基于网络药理学研究心舒宝片抗炎、血管舒张和心肌保护作用机制

Mechanism of Xinshubao Tablets in exerting anti-inflammatory,vasodilation,and cardioprotective effects based on network pharmacology

通过实验研究心舒宝片全方及其单味药肠吸收液对抗炎、血管舒张和心肌保护的影响,并结合网络药理学阐明其潜在作用机制,Western blot进一步验证潜在核心靶点的表达情况。脂多糖(lipopolysaccharide,LPS)刺激RAW264.7细胞产生炎症因子观察抗炎作用,离体微血管活性实验观察血管舒张作用,氧糖剥夺(oxygen-glucose deprivation,OGD)损伤H9c2细胞实验观察心肌保护作用。使用Herb数据库结合心舒宝片肠吸收液成分信息,获得心舒宝片化学成分,SwissTargetPrediction数据库查找成分作用靶点;GeneCards数据库查询抗炎、血管舒张和心肌保护相关的靶点。药物靶点分别与各疾病靶点以及疾病总靶点取交集后,进行蛋白-蛋白互作(protein-protein interaction,PPI)分析,得到核心靶点,最后将四者交集核心靶点导入Cytoscape 3.9.1进行基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析。抗炎实验表明心舒宝片全方和各单味药均有抗炎作用,以郁金抑制肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的作用最强,以丹参抑制白细胞介素-6(interleukin-6,IL-6)的作用最强。离体微血管舒张实验表明心舒宝片、郁金和山楂均有血管舒张作用,单味药以山楂最强。心肌保护实验表明心舒宝片全方、丹参、刺五加和白芍均具有心肌保护作用,单味药以丹参最强。网络药理学研究结果表明除全方外,抗炎作用以丹参活性成分最多,血管舒张和心肌保护作用以郁金活性成分最多,丹参次之。心舒宝片发挥抗炎、血管舒张和心肌保护作用的核心靶点为内皮型一氧化氮合酶3(nitric oxide synthase 3,NOS3),并用Western blot实验验证表明心舒宝片可显著增加OGD损伤H9c2细胞NOS3表达水平。GO生物学过程分析主要与细胞脂质输出、血压调节和炎症因子等有关;KEGG通路富集分析主要涉及AGE-RAGE信号通路和HIF-1信号通路等。

This study aims to explore the anti-inflammatory,vasodilation,and cardioprotective effects of the intestinal absorption liquids containing Xinshubao Tablets or single herbs,and to elucidate the potential mechanism based on network pharmacology.Western blot was then conducted to validate the expression changes of core proteins.Lipopolysaccharide(LPS)-stimulated RAW264.7 cells were used to observe the anti-inflammatory effect.The vasodilation activity was examined by the microvessel relaxation assay in vitro.Oxygen-glucose deprivation(OGD)-induced H9c2 cells were used to investigate the cardioprotective effect.The chemical components were retrieved from Herb databases and composition of Xinshubao Tablets drug-containing intestinal absorption solution.Drug targets were retrieved from SwissTargetPrediction databases.GeneCards was searched for the targets associated with the anti-inflammatory,vasodilation,and cardioprotective effects.The common targets shared by the drug and the effects were used to establish the protein-protein interaction(PPI) network,from which the core targets were obtained.Finally,the core targets were imported into Cytoscape 3.9.1 for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analyses.The anti-inflammatory experiment showed that both Xinshubao Tablets and the single herbs constituting this formula had anti-inflammatory effects.Curcumae Radix had the strongest inhibitory effect on the production of tumor necrosis factor-α(TNF-α),and Salviae Miltiorrhizae Radix et Rhizoma had the strongest inhibitory effect on the generation of interleukin-6(IL-6).Xinshubao Tablets,Curcumae Radix,and Crataegi Fructus had vasodilation effect,and Crataegi Fructus had the strongest effect.Xinshubao Tablets,Salviae Miltiorrhizae Radix et Rhizoma,Acanthopanacis Senticosi Radix et Rhizoma seu Caulis,and Paeoniae Radix Alba had cardioprotective effects,and Salviae Miltiorrhizae Radix et Rhizoma had the strongest cardioprotective effect.Network pharmacology results demonstrated that except the whole formula,Salviae Miltiorrhizae Radix et Rhizoma had the most components with anti-inflammatory effect,and Curcumae Radix had the most components with vasodilation and cardioprotective effects,followed by Salviae Miltiorrhizae Radix et Rhizoma.The nitric oxide synthase 3(NOS3) was predicted as the core target for the anti-inflammatory,vasodilation,and cardioprotective effects.Western blot results showed that Xinshubao Tablets significantly up-regulated the expression of NOS3 in OGD-induced H9c2 cells.GO enrichment analysis showed that the effects were mainly related to lipid exported from cell,regulation of blood pressure,and inflammatory response.KEGG pathway enrichment predicted AGE-RAGE and HIF-1 signaling pathways as the key pathways.