ObjectiveThis 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.

MethodsThe 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 DrugBank 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 PI3K, AKT1, BAX, and Bcl-2, respectively.

ResultsA total of 4 123 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 mRNA levels of PI3K, Akt, and Bax and upregulated the expression of the protein and mRNA levels of Bcl-2, reduced plasma renin, Ang II, and Ald levels, improved the hyperactivity of RAAS, and significantly reduced SBP in SHRs.

ConclusionPYJYF is effective for hypertension therapy that acts through multiple compounds and targets. The possible underlying molecular mechanism includes regulating the PI3K/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.