The Network Pharmacological Study and Experimental Verification of Huanglong Antitussive Granule in the Treatment of Asthma
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摘要:
目的 结合网络药理学方法和实验验证探讨黄龙止咳颗粒治疗哮喘的作用靶点及潜在机制。 方法 借助TCMSP、TCMID、Swiss Target Prediction等数据库检索黄龙止咳颗粒的化学成分和作用靶点。通过GeneCards、OMIM、Disgenet、Drugbank数据库筛选出哮喘的疾病靶点。使用Cytoscape软件构建“药物-成分-靶点-疾病”的网络图和潜在靶点的相互作用关系, 通过富集分析预测作用机制。构建哮喘小鼠模型, 通过病理染色, ELISA检测, qPCR及Western blot来验证网络药理学富集分析结果。 结果 共获得黄龙止咳颗粒中175种活性成分、1 026个药物靶点, 哮喘900个疾病靶点, 得到药物-疾病共同靶点153个, GO富集分析共得到条目1 967个, KEGG通路富集筛选出包括TNF信号通路在内的共计84条信号通路。动物实验表明, 黄龙止咳颗粒能有效改善哮喘小鼠的气道炎症, 并通过影响TNF信号通路从而抑制MAPK和NF-κB信号通路。 结论 该研究初步揭示了黄龙止咳颗粒治疗哮喘的作用机制, 为临床用药和后续研究提供参考依据。 Abstract:OBJECTIVE Combining network pharmacology methods and experimental verification to explore the target and potential mechanism of Huanglong Antitussive Granule in treating asthma. METHODS With the help of TCMSP, TCMID, Swiss Target Prediction and other databases, the chemical components and targets of Huanglong Antitussive Granule are searched. Through GeneCards, OMIM, Disgenet, Drugbank database to screen out the disease targets of asthma. Use Cytoscape software to construct a network diagram of "drug-component-target-disease" and the interaction relationship between potential targets, and predict the mechanism of action through enrichment analysis. Mice model of asthma was constructed, and the results of network pharmacology enrichment analysis were verified by pathological staining, Elisa detection, q-PCR and Western Blot. RESULTS A total of 175 active ingredients, 1 026 drug targets, 900 disease targets for asthma and 153 common drug-disease targets were obtained in Huanglong Antitussive Granule. A total of 1 967 entries were obtained by GO enrichment analysis, and KEGG pathway enrichment screened out a total of 84 signaling pathways including TNF signaling pathways. Animal experiments show that Huanglong Antitussive Granule can effectively improve airway inflammation in asthmatic mice, and inhibit the MAPK and NF-κB signaling pathways by affecting the TNF signaling pathway. CONCLUSION This study initially revealed the mechanism of action of Huanglong Antitussive Granule in treating asthma, providing a reference for clinical medication and follow-up research. -
Key words:
- Huanglong Antitussive Granule /
- asthma /
- network pharmacology /
- molecular mechanism
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图 2 药物-成分-靶点-疾病相互作用的网络图
注: ◇.疾病; ○.靶点; □.药物; △.成分
Figure 2. Drugs-components-targets-disease interaction network
图 5 GO生物学过程分析、细胞组分分析、分子功能分析(A)和KEGG富集分析(B)
Figure 5. GO biological process、cellular component, molecular function(A) and KEGG enrichment analysis (B)
图 7 各组小鼠血清IL-5、TNF-α水平变化
注: 与空白组比较, ###P < 0.001;与模型组比较, *P < 0.05，**P < 0.01, ***P < 0.001。x±s, n=5。
Figure 7. IL-5 and TNF-α in mouse serum
图 8 各组小鼠肺组织中关键靶基因转录的变化
注: 与空白组比较, #P < 0.05，##P < 0.01，###P < 0.001;与模型组比较, *P < 0.05，**P < 0.01，***P < 0.001。x±s, n=3。
Figure 8. Changes in the transcription of key target genes in the lung tissues of each group of mice
图 9 各组小鼠肺组织关键蛋白表达
注: 与空白组比较, ##P < 0.01, ###P < 0.001;与模型组比较, *P < 0.05, **P < 0.01。x±s, n=3。
Figure 9. Expression of key proteins in lung tissue of mice in each group
表 1 引物序列
Table 1. Primer sequence
基因名 上游引物 下游引物 IL-6 CTCCCAACAGACCTGTCTATAC CCATTGCACAACTCTTTTCTCA TNF ATGTCTCAGCCTCTTCTCATTC CGATCACCCCGAAGTTCAGTAG MAPK3 CAGCTCAACCACATTCTAGGTA TCAAGAGCTTTGGAGTCAGATT PTGS2 ATTCCAAACCAGCAGACTCATA CTTGAGTTTGAAGTGGTAACCG IL-1β GCCAGTGAAATGATGGCTTATT AGGAGCACTTCATCTGTTTAGG TLR4 GCCATCATTATGAGTGCCAATT AGGGATAAGAACGCTGAGAATT MMP9 CAAAGACCTGAAAACCTCCAAC GACTGCTTCTCTCCCATCATC ICAM1 CTGAAAGATGAGCTCGAGAGTG AAACGAATACACGGTGATGGTA MPO CAGGAACAACATCACCATTCG CTTGGAAGCGTGTATTGATAGC GAPDH AGGTCGGTGTGAACGGATTTG GGGGTCGTTGATGGCAACA 
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表 2 中药-成分-靶点基本信息统计表
Table 2. Chinese medicines-components-targets statistics
药物名称 成分数量 预测靶点数量 地龙 6 186 黄芪 21 450 桔梗 8 278 麻黄 25 365 山楂 74 694 射干 17 445 葶苈子 12 262 淫羊藿 23 486 鱼腥草 7 278
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[1] SOCKRIDER M, FUSSNER L. What Is Asthma?[J]. Am J Respir Crit Care Med, 2020, 202(9): 25-26. doi: 10.1164/rccm.2029P25 [2] CASTILLO JR, PETERS SP, BUSSE WW. Asthma exacerbations: Pathogenesis, prevention, and treatment[J]. J Allergy Clin Immunol Pract, 2017, 5(4): 918-927. doi: 10.1016/j.jaip.2017.05.001 [3] RUSSELL RJ, BRIGHTLING C. Pathogenesis of asthma: Implications for precision medicine[J]. Clin Sci, 2017, 131(14): 1723-1735. doi: 10.1042/CS20160253 [4] 严花, 单进军, 赵霞. 代谢组学和脂质组学在哮喘研究中的应用进展[J]. 南京中医药大学学报, 2019, 35(5): 552-561. http://xb.njucm.edu.cn/article/id/zr20190514YAN H, SHAN JJ, ZHAO X. Application and prospect on metabolomics and lipidomics in asthma[J]. J Nanjing Univ Tradit Chin Med, 2019, 35(5): 552-561. http://xb.njucm.edu.cn/article/id/zr20190514 [5] HUANG KW, YANG T, XU JY, et al. Prevalence, risk factors, and management of asthma in China: A national cross-sectional study[J]. Lancet, 2019, 394(10196): 407-418. doi: 10.1016/S0140-6736(19)31147-X [6] BEASLEY R, SEMPRINI A, MITCHELL EA. Risk factors for asthma: Is prevention possible?[J]. Lancet, 2015, 386(9998): 1075-1085. doi: 10.1016/S0140-6736(15)00156-7 [7] LIN JT, WANG WY, CHEN P, et al. Prevalence and risk factors of asthma in mainland China: The CARE study[J]. Respir Med, 2018, 137: 48-54. doi: 10.1016/j.rmed.2018.02.010 [8] HALES CM, KIT BK, GU QP, et al. Trends in prescription medication use among children and adolescents: United States, 1999-2014[J]. JAMA, 2018, 319(19): 2009. doi: 10.1001/jama.2018.5690 [9] BENARD B, BASTIEN V, VINET B, et al. Neuropsychiatric adverse drug reactions in children initiated on montelukast in real-life practice[J]. Eur Respir J, 2017, 50(2): 1700148. doi: 10.1183/13993003.00148-2017 [10] ALDEA PERONA A, GARCÍA-SÁIZ M, SANZ ÁLVAREZ E. Psychiatric disorders and montelukast in children: A disproportionality analysis of the VigiBase[J]. Drug Saf, 2016, 39(1): 69-78. doi: 10.1007/s40264-015-0360-2 [11] YAN H, QIAN GY, YANG R, et al. Huanglong antitussive granule relieves acute asthma through regulating pulmonary lipid homeostasis[J]. Front Pharmacol, 2021, 12: 656756. doi: 10.3389/fphar.2021.656756 [12] YANG XF, WANG FD. The effect of astragaloside Ⅳ on JAK2-STAT6 signalling pathway in mouse model of ovalbumin-induced asthma[J]. J Animal Physiol Animal Nutr, 2019, 103(5): 1578-1584. doi: 10.1111/jpn.13114 [13] ZHANG TZ, YANG SH, DU J, et al. Platycodin D attenuates airway inflammation in a mouse model of allergic asthma by regulation NF-κB pathway[J]. Inflammation, 2015, 38(3): 1221-1228. doi: 10.1007/s10753-014-0089-6 [14] 刘赜, 石倩, 杨洋, 等. 麻黄碱与伪麻黄碱平喘效果及机制比较研究[J]. 中草药, 2009, 40(5): 771-774. doi: 10.3321/j.issn:0253-2670.2009.05.031LIU Z, SHI Q, YANG Y, et al. A comparative study on the antiasthmatic effect and mechanism of ephedrine and pseudoephedrine[J]. Chin Tradit Herb Drugs, 2009, 40(5): 771-774. doi: 10.3321/j.issn:0253-2670.2009.05.031 [15] XUE KJ, RUAN LY, HU J, et al. Panax notoginseng saponin R1 modulates TNF-α/NF-κB signaling and attenuates allergic airway inflammation in asthma[J]. Int Immunopharmacol, 2020, 88: 106860. doi: 10.1016/j.intimp.2020.106860 [16] KIST M, KMVES LG, GONCHAROV T, et al. Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death[J]. Cell Death Differ, 2021, 28(3): 985-1000. doi: 10.1038/s41418-020-00629-3 [17] VARFOLOMEEV E, GONCHAROV T, MAECKER H, et al. Cellular inhibitors of apoptosis are global regulators of NF-κB and MAPK activation by members of the TNF family of receptors[J]. Sci Signal, 2012, 5(216): ra22. [18] ZHENG LW, WANG WC, MAO XZ, et al. TNF-α regulates the early development of avascular necrosis of the femoral head by mediating osteoblast autophagy and apoptosis via the p38 MAPK/NF-κB signaling pathway[J]. Cell Biol Int, 2020, 44(9): 1881-1889. doi: 10.1002/cbin.11394 [19] SEDGER LM, MCDERMOTT MF. TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants-past, present and future[J]. Cytokine Growth Factor Rev, 2014, 25(4): 453-472. doi: 10.1016/j.cytogfr.2014.07.016 [20] DOSTERT C, GRUSDAT M, LETELLIER E, et al. The TNF family of ligands and receptors: Communication modules in the immune system and beyond[J]. Physiol Rev, 2019, 99(1): 115-160. doi: 10.1152/physrev.00045.2017 [21] PROUDFOOT A, BAYLIFFE A, O'KANE CM, et al. Novel anti-tumour necrosis factor receptor-1 (TNFR1) domain antibody prevents pulmonary inflammation in experimental acute lung injury[J]. Thorax, 2018, 73(8): 723-730. doi: 10.1136/thoraxjnl-2017-210305 [22] VANAMEE E, FAUSTMAN D. TNFR2: A novel target for cancer immunotherapy[J]. Trends Mol Med, 2017, 23(11): 1037-1046. doi: 10.1016/j.molmed.2017.09.007 [23] LYU F, YANG LB, WANG JX, et al. Inhibition of TNFR1 attenuates LPS induced apoptosis and inflammation in human nucleus pulposus cells by regulating the NF-κB and MAPK signalling pathway[J]. Neurochem Res, 2021, 46(6): 1390-1399. doi: 10.1007/s11064-021-03278-1 [24] RUSPI G, SCHMIDT EM, MCCANN F, et al. TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages[J]. Cell Signal, 2014, 26(4): 683-690. doi: 10.1016/j.cellsig.2013.12.009 [25] TANG XJ, SUN L, WANG G, et al. RUNX1: A regulator of NF-κB signaling in pulmonary diseases[J]. Curr Protein Pept Sci, 2018, 19(2): 172-178. [26] VALOVKA T, HOTTIGER MO. p65 controls NF-κB activity by regulating cellular localization of IκBβ[J]. Biochem J, 2011, 434(2): 253-263. doi: 10.1042/BJ20101220 [27] GIRIDHARAN S, SRINIVASAN M. Mechanisms of NF-κB p65 and strategies for therapeutic manipulation[J]. J Inflamm Res, 2018, 11: 407-419. doi: 10.2147/JIR.S140188 [28] RIEDLINGER T, LIEFKE R, MEIER-SOELCH J, et al. NF-κB p65 dimerization and DNA-binding is important for inflammatory gene expression[J]. FASEB J, 2019, 33(3): 4188-4202. doi: 10.1096/fj.201801638R [29] MIN Y, KIM MJ, LEE SN, et al. Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation[J]. Autophagy, 2018, 14(8): 1347-1358. doi: 10.1080/15548627.2018.1474995 [30] VAN QUICKELBERGHE E, DE SUTTER D, VAN LOO G, et al. A protein-protein interaction map of the TNF-induced NF-κB signal transduction pathway[J]. Sci Data, 2018, 5: 180289. doi: 10.1038/sdata.2018.289 [31] MISHRA V, BANGA J, SILVEYRA P. Oxidative stress and cellular pathways of asthma and inflammation: Therapeutic strategies and pharmacological targets[J]. Pharmacol Ther, 2018, 181: 169-182. doi: 10.1016/j.pharmthera.2017.08.011 [32] KANDHARE AD, LIU ZH, MUKHERJEE AA, et al. Therapeutic potential of morin in ovalbumin-induced allergic asthma via modulation of SUMF2/IL-13 and BLT2/NF-kB signaling pathway[J]. Curr Mol Pharmacol, 2019, 12(2): 122-138. doi: 10.2174/1874467212666190102105052 [33] LIU XM, YI MJ, JIN R, et al. Correlation between oxidative stress and NF-κB signaling pathway in the obesity-asthma mice[J]. Mol Biol Rep, 2020, 47(5): 3735-3744. doi: 10.1007/s11033-020-05466-8 [34] ZHANG DW, WEI YY, JI S, et al. Correlation between sestrin2 expression and airway remodeling in COPD[J]. BMC Pulm Med, 2020, 20: 297. doi: 10.1186/s12890-020-01329-x [35] KHORASANIZADEH M, ESKIAN M, GELFAND EW, et al. Mitogen-activated protein kinases as therapeutic targets for asthma[J]. Pharmacol Ther, 2017, 174: 112-126. doi: 10.1016/j.pharmthera.2017.02.024 [36] PELAIA C, VATRELLA A, CRIMI C, et al. Clinical relevance of understanding mitogen-activated protein kinases involved in asthma[J]. Expert Rev Respir Med, 2020, 14(5): 501-510. doi: 10.1080/17476348.2020.1735365 [37] SU XQ, PAN J, BAI FX, et al. IL-27 attenuates airway inflammation in a mouse asthma model via the STAT1 and GADD45γ/p38 MAPK pathways[J]. J Transl Med, 2016, 14: 283. doi: 10.1186/s12967-016-1039-x -
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