Uncovering the Effect and Mechanism of Rhizoma Corydalis on Myocardial Infarction Through an Integrated Network Pharmacology Approach and Experimental Verification

Abstract

Background: Myocardial infarction (MI), characterized by reduced blood flow to the heart, is a coronary artery disorder with the highest morbidity and mortality among cardiovascular diseases. Consequently, there is an urgent need to identify effective drugs to treat MI. Rhizoma Corydalis (RC) is the dry tuber of Corydalis yanhusuo W.T. Wang, and is extensively applied in treating MI clinically in China. Its underlying pharmacological mechanism remains unknown. This study aims to clarify the molecular mechanism of RC on MI by utilizing network pharmacology and experimental verification. Methods: Based on network pharmacology, the potential targets of the RC ingredients and MI-related targets were collected from the databases. Furthermore, core targets of RC on MI were identified by the protein-protein interaction (PPI) network and analyzed with Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was used to validate the binding affinity between the core targets and the bioactive components. Oxygen-glucose deprivation (OGD) was performed on H9c2 cells to mimic MI in vitro. A Cell Counting Kit-8 assay was used to assess the cardioprotective effect of the active ingredient against OGD. Western blot analysis and RT-qPCR were used to measure the cell apoptosis and inflammation level of H9c2 cells. Results: The network pharmacology obtained 60 bioactive components of RC, 431 potential targets, and 1131 MI-related targets. In total, 126 core targets were screened according to topological analysis. KEGG results showed that RC was closely related to the phosphatidylinositol 3-kinase (PI3K)/Protein kinase B (PKB, also called Akt) signaling pathway. The experimental validation data showed that tetrahydropalmatine (THP) pretreatment preserved cell viability after OGD exposure. THP suppressed cardiomyocyte apoptosis and inflammation induced by OGD, while LY294002 blocked the inhibition effect of THP on OGD-induced H9c2 cell injury. Moreover, the molecular docking results indicated that THP had the strongest binding affinity with Akt over berberine, coptisine, palmatine, and quercetin. Conclusion: THP, the active ingredient of RC, can suppress OGD-induced H9c2 cell injury by activating the PI3K/Akt pathway, which in turn provides a scientific basis for a novel strategy for MI therapy and RC application.

Keywords: Corydalis yanhusuo; PI3k/Akt signaling pathway; apoptosis; myocardial infarction; network pharmacology; tetrahydropalmatine.

FIGURE 1

Diagram of the study strategy. Flowchart showing the combination of network pharmacology technology, experimental validation, and molecular docking used in this study.

FIGURE 2

Topological screening process of PPI network. (A) PPI network of the potential targets of the RC ingredients. 8,287 nodes represent the interactive proteins of RC, 2,96,987 edges represent the interactive relationship. (B) PPI network of the MI-related targets. 11,735 nodes represent the interactive proteins of MI, 3,91,369 edges represent the interactive relationship. (C) PPI network of merged RC and MI. 7,462 nodes represent the interactive proteins of the merged network, 2,79,264 edges represent the interactive relationship. (D) PPI network of merged RC and MI with DC ≥ 92. 1,587 nodes represent the interactive proteins of the merged network with DC ≥ 92, 76,869 edges represent the interactive relationship. (E) Central PPI network of merged RC and MI. 126 nodes represent the core proteins of the merged network, 3,245 edges represent the interactive relationship.

FIGURE 3

Component-core target network of RC and 12 core targets. 12 of the 126 core targets were RC-targets that correlated with 60 total bioactive components. The orange nodes represent the potential active ingredients, and the violet nodes represent the core targets.

FIGURE 4

Enrichment of GO and KEGG analysis of the 126 key genes targeted by RC against MI. (A) The GO enrichment results of biological processes, cellular components, and molecular function. (B) Enriched KEGG pathways of potential key targets. The top 10 major enriched pathways are shown.

FIGURE 5

Core targets enriched in the PI3K/Akt pathway. The red nodes represent 15 core targets enriched in the PI3K/Akt pathway.

FIGURE 6

Molecular docking diagram of Akt with THP. (A) Structure diagram of Akt docked with THP. Yellow lines represent hydrogen bond. (B) Surface diagram of Akt docked with THP. (C) The binding energy for THP docked into the Akt crystal structure.

FIGURE 7

THP had no cellular toxicity on H9c2 cells under 240 μM and protected H9c2 against OGD-induced injury. (A) Chemical structure of THP. (B) H9c2 cells were incubated with THP for the indicated concentrations ranging from 3.75 to 240 μM for 24 h. (C,D) The effect of THP on the OGD-induced H9c2 cells. H9c2 cells were treated with different concentrations of THP or diazoxide for 48 h following OGD exposure. Then the H9c2 cells were cultured under OGD condition for the indicated time. A CCK-8 assay was used to determine cell viability. Data are presented as the mean ± SD. **p < 0.01, ***p < 0.001 vs. control, ^## p < 0.01 vs. OGD. n = 3 (three independent replicates).

FIGURE 8

THP was able to suppress apoptosis and the expressions of inflammatory factors induced by OGD in H9c2 cells. The cultured cells were treated with different concentrations of THP or diazoxide and stimulated with OGD for 3 h (A–C) The protein expressions of p-Akt, total Akt, cleaved caspase-3, and caspase-3. (D–F) The mRNA expressions of apoptosis factors (Bax, Bcl-2, and p53). (G–I) The mRNA expressions of inflammatory factors (IL-6, IL-1β, and TNF-α). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. OGD. n = 3 (three independent replicates).

FIGURE 9

THP exhibited anti-apoptotic effect and suppressed the expression of inflammatory factors via regulating the PI3K/Akt signaling pathway. H9c2 cells were incubated with LY294002 (10 μM, diluted in DMSO), an inhibitor of the PI3K/Akt pathway or treated with THP (60 μM) for 48 h, and then the cells were stimulated with OGD. (A–C) The protein expressions of p-Akt, total Akt, cleaved caspase-3, and caspase-3. (D–F) The mRNA expressions of apoptosis factors (Bax, Bcl-2, and p53). (G–I) The mRNA expressions of inflammatory factors (IL-6, IL-1β, and TNF-α). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. OGD, ^@ p < 0.05, ^@@ p < 0.01, ^@@@ p < 0.001 vs. OGD + THP. n = 3 (three independent replicates).

FIGURE 10

THP decreased H9c2 cells apoptosis by activating the PI3K/Akt signaling pathway. H9c2 cells were incubated with LY294002 (10 μM, diluted in DMSO) or treated with THP (60 μM) for 48 h, and then the cells were stimulated with OGD. (A) H9c2 cells apoptosis was assessed through TUNEL assay (scale bar = 25 μm).