Title : Deciphering the cardioprotective mechanism of a natural compound in post-infarct mice: an integrative approach combining network pharmacology, in vitro, and in vivo validation
Abstract:
Herbs have long served as foundational sources of medicinal agents and continue to play a pivotal role in both traditional and modern pharmaceutical practices. Among these, several naturally derived compounds exhibit promising therapeutic potential against cardiovascular diseases. This study investigates the cardioprotective effects of a natural compound against acute myocardial infarction (AMI), elucidating its molecular mechanisms through a comprehensive integrative approach combining insilico, invitro, and invivo analyses. cardioprotective role of a natural compound in AMI by identifying its key targets and signaling pathways using a network pharmacology framework supported by experimental validation. A network pharmacology-based analysis was conducted using public databases such as PubChem, IMPPAT, SwissADME, SwissTargetPrediction, GeneCards, DisGeNET, and PubMed to identify bioactive compounds, predict biological targets, and associate them with cardiovascular diseases. Visualization and integration of compound-target-disease networks were performed using Cytoscape. Functional annotation through Gene Ontology (GO) and KEGG pathway enrichment analyses was done using the DAVID database. Protein–protein interaction (PPI) networks were constructed via STRING, and key targets were selected for molecular docking using AutoDock 4.2.6. Molecular dynamics simulations over 100 ns were performed to validate the stability of ligand-target interactions.
In vitro assays using H9c2 cardiomyoblast cells included MTT assay for cytotoxicity, ROS assay for oxidative stress, JC-1 dye for mitochondrial membrane potential (MMP), Annexin V/PI staining for apoptosis, and untargeted metabolomic analysis via LC-MS to identify metabolic alterations under treatment.
In vivo validation was carried out using a mouse model of isoproterenol-induced myocardial infarction. Cardioprotective effects were evaluated by echocardiography to assess cardiac function, histopathological examination to assess tissue architecture, immunohistochemistry (IHC) for localization of key proteins, and Western blotting to quantify expression of signaling proteins involved in apoptosis and MAPK pathways (e.g., p-ERK, p-JNK, p-P38, caspase-3).
The network pharmacology analysis revealed 328 potential targets associated with the natural compound. Key proteins included TNF, AKT1, EGFR, ERK, JNK, HIF1A, and P38 MAPK. Molecular docking confirmed strong binding affinities with these targets, and molecular dynamics simulations verified the stability of interactions. In vitro results showed that the compound significantly improved cell viability, reduced ROS generation, maintained mitochondrial integrity, and decreased apoptotic cell populations. Metabolomic profiling indicated modulation of metabolic pathways related to energy metabolism and oxidative stress. In vivo findings demonstrated improved cardiac function via echocardiography and reduced tissue injury and fibrosis. Western blot and IHC analyses confirmed regulation of signaling pathways involved in inflammation, apoptosis, and cell survival. This integrative study demonstrates the cardioprotective efficacy of a natural compound through modulation of multiple molecular pathways associated with myocardial infarction. The synergistic use of computational modeling, in vitro cell-based assays, and in vivo animal studies provides robust evidence supporting its potential as a therapeutic agent for cardiovascular diseases.
