网络药理学预测及代谢组学验证 凤仙花抗高脂血症的机制
1School of Health Preservation and Rehabilitation,Chengdu University of Traditional Chinese Medicine, 2State Administration of Traditional Chinese Medicine Key Laboratory of Traditional Chinese Medicine, Regimen and Health,Chengdu University of Traditional Chinese Medicine, 3Key Laboratory of Traditional Chinese Medicine Regimen and Health of Sichuan Province
Summary
本协议描述了一种基于网络药理学预测和代谢组学验证的探索叶藻对抗高脂血症的关键靶点和机制的综合策略。
Abstract
高脂血症已成为全球心血管疾病和肝损伤的主要危险因素。在中医和印度医学理论中，Fructus Phyllanthi（FP）是一种有效的抗高脂血症药物，但其潜在机制有待进一步探索。本研究旨在基于网络药理学预测与代谢组学验证相结合的综合策略，揭示FP对抗高脂血症的机制。通过评估血浆脂质水平，包括总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）和高密度脂蛋白胆固醇（HDL-C），建立了高脂肪饮食（HFD）诱导的小鼠模型。应用网络药理学找出FP的有效成分和抗高脂血症的潜在靶点。进行血浆和肝脏代谢组学鉴定正常组、模型组和干预组的差异代谢产物及其对应通路。进一步构建网络药理学与代谢组学的关系，全面了解FP抗高脂血症的过程。通过分子对接验证获得的关键靶蛋白。这些结果表明，FP改善了HFD诱导的高脂血症的血脂水平和肝损伤。没食子酸、槲皮素和β-谷甾醇在FP中被证明是关键的活性化合物。通过代谢组学发现血浆和肝脏中分别有16种和6种潜在差异代谢物参与FP对高脂血症的治疗效果。此外，整合分析表明，干预效果与CYP1A1、AChE和MGAM以及L-犬尿氨酸、皮质酮、乙酰胆碱和棉子糖的调节有关，主要涉及色氨酸代谢途径。分子对接确保了上述作用于高脂血症相关蛋白靶标的成分在降低脂质中发挥关键作用。综上所述，本研究为防治高脂血症提供了新的可能性。
Introduction
高脂血症是一种常见的代谢性疾病，严重影响人体健康，也是心血管疾病的主要危险因素1。最近，这种疾病出现了与年龄相关的下降趋势，由于长期不规律的生活方式和不健康的饮食习惯，年轻人变得更加易感2。在临床上，各种药物已被用于治疗高脂血症。例如，高脂血症和相关动脉粥样硬化性疾病患者最常用的药物之一是他汀类药物。然而，长期使用他汀类药物具有不容忽视的副作用，导致预后不良，例如不耐受，治疗耐药性和不良事件3，4。这些缺点已成为高脂血症患者的额外痛点。因此，应提出稳定的降脂效果和较少副作用的新疗法。
中医（TCM）因其疗效好、副作用少而被广泛用于治疗疾病5。Fructus Phyllanthi （FP），余甘子林恩的干果。（俗称余甘子或印度醋栗），是中印中药6、7的著名药食同源材料。根据中医理论8，该药已被用于清热，冷却血液和促进消化。现代药理研究表明，FP富含没食子酸、鞣花酸、槲皮素9等生物活性化合物，它们通过作为抗氧化剂、抗炎、护肝、抗降血脂等，具有一系列多方面的生物学特性10。最近的研究也表明，FP可以有效调节高脂血症患者的血脂。例如，Variya等人11已经证明，FP果汁及其主要化学成分没食子酸可以降低血浆胆固醇，减少肝脏和主动脉中的油脂浸润。治疗效果与FP调节增加过氧化物酶体增殖物激活受体α的表达和降低肝质活性有关。然而，FP改善高脂血症的潜在机制有待进一步研究，因为它的生物活性成分相当广泛。我们试图探索FP治疗效果的潜在机制，这可能有利于该药物的进一步开发和利用。
目前，网络药理学被认为是研究中医治疗机制的一种整体有效的技术。构建完整的药物-成分-基因-疾病网络，寻找多成分药物综合治疗的多靶点机制，而不是寻找单一的致病基因和治疗单个靶点的药物12。这种技术特别适用于中医，因为它们的化学成分很大。不幸的是，网络药理学在理论上只能用于预测受化学成分影响的靶点。应观察疾病模型中的内源性代谢物，以验证网络药理学的有效性。随着系统生物学的发展而出现的代谢组学方法是监测内源性代谢物变化的重要工具13。代谢产物的变化反映了宿主的稳态变化，也是研究内部机制的重要指标。一些研究人员成功地整合了网络药理学和代谢组学，探索了药物与疾病之间的相互作用机制14，15。
本文通过整合网络药理学和代谢组学技术，探讨FP对抗高脂血症的机制基础。应用网络药理学分析FP中主要活性成分与高脂血症分子靶点的关系。随后，进行代谢组学观察动物模型中内源性代谢物的变化，这可以解释药物在代谢水平的作用。与单纯应用网络药理学或代谢组学相比，这种综合分析提供了更具体、更全面的研究机制。此外，分子对接策略用于分析活性成分与关键蛋白质之间的相互作用。总的来说，这种综合方法可以弥补网络药理学实验证据的缺乏和代谢组学方法缺乏内源性机制的不足，可用于天然药物的治疗机制分析。该协议的主要原理图流程图如图 1所示。
Protocol
所有涉及动物处理的程序均按照《成都中医药大学实验动物护理和使用指南》进行，并经成都中医药大学机构伦理委员会批准（协议编号2020-36）。雄性C57BL / 6小鼠（20±2g）用于本研究。小鼠是从商业来源获得的（见 材料表）。 1. 基于网络药理学的预测 注：网络药理学用于预测FP对抗高脂血症的活性成分及其关键靶点。 ?…
Representative Results
网络药理学根据数据库和LC-MS分析的药代动力学和药效学特性，共筛选了FP中的18种潜在成分（总离子色谱图见 补充图1）。通过相关文献，没食子酸的含量远高于其他成分，可有效降低脂质9，11。因此，这种成分也被认为是一种潜在的成分。总共确定了19个成分和134个与FP相关的成分目标。所有19种成分如 表1所示…
Discussion
近年来，高脂血症的发病率一直在上升，主要是由于长期不健康的饮食习惯。中药及其化学成分具有多种药理活性，近年来已被广泛研究37，38。FP是一种水果资源，既可用作药物，又可用作食品，具有治疗高脂血症的重要潜力。然而，FP对抗高脂血症的潜在治疗机制有待进一步研究。
网络药理学在分子水平上评估药物多药理作用，?…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究得到了中医养生康复产品开发创新团队（2022C005）和“养生康复+”新业务跨界融合研究的支持。
Materials
| 101-3B Oven | Luyue Instrument and Equipment Factory | ||
| 80312/80302 Glass Slide | Jiangsu Sitai Experimental Equipment Co., LTD | ||
| 80340-1630 Cover Slip | Jiangsu Sitai Experimental Equipment Co., LTD | ||
| AccucoreTM C18 (3 mm × 100 mm, 2. 6 μm) | Thermo Fisher Scientific | ||
| Acetonitrile | Fisher Chemical | A998 | Version 1.5.6 |
| ACQUITY UPLC HSS T3 Column (2.1 mm × 100 mm, 1.8 μm) | Thermo Fisher Scientific | ||
| Aethanol | Fisher Chemical | A995 | Version 3.0 |
| Ammonia Solution | Chengdu Cologne Chemicals Co., LTD | 1336-21-6 | Version 3.9.1 |
| AutoDockTools | Scripps Institution of Oceanography | ||
| BS-240VT Full-automatic Animal Biochemical Detection System | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | ||
| Compound Discoverer | Thermo Fisher Scientific | ||
| Cytoscape | Cytoscape Consortium | ||
| DM500 Optical Microscope | Leica | ||
| DV215CD Electronic Balance | Ohaus Corporation ., Ltd | T15A63 | |
| Ethyl Alcohol | Chengdu Cologne Chemicals Co., LTD | 64-17-5 | |
| Formic Acid | Fisher Chemical | A118 | |
| HDL-C Assay Kit | Nanjing Jiancheng Bioengineering Institute | A112-1-1 | |
| Hematoxylin Staining Solution | Biosharp | BL700B | |
| High Fat Diet | ENSIWEIER | 202211091031 | |
| Hitachi CT15E/CT15RE Centrifuge | Hitachi., Ltd. | ||
| Homogenizer | Oulaibo Technology Co., Ltd | ||
| Hydrochloric Acid | Chengdu Cologne Chemicals Co., LTD | 7647-01-0 | |
| Image-forming System | LIOO | ||
| JB-L5 Freezer | Wuhan Junjie Electronics Co., Ltd | ||
| JB-L5 Tissue Embedder | Wuhan Junjie Electronics Co., Ltd | ||
| JK-5/6 Microtome | Wuhan Junjie Electronics Co., Ltd | ||
| JT-12S Hydroextractor | Wuhan Junjie Electronics Co., Ltd | ||
| KQ3200E Ultrasonic Cleaner | Kun Shan Ultrasonic Instruments Co., Ltd | ||
| LDL-C Assay Kit | Nanjing Jiancheng Bioengineering Institute | A113-1-1 | |
| Male C57BL/6 Mice | SBF Biotechnology Co., Ltd. | Version 2.3.2 | |
| Neutral Balsam | Shanghai Yiyang Instrument Co., Ltd | 10021190865934 | |
| Pure Water | Guangzhou Watson's Food & Beverage Co., Ltd | GB19298 | |
| PyMOL | DeLano Scientific LLC | Version 14.1 | |
| RE-3000 Rotary Evaporator | Yarong Biochemical Instrument Factory ., Ltd | ||
| RM2016 Pathological Microtome | Shanghai Leica Instruments Co., Ltd | Version 26.0 | |
| SIMCA-P | Umetrics AB | ||
| Simvastatin | Merck Sharp & Dohme., Ltd | 14202220051 | |
| SPSS | International Business Machines Corporation | ||
| TC Assay Kit | Nanjing Jiancheng Bioengineering Institute | A111-1-1 | |
| TG Assay Kit | Nanjing Jiancheng Bioengineering Institute | A110-1-1 | |
| UPLC-Q-Exactive Quadrupole Electrostatic Field Orbital Hydrazine High Resolution Mass Spectrometry | Thermo Fisher Scientific | ||
| Vortex Vibrator | Beijing PowerStar Technology Co., Ltd. | LC-Vortex-P1 | |
| Xylene | Chengdu Cologne Chemicals Co., LTD | 1330-20-7 |
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Citar este artigo
Zeng, B., Qi, L., Wu, S., Liu, N., Wang, J., Nie, K., Xia, L., Yu, S. Network Pharmacology Prediction and Metabolomics Validation of the Mechanism of Fructus Phyllanthi against Hyperlipidemia. J. Vis. Exp. (194), e65071, doi:10.3791/65071 (2023).