基于网络药理学方法研究玉屏风散治疗哮喘的作用机理

发布时间：2017-12-27 05:14

本文关键词：基于网络药理学方法研究玉屏风散治疗哮喘的作用机理　出处：《西南交通大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本研究目的在于通过网络药理学的方法预测玉屏风散治疗哮喘的作用机制,并通过细胞实验和动物实验对预测的机制进行进一步的验证。方法:1.采用网络药理学的方法对玉屏风散调控哮喘的作用方式及作用机制的分析步骤如下:通过TCMSP数据库分别查询黄芪、白术和防风的成份并筛选其中OB≥30%或者DL≥0.18的化合物。合并3种药物的成分,删除冗杂,通过TCMSP数据库查询每种成分对应的靶标,运用UniProt数据库查询靶蛋白对应的Gene Name并排除非人靶蛋白最终得到玉屏风散调控的靶蛋白。人的哮喘基因通过NCBI Gene数据库查询得到。将玉屏风散靶基因和哮喘靶基因相映射,映射所得的基因通过相互作用数据库String进行相互作用蛋白查询并构建蛋白质相互作用网络。采用Cytoscape的BiNGO分析工具对相互作用基因进行GO分子功能分析和生物学过程分析,构建相互作用基因的GO分子功能分类的层次网络和GO生物学过程分类的层次网络。采用DAVID数据库对相互作用蛋白进行通路富集分析,排除宽泛的通路,提取排名前五的通路并对排名第一的NOD样受体信号通路的相匹配基因构建相互作用网络进一步挖掘玉屏风散调控哮喘的具体作用机制。2.基于网络药理学分析结果,利用细胞实验探究玉屏风散对NLRP3炎症小体的调节作用,具体方法如下:利用LPS刺激PMA诱导的U937贴壁细胞建立炎症巨噬细胞模型,实验设正常对照组(Control)、模型组(LPS,100 ng/ml)和给药组(YPFS,25μg/ml)共三组。CCK-8法检测细胞活性后,六孔板中接种2 ml含1×106个细胞的U937细胞混悬液,同时加入终浓度为10 ng/mLPMA诱导培养。孵育48 h后,给药组中加入终浓度为25 μg/ml的玉屏风散的新鲜完全培养基,正常组和模型组更换等量的新鲜完全培养基进行培养,每组设置三个复孔。孵育2 h后,除正常对照组外,其余两组加入终浓度为100 ng/ml的LPS刺激剂,作用48 h后收集各组细胞上清和细胞。利用ELISA法检测细胞上清中细胞炎症因子IL-1β、TNF-α和IL-6的含量;采用Real-time PCR和Western Blot 法分别检测 IL-1β 及 NLRP3 炎症小体中 NLRP3、Caspase-1 和 ASC 的 mRNA相对表达水平和蛋白表达水平。同样的操作步骤重复实验三次。3.采用动物实验进一步验证玉屏风散对NLRP3炎症小体的调控作用。具体实验步骤如下:使用OVA致敏Balb/c小鼠构建哮喘动物模型,共分为4组:正常组、模型组、地塞米松组(1 mg/kg/d)和玉屏风散组(13 g/kg/d),每组10只。从雾化期开始每次雾化前半小时灌胃玉屏风散或注射地塞米松,连续给药12天。每2天称取一次小鼠的体重并观察在雾化期小鼠的一般情况。治疗结束后,采用ELISA法检测小鼠血清中IL-1β、TNF-α、IL-6的含量;采用HE染色法观察左肺上叶的病理学变化;采用AB-PAS染色法观察支气管中粘液分泌的情况;采用Real-time PCR和Western Blot法分别检测右肺上叶中NLRP3、Caspase-1、ASC和IL-1β的mRNA和蛋白的表达水平。结果:1.网络药理学分析结果:共得到OB≥30%或者DL≥0.18的玉屏风散人源靶蛋白372个(黄芪71个、白术39个、防风120个),哮喘相关的人源基因793个。采用String数据库构建了玉屏风散对抗哮喘的体内反应网络。采用Cytoscape的BiNGO分析工具对相互作用基因构建了 GO分子功能分类的层次网络和GO生物学过程分类的层次网络。发现玉屏风散可通过调节分子转导活性、酶调节剂活性、结合、抗氧化活性等分子功能及调节刺激的应答、免疫系统过程、生物调节、代谢调节、信号过程等生物学过程调控哮喘。DAVID数据库通路富集分析得出了玉屏风散调控哮喘的排名前五的通路分别是NOD样受体信号通路、TNF信号通路、PI3K-AKT信号通路、HIF-1信号通路和NF-κB信号通路。采用String数据库对排名第一的NOD样受体信号通路的匹配基因进行了可视化研究发现NLRP3炎症小体在玉屏风散调控哮喘可能发挥重要的作用。2.细胞实验结果:毒性实验结果表明,3.125,6.25 12.5和25 μg/ml浓度的玉屏风散对细胞活力没有显著影响(P0.05),因此最大无毒浓度(25μg/ml)用作后续实验指定浓度。ELISA结果显示,模型组和给药组细胞上清中IL-1β、IL-6和TNF-α的表达明显增高,和模型组相比,给药组能明显降低这三种炎症细胞因子的表达(P0.01);Real-time PCR和Western Blot结果显示,玉屏风散均能抑制细胞中NLRP3、Caspase-1、ASC和IL-1β的mRNA相对表达水平和蛋白表达水平,和模型组相比具有显著性差异(P0.05 或 P0.01)。3.动物实验结果:体重数据变化显示玉屏风散治疗能有效减缓小鼠的体重下降速度(P0.01);肺组织炎症评分和粘液评分显示玉屏风能显著降低给药组小鼠的炎细胞浸润和粘液的分泌(P0.01);ELISA结果表明玉屏风散能明显降低哮喘小鼠血清中炎症因子IL-1β、TNF-α和IL-6的含量(P0.01);Real-time PCR结果显示玉屏风能显著降低小鼠肺组织中NLRP3、Caspase-1、ASC和IL-1β的表达水平(P0.05或P0.01);Western Blot结果表明玉屏风散能显著减少小鼠肺组织中NLRP3、Caspase-1、Pro-Caspase-1、ASC 和 IL-1β 蛋白的含量(P0.05 或 P0.01)。结论:1.网络药理学方法分析预测得到炎症小体在玉屏风散治疗哮喘时扮演着重要的作用。2.体内实验证实玉屏风散对OVA诱导的哮喘小鼠有一定治疗效果。3.体内外实验证实玉屏风散可能通过下调炎症小体中关键的蛋白NLRP3、Caspase-1、ASC和IL-1β的表达来发挥治疗作用。
[Abstract]:Objective: the purpose of this study is to predict the mechanism of Yuping wind powder in treating asthma by network pharmacology, and further verify the prediction mechanism through cell experiments and animal experiments. Methods: to analyze the mode of action of 1. steps using network pharmacology methods regulating asthma in Yuping wind and mechanism are as follows: through the TCMSP database query Huangqi and Baizhu and windproof ingredients and screening wherein OB = 30% or DL = 0.18 compounds. With 3 kinds of drug ingredients, remove the jumbled, through the TCMSP database query for each component corresponding to the target, using the UniProt database query target protein corresponding to the Gene Name and the exclusion of non target protein obtained target protein Yuping Fengsan regulation. The human asthma gene was queried by the NCBI Gene database. Mapping the Yuping wind target gene with asthma target gene, mapping the obtained genes to interact protein queries through interaction database String, and constructing protein interaction network. Cytoscape BiNGO analysis tools were used to analyze GO gene function and biological process of interacting genes, and construct hierarchical network of GO molecular function classification of interaction genes and hierarchical network of GO biological process classification. Using DAVID database pathway enrichment analysis on interaction of protein, exclude broad pathway pathway extraction top five and NOD like receptor signaling pathway ranked first by matching the gene interaction network to further tap the specific mechanism of Yuping wind control asthma. 2. based on the analysis results of network pharmacology, Yuping wind and regulation of NLRP3 inflammasome by cell experiment, specific methods are as follows: the use of LPS PMA induced U937 adherent cells to establish inflammatory macrophage model, rats were divided into control group (Control), model group (LPS, 100 ng/ml) and drug group (YPFS a total of three, 25 g/ml) group. Cell activity was detected by CCK-8 after U937 cells were inoculated in 6-well plates containing 1 * 106 2 ml cell suspension, while adding a final concentration of 10 ng/mLPMA induced culture. After incubation for 48 h, the fresh medium of Yuping wind powder with a final concentration of 25 g/ml was added to the drug delivery group. The fresh and complete medium was replaced by the same group and the model group, and three holes were set up in each group. After 2 h incubation, except the normal control group, the other two groups were added to the LPS stimulant with a final concentration of 100 ng/ml, and the cell supernatant and cell were collected after the action of 48 h. ELISA assay was used to detect the contents of IL-1, TNF- and IL-6 in cell supernatants. Real-time PCR and Western Blot were used to detect the relative expression level and protein expression of IL-1, beta and NLRP3 in inflammatory cells. The same procedure was repeated three times. 3. animal experiments were used to further verify the regulation of Yuping Feng powder on NLRP3 inflammatory corpuscles. The specific experimental steps are as follows: using OVA sensitized Balb/c mice to establish asthma animal models, they were divided into 4 groups: normal group, model group, dexamethasone group (1 mg/kg/d) and Yuping wind powder group (13 g/kg/d), 10 rats in each group. From the beginning of atomization, Yuping wind or dexamethasone was injected at half an hour before each atomization, and the drug was given for 12 days. The mice were weighed every 2 days and the general condition of the mice during the nebulization period was observed. After the end of treatment, the content of ELISA was used to detect serum IL-1 beta, TNF- alpha, IL-6 was observed by HE staining; the upper lobe of the left lung pathological changes observed by AB-PAS staining; bronchial mucus secretion; the expression level of mRNA and protein were detected in the upper lobe of the right lung in NLRP3, Caspase-1, ASC using Real-time and IL-1 beta PCR and Western by Blot. Results: 1. network pharmacology analysis results were obtained: OB = 30% or DL = 0.18 in the Yuping wind source target protein 372 (Huangqi 71, 39, 120 Atractylodes wind), asthma related human source gene 793. The body reaction network of Yuping wind powder was constructed by using the String database. The hierarchical network of the hierarchical network of GO molecular function classification and the classification of the biological process of GO were constructed by the BiNGO analysis tools of Cytoscape. It is found that Yuping wind powder can regulate asthma through regulating the molecular functions such as molecular transduction activity, enzyme regulator activity, binding, antioxidant activity and other biological functions, such as regulating the response of stimuli, immune system process, biological regulation, metabolic regulation, signal process and other biological processes. DAVID database enrichment analysis showed that the top five pathways of Yuping wind scattered asthma control were NOD like receptor signaling pathway, TNF signaling pathway, PI3K-AKT signaling pathway, HIF-1 signaling pathway and NF- kappa B signaling pathway. The String database was used to visualize the matching gene of the first NOD like receptor signaling pathway. It was found that NLRP3 inflammable corpuscle may play an important role in the regulation of asthma by Yuping wind powder. 2. cell experiment results: toxicity test results showed that 3.125,6.25 12.5 and 25 g/ml concentration of Yuping wind powder had no significant effect on cell viability (P0.05), so the maximum non-toxic concentration (25 g/ml) was used as the specified concentration in subsequent experiments. The results of ELISA showed that the expression of IL-1 in model group and medicine group in the culture supernatant, IL-6 beta and TNF- alpha was significantly increased, compared with the model group, treatment group can significantly reduce the expression of the three cytokines (P0.01); Real-time PCR and Western Blot results showed that Yuping Fengsan inhibited cells NLRP3, Caspase-1, ASC and IL-1 beta mRNA expression and protein expression level, compared with the model group with significant difference (P0.05 or P0.01). 3. animal experiment results: weight data change showed that Yuping wind powder treatment can effectively slow down the weight loss rate of mice (P0.01), lung tissue inflammation score and mucus score showed that Yuping wind energy was significantly reduced.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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