本文關(guān)鍵詞：基于網(wǎng)絡(luò)藥理學(xué)方法研究玉屏風(fēng)散治療哮喘的作用機理　出處：《西南交通大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 玉屏風(fēng)散 哮喘 網(wǎng)絡(luò)藥理學(xué) 作用機制 NLRP3炎癥小體

【摘要】：目的:本研究目的在于通過網(wǎng)絡(luò)藥理學(xué)的方法預(yù)測玉屏風(fēng)散治療哮喘的作用機制,并通過細胞實驗和動物實驗對預(yù)測的機制進行進一步的驗證。方法:1.采用網(wǎng)絡(luò)藥理學(xué)的方法對玉屏風(fēng)散調(diào)控哮喘的作用方式及作用機制的分析步驟如下:通過TCMSP數(shù)據(jù)庫分別查詢黃芪、白術(shù)和防風(fēng)的成份并篩選其中OB≥30%或者DL≥0.18的化合物。合并3種藥物的成分,刪除冗雜,通過TCMSP數(shù)據(jù)庫查詢每種成分對應(yīng)的靶標(biāo),運用UniProt數(shù)據(jù)庫查詢靶蛋白對應(yīng)的Gene Name并排除非人靶蛋白最終得到玉屏風(fēng)散調(diào)控的靶蛋白。人的哮喘基因通過NCBI Gene數(shù)據(jù)庫查詢得到。將玉屏風(fēng)散靶基因和哮喘靶基因相映射,映射所得的基因通過相互作用數(shù)據(jù)庫String進行相互作用蛋白查詢并構(gòu)建蛋白質(zhì)相互作用網(wǎng)絡(luò)。采用Cytoscape的BiNGO分析工具對相互作用基因進行GO分子功能分析和生物學(xué)過程分析,構(gòu)建相互作用基因的GO分子功能分類的層次網(wǎng)絡(luò)和GO生物學(xué)過程分類的層次網(wǎng)絡(luò)。采用DAVID數(shù)據(jù)庫對相互作用蛋白進行通路富集分析,排除寬泛的通路,提取排名前五的通路并對排名第一的NOD樣受體信號通路的相匹配基因構(gòu)建相互作用網(wǎng)絡(luò)進一步挖掘玉屏風(fēng)散調(diào)控哮喘的具體作用機制。2.基于網(wǎng)絡(luò)藥理學(xué)分析結(jié)果,利用細胞實驗探究玉屏風(fēng)散對NLRP3炎癥小體的調(diào)節(jié)作用,具體方法如下:利用LPS刺激PMA誘導(dǎo)的U937貼壁細胞建立炎癥巨噬細胞模型,實驗設(shè)正常對照組(Control)、模型組(LPS,100 ng/ml)和給藥組(YPFS,25μg/ml)共三組。CCK-8法檢測細胞活性后,六孔板中接種2 ml含1×106個細胞的U937細胞混懸液,同時加入終濃度為10 ng/mLPMA誘導(dǎo)培養(yǎng)。孵育48 h后,給藥組中加入終濃度為25 μg/ml的玉屏風(fēng)散的新鮮完全培養(yǎng)基,正常組和模型組更換等量的新鮮完全培養(yǎng)基進行培養(yǎng),每組設(shè)置三個復(fù)孔。孵育2 h后,除正常對照組外,其余兩組加入終濃度為100 ng/ml的LPS刺激劑,作用48 h后收集各組細胞上清和細胞。利用ELISA法檢測細胞上清中細胞炎癥因子IL-1β、TNF-α和IL-6的含量;采用Real-time PCR和Western Blot 法分別檢測 IL-1β 及 NLRP3 炎癥小體中 NLRP3、Caspase-1 和 ASC 的 mRNA相對表達水平和蛋白表達水平。同樣的操作步驟重復(fù)實驗三次。3.采用動物實驗進一步驗證玉屏風(fēng)散對NLRP3炎癥小體的調(diào)控作用。具體實驗步驟如下:使用OVA致敏Balb/c小鼠構(gòu)建哮喘動物模型,共分為4組:正常組、模型組、地塞米松組(1 mg/kg/d)和玉屏風(fēng)散組(13 g/kg/d),每組10只。從霧化期開始每次霧化前半小時灌胃玉屏風(fēng)散或注射地塞米松,連續(xù)給藥12天。每2天稱取一次小鼠的體重并觀察在霧化期小鼠的一般情況。治療結(jié)束后,采用ELISA法檢測小鼠血清中IL-1β、TNF-α、IL-6的含量;采用HE染色法觀察左肺上葉的病理學(xué)變化;采用AB-PAS染色法觀察支氣管中粘液分泌的情況;采用Real-time PCR和Western Blot法分別檢測右肺上葉中NLRP3、Caspase-1、ASC和IL-1β的mRNA和蛋白的表達水平。結(jié)果:1.網(wǎng)絡(luò)藥理學(xué)分析結(jié)果:共得到OB≥30%或者DL≥0.18的玉屏風(fēng)散人源靶蛋白372個(黃芪71個、白術(shù)39個、防風(fēng)120個),哮喘相關(guān)的人源基因793個。采用String數(shù)據(jù)庫構(gòu)建了玉屏風(fēng)散對抗哮喘的體內(nèi)反應(yīng)網(wǎng)絡(luò)。采用Cytoscape的BiNGO分析工具對相互作用基因構(gòu)建了 GO分子功能分類的層次網(wǎng)絡(luò)和GO生物學(xué)過程分類的層次網(wǎng)絡(luò)。發(fā)現(xiàn)玉屏風(fēng)散可通過調(diào)節(jié)分子轉(zhuǎn)導(dǎo)活性、酶調(diào)節(jié)劑活性、結(jié)合、抗氧化活性等分子功能及調(diào)節(jié)刺激的應(yīng)答、免疫系統(tǒng)過程、生物調(diào)節(jié)、代謝調(diào)節(jié)、信號過程等生物學(xué)過程調(diào)控哮喘。DAVID數(shù)據(jù)庫通路富集分析得出了玉屏風(fēng)散調(diào)控哮喘的排名前五的通路分別是NOD樣受體信號通路、TNF信號通路、PI3K-AKT信號通路、HIF-1信號通路和NF-κB信號通路。采用String數(shù)據(jù)庫對排名第一的NOD樣受體信號通路的匹配基因進行了可視化研究發(fā)現(xiàn)NLRP3炎癥小體在玉屏風(fēng)散調(diào)控哮喘可能發(fā)揮重要的作用。2.細胞實驗結(jié)果:毒性實驗結(jié)果表明,3.125,6.25 12.5和25 μg/ml濃度的玉屏風(fēng)散對細胞活力沒有顯著影響(P0.05),因此最大無毒濃度(25μg/ml)用作后續(xù)實驗指定濃度。ELISA結(jié)果顯示,模型組和給藥組細胞上清中IL-1β、IL-6和TNF-α的表達明顯增高,和模型組相比,給藥組能明顯降低這三種炎癥細胞因子的表達(P0.01);Real-time PCR和Western Blot結(jié)果顯示,玉屏風(fēng)散均能抑制細胞中NLRP3、Caspase-1、ASC和IL-1β的mRNA相對表達水平和蛋白表達水平,和模型組相比具有顯著性差異(P0.05 或 P0.01)。3.動物實驗結(jié)果:體重數(shù)據(jù)變化顯示玉屏風(fēng)散治療能有效減緩小鼠的體重下降速度(P0.01);肺組織炎癥評分和粘液評分顯示玉屏風(fēng)能顯著降低給藥組小鼠的炎細胞浸潤和粘液的分泌(P0.01);ELISA結(jié)果表明玉屏風(fēng)散能明顯降低哮喘小鼠血清中炎癥因子IL-1β、TNF-α和IL-6的含量(P0.01);Real-time PCR結(jié)果顯示玉屏風(fēng)能顯著降低小鼠肺組織中NLRP3、Caspase-1、ASC和IL-1β的表達水平(P0.05或P0.01);Western Blot結(jié)果表明玉屏風(fēng)散能顯著減少小鼠肺組織中NLRP3、Caspase-1、Pro-Caspase-1、ASC 和 IL-1β 蛋白的含量(P0.05 或 P0.01)。結(jié)論:1.網(wǎng)絡(luò)藥理學(xué)方法分析預(yù)測得到炎癥小體在玉屏風(fēng)散治療哮喘時扮演著重要的作用。2.體內(nèi)實驗證實玉屏風(fēng)散對OVA誘導(dǎo)的哮喘小鼠有一定治療效果。3.體內(nèi)外實驗證實玉屏風(fēng)散可能通過下調(diào)炎癥小體中關(guān)鍵的蛋白NLRP3、Caspase-1、ASC和IL-1β的表達來發(fā)揮治療作用。
[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.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5

【參考文獻】

相關(guān)期刊論文 前10條

1 王華;王嵩;丁肖媛;陸磊;蔡紅;王巧娥;沈朝斌;;玉屏風(fēng)散對哮喘模型小鼠氣道上皮細胞表達TSLP的影響[J];上海中醫(yī)藥雜志;2017年02期

2 高月娟;劉金麗;王景欣;孫琳林;宋懿玲;田桂元;何帥;佟雷;;玉屏風(fēng)顆粒對過敏性哮喘大鼠氣道炎癥因子的影響[J];中成藥;2016年11期

3 李泮霖;蘇薇薇;;網(wǎng)絡(luò)藥理學(xué)在中藥研究中的最新應(yīng)用進展[J];中草藥;2016年16期

4 馬舉斌;馬英;趙振軍;;黃芪桂枝五物湯合玉屏風(fēng)散治療類風(fēng)濕性關(guān)節(jié)炎臨床研究[J];新中醫(yī);2016年07期

5 趙彩霞;;支氣管哮喘發(fā)病機制的研究進展[J];醫(yī)學(xué)理論與實踐;2016年07期

6 李紅念;梅全喜;戴衛(wèi)波;董鵬鵬;;玉屏風(fēng)散的臨床應(yīng)用與藥理作用研究進展[J];廣州中醫(yī)藥大學(xué)學(xué)報;2016年02期

7 孟繁璞;王志飛;謝雁鳴;黎元元;;基于文本挖掘的中醫(yī)藥治療哮喘處方規(guī)律的現(xiàn)代文獻研究[J];中華中醫(yī)藥雜志;2015年10期

8 薛瀟春;胡晉紅;;網(wǎng)絡(luò)藥理學(xué)的研究方法與應(yīng)用進展[J];藥學(xué)實踐雜志;2015年05期

9 蘇立明;池永學(xué);金正勇;林蓮花;;促紅細胞生成素對哮喘模型小鼠肺部炎癥改變及細胞因子的影響[J];中國婦幼保健;2015年12期

10 劉，

本文編號：1340383