基于动物实验与计算机验证探讨人参-三七-川芎药对延缓心脏衰老的作用机制

Mechanism of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma medicinal pair in delaying heart aging based on animal experiments and computer verification

目的利用动物实验、网络药理学与分子对接技术研究人参-三七-川芎药对延缓心脏衰老的作用机制。方法将小鼠按随机数字表法分为空白对照组、衰老模型组、二甲双胍组、中药组。除空白对照组外,其余各组小鼠采用皮下注射D-半乳糖诱导亚急性衰老小鼠模型。2周后,二甲双胍组灌胃二甲双胍混悬液150 mg/kg,中药组灌胃人参、三七、川芎冻干粉溶液650 mg/kg,空白对照组与衰老模型组灌胃等体积超纯水,1次/d,6次/周,连续灌胃10周。采用PCR检测心脏组织端粒酶蛋白催化亚基(TERT)mRNA水平,采用免疫组化染色观察心脏组织p53表达,采用HE染色观察心脏组织形态。检索TCMSP、Swiss Target Prediciton数据库中人参、三七、川芎的活性成分和靶点;利用TTD、OMIM、Gene、HAGR、DisGeNET数据库筛选心脏衰老靶点;将药物与疾病靶点取交集后得到人参-三七-川芎活性成分作用心脏衰老靶点;采用STRING数据库、Cytoscape 3.8.0软件构建交集靶点PPI网络,筛选核心靶点;利用FunRich软件对核心靶点进行细胞组分、分子功能、生物过程及通路富集分析;运用Schrödinger Maestro软件对药物活性成分与核心靶点进行分子对接,使用PyMOL2.1软件将对接结果可视化。结果实验结果表明,人参-三七-川芎可明显改善心脏衰老小鼠心脏组织损伤,升高TERT mRNA水平,降低p53阳性表达。共获得人参-三七-川芎活性成分32个,共对应637个靶点基因;心脏衰老靶点263个,药物活性成分靶点与心脏衰老交集靶点67个,筛选得到核心靶点31个。富集分析显示,分子功能与转录因子活性、蛋白酪氨酸激酶活性有关;生物过程涉及信号转导、细胞交流;信号通路涉及PDGFR-beta、PI3K-Akt、S1P1、Glypican、TRAIL、Glypican 1通路等。分子对接显示,人参-三七-川芎药对中山柰酚、苏齐内酯、人参皂苷Rg5和p53结合能力较强。结论人参-三七-川芎可通过抑制p53表达延缓心脏衰老,可为益气活血药延缓心脏衰老的作用机制研究提供依据。

Objective To explore the mechanism of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma medicinal pair in delaying heart aging based on animal experiments,network pharmacology and molecular docking.Methods Mice were divided into control group,aging group,metformin group and TCM group according to random number table method.All the groups were injected subcutaneously by D-galactose except the control group to build the subacute aging model.Two weeks later,the metformin group was given metformin suspension(150 mg/kg),the TCM group was given Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma lyophilized powder solution(650 mg/kg),and the control group and aging group were given an equivalent volume of ultrapure water by gastric gavage,once a day,six times a week,for 10 weeks.The level of heart TERT mRNA was detected by PCR;the expression of heart p53 was observed by immunohistochemical staining;the morphology of heart tissue was observed by HE staining.TCMSP and SwissTargetPrediciton databases were used to retrieve the active components and targets of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma medicinal pair;TTD,OMIM,Gene,HAGR,DisGeNET and other data platforms were used to screen the targets of heart aging;after the drug and disease targets were intersected,the active components of them were collected;STRING database,Cytoscape 3.8.0 software,etc.were used to make PPI of the intersection targets,and screen out the key targets;FunRich was used to perform enrichment analysis of cellular components,molecular functions,biological processes,and biological signal pathways for key targets;Schrödinger Maestro software was used to do the molecular docking of the screened active components and key targets,and docking results were visualized via PyMOL 2.1 software.Results Experiment results showed that Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma could significantly ameliorate the damage of aging heart tissues,elevate TERT mRNA level,while significantly reducing the positive expression of p53.A total of 32 active components from the medicinal pair were screened,corresponding to 637 target genes.There were 263 targets for heart aging,and 67 intersection targets of drug active component targets and heart aging targets.31 key targets were obtained after screening.Enrichment analysis showed that molecular functions were related to transcription factor activity and protein-tyrosine kinase activity.Biological processes involved signal transduction and cell communication.Signaling pathways mainly involved PDGFR-beta,PI3K-Akt,S1P1,Glypican,TRAIL,and Glypican 1.The molecular docking results showed that kaempferol,suchilactone,and ginsenoside Rg5_qt in the medicinal pair had a strong binding ability to p53.Conclusion Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma may achieve the effect of delaying heart aging by inhibiting p53 expression,providing a foundation for further research on mechanism of invigorating qi and activating blood circulation drugs to delay heart aging.