Network Pharmacology-Based Combined with Experimental Validation Study to Explore the Underlying Mechanism of Agrimonia pilosa Ledeb. Extract in Treating Acute Myocardial Infarction

Abstract

Purpose: The network pharmacology approach and validation experiment were performed to investigate the potential mechanisms of Agrimonia pilosa Ledeb. (APL) extract against acute myocardial infarction (AMI).

Methods: The primary compounds of APL extract were identified by High-Performance Liquid Chromatography (HPLC) analysis. The intersecting targets of active compounds and AMI were determined via network pharmacology analysis. A mouse model of AMI was established by subcutaneous injection of isoproterenol (Iso). Mice were treated with APL extract by intragastric administration. We assessed the effects of APL extract on the electrocardiography (ECG), cardiac representative markers, representative indicators of oxidative stress, pathological changes, and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, as well as apoptosis-related indicators in the mice.

Results: Five candidate compounds were identified in APL extract. Enrichment analyses indicated that APL extract could exert myocardial protective effects via the PI3K/Akt pathway. ST segment elevation and increased heart rate were obviously reversed in APL extract groups compared to Iso group. We also detected significant decreases in lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase MB (CK-MB), malondialdehyde (MDA), and reactive oxygen species (ROS), as well as a significant increase in superoxide dismutase activities (SOD) after APL extract treatment. In addition, APL extract markedly decreased the number of apoptotic cardiomyocytes after AMI. In the APL extract groups of AMI mice, there were increased expression levels of p-PI3K, p-Akt, and B-cell lymphoma-2 (Bcl-2) protein, and there were decreases in Bcl-2-associated X (Bax), cysteinyl aspartate-specific proteases-3 (caspase-3), and cleaved-caspase-3 protein expression levels, as well as the Bax/Bcl-2 ratio.

Conclusion: APL extract had a protective effect against Iso-induced AMI. APL extract could ameliorate AMI through antioxidant and anti-apoptosis actions which may be associated with the activation of the PI3K/Akt signaling pathway.

Keywords: APL extract; PI3K/Akt; acute myocardial infarction; apoptosis; isoproterenol; oxidative stress.

Figure 1

APL extract. (A) Standard chromatograms. (B) Chemically active ingredients. 1. (+)-catechin, 2. ellagic acid, 3. taxifolin, 4. quercitrin, 5. quercetin.

Figure 2

Target screening and network construction. (A) Drug-compound-target visualization network. (B) Venn diagram of the targets of APL extract’s active compounds and AMI. (C) PPI network of the targets of APL extract’s active compounds and AMI. The green rectangle represents the drug, the blue hexagon represents the candidate components of APL extract for the treatment of AMI, the purple circle represents the targets of APL extract active components, and the edges between targets represent their connections.

Figure 3

KEGG pathway and GO enrichment analyses. (A) KEGG pathway analysis of candidate targets of APL extract against AMI. (B) GO terms of candidate targets of APL extract against AMI.

Figure 4

APL extract decreased HR (A) and Degree of ST segment elevation (B) in Iso-induced AMI mice. Values are displayed as mean ± SEM (n=10). Significant differences are presented as **P < 0.01 in comparison to the Con group. ^#P < 0.05, ^##P < 0.01, in comparison to the Iso group.

Figure 5

APL extract reduced LDH (A), CK (B), and CK-MB (C) activities in Iso-induced AMI mice. Values are displayed as mean ± SEM (n=10). Significant differences are displayed as **P < 0.01 in comparison with the Con group. ^#P < 0.05, ^##P < 0.01 in comparison with the Iso group.

Figure 6

APL extract mitigated Iso-induced AMI in mice. Effects of APL extract on cardiac histopathologic in Iso-treated mice. Observation of histopathological changes in the heart using typical H&E staining images (magnification 400 ×). Red arrow indicates edema, black arrow indicates cell necrosis, and yellow arrow indicates inflammatory infiltration.

Figure 7

APL extract mitigated Iso-induced excessive oxidative stress in mice. ROS (A and B) (magnification 200 ×), SOD (C), and MDA (D) levels were detected in mice. Values are displayed as mean ± SEM (n=10). Significant differences are displayed as **P < 0.01 in comparison with the Con group. ^#P < 0.05, ^##P < 0.01 in comparison with the Iso group.

Figure 8

APL extract decreased cardiomyocyte apoptosis in AMI mice. Typical pictures of immunofluorescent TUNEL staining (magnification 200 ×).

Figure 9

APL extract inhibited cardiomyocyte apoptosis of Iso-induced mice. The expression levels of apoptosis-related proteins were tested by Western blotting assay (A). Bcl-2 (B), Bax (C), Bax/Bcl-2 (D), caspase-3 (E), and cleaved-caspase-3 (F) levels were calculated by grayscale analysis. Values are displayed as mean ± SEM (n=3). Significant differences are shown as *P < 0.05, **P < 0.01 in comparison to the Con group. ^#P < 0.05, ^##P < 0.01 in comparison to the Iso group.

Figure 10

APL extract upregulated PI3K/Akt signaling of Iso-induced mice. The expression levels of PI3K, p-PI3K, Akt, and p-Akt protein were inspected by Western blotting assay (A), and p-PI3K (B) and p-Akt (C) were calculated by densitometric analysis. Values are shown as mean ± SEM (n=3). Significant differences are presented as **P < 0.01 in comparison with the Con group. ^#P < 0.05, ^##P < 0.01 in comparison with the Iso group.

Figure 11

Diagram of the likely protective mechanism of APL extract against Iso-induced AMI.