Network Pharmacology Analysis and Experimental Verification Strategies Reveal the Action Mechanism of Danshen Decoction in Treating Ischemic Cardiomyopathy

Abstract

Background: Danshen Decoction comprises Salvia miltiorrhiza, Santalum album, and Amomum villosum. It can promote blood circulation and remove blood stasis, and is commonly used in the treatment of gastric and duodenal ulcers, coronary heart disease, angina pectoris, etc. This research is based on network pharmacology and is experimentally verified to explore the potential mechanism of Danshen Decoction in the treatment of ischemic cardiomyopathy (ICM).

Methods: The effective components and targets of Danshen Decoction were firstly extracted from Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform, the drug-component-target-disease network was then constructed, the protein-protein interaction (PPI) network was constructed, the Gene Ontology (GO) enrichment analysis was carried out, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was analyzed in order to find the potential active components and therapeutic mechanisms. Finally, the in vitro hypoxia/reoxygenation model in H9c2 cells was established to verify the predicted active components and therapeutic mechanisms.

Results: The results showed that Danshen Decoction has 67 potential active components and 109 therapeutic targets in treating ICM. These targets were rich in a variety of gene functions and different signaling pathways; the main gene targets include TP53, c-Jun, and Akt1. Go enrichment analysis showed that response to drug, membrane raft, and G protein-coupled amine receiver activity rank first in each process, and the main signaling pathways include PI3K-Akt signaling pathway. Through molecular docking and experimental verification of the major active components and core therapeutic targets, the active components of Danshen Decoction demonstrated an ability to reduce the cell damage caused by hypoxia/reoxygenation in H9c2 cells by regulating the core therapeutic target including Akt1, c-Jun, and TP53.

Conclusion: Danshen Decoction has the effect of treating ICM in multiple ways, which is consistent with the results of network pharmacology. This laid a foundation for further study in exploring the active principles and pharmacological mechanism of Danshen Decoction.

Figure 1

Workflow for dissecting the mechanisms of Danshen Decoction in the treatment of ischemic cardiomyopathy. The first step was to screen the target, the second step was to establish the relationship between the compound and the target, the third step was core gene screening and molecular docking, and finally in vitro experiments were carried out.

Figure 2

Ingredient–target network. Yellow represents the disease target, blue represents the compound, red represents Salvia miltiorrhiza, cyan represents Santalum album, purple represents Amomum villosum, and the connection of lines represents their correlation.

Figure 3

Overlapped genes interaction. (a) PPI network showing interactions between the involved genes. The more connections between each gene indicates that they are more closely related. (b) Core gene target screening. Yellow represents the selected genes.

Figure 4

GO enrichment analyses. (a) Box plot of GO enrichment. (b) Dot plot of GO enrichment. The color changes from red to blue, indicating that the P value of the path changes from large to small; and the larger the surface area, the greater the enrichment degree.

Figure 5

KEGG enrichment analyses. (a) Box plot of KEGG enrichment. (b) Dot plot of KEGG enrichment. The color changes from red to blue, indicating that the P value of the path changes from large to small; and the larger the surface area, the greater the enrichment degree.

Figure 6

The effect of predicted compounds on cell viability. (a) Different concentrations of active compounds on cell viability. (b) Exploration of optimal dosage of active compounds on improvement rate (n = 5). ^∗∗P < 0.01 vs. Model group. (c) Alterations of cellular morphology. The cells were placed under an inverted microscope for observation (×200).

Figure 7

The effect of predicted compounds on improving cell hypoxia in multiple ways. (a) The expressions of LDH and MDA from different groups (n = 3). (b) Western blot bands. (c) The expressions of TP53, p-c-Jun, and p-Akt from different groups were detected by Western blot assay (n = 3). (d) The expression of Akt, c-Jun, and TP53 mRNA in different groups was detected by PCR (n = 6). The results were presented as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01 vs. Model group.