基于网络药理学分析复方利血平氨苯蝶啶片降压作用机制与验证研究

Use of network pharmacology to analyze compound reserpine and triamterene tablets in the treatment of hypertension

复方利血平氨苯蝶啶片是由我国学者研发的复方降压药,至今仍在临床广泛使用,但其降压作用机制仍有待阐释。本文拟基于网络药理学分析其降压作用机制并在细胞水平进行初步研究验证。本文通过Swiss Target Prediction数据库收集复方利血平氨苯蝶啶片中4种化学成分的作用靶点;通过TTD、OMIM数据库选取高血压病的相关蛋白靶标;利用STRING数据库构建复方利血平氨苯蝶啶片-高血压病网络模型;通过Metascape数据库对交集靶点进行GO富集分析和通路富集分析;采用人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)和血管平滑肌细胞(vascular smooth muscle cells,VSMC)分别经1μmol·L^(-1)血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导处理,低中高浓度复方利血平氨苯蝶啶片原料(0.01、0.1和1μmol·L^(-1))进行干预,Western blot法检测HUVEC中磷酯酰肌醇-3激酶/蛋白激酶B/内皮型一氧化氮合酶(phosphatidylinositol-3-kinase/protein serine threonine kinase/endothelial nitric oxide synthase,PI3K/Akt/eNOS)通路和VSMC中环磷酸鸟苷酸/环磷酸鸟苷酸依赖的蛋白激酶G(cyclic guanosinc monophosphate/cGMP-dependent protein kinase,cGMP/PKG)通路的变化。网络药理学分析揭示,复方利血平氨苯蝶啶片降压作用与血管张力调节、肾上腺素受体激活、蛋白激酶活性等密切相关,其中涉及cGMP/PKG信号通路、血管平滑肌收缩、调控神经活动的配体-受体相互作用、心肌细胞肾上腺素信号传导、钙信号通路等信号通路。细胞水平研究证实,复方利血平氨苯蝶啶片处理升高HUVEC中磷酸化磷酯酰肌醇-3激酶(phosphorylated phosphatidylinositol-3-kinase,p-PI3K)、磷酸化蛋白激酶B(phosphorylated protein serine threonine kinase,p-Akt)、磷酸化内皮型一氧化氮合酶(phosphorylated endothelial nitric oxide synthase,p-eNOS)以及VSMC中eNOS、磷酸化血管舒张剂刺激磷蛋白(phosphorylated vasodilator-stimulated phosphoprotein,p-VASP)、PKG蛋白水平。复方利血平氨苯蝶啶片通过多靶点、多通路发挥降压作用,进一步研究证实,激活PI3K/Akt/eNOS通路诱导内皮依赖的NO/cGMP/PKG信号,从而舒张血管可能是其重要作用机制之一。

Compound reserpine and triamterene tablets(CRTT),a compound antihypertensive drug developed by Chinese scientists,is still widely used in clinical practice.However,the mechanisms by which CRTT treats hypertension remain to be fully understood.This study used network pharmacology to analyze CRTT’s antihypertensive mechanisms with in vitro experiments.The targets of the four chemical components of CRTT were collected from the Swiss Target Prediction database;1828 protein targets related to hypertension were collected from the Therapeutic Target Database(TTD)and Online Mendelian Inheritance in Man(OMIM)database.The CRTT-hypertension network model was constructed using a search tool for recurring instances of neighbouring genes(STRING).Gene ontology(GO)and pathway enrichment analysis of targets of interest was conducted with the Metascape database.In the in vitro study,human umbilical vein endothelial cells(HUVEC)and vascular smooth muscle cells(VSMC)were treated with 1μmol·L^(-1)angiotensinⅡ(AngⅡ)and CRTT was administered at concentrations of 0.01,0.1,and 1μmol·L^(-1).Changes in the phosphatidylinositol-3-kinase/protein serine threonine kinase/endothelial nitric oxide synthase(PI3 K/Akt/eNOS)pathway in HUVEC and the cyclic guanosine monophosphate/cGMP-dependent protein kinase(cGMP/PKG)pathway in VSMC were determined by Western blot.Network pharmacological analysis revealed that the antihypertensive effect of CRTT is closely associated with biological pathways such as vascular tone regulation,adrenergic receptor activation,protein kinase activity and signaling pathways such as the cGMP/PKG signaling pathway,vascular smooth muscle contraction,neuroactive ligand-receptor interaction,adrenergic signaling in cardiomyocytes and calcium signaling pathways.The in vitro study confirmed that CRTT increased the levels of phosphorylated phosphatidylinositol-3-kinase(p-PI3 K),phosphorylated protein serine threonine kinase(p-Akt),phosphorylated endothelial nitric oxide synthase(p-eNOS)in HUVEC and the levels of eNOS,phosphorylated vasodilator-stimulated phosphoprotein(p-VASP),and PKG in VSMC through multiple targets and pathways.These results suggest that the activation of PI3 K/Akt/eNOS pathway and endothelialdependent NO/cGMP signaling may be involved in the CRTT-mediated hypotensive effect.