摘要
目的:利用网络药理学探究附子-淫羊藿药对治疗慢性心功能不全的作用机制,并通过心肌细胞H9c2和斑马鱼模型进行验证。方法:TCMSP采集搜索附子、淫羊藿的目标活性成分,UniProt得到相对应的靶点基因。OMIM,GeneCards获取与慢性心功能不全相关的靶点信息。Cytoscape 3.6.1软件,生成靶点网络图。MetaScape数据库进行蛋白质-蛋白质相互作用(PPI)的分析,基因本体(GO)生物过程,分子功能,细胞组成,京都基因与基因组百科全书(KEGG)通路富集分析。建立心肌细胞H9c2缺氧复氧模型,噻唑蓝(MTT)法测定促增殖效果,蛋白免疫印迹法(Western blot)测定B淋巴细胞瘤-2相关的X蛋白(Bax),B淋巴细胞瘤-2(Bcl-2),半胱氨酸蛋白酶-3(Caspase-3),蛋白激酶B(PKB/Akt),磷酸化蛋白激酶B(p-Akt),细胞外调节蛋白激酶(ERK),磷酸化细胞外调节蛋白激酶(p-ERK),聚腺苷二磷酸核糖聚合酶(PARP)蛋白表达。建立血管内皮生长因子酪氨酸酶抑制剂Ⅱ(VRI)诱导的斑马鱼节间血管损伤模型,验证促血管新生作用。结果:得到药对活性成分28个,209个靶点,1 296个疾病靶点,药对与疾病共同基因靶点94个。PPI聚类提示药对可能通过细胞分化、细胞代谢、血管生成等途径干预慢性心功能不全。GO富集提示药对可能通过改变细胞迁移,血管发育和生成等生物过程治疗慢性心功能不全。10 mg·L^(-1)的淫羊藿,10 mg·L^(-1)的药对能够促进H9c2的增殖(P<0.05),10 mg·L^(-1)的药对能够上调H9c2的Bcl-2,Bcl-2/Bax,PARP的表达(P<0.05),降低Caspase-3,Akt,ERK的表达(P<0.05)。药对在0.3,0.1 g·L^(-1)的质量浓度下有明显的促血管新生的作用(P<0.05)。结论:附子-淫羊藿药对可以通过提高Bcl-2,PARP,提高Bcl-2/Bax的比值,降低Caspase-3,Akt,ERK的表达,实现H9c2的缺氧条件下的促增殖作用,同时可以保护斑马鱼损伤的血管,从而通过多成分、多靶点、多途径的治疗慢性心功能不全。
Objective:To explore the mechanism of the prescription consisting Aconiti Lateralis Radix Praeparata and Epimedii Folium in the treatment of chronic heart failure(CHF) based on network pharmacology,followed by verification in H9 c2 myocardial cells with hypoxia-reoxygenation injury in vitro and in zebrafish with vascular endothelial growth factor receptor(VEGFR)tyrosine kinase inhibitor Ⅱ(VRI)-induced vascular insufficiency. Method: The active ingredients in Aconiti Lateralis Radix Praeparata and Epimedii Folium were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),the corresponding target genes from the Universal Protein Resource(UniProt),and the CHF-related targets from Online Mendelian Inheritance in Man(OMIM)and GeneCards. Both the active ingredient-potential target network and the active ingredient-CHF-related target network were generated using Cytoscape 3.6.1,followed by the protein-protein interaction(PPI)network construction and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genome(KEGG)enrichment analysis based on MetaScape. H9 c2 myocardial cells exposed to hypoxia-reoxygenation were selected for determining the proliferation-promoting effect by methyl thiazolyl tetrazolium(MTT)assay. The protein expression of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cysteinyl aspartate-specific protease-3(Caspase-3),protein kinase B(PKB/Akt),phosphorylated protein kinase B(p-Akt),phosphorylated extracellular signal-regulated kinases 1/2(p-ERK1/2),extracellular signal-regulated kinase 1/2(ERK1/2),and poly adenosine diphosphate ribose polymerase(PARP)was detected by Western blotting. The efficacy of the prescription in promoting angiogenesis was verified in a zebrafish model of VRI-induced vascular injury. Result: There were 28 active ingredients for the prescription,209 corresponding targets,1 296 CHF-related targets,and 94 common gene targets shared by the prescription and CHF. PPI network clustering suggested that Aconiti Lateralis Radix Praeparata and Epimedii Folium alleviated CHF by interfering with cell differentiation and metabolism and angiogenesis. GO analysis revealed that CHF relief was achieved via the intervention in such biological processes as cell migration,vascular development,and angiogenesis. Pharmacodynamic experiments verified that Epimedii Folium(10 mg·L^(-1))alone and the prescription(10 mg·L^(-1))both enhanced the proliferation of H9 c2 myocardial cells under the hypoxia-reoxygenation condition(P<0.05),while the latter also increased the expression of Bcl-2,Bcl-2/Bax,and PARP(P<0.05)and reduced the expression of Caspase-3,Akt,and ERK(P<0.05). The prescription at the concentrations of 0.3 and 0.1 g·L^(-1) promoted angiogenesis(P<0.05). Conclusion: Aconiti Lateralis Radix Praeparata and Epimedii Folium exert the therapeutic effect against CHF via multiple ingredients,multiple targets,and multiple channels. Such combination promotes the proliferation of H9 c2 myocardial cells under hypoxic condition and protects zebrafish from vascular injury by up-regulating the expression of Bcl-2 and PARP,increasing Bcl-2/Bax ratio,and down-regulating the expression of Caspase-3,Akt,and ERK.
作者
黄嘉滢
马子霖
程天翊
周忠焱
诸晨
唐靖一
HUANG Jia-ying;MA Zi-lin;CHENG Tian-yi;ZHOU Zhong-yan;ZHU Chen;TANG Jing-yi(Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第10期142-151,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
上海市中医药事业发展三年行动计划项目(ZY-(2018-2020)-FWTX-8001)
中医药文化背景下的医养结合示范工程建设项目(A1-N19205010302)
上海市加强公共卫生体系建设三年行动计划项目(GWIV-28)。
关键词
网络药理学
慢性心功能不全
血管新生
附子
淫羊藿
network pharmacology
chronic heart failure(CHF)
angiogenesis
Aconiti Lateralis Radix Praeparata
Epimedii Folium
