【作者】 程彬峰；

【导师】 白钢；

【作者基本信息】 南开大学 ， 微生物学， 2013， 博士

【摘要】 哮喘和慢性阻塞性肺疾病是气道炎症的主要表现形式,是多种刺激因素、多种细胞、多种炎症介质、多种靶点和通路共同参与构成的复杂炎症网络。目前这类疾病困扰着世界上3亿人口,位于疾病死亡率的第四位。中药对哮喘有较多的治疗经验,中药复方集中体现了中医方剂理论和组方原则,其防病治病的有效性已被几千年的临床实践所证实。而中药具有多成分、多靶点的多样性特点,是一个十分复杂的系统,大部分中药复方的药效物质基础和药理机制尚不清晰,本研究选取治疗呼吸道炎症的中药复方-清肺消炎丸作为研究对象,对其多组分协同作用的治愈效果及分子作用机理进行了研究。在本研究中,首先采用UPLC/Q-TOF分析技术,对清肺消炎丸的整体化学物质基础进行研究,共鉴定出了55种化学成分,对鉴定出的物质利用Molinspiration、PharmMapper和KEGG等生物信息学手段对其进行吸收、靶点及作用通路的预测分析,预测结果有27种成分可能被吸收,其中19种成分可能通过HRAS、PDPK1等11个靶点起到抗炎作用,主要为硫炔类、木脂内酯类、酚酸类和类固醇类化合物。建立了铜绿假单胞菌PAK菌株诱导的小鼠急性肺炎模型和TNF-a诱导人支气管上皮BEAS-2B的细胞炎症模型,在体内、外(in vivo和in vitro)对清肺消炎丸的抗炎效果进行研究。高、中剂量(18或6g／k曲的清肺消炎丸预给药1周后,能显著提高小鼠的存活率,降低TNF-α、IL-6、IL-8和RANTES等炎症因子的表达,减少肺部细胞因子及免疫细胞的浸润,改善肺部组织的水肿、坏死脱落；2.5或0.5mg/mL的清肺消炎丸干预能显著地抑制BEAS-2B细胞中TNF-α诱导的IL-6和IL-8的增高,NF-κB荧光活性及Western blot分析显示,清肺消炎丸通过抑制IκB的降解和NF-κB的激活进而起到抗炎的作用。建立了一种简便、快速的UPLC/Q-TOF-MS结合双荧光素酶报告基因系统的筛选NF-κB抑制剂的分析方法,对清肺消炎丸中的抗炎成分进行筛选鉴定,共筛选出4种结构类型的NF-κB抑制剂,分别为：牛蒡子苷元类(络石苷、牛蒡子苷元)、胆酸类(甘氨胆酸、胆酸、脱氧胆酸、脱氧甘氨胆酸)、芥子酸和绿原酸；采用EILISA法对筛选获得的单体化合物的抗炎活性进行了验证,结果表明,牛蒡子苷元类、胆酸、绿原酸和芥子酸能显著地降低BEAS-2B细胞中TNF-a诱导的IL-6和IL-8的表达,进一步确证了清肺消炎丸中NF-κB抑制剂的抗炎效果。采用TNF-a刺激的人支气管上皮细胞BEAS-2B炎症模型,研究了清肺消炎丸中4种抗炎成分的协同作用关系及作用机理。NF-κB荧光素酶分析结果显示,牛蒡子苷元与胆酸、绿原酸和芥子酸等两两配伍合用时,比各自单独使用有更强的NF-κB抑制效果；进一步以牛蒡子苷元为例,利用real-time PCR技术,检测炎症反应相关通路中关键基因的表达,来研究协同抗炎的作用机理。结果显示,牛蒡子苷元除了影响p38、JNK, ERK1、FAK等基因外,还能显著影响炎症网络较上游的JAK和PKC的表达,而且ELISA检测结果显示,牛蒡子苷元的配伍协同作用主要通过抑制p38、JNK和ERK等蛋白的表达而起到协同增效作用,而且不同的配伍所干预的抑制MAPK的途径也不同,牛蒡子苷元与绿原酸和芥子酸配伍主要干预JNK途径,而牛蒡子苷元与胆酸配伍侧偏重干预ERK途径。在本研究中,我们建立了一种UPLC/Q-TOF-MS结合双荧光素酶报告基因系统的筛选β2-AR激动剂的筛选方法,对清肺消炎丸中的平喘相关成分进行筛选鉴定,共筛选出1个β-AR激动剂麻黄碱和牛蒡子苷、牛蒡子苷元、南葶苈子苷和南葶苈内酯B等4种牛蒡子苷元结构类型的协同麻黄碱增效的成分。采用豚鼠离体平滑肌实验对牛蒡子苷元与麻黄碱协同平喘效果进行确认,结果显示,牛蒡子苷元和麻黄碱各自均能较好地抑制乙酰胆碱(ACh)诱导的气管平滑肌的收缩,两者联用时,平喘效果显著增强,β-AR拮抗剂普萘洛尔(Pro)能阻断麻黄碱与牛蒡子苷元的协同增强作用。利用双荧光素酶报告基因的β2AR活性评价系统,研究了特异性β2AR阻断剂ICI118551、通透细胞膜的AC激活剂forskolin对牛蒡子苷元协同作用的影响。ICI118551可阻断牛蒡子苷元与麻黄碱的协同作用,10-5mol/L的牛蒡子苷元能显著增强forskolin的激动效应,结果表明,牛蒡子苷元的协同作用建立在p2-AR通路信号激活的基础之上,但并不是β2-AR依赖性的,其作用位点应位于细胞内AC信号的下游。综上所述,我们基本确定了清肺消炎丸中的抗炎、平喘的有效化学成分,初步阐释了清肺消炎丸有效成分之间的协同增效关系及其作用机理,揭示了中医清肺消炎多成分、多靶点、多途径的网络作用特点,对于诠释中医治肺理论有一定的借鉴意义。更多还原

【Abstract】 Airway inflammation is a central problem in allergic asthma and chronic obstructive pulmonary disease (COPD). Airway inflammation is a consequence of complex interactions between multiple cell types, cytokines, and mediators in an inflammatory network. A single-target approach is unlikely to be effective for the treatment of inflammatory lung diseases. Multiple-drug therapy, which maintains efficacy and diminishes the risk of toxicity from individual drugs, has become widely recognized in recent years. Traditional Chinese medicine (TCM) preparations have been used as an effective multi-target strategy for the treatment of complex diseases; however, their bioactive constituents are difficult to identify and the mechanisms are undefined.In this study, the global chemome of Qingfei Xiaoyan Wan (QFXY) was illustrated by using the ultra-performance liquid chromatography/quadrupole time-of-flight (UPLC/Q-TOF) mass spectrum, and55compounds were identified. Chemical constituents of QFXY were submit to Molinspiration, PharmMapper and KEGG bioinformatics softwares for predicting their absorption parameters, target proteins and related pathways, respectively. The predicted results showed that19of the27absorbable constituents, which are sulfur alkynes, lignan lactones, phenolic acids and steroid compounds, may be involved in inflammation through11protein targets such as HRAS and PDPK1.We investigate anti-inflammatory effects of QFXY in vivo and in vitro. QFXY was administered orally to KM mice twice per day for one week, followed by challenge with an intratracheal Pseudomonas aeruginosa suspension. Mortality, lung histological features, and inflammatory cytokines were evaluated" Cytokines were also evaluated in TNF-a stimulated bronchial epithelial cells (BEAS-2B) incubated with QFXY, and NF-κB activation was evaluated using luciferase assay and Western blot. QFXY treatment significantly reduced Pseudomonas aeruginosa-induced mortality and also reduced cytokine production in lung tissue and plasma (TNF-a, IL-6, IL-8, and RANTES) and in TNF-a stimulated BEAS-2B cells (IL-6and IL-8). Moreover, in bronchial epithelial cells, an NF-κB luciferase assay and Western blot analysis demonstrated that QFXY inhibited NF-κB activation and IκB-α degradation.A simple method based on UPLC/Q-TOF MS combined NF-κB dual-luciferase reporter assay systems was developed for the rapid determination of anti-inflammatory compounds of TCM preparations.8potential NF-κB inhibitors were characterized and they could be classified into four types according to their chemical structures:arctigenin derivatives (tracheloside and arctigenin), cholic acid derivatives (cholic acid, glycocholic acid, deoxycholic acid, and deoxyglycocholic acid), chlorogenic acid, and sinapic acid. Tracheloside was considered a new NF-κB inhibitor. Further cytokine and chemokine (IL-6and IL-8) detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors.TNF-a induced inflammation model in human bronchial epithelial cells BEAS-2B was used to investigate synergistic anti-inflammatory effects and mechanisms of the four NF-κB inhibitors in QFXY. NF-κB luciferase analysis revealed that paired combination with arctigenin showing stronger inhibition effect than the single use. Real-time PCR was used to detect the expression of key inflammatory genes for the study of synergistic anti-inflammatory mechanism of arctigenin. The results showed that paired combination with arctigenin not only affect the expression of p38, JNK, ERK1, FAK, but also significantly affect the expression of JAK and PKC. The results of ELISA test showed that synergistic anti-inflammatory mechanism of paired combination with arctigenin mainly focused on the intervention of the levels of p38, JNK and ERK proteins.We developed a simple method based on UPLC/Q-TOF MS combined β2-adrenergic receptor (β2-AR) dual-luciferase reporter assay systems for the rapid determination of spasmolytic constituents in TCM preparations. One β2-AR agonist ephedrine and four synergistic constituents (arctiin, arctigenin, descurainoside and descurainolide B) were characterized. The four synergistic compounds could be classified into one type according to their chemical structures:arctigenin derivatives. Ephedrine and arctigenin combination strongerly relax the isolated guinea pig trachea strip precontraction with acetylcholine (ACh) than the single use, and the β-AR antagonist propranolol (Pro) could block the synergistic effect. From the β2-AR dual luciferase reporter gene activity analysis, specific the β2-AR blocking agent ICI118551blocked the synergistic effect of ephedrine and arctigenin combination, and10-5mol/L of arctigenin can significantly enhance the effects of the AC activator forskolin. These results indicated that β2-AR signal is necessary for the synergistic effect, and it is not β2-AR dependent.In this study, we demonstrate the anti-inflammatory and anti-asthmatic ingredients, the synergistic effects of active compounds and their mechanism of action. These results reveal multiple active components, multiple targets and synergistic effects in the mechanism of action of a TCM.更多还原