苦豆子抗炎机制的网络药理学分析及试验验证

doi:10.16431/j.cnki.1671-7236.2023.07.035

摘要/Abstract

摘要： 【目的】基于网络药理学和试验验证探究苦豆子的抗炎作用机制。【方法】依托HERB数据库筛选苦豆子有效成分并利用SIB在线工具检索相应靶点，利用Cytoscape 3.6.1软件构建有效成分-靶点网络图；结合NCBI、GeneCards、OMIM等数据库检索苦豆子抗炎相关靶点，上传至STRING平台，构建蛋白-蛋白互作（PPI）网络图；利用DAVID数据库对关键靶点进行GO功能和KEGG通路富集分析；选择PPI中度值最高的靶点与关键药效成分进行分子对接；通过体外试验检测苦豆子关键活性成分（槐定碱、苦参碱）对小鼠巨噬细胞RAW264.7的细胞毒性，以及对脂多糖（LPS）刺激RAW264.7细胞后一氧化氮（NO）相对产生量的影响。【结果】苦豆子中主要含有槐定碱、苦参碱、金雀花碱、槐胺碱、槐果碱等10种有效成分，可作用于核因子κB激酶亚基β抑制因子（IKBKB）、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、丝裂原活化蛋白激酶14（MAPK14）和基质金属蛋白酶2（MMP2）等55个关键抗炎靶点。GO功能富集分析得到121个GO条目（P<0.01），其中与生物过程相关的条目61个，包括信号转导、蛋白质磷酸化、对药物的反应、炎症反应等；与分子功能相关的条目37个，包括蛋白质结合、蛋白激酶活性、酶结合等；与细胞组分相关的条目23个，包括质膜、胞浆、细胞表面、大分子复合物等；涉及KEGG信号通路104条，可通过调节磷脂酰激醇3-激酶（PI3K）/蛋白激酶B （Akt）、促分裂素原活化蛋白激酶（MAPK）、核因子κB （NF-κB）、肿瘤坏死因子（TNF）、趋化因子等信号通路发挥抗炎作用。分子对接结果显示，槐定碱与热休克蛋白HSP90α（HSP90AA1）靶点、苦参碱与组蛋白去乙酰化酶11（HDAC11）靶点紧密结合。细胞毒性试验结果显示，0.02~1.28 mg/mL的槐定碱和苦参碱对RAW264.7细胞无毒副作用。炎症细胞模型试验结果显示，槐定碱和苦参碱可抑制由LPS刺激的RAW264.7细胞中NO的产生。【结论】苦豆子可能通过槐定碱、苦参碱等活性成分作用于IKBKB、NLRP3、MAPK14和MMP2等关键靶标，调控PI3K-Akt、MAPK、NF-κB、TNF等信号通路发挥抗炎作用，这为进一步深入阐明苦豆子的抗炎作用机制提供依据。

关键词: 苦豆子; 网络药理学; 抗炎活性; RAW264.7细胞; 细胞毒性

Abstract: 【Objective】 The aim of this study was to analyze the anti-inflammatory ingredients and mechanism of Sophora alopecuroides (S.alopecuroides) based on network pharmacology and experimental verification.【Method】 The effective ingredients of S.alopecuroides were screened from the HERB database and the corresponding targets were retrieved through SIB online tool.Cytoscape 3.6.1 software was used to construct an active ingredient-target network diagram.The anti-inflammatory related targets of S.alopecuroides were obtained from NCBI, GeneCards and OMIM databases, and the core target genes were uploaded to STRING platform to build a protein-protein interaction (PPI) network.Finally, DAVID database was used to analyze key targets by GO function and KEGG pathway enrichment.The target with the highest degree of PPI was selected for molecular docking with key pharmacodynamic components.The cytotoxicity of sophoridine and matrine of S.alopecuroides to RAW264.7 cells was detected, and the effects of sophoridine and matrine on the relative production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells were verified.【Result】 The results showed that there were ten active ingredients in S.alopecuroides, including sophoridine, matrine, cytisine, sophoramine, sophocarpine, etc.They could act on 55 key anti-inflammatory targets, including inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), NLR family pyrin domain containing 3 (NLRP3), mitogen-activated protein kinase 14(MAPK14), matrix metallopeptidase 2 (MMP2), etc.GO function enrichment analysis yielded 121 items (P<0.01), involving 61 biological processes, such as signal transduction, protein phosphorylation, response to drug, inflammatory response, etc.;37 molecular functions, such as protein binding, protein kinase activity, enzyme binding, etc.;And 23 cellular components, such as plasma membrane, cell surface, macromolecular complex, etc.104 related signaling pathways mainly included PI3K-Akt, MAPK, NF-kappa B (NF-κB), TNF and chemokine, etc.Molecular docking showed that sophoridine could bind tightly to target HSP90AA1 and matrine to target HDAC11.The cytotoxicity results showed that 0.02-1.28 mg/mL of sophoridine and matrine had no toxicity to RAW264.7 cells.Moreover, different concentrations of sophoranine and matrine could inhibit relative NO production in LPS-stimulated RAW264.7 cells.【Conclusion】 Sophoridine, matrine and other active ingredients of S.alopecuroides might have anti-inflammatory activity by regulating multiple targets such as IKBKB, NLRP3, MAPK14, MMP2, etc., and pathways such as PI3K-Akt, MAPK, NF-κB, TNF and other signaling pathways, etc., which provided a reference for further elucidation the anti-inflammatory mechanism of S.alopecuroides.

Key words: Sophora alopecuroides; network pharmacology; anti-inflammatory activity; RAW264.7 cells; cytotoxicity

中图分类号:

S859.7

范秋雨, 武建文, 李春晓, 王金全, 李焕荣, 王秀敏. 苦豆子抗炎机制的网络药理学分析及试验验证[J]. 中国畜牧兽医, 2023, 50(7): 2951-2965.

FAN Qiuyu, WU Jianwen, LI Chunxiao, WANG Jinquan, LI Huanrong, WANG Xiumin. Network Pharmacology Analysis of Anti-inflammatory Mechanism of Sophora alopecuroides and Experimental Validation[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2951-2965.

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