基于网络药理学探讨桂枝甘草汤(Guizhi Gancao Decoction,GGD)对心肌缺血再灌注损伤(myocardial ischemia-reper-fusion injury,MI/RI)大鼠的保护作用及可能的药理机制.首先通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)、SwissTargetPrediction 数据库和文献检索获取 GGD 抗MI/RI的化学成分及作用靶点,采用STRING数据库和Cytoscape 3.7.2软件对交集靶点进行蛋白-蛋白相互作用(protein-pro-tein interaction,PPI)网络分析,对核心靶点进行基因本体(Gene Ontology,GO)和京都基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,并构建"药物-核心成分-靶点-通路"网络,采用AutoDock Vina软件对主要活性成分与关键靶点进行分子对接验证.然后构建MI/RI大鼠模型,观察大鼠心肌梗死面积变化,苏木素-伊红(hematoxylin-eosin,HE)染色和透射电子显微镜(transmission electron microscope,TEM)检测心肌细胞病理和超微结构改变,蛋白免疫印迹(Western blot,WB)检测心肌组织相关蛋白表达.从GGD中筛选得到75个活性成分,对应318个治疗MI/RI靶点.PPI筛选得到肿瘤蛋白p53 基因(tumor protein p53,TP53)、丝氨酸/苏氨酸激酶 3(serine/threonine kinase 1,AKT1)、信号传导及转录激活蛋白(signal transducer and activator of transcription,STAT3)、非受体酪氨酸激酶(non-receptor tyrosine kinase,SRC)、丝裂原活化蛋白激酶 1(mitogen-activated protein kinase 1,MAPK1)、丝裂原活化蛋白激酶 3(mitogen-activated protein kinase 3,MAPK3)、肿瘤坏死因子(tumor necrosis factor,TNF)等46个核心靶点.GO和KEGG富集分析得知,核心靶点主要影响细胞增殖和迁移、信号转导、细胞凋亡、转录过程,涉及癌症、糖尿病并发症中的高级糖基化终末产物-受体(advanced glycation end products-receptor,AGE-RAGE)、MAPK等信号通路.分子对接结果显示,GGD核心成分肉桂醛、儿茶素和甘草查尔酮A与MAPK1、MAPK3等核心靶点均具有较好的结合活性.动物实验结果显示,GGD能够显著减少心肌梗死面积,提高MI/RI大鼠超氧化物歧化酶(supero-xide dismutase,SOD)水平,同时降低丙二醛(malondialdehyde,MDA)、乳酸脱氢酶(lactatedehydrogenase,LDH)、肌酸激酶同工酶(creatine kinase isoenzymes,CK-MB)水平;HE染色和TEM结果显示,模型组大鼠经GGD预处理后心肌细胞结构恢复正常,病理状态和线粒体超微结构损伤有所改善,且两药合用效果优于单用.WB结果显示GGD可显著下调心肌组织c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、酪氨酸磷酸蛋白激酶(tyrosine phosphoprotein kinase,p38)和上调细胞外调节蛋白激酶1/2(extracellular regulated protein kinases 1/2,ERK1/2)蛋白的磷酸化水平.结果表明,GGD可能通过桂枝、甘草的协同作用调控MAPK信号通路,发挥抗MI/RI作用.
Mechanism of Guizhi Gancao Decoction against myocardial ischemia-reperfusion injury based on network pharmacology and experimental verification
This study employed network pharmacology to investigate the effect of Guizhi Gancao Decoction(GGD)on myocardial ischemia-reperfusion injury(MI/RI)in rats and decipher the underlying mechanism.Firstly,the chemical components and targets of GGD against MI/RI were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),SwissTargetPrediction,and available articles.STRING and Cytoscape 3.7.2 were used to establish the protein-protein interaction(PPI)network for the common targets,and then Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were carried out for the core targets.The"drug-active component-target-pathway"network was built.Furthermore,molecular docking between key active components and targets was conducted in AutoDock Vina.Finally,the rat model of MI/RI was established,and the myocardial infarction area was measured.Hematoxylin-eosin(HE)staining and transmission electron microscopy(TEM)were employed to detect cardiomyocyte pathology and ultrastructural changes.Western blot was employed to determine the expression of related proteins in the myocardial tissue.A total of 75 chemical components of GGD were screened out,corresponding to 318 targets.The PPI network revealed 46 core targets such as tumor protein p53(TP53),serine/threonine kinase 1(AKT1),signal transducer and activator of transcription 3(STAT3),non-receptor tyrosine kinase(SRC),mitogen-activated protein kinase 1(MAPK1),MAPK3,and tumor necrosis factor(TNF).According to GO and KEGG enrichment analyses,the core targets mainly affected the cell proliferation and migration,signal transduction,apoptosis,and transcription,involving advanced glycation end products-receptor(AGE-RAGE),MAPK and other signaling pathways in cancers and diabetes complications.The molecular docking results showed that the core components of GGD,such as licochalcone A,(+)-catechin,and cinnamaldehyde,had strong binding activities with the core target proteins,such as MAPK1 and MAPK3.The results of animal experiments showed that compared with the model group,GGD significantly increase superoxide dismutase,decreased malondialdehyde,lactate dehydrogenase,and creatine kinase-MB,and reduced the area of myocardial infarction.HE staining and TEM results showed that GGD pretreatment restored the structure of cardiomyocytes and alleviated the pathological changes and ultrastructural damage of mitochondria in the model group.In addition,GGD significantly down-regulated the phosphorylation of c-Jun N-terminal kinase and p38 and up-regulate that of extracellular regulated kinases 1/2 in the myocardial tissue.The results suggested that GGD may exert the anti-MI/RI effect by regulating the MAPK signaling pathway via the synergistic effects of Cinnamomi Ramulus and Glycyrrhizae Radix et Rhizoma.
万方数据
维普
