Mechanism of Pingyang Jiangya Formula in treating hypertension based on network pharmacology and in vivo study

平阳降压方治疗高血压的网络药理学研究及体内实验验证

Objective This study aimed to analyze the mechanism of action of the Pingyang Jiangya Formula(平阳降压方,PYJYF)in treating hypertension,based on network pharmacology,and to verify the subsequent predictions through animal experiments.Methods The active components and related target genes of PYJYF were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM),Encyclopedia of Traditional Chinese Medicine(ETCM),and Drug Bank databases and available literature.The hypertension target genes were screened based on Therapeutic Target Database(TTD),GeneCards,Online Mendelian Inheritance in Man(OMIM),UniProt,and relevant literature.The component-disease-target network intersection target genes were inputted into the STRING database,and the key target genes were selected according to the degree algorithm.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed to explore the multitarget mechanism of action and molecular regulatory network of PYJYF in the treatment of hypertension.To verify this prediction,we used PYJYF to intervene in spontaneously hypertensive rats(SHRs)and Wistar–Kyoto rats(WKY)as normal control,and the noninvasive tail artery manometry method was used to measure systolic blood pressure(SBP)in the rat tail before PYJYF intervention.After drug intervention,the SBP of each group rats were measured and compared every week.Enzyme-linked immunosorbent assay(ELISA)was used to test plasma renin,angiotensin II(Ang II),and aldosterone(Ald)levels,and hematoxylin-eosin(HE)staining was used to observe pathological damage to the renal vessels in each group of rats.Western blot and reverse transcription real-time quantitative PCR(RT-PCR)were used to detect the protein and mRNA expression levels of PI3 K,AKT1,BAX,and Bcl-2,respectively.Results A total of 4123 hypertension targets were obtained from related databases.From the TCMSP and chemical databases,78 active components of PYJYF and the corresponding 401 drug targets were retrieved.Data analysis revealed that 208 drug targets directly interacted with the hypertension targets in PYJYF.The 10 targets most closely related to hypertension target proteins in PYJYF were directly retrieved from relevant databases.GO analysis revealed that 10 direct target proteins were involved in all aspects of the antihypertensive effects of PYJYF,as well as molecular biological processes,such as the regulation of blood pressure,renin-angiotensin-aldosterone system(RAAS),angiotensin-mediated ligand reactions,and biological stimulation of cardiomyocyte apoptosis.KEGG pathway enrichment analysis revealed that PYJYF directly affected 20 signaling pathways associated with hypertension.In animal experiments,PYJYF reduced the protein and m RNA levels of PI3 K,Akt,and Bax and upregulated the expression of the protein and m RNA levels of Bcl-2,reduced plasma renin,Ang II,and Ald levels,improved the hyperactivity of RAAS,and significantly reduced SBP in SHRs.Conclusion PYJYF is effective for hypertension therapy that acts through multiple compounds and targets.The possible underlying molecular mechanism includes regulating the PI3 K/Akt signaling pathway to suppress RAAS,increasing the ratio of Bcl-2/Bax proteins,and inhibiting apoptosis,thereby mediating the repair of renal and renal vascular damage caused by hypertension.These findings warrant further research for use in clinical settings.

目的本研究旨在基于网络药理学分析平阳降压方(PYJYF)治疗高血压的生物学机制,并通过动物实验进行验证。方法借助TCMSP、BATMAN-TCM、ETCM、Drug Bank数据库及文献查找筛选PYJYF的活性成分和相关靶基因,检索TTD、Gene Cards、OMIM、Uniprot数据库及文献查找筛选高血压靶基因。将"PYJYF-高血压"交集靶基因输入到STRING数据库,依据Degree算法筛选出核心靶基因。通过GO及KEGG富集分析探讨PYJYF在高血压治疗中的多靶点作用机制和分子调控网络。为验证上述预测,用PYJYF干预自发性高血压大鼠(SHRs),以WKY大鼠作为正常对照,采用无创尾动脉压力测量法测量PYJYF干预前大鼠尾动脉收缩压(SBP)。药物干预后,每周测量并比较各组大鼠SBP。采用酶联免疫吸附法(ELISA)检测血浆肾素、血管紧张素II(Ang II),醛固酮(Ald)水平。采用苏木精-伊红(HE)染色观察各组大鼠肾脏,肾血管和骨骼肌的病理损伤。采用Western blot和实时荧光定量PCR(RT-PCR)分别检测PI3K,AKT1,BAX,Bcl-2蛋白及其m RNA表达水平。结果通过相关数据库共获得PYJYF的78种活性组分和相应的401种药物靶标,4123个高血压靶标,208个共同靶点。GO分析显示10个生物学过程参与了PYJYF治疗高血压的分子生物学过程,包括调节血压,肾素-血管紧张素-醛固酮系统(RASS),血管紧张素介导的配体反应,生物刺激心肌细胞凋亡等,KEGG通路富集分析显示PYJYF直接影响20条与高血压相关的信号通路。动物实验结果显示,PYJYF可降低PI3K,Akt,Bax蛋白和m RNA的表达水平,上调Bcl-2蛋白和m RNA的表达水平;降低血浆肾素,Ang II和Ald的含量,改善RASS系统机能亢进,并显著降低SHRs的SBP数值。结论PYJYF可通过多靶点、多途径治疗高血压,其可能的潜在分子机制包括调节PI3K/Akt信号通路抑制RASS,增加Bcl-2/Bax比值,抑制细胞凋亡,从而修复高血压引起的肾脏和肾血管损伤,这些发现将有利于PYJYF在临床中的进一步研究及应用。