Network pharmacology and in vitro studies show the pharmacological effects and molecular mechanisms of salidroside against coronary artery disease
Authors: Jiang Bing , Feng Xin , Wang Zheng , Xu Jin , Yang Tao , Feng Longfei , Zhou Xuan , Wang Linlin , Su Haixiang , Zhang Zheng 
Published: November 2022

Abstract
Background
Salidroside (Sal) has many pharmacological effects, including anti-fatigue, anti-aging, immunoregulation and so on. However, its effects and related regulatory mechanisms on coronary artery disease (CAD) are elusive. We hypothesize that Sal may play a role in CAD through autophagic pathway. Herein, we used autophagy agonist (sirolimus, Sir) and inhibitor (chloroquine diphosphate, CQ) as the autophagic control group. And network pharmacology prediction was employed to explore the pharmacological effects and molecular mechanisms of Sal on CAD using network pharmacology prediction, and validated the results through in vitro experiments.
Methods
Through a search in the Swisstarget Prediction database, chemical composition and targets were retrieved. The related-autophagic targets for CAD were then obtained from the GEO and human autophagy databases. The network was constructed that depicted the interactions between putative drugs (Sal, Sir and CQ) and known related-autophagic targets for CAD using Cytoscape 3.8.0. Analysis of protein-protein interaction (PPI) was achieved via STRING software, followed by gene ontology (GO) functional enrichment and kyoto enrichment of gene and genome (KEGG) pathway analyses. The 3D structure of target protein was downloaded from RSCB PDB database, the 2D structure of drug was obtained from PubChem database, then the molecular docking was constructed by using AutoDockTools 1.5.6 and AutoDock Vina software, and the results were visualized by using PyMoL sofeware. To validate the computer-predicted results, in vitro experiments based on an anoxic injury model were designed by using CoCl2-exposed H9C2 cells, and treated with various concentrations of Sal/Sir/CQ. The effects of drugs on cells were detected by using the CCK-8, inverted microscope (IM), transmission electron microscope (TEM), MDC staining and AO staining. Moreover, the expression profiles of related-proteins were analyzed via western blot.
Results
Three compounds were identified a total of 399 targets, GEO revealed a total of 576 CAD-related genes, and 36 overlapping genes of drug-disease were obtained. The PPI network was employed for the analysis of 36 target proteins, including CASP3, mTOR, VEGFA and EGFR. The enrichment analysis demonstrated candidate targets of three compounds were more frequently involved in inflammation, lipid response, oxidative stress, autophagy, apoptosis, diabetic cardiomyopathy and estrogen signaling pathways. In vitro experiments revealed that Sal could induce autophagy in CoCl2-exposed H9C2 cells, significantly reverse cell death in the early period, and decrease the levels of Caspase-3 protein in CoCl2-exposed H9C2 cells. However, Sal-induced autophagy also could promoted the process of CoCl2-exposed H9C2 cells death in the late period.
Conclusions
This study revealed that Sal existed the effect of treating CAD by regulating multi-targets and multi-channels through the method of network pharmacology. Furthermore, in vitro results confirmed that Sal-induced autophagy played a protective role in the early period of CAD, while aggravated the condition in the late period of CAD.