋白互作关系,投注于GAD数据库中哮喘相关的基因网络中,获得清肺消炎丸靶点与哮喘相关基因互作网络关系图,共含有1214个节点和1886个互作关系,表明清肺消炎丸的作用靶点与哮喘疾病相关基因有直接和间接的相互作用关系,对于探讨清肺消炎丸治疗哮喘、肺部炎症等疾病的作用机制提供有意义参考。
基于前期对于清肺消炎丸化学物质组学及药物作用通路的分析发现,牛蒡子苷元是清肺消炎丸中木质素类的代表成分,可能通过ERK/MAPK通路发挥抗炎作用。本研究进一步选择了牛蒡子苷元单体成分,对其缓解气道收缩、对气道平滑肌以及平滑肌细胞内的钙离子的影响、以及电生理方面的影响进行探索研究。
在牛蒡子舒张气道平滑肌的研究中,首先通过气管条张力测定的实验详细考察了牛蒡子苷元对缓解不同收缩剂致敏的豚鼠气管条的收缩现象,并进一步通过膜片钳记录牛蒡子苷元对L-型钙离子通道的影响和激光共聚焦显微镜(Confocal)来测定牛蒡子苷元影响支气管平滑肌细胞内钙离子浓度的变化,初步探索了其气管舒张作用的机制。结果发现,在正常K-H液中,牛蒡子苷元(0.1μM-1mM)可缓解10μM乙酰胆碱、10μM组胺、10mM氯化钾、30mM氯化钙不同预收缩剂所导致气管条的收缩,预孵育0.1mM牛蒡子苷元也可缓解气管条对收缩剂的反应,提示牛蒡子苷元可能通过不同机制缓解气管条收缩。在无钙K-H液中,牛蒡子苷元能显著降低气管条收缩,提示牛蒡子苷元能降低细胞内钙离子浓度,可能促进胞浆中的钙离子进入肌浆网或者促进钙离子排出胞外。膜片钳检测发现0.01mM牛蒡子苷元可降低人支气管平滑肌细胞(HBSMC)膜钙离子通道电流。Confocal显示牛蒡子苷元能促进细胞内钙离子排出和抑制细胞外钙离子入胞,进而降低细胞内钙离子浓度。推测牛蒡子苷元通过影响L-型钙离子通道而降低细胞内钙离子浓度可能是其中一种作用机制。
综上所述,我们通过实验筛选及验证了清肺消炎丸可能的作用靶点,并构建了与哮喘相关基因的相互作用关系网络,揭示了清肺消炎丸多成分、多靶点、多途径的网络作用特点,并进一步考察了复方中的一种单体成分牛蒡子苷元,舒张支气管平滑肌、调节细胞膜离子通道的作用机制,对于诠释中医药缓解哮喘有一定的借鉴意义。
Asthma, as defined in2008by the Global Initiative for Asthma (GINA), is an inflammatory disorder of the airways in which many cells and cellular elements play roles. Bronchial hyperactivity associates with inflammation that together with an external or environmental insult, on vulnerable bronchial epithelial structures, generates tissue remodelling and respiratory functional impairment. Asthma is not a curable disease at the present time. However, with proper treatments, the risk of mortality for asthmatic individuals could be comparable to that of the general population. Presently, the treatment of asthma includes a dual focus:the short-term treatment of acute symptoms with bronchodilators, and together with the prevention or eventual reversal of chronic inflammation using anti-inflammatory drugs.
Qingfei Xiaoyan Wan (QFXY) is originated from a famous Traditional Chinese Medicine (TCM) formula "Maxing Shigan Decoction". It has been experimentally improved, consisting of eight materia medicas, Ephedra Herba, Saigae Tataricae Cornu, Pheretima, Arctii Fructus, Lepidii Semen, Bovis Calculus Artifactus, Armeniacae Semen Amarum and Gypsum Fibrosum. Since decades of extensive clinical practice, QFXY has shown significantly therapeutic effects on dissolving phlegm as well as relieving cough, asthma, upper respiratory tract infection, bronchitis, pneumonia, and etc. Comparing with other anti-asthma drugs, it is characterised with moderate and persistent efficacy as well as few side effects, however, the underlying action mechanism still remains elusive.
We first established asthma model induced by histamine phosphate and acetylcholine chloride (His&Ach) in guinea pigs, which then were administered orally with QFXY (132.5mg/100g). Asthma prolonged time and Hematoxylin-Eosin staining sections were applied for evaluating QFXY effect. Comparing with the model group, in the QFXY group, the asthma onset time was significantly prolonged and HE sections presented anti-inflammation and anti-remodelling effects of QFXY. In both Model and QFXY groups, customized microarrays and2D electrophoresis were adopted to detect differentially expressed genes (diff genes) and proteins (diff proteins) respectively, and some diff proteins were identified with MALDI-TOF/MS. As a result,55diff genes (27human homologs after Blasting) and6diff proteins were identified in QFXY group. Validation by qPCR and Western blot showed the microarray and2D data reliable. The checked diff genes and proteins underwent Cluster, GO and KEGG analysis, which focused on signaling pathways in cytoskeleton and extracellular matrix regulation, smooth muscle contraction as well as inflammation response,etc. Based on GAD and HPRD databases, QFXY-asthma target regulation network was constructed, containing1214nodes and1886interactions.
Based on the combination of Chemomics and Systems biology research, after the formula study, we next focused on Arctigenin, an active ingredient in QFXY, is reported with anti-inflammation, antioxidation and vasodilator effects. However, its effects on airway smooth muscle and intracellular calcium, as well as ion channels remain eclusive.
To investigate how arctigenin regulates bronchus tone and calcium ion (Ca2+) flow. Trachea strips of guinea pigs were prepared for testing relaxation effect of arctigenin (0.1μM-1mM) to10μM acetylcholine,10μM histamine,10mM KCl and30mM CaCl2, respectively. Furthermore, L-type calcium channel currents were detected by patch-clamp and intracellular Ca2+concentration treated with arctigenin in human bronchial smooth muscle cells (HBSMC) was detected by confocal microscopy. The results showed that arctigenin exhibited relaxation effect on tracheae to different constrictors, and this was related to decreasing cytoplasmic Ca2+concentration by inhibiting Ca2+influx partly through L-type calcium channel as well as promoting Ca2+efflux.
Hereby, in the first part of the study, a primarily combined genomic and proteomic screening of QFXY targets displayed a series of candidate genes and proteins, which indicated that the effect of QFXY relied on the combined mechanism, anti-inflammation and anti-remodelling, as well as influencing signal transduction in vivo, demonstrating QFXY multi-target network regulation as an asthma controller. ASM related study provides new insight into the mechanisms by which arctigenin exhibits relaxation effect on bronchus and suggests its potential use for airway disease therapy. All together, these results reveal multiple active components, multiple targets and synergistic effects in the mechanism of action of a TCM.
清肺消炎丸是根据医圣张仲景所著《伤寒论》中的经典名方“麻杏石甘汤”为基础的复方丸剂,有清肺化痰、止咳平喘之功效。临床用于痰热阻肺、咳嗽气喘、胸胁胀痛、吐痰黄稠；以及上呼吸道感染、急性支气管炎、慢性支气管炎急性发作等证候者。我们前期研究中发现,清肺消炎丸对组胺致喘豚鼠的哮喘潜伏期有明显的延长作用,能有效减少豚鼠的咳嗽次数；对豚鼠离体气管收缩的抑制效果较好,但其平喘作用机理方面的研究尚不够深入。本课题旨在从系统生物学角度探讨清肺消炎丸缓解哮喘的作用机制。
在本研究中的清肺消炎丸的系统生物学研究部分,首先建立豚鼠哮喘模型,并给予清肺消炎丸干预治疗；发现清肺消炎丸给药组明显延缓哮喘发作开始时间,并且支气管和肺组织病理切片显示给药后气道和肺组织炎症、水肿、嗜酸性粒细胞浸润、气道重塑现象都明显减轻,表明清肺消炎丸缓解哮喘的药效良好。然后设计并定制Agilent4x44K双通道豚鼠全基因组表达谱芯片对模型组和给药组的肺组织进行基因表达差异的筛查,共筛选豚鼠差异基因55条。对差异表达基因的Cluster聚类分析结果显示,清肺消炎丸组的基因表达特征与正常对照组样本较为接近,提示给药后整体基因表达水平有恢复正常的趋势。经Blast比对获得相应的人源基因27条,参与了炎症信号传递、气道平滑肌细胞凋亡、平滑肌重构、基质蛋白分泌等生物学过程。其中MAPK3,RHO,VIM,CLU, GNB1,ENO1(3)基因的荧光定量PCR验证结果与芯片实验结果基本相符,表明了芯片实验结果可靠性。
而后,采用蛋白质二维电泳-基质辅助激光解析电离飞行时间质谱技术对模型组和给药组样本进行了蛋白质组学研究。通过二维电泳筛选差异蛋白,并对其中6条差异蛋白进行质谱鉴定,发现其与炎症、哮喘、机体对药物敏感性、肺组织重构等相关,并对其中Hsp90的基因和蛋白表达水平进行了验证。将上述差异基因及差异蛋白对应基因进行GO基因功能注释、KEGG信号通路分析,发现各差异分子分别定位于多种细胞通路,共同参与炎症反应、细胞骨架及胞外基质调控、平滑肌收缩等信号通路。将实验确定的总的差异基因,基于HPRD数据库蛋白互作关系,投注于GAD数据库中哮喘相关的基因网络中,获得清肺消炎丸靶点与哮喘相关基因互作网络关系图,共含有1214个节点和1886个互作关系,表明清肺消炎丸的作用靶点与哮喘疾病相关基因有直接和间接的相互作用关系,对于探讨清肺消炎丸治疗哮喘、肺部炎症等疾病的作用机制提供有意义参考。
基于前期对于清肺消炎丸化学物质组学及药物作用通路的分析发现,牛蒡子苷元是清肺消炎丸中木质素类的代表成分,可能通过ERK/MAPK通路发挥抗炎作用。本研究进一步选择了牛蒡子苷元单体成分,对其缓解气道收缩、对气道平滑肌以及平滑肌细胞内的钙离子的影响、以及电生理方面的影响进行探索研究。
在牛蒡子舒张气道平滑肌的研究中,首先通过气管条张力测定的实验详细考察了牛蒡子苷元对缓解不同收缩剂致敏的豚鼠气管条的收缩现象,并进一步通过膜片钳记录牛蒡子苷元对L-型钙离子通道的影响和激光共聚焦显微镜(Confocal)来测定牛蒡子苷元影响支气管平滑肌细胞内钙离子浓度的变化,初步探索了其气管舒张作用的机制。结果发现,在正常K-H液中,牛蒡子苷元(0.1μM-1mM)可缓解10μM乙酰胆碱、10μM组胺、10mM氯化钾、30mM氯化钙不同预收缩剂所导致气管条的收缩,预孵育0.1mM牛蒡子苷元也可缓解气管条对收缩剂的反应,提示牛蒡子苷元可能通过不同机制缓解气管条收缩。在无钙K-H液中,牛蒡子苷元能显著降低气管条收缩,提示牛蒡子苷元能降低细胞内钙离子浓度,可能促进胞浆中的钙离子进入肌浆网或者促进钙离子排出胞外。膜片钳检测发现0.01mM牛蒡子苷元可降低人支气管平滑肌细胞(HBSMC)膜钙离子通道电流。Confocal显示牛蒡子苷元能促进细胞内钙离子排出和抑制细胞外钙离子入胞,进而降低细胞内钙离子浓度。推测牛蒡子苷元通过影响L-型钙离子通道而降低细胞内钙离子浓度可能是其中一种作用机制。
综上所述,我们通过实验筛选及验证了清肺消炎丸可能的作用靶点,并构建了与哮喘相关基因的相互作用关系网络,揭示了清肺消炎丸多成分、多靶点、多途径的网络作用特点,并进一步考察了复方中的一种单体成分牛蒡子苷元,舒张支气管平滑肌、调节细胞膜离子通道的作用机制,对于诠释中医药缓解哮喘有一定的借鉴意义。
Asthma, as defined in2008by the Global Initiative for Asthma (GINA), is an inflammatory disorder of the airways in which many cells and cellular elements play roles. Bronchial hyperactivity associates with inflammation that together with an external or environmental insult, on vulnerable bronchial epithelial structures, generates tissue remodelling and respiratory functional impairment. Asthma is not a curable disease at the present time. However, with proper treatments, the risk of mortality for asthmatic individuals could be comparable to that of the general population. Presently, the treatment of asthma includes a dual focus:the short-term treatment of acute symptoms with bronchodilators, and together with the prevention or eventual reversal of chronic inflammation using anti-inflammatory drugs.
Qingfei Xiaoyan Wan (QFXY) is originated from a famous Traditional Chinese Medicine (TCM) formula "Maxing Shigan Decoction". It has been experimentally improved, consisting of eight materia medicas, Ephedra Herba, Saigae Tataricae Cornu, Pheretima, Arctii Fructus, Lepidii Semen, Bovis Calculus Artifactus, Armeniacae Semen Amarum and Gypsum Fibrosum. Since decades of extensive clinical practice, QFXY has shown significantly therapeutic effects on dissolving phlegm as well as relieving cough, asthma, upper respiratory tract infection, bronchitis, pneumonia, and etc. Comparing with other anti-asthma drugs, it is characterised with moderate and persistent efficacy as well as few side effects, however, the underlying action mechanism still remains elusive.
We first established asthma model induced by histamine phosphate and acetylcholine chloride (His&Ach) in guinea pigs, which then were administered orally with QFXY (132.5mg/100g). Asthma prolonged time and Hematoxylin-Eosin staining sections were applied for evaluating QFXY effect. Comparing with the model group, in the QFXY group, the asthma onset time was significantly prolonged and HE sections presented anti-inflammation and anti-remodelling effects of QFXY. In both Model and QFXY groups, customized microarrays and2D electrophoresis were adopted to detect differentially expressed genes (diff genes) and proteins (diff proteins) respectively, and some diff proteins were identified with MALDI-TOF/MS. As a result,55diff genes (27human homologs after Blasting) and6diff proteins were identified in QFXY group. Validation by qPCR and Western blot showed the microarray and2D data reliable. The checked diff genes and proteins underwent Cluster, GO and KEGG analysis, which focused on signaling pathways in cytoskeleton and extracellular matrix regulation, smooth muscle contraction as well as inflammation response,etc. Based on GAD and HPRD databases, QFXY-asthma target regulation network was constructed, containing1214nodes and1886interactions.
Based on the combination of Chemomics and Systems biology research, after the formula study, we next focused on Arctigenin, an active ingredient in QFXY, is reported with anti-inflammation, antioxidation and vasodilator effects. However, its effects on airway smooth muscle and intracellular calcium, as well as ion channels remain eclusive.
To investigate how arctigenin regulates bronchus tone and calcium ion (Ca2+) flow. Trachea strips of guinea pigs were prepared for testing relaxation effect of arctigenin (0.1μM-1mM) to10μM acetylcholine,10μM histamine,10mM KCl and30mM CaCl2, respectively. Furthermore, L-type calcium channel currents were detected by patch-clamp and intracellular Ca2+concentration treated with arctigenin in human bronchial smooth muscle cells (HBSMC) was detected by confocal microscopy. The results showed that arctigenin exhibited relaxation effect on tracheae to different constrictors, and this was related to decreasing cytoplasmic Ca2+concentration by inhibiting Ca2+influx partly through L-type calcium channel as well as promoting Ca2+efflux.
Hereby, in the first part of the study, a primarily combined genomic and proteomic screening of QFXY targets displayed a series of candidate genes and proteins, which indicated that the effect of QFXY relied on the combined mechanism, anti-inflammation and anti-remodelling, as well as influencing signal transduction in vivo, demonstrating QFXY multi-target network regulation as an asthma controller. ASM related study provides new insight into the mechanisms by which arctigenin exhibits relaxation effect on bronchus and suggests its potential use for airway disease therapy. All together, these results reveal multiple active components, multiple targets and synergistic effects in the mechanism of action of a TCM.
引文
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