基于网络药理学和动物实验探讨胸痹片治疗动脉粥样硬化的作用机制

Exploration on the Mechanism of Action of Xiongbi Tablets in the Treatment of Atherosclerosis Based on Network Pharmacology and Animal Experiments

摘要:

目的：基于网络药理学和动物实验探讨胸痹片治疗动脉粥样硬化（AS） 的作用机制。方法：通过TCMSP和HERB2.0筛选胸痹片的活性成分和作用靶点，从GeneCards、NCBI、OMIM数据库获得AS的疾病靶点，并用Venny2.1.0平台取交集获得胸痹片治疗AS的潜在作用靶点。利用STRING数据库构建蛋白互作（PPI）网络，Cytoscape3.10.3软件构建“中药-活性成分-靶点-疾病”网络，筛选胸痹片治疗AS的关键活性成分和关键靶点。使用DAVID数据库对潜在作用靶点进行基因本体（GO） 功能和京都基因与基因组百科全书（KEGG）通路富集分析。使用AutoDock验证关键活性成分与关键靶点的结合能力。采用高脂饲料喂养ApoE-/-小鼠建立AS 小鼠模型。观察小鼠主动脉窦血管壁病理变化， 检测血浆脂蛋白相关磷脂酶A2 （LP-PLA2）、丙二醛（MDA）、超氧化物歧化酶（SOD） 浓度，以及磷脂酰肌醇3激酶（PI3K） /蛋白激酶B（Akt） /哺乳动物雷帕霉素靶蛋白（mTOR） 信号通路相关蛋白及其mRNA表达水平。结果：获得胸痹片对应靶点572个，AS疾病靶点6 197 个，交集靶点405 个。胸痹片治疗AS 的关键活性成分有槲皮素、7-羟基雅特因、L-牛蒡子苷元、（+） -扁柏脂素、当归酰鬼臼毒素、二氢脱水鬼臼树脂醇等，关键靶点为磷脂酰肌醇-4，5-二磷酸肌醇-3-激酶（PIK3CA）、SOD1、蛋白激酶Bα（AKT1）、磷脂酶A2第Ⅶ组（PLA2G7）、mTOR；潜在作用靶点共富集到193条信号通路，其中PI3K/Akt、丝裂原活化蛋白激酶（MAPK）、糖尿病并发症中的晚期糖基化终末产物-受体（AGE-RAGE） 为主要信号通路。关键活性成分与关键靶点的分子对接结合能均＜0 kcal/mol。动物实验结果显示：胸痹片能降低AS小鼠LP-PLA2、MDA水平（P＜0.05），升高SOD水平（P＜0.05），使PI3K、Akt、mTOR蛋白表达减少（P＜0.05），同时能使小鼠主动脉窦斑块、坏死及炎性细胞浸润的范围缩小。结论：胸痹片能减轻动脉粥样硬化小鼠氧化应激损伤，降低血清中LP-PLA2、MDA 水平，升高SOD 水平，同时抑制PI3K、Akt、mTOR蛋白表达，其机制可能与抑制PI3K/Akt/mTOR信号通路活化有关。

Abstract:

Abstract： Objective： To explore the mechanism of action of Xiongbi Tablets （XBP） in the treatment of atherosclerosis （AS） based on network pharmacology and animal experiments. Methods： The active components and therapeutic targets of XBP were screened using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and HERB2.0. AS-related disease targets were obtained from the Human Gene Database （GeneCards），National Center for Biotechnology Information （NCBI），and Online Mendelian Inheritance in Man （OMIM） databases. The Venny2.1.0 platform was used to identify the intersection， thus yielding potential therapeutic targets of XBP for AS. A protein-protein interaction （PPI） network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins （STRING） database， and a "Chinese herbal medicine-active componenttarget- disease" network was built using Cytoscape3.10.3 software to screen core active components and core targets of XBP for AS. Gene Ontology （GO） function and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses of potential targets were performed using the Database for Annotation， Visualization， and Integrated Discovery （DAVID）. AutoDock was used to validate the binding affinity between core active components and key targets. An AS mouse model was established by feeding ApoE-/- mice with a high-fat diet. Pathological changes in the aortic sinus vascular wall were observed. Plasma concentrations of lipoprotein-associated phospholipase A2（ LP-PLA2）， malondialdehyde （MDA），and superoxide dismutase （SOD），as well as the expression levels of phosphatidylinositol- 3-kinase/protein kinase B （PI3K/Akt）/mammalian target of rapamycin （mTOR） signaling pathway-related proteins and their mRNAs， were measured. Results： A total of 572 targets for XBP and 6 197 AS-related disease targets were obtained， yielding 405 overlapping targets. The core active components of XBP for treating AS included quercetin， 7-hydroxyyatein，L-arcigenin，（+）-hinokinin，angeloylpodophyllotoxin，and dimethylmatairesinol，among others. The key targets of XBP in the treatment of AS were phosphatidylinositol-4， 5-bisphosphate 3-kinase （PIK3CA）， SOD1，protein kinase B alpha （AKT1），phospholipase A2 group Ⅶ （PLA2G7），and mTOR. A total of 193 signaling pathways were enriched in the potential therapeutic targets， among which the PI3K/Akt， mitogen-activated protein kinase （MAPK）， and advanced glycation end product-advanced glycation end product receptor （AGE-RAGE） pathways were the main ones. The binding energies of molecular docking between core active components and key targets were all less than 0 kcal/mol. Animal experiment results showed that XBP reduced the levels of LP-PLA2 and MDA（ P＜ 0.05），increased the level of SOD （P＜0.05），and decreased the protein expression of PI3K，Akt，and mTOR （P＜ 0.05） in AS mice. Additionally，XBP reduced the scope of aortic sinus plaque formation，necrosis，and inflammatory cell infiltration in mice. Conclusion： XBP can alleviate oxidative stress injury in AS mice， reduce serum levels of LP-PLA2 and MDA，increase serum levels of SOD，and inhibit the protein expression of PI3K，Akt，and mTOR. The mechanism may be related to the inhibition of the activation of PI3K/Akt/mTOR signaling pathway.