基于网络药理学及实验验证养心汤抗动脉粥样硬化作用机制研究

Based on Network Pharmacology and Experimental Verification to Explore the Mechanism of Yangxin Decoction Anti-atherosclerosis

目的基于网络药理学及体内实验探讨养心汤治疗动脉粥样硬作用机制。方法网络药理学部分利用TCMSP数据库筛选养心汤的药物活性成分及对应靶点,Gene Cards、DisGeNet、OMIM、TTD数据库筛选动脉粥样硬化疾病靶点,EVenn平台交互映射得到药物-疾病交集靶点,利用Cytoscape 3.7.2软件构建药物-活性成分-核心靶点-疾病互作网络图,交集靶点导入STRING数据库获取PPI网络图,同时Cytoscape软件可视化处理,利用Metascape v3.5平台进行GO、KEGG富集分析,微生信平台可视化处理。体内实验部分:ApoE^(-/-)小鼠通过高脂饮食建立动脉粥样硬化动物模型,成模小鼠随机分为模型组(Model)、养心汤低剂量组(YXT-L)、养心汤高剂量组(YXT-H),C57BL/6小鼠为Control组,YXT-L组、YXT-H组分别灌胃养心汤浸膏水溶液2.6 g·kg^(-1)·d^(-1)和5.2 g·kg^(-1)·d^(-1),Control组、Model组灌胃等体积蒸馏水,灌胃4周。油红O染色观察主动脉窦斑块面积,ELISA检测血清IL-6、IL-1β、Caspase-3、VEGF水平,全自动生化仪检测血脂TG、TC、LDL-C、HDL-C水平,Western blot检测主动脉NF-κB p65、TNF-α、IKKβ蛋白的表达。结果网络药理学部分:共筛选出养心汤活性成分105个,对应靶点535个,动脉粥样硬疾病靶点4921个,药物-疾病交集靶点162个,前10位的靶点包括AKT1、TNF、IL-6、VEGFA、IL-1β、TP53、JUN、CASP3、PPARG、PTGS2,GO分析共得到2224个条目,其中生物学过程1946条,细胞成分100条,分子功能178条。KEGG信号通路富集分析共得到信号通路216个,主要涉及流体剪切应力与动脉粥样硬化、NF-κB信号通路、cAMP信号通路、糖尿病心肌病、半胱氨酸和蛋氨酸代谢、VEGF信号通路、钙信号通路等。体内实验部分:养心汤呈剂量依赖性降低ApoE^(-/-)动脉粥样硬化小鼠血清TG、TC、LDL-C增加HDL-C水平,减少主动脉窦斑块面积,降低血清IL-6、IL-1β、Caspase-3、VEGF水平;抑制主动脉NF-κB p65、TNF-α、IKKβ蛋白表达。结论养心汤可能通过抑制TNF-α/IKKβ/NF-κB信号通路,调节脂质代谢、抑制炎症反应、调控细胞凋亡等途径发挥抗动脉粥样硬化的作用。

Objective Based on network pharmacology and in vivo experiments,to explore the mechanism of Yangxin Decoction in treating arterial atherosclerosis.Methods In the network pharmacology part,TCMSP database is used to screen the drug active ingredients and corresponding targets of Yangxin Decoction,Gene Cards,DisGeNet,OMIM,TTD database is used to screen atherosclerosis disease targets,the Evenn platform for interactive mapping to obtain drug disease intersection targets.Cytoscape 3.7.2 software is used to build a drug active ingredient core target disease interaction network diagram,The intersection target points are imported into STRING database to obtain PPI network diagram,and the Cytascape software is used for visualization processing.The metascape v3.5 platform is used for GO and KEGG enrichment analysis,and the micro-signal platform is used for visualization processing.In vivo experiment:ApoE^(-/-)mice established atherosclerosis animal models through high-fat diet.The model mice were randomly divided into model group(Model),low-dose Yangxin Decoction group(YXT-L),and high-dose Yangxin Decoction group(YXT-H).C57BL/6 mice were taken as the control group,YXT-L group and YXT-H group were respectively given 2.6 g·kg^(-1)·d^(-1)and 5.2 g·kg^(-1)·d^(-1)of Yangxin Decoction extract aqueous solution,and the control group and model group were given equal volume distilled water for 4 weeks.Oil red O staining was used to observe the plaque area of aortic sinus,and ELISA was used to detect serum IL-6 and IL-1β,Caspase-3,VEGF levels,TG,TC,LDL-C,HDL-C levels of blood lipids detected by automatic biochemical instrument,and NF-κB p65,TNF-α,IKKβProtein expression of aorta detected by Western blot.Results Network pharmacology:105 active ingredients of Yangxin Decoction were screened out,including 535 corresponding targets,4921 atherosclerotic disease targets,162 drug disease intersection targets,and the top 10 targets include AKT1,TNF,IL-6,VEGFA,IL-1β,TP53,JUN,CASP3,PPARG,PTGS2.A total of 2224 items were obtained from and GO analysis,including 1946 biological processes,100 cell components and 178 molecular functions.A total of 216 signal pathways were obtained by KEGG signal pathway enrichment analysis,mainly involving fluid shear stress,atherosclerosis,NF-κB signaling pathway,cAMP signaling pathway,diabetes cardiomyopathy,cysteine and methionine metabolism,VEGF signaling pathway,calcium signaling pathway,etc.In vitro experiment:Yangxin Decoction reduces serum TG,TC,LDL-C in ApoE^(-/-)atherosclerosis mice in a dose-dependent manner,increases HDLC level,reduces aortic sinus plaque area,and reduces serum IL-6,IL-1β,Caspase-3 and VEGF level;Inhibition of aortic NF-κB p65,TNF-α,IKKβProtein expression.Conclusion Yangxin Decoction may inhibit TNF-α/IKKβ/NF-κB signaling pathway plays an anti-atherosclerosis role by regulating lipid metabolism,inhibiting inflammatory reaction,regulating cell apoptosis,etc.