Exploring the Cardioprotective Mechanisms of Ligusticum wallichii in Myocardial Infarction Through Network Pharmacology and Experimental Validation

Abstract

Background: Myocardial infarction represents a coronary artery ailment with the highest incidence and fatality rates among cardiovascular conditions. However, effective pharmacological interventions remain elusive. This study seeks to elucidate the molecular mechanisms underlying the effects of Ligusticum wallichii on myocardial infarction through network pharmacology and experimental validation.

Methods: Initially, potential targets of Ligusticum wallichii's active ingredients and myocardial infarction-related targets were retrieved from databases. Subsequently, core targets of Ligusticum wallichii on myocardial infarction were identified via the PPI network analysis and subjected to GO and KEGG pathway enrichment analyses. Molecular docking was employed to validate the binding affinities between the core targets and the bioactive components. The findings from network pharmacology analysis were corroborated through in vitro and in vivo experiments.

Results: Seven active ingredients from Ligusticum wallichii were identified, corresponding to 122 targets. Molecular docking revealed robust binding affinities of Myricanone, Senkyunone, and Sitosterol to key target proteins (EGFR, STAT3, and SRC). In vitro, experiments demonstrated that pretreatment with the active components of Ligusticum wallichii protected myocardial cells from OGD exposure and modulated the expression of their key target genes. In vivo, experiments showed that the active components of Ligusticum wallichii significantly improved myocardial infarction via alleviating myocardial fibrosis and oxidative stress and did not elicit toxic effects in mice.

Conclusion: The collective findings suggest that Ligusticum wallichii shows promising potential for myocardial infarction treatment by regulating key target proteins (EGFR, STAT3, and SRC), which play roles in oxidative stress and myocardial fibrosis.

Keywords: Ligusticum wallichii; Molecular docking; experimental validation; myocardial infarction; network pharmacology.

Figure 1

The overlap of target genes between the active constituents of Ligusticum wallichii and those associated with myocardial infarction.

Figure 2

The construction of the target network involves identifying active ingredients. The Orange circular node represents the active ingredients found in Ligusticum wallichii, while the other nodes represent their corresponding targets.

Figure 3

The protein-protein interaction (PPI) network of Ligusticum wallichii in myocardial infarction treatment was analyzed. (A) PPI network generated using STRING. (B) PPI network constructed via Cytoscape 3.6.0.

Figure 4

Frequency histograms depicting the top 10 genes according to their degree values in the PPI network.

Figure 5

Target selection process: (A) Identification of key targets through the intersection of centrality measures (Closeness, Betweenness, and Degree). (B) Heat map visualization showcasing the binding affinities between active ingredients and selected key targets as determined by molecular docking.

Figure 6

GO enrichment analysis.

Figure 7

Enrichment analysis of KEGG pathways.

Figure 8

Diagram illustrating the network between targets and pathways.

Figure 9

Molecular docking analysis of active compounds with EGFR. (A) Myricanone-EGFR; (B) Senkyunone-EGFR; (C) Sitosterol-EGFR.

Figure 10

Molecular docking analysis of active compounds with SRC. (A) Myricanone-SRC; (B) Senkyunone-SRC; (C) Sitosterol-SRC.

Figure 11

Molecular docking analysis of active compounds with STAT3. (A) Myricanone-STAT3; (B) Senkyunone-STAT3; (C) Sitosterol-STAT3.

Figure 12

The active components derived from Ligusticum wallichii exhibited no cytotoxic effects on H9c2 cells at concentrations up to 160 μM and demonstrated protective effects against OGD-induced injury in H9c2 cells. (A) The planar structures of these active ingredients were analyzed in silico. (B) H9c2 cells were treated with various concentrations of Ligusticum wallichii’s active ingredients, ranging from 10 to 160 μM, for 24 hours. (C, D) The impact of these active ingredients on H9c2 cells subjected to OGD was assessed. (E, F) Following OGD exposure, H9c2 cells were treated with different concentrations of the active ingredients or diazoxide for 48 hours. Subsequently, cell viability was assessed using a CCK-8 assay. The data are presented as mean ± SD. ***p < 0.001, **p < 0.01 vs control; ###p < 0.001, ##p < 0.01, #p < 0.05 vs OGD. n = 5.

Figure 13

The active constituents of Ligusticum wallichii improved myocardial infarction in vivo and modulated the expression of pivotal target genes. Myocardial infarction was induced by ligation of the proximal left anterior descending (LAD) coronary artery in C57BL/6 mice, while the Sham group underwent the identical surgical procedure without the LAD being ligated. Then, different treatments or an equal volume of PBS were administered both before and after the myocardial infarction. (A-B) The myocardial infarct area was detected by TTC among myocardial infarction mice (n = 6). (C-E) Histopathological changes and myocardial fibrosis in the cardiac apex were subsequently analyzed using HE and Masson staining (200× magnification, scale bar = 100 µm) (n = 6). (F-H) The levels of serum marker enzymes CK-MB, AST, and LDH were measured by biochemistry assays (n = 6). Following treatment with various concentrations of these active components (Myricanone, Senkyunone, and Sitosterol) or diazoxide, cultured cells were subjected to OGD stimulation for 3 hours. (I-K) The mRNA levels of key target genes (EGFR, SRC, and STAT3) in H9c2 cells were evaluated. ****p < 0.0001, ***p < 0.001, **p < 0.01 vs control or model; ####p < 0.0001, ###p < 0.001, ##p < 0.01 vs OGD. n = 6.