液相色谱-质谱结合网络药理学及实验验证新疆樱桃李花色苷改善动脉粥样硬化的作用机制

目的:研究新疆樱桃李花色苷（XJP-ACY）抗动脉粥样硬化（AS）作用的活性成分及作用机制。方法：通过液相色谱-质谱（LC-MS）鉴定的新疆樱桃李花色苷主要成分，从XJP-ACY主要成分和AS角度，应用网络药理学方法，筛选其潜在活性成分潜在靶点，构建“成分-疾病-靶点”网络，并预测XJP-ACY抗AS作用信号通路。观察XJP-ACY对氧化型低密度脂蛋白（ox-LDL）诱导EA.hy926内皮细胞损伤模型作用，分析内皮细胞活力、测定抗氧化酶活力，细胞凋亡以及内皮细胞黏附等的影响。蛋白质印迹检测相关蛋白表达，初步验证网络药理学的预测结果。结果：从新疆樱桃李花色苷中鉴定成分4个，预测得到其潜在抗AS作用靶点21个；聚类分析得到2个基因簇和2个核心基因，核心基因为FGF2、ADRA2C。基因本体（GO）分析结果表明筛选得到的靶点主要涉及炎症反应、MAPK的正调控等生物过程。京都基因和基因组百科全书（KEGG）通路富集分析得到28条信号通路，涉及PI3K-AKT信号通路、糖尿病并发症中的AGE-RAGE信号通路、C型凝集素受体信号通路等相关通路。细胞实验表明，ox-LDL诱导EA.hy926建立内皮细胞损伤模型：100 μmol/L ox-LDL作用24 h，为适合本研究的最优条件。XJP-ACY可提高ox-LDL诱导的EA.hy926细胞存活率，增加一氧化氮（NO）水平和超氧化物歧化酶（SOD）活力，降低乳酸脱氢酶(LDH)、丙二醛(MDA)含量和内皮素-1(ET-1)表达水平，降低细胞活性氧（ROS）水平，抑制细胞间黏附分子-1（ICAM-1）的表达，减少炎症介质分泌；XJP-ACY可抑制ox-LDL诱导的EA.hy926细胞凋亡，其机制与其提高B淋巴细胞瘤-2（Bcl-2）蛋白表达相关。结论：本研究初步阐释XJP-ACY治疗AS多成分、多靶点、多途径的作用特点在于参与抑制炎症反应、促进细胞凋亡。

LC-MS Combined with Network Pharmacology and Experimental Validation Reveals Mechanism of Anthocyanins from Prunus cerasifera in Xinjiang in Treating Atherosclerosis

To study the active components and mechanism of anthocyanins from Prunus cerasifera in Xinjiang(XJP-ACY) in treating atherosclerosis.Methods:The main components were identified by liquid chromatography-mass spectrometry(LC-MS)，and network pharmacology was employed to screen the potential targets of the main components of XJP-ACY and atherosclerosis.A “component-disease-target” network was established to predict the potential signaling pathways involved in the treatment of atherosclerosis with XJP-ACY.The EA.hy926 endothelial cell model of cell injury was established with oxidized low density lipoprotein(ox-LDL) and treated with XJP-ACY.The effects of XJP-ACY on the viability，activities of antioxidant enzymes，apoptosis，and adhesion of the cells were examined.Western blotting was employed to determine the expression of related proteins，on the basis of which the prediction results of network pharmacology were validated.Results:Four components and 21 potential antiatherogenic targets of XJP-ACY were predicted.Two gene clusters and two core genes(FGF2 and ADRA2C) were yielded by the cluster analysis.The gene ontology analysis results showed that the selected targets were mainly involved in biological processes such as inflammatory response and positive regulation of MAPK.The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed 28 signaling pathways，including the PI3K-AKT signaling pathway，AGE-RAGE signaling pathway in diabetic complications，and C-type lectin receptor signaling pathway.The cell experiment results showed that the suitable modeling conditions were incubation with 100 μmol/L ox-LDL for 24 h.XJP-ACY increased the survival rate，elevated the nitric oxide and superoxide dismutase levels，lowered the lactate dehydrogenase，malondialdehyde，endothelin-1，and reactive oxygen species levels，inhibited intercellular adhesion molecule-1 expression，and reduced inflammatory cytokine secretion in the EA.hy926 cells treated with ox-LDL.In addition，XJP-ACY inhibited the apoptosis of EA.hy926 cells exposed to ox-LDL by upregulating the expression of B-cell lymphoma-2.Conclusion:XJP-ACY can treat atherosclerosis by inhibiting inflammation and promoting apoptosis in a multi-component，multi-target，and multi-pathway manner.