Notoginsenoside R1 Regulates Ischemic Myocardial Lipid Metabolism by Activating the AKT/mTOR Signaling Pathway

Abstract

Ischemic heart diseases are responsible for more than one-third of all deaths worldwide. Radix notoginseng is widely used to treat ischemic heart disease in China and other Asian countries, and notoginsenoside R1 (NGR1) is its characteristic and large-amount ingredient. However, the potential molecular mechanisms of NGR1 in improving ischemic heart diseases are unclear. In this study, we combined pharmacological evaluation with network pharmacology, myocardial proteomics, and conventional molecular dynamics (MD) simulation to explore the cardio-protection mechanisms of NGR1. Our results revealed that NGR1 improved the echocardiographic, tissue pathological, and serum biochemical perturbations in myocardial ischemic rats. The network pharmacology studies indicated that NGR1 mainly regulated smooth muscle cell proliferation, vasculature development, and lipid metabolism signaling, especially in the PI3K/AKT pathway. Myocardial proteomics revealed that the function of NGR1 was focused on regulating metabolic and energy supply processes. The research combined reverse-docked targets with differential proteins and demonstrated that NGR1 modulated lipid metabolism in ischemic myocardia by interacting with mTOR and AKT. Conventional MD simulation was applied to investigate the influence of NGR1 on the structural stabilization of the mTOR and AKT complex. The results suggested that NGR1 can strengthen the affinity stabilization of mTOR and AKT. Our study first revealed that NGR1 enhanced the affinity stabilization of mTOR and AKT, thus promoting the activation of the AKT/mTOR pathway and improving lipid metabolic abnormity in myocardial ischemic rats.

Keywords: Akt/mTOR pathway; cardio-protection; ischemic heart disease; lipid metabolism; notoginsenoside R1.

FIGURE 1

NGR1 alleviates LADCA ligation–induced ventricular dysfunctions, myocardial infarction, and fibrosis. (A) Representative electrocardiogram and (B) values of EF (left ventricular ejection fraction), (C) values of FS (left ventricular fraction shortening), and (D) values of SV (stroke volume). (E) Representative photos of rat heart slices by TTC staining and quantitative analysis of infarct size. (F) Representative images of rat heart slices by Masson’s staining and quantitative analysis of collagen content. Scale bar = 2000 μm. Values expressed as the mean ± SD. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001 vs. the MI group; ^# p < 0.05, ^## p < 0.01, and ^#### p < 0.001 vs. the sham group (n = 6 for sham, MI, Met, NGR1-L, and NGR1-H groups).

FIGURE 2

Representative images of histopathology and TUNEL staining. (A) Representative photomicrographs of cardiac tissue sections with H & E staining and quantitative analysis of the cross-sectional area. Scale bar = 40 μm. (B) TUNEL assay of apoptotic cardiomyocytes. Scale bar = 40 μm. Values expressed as the mean ± SD. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001 vs. the MI group; ^# p < 0.05, ^## p < 0.01, and ^#### p < 0.001 vs. the sham group (n = 6 for sham, MI, Met, NGR1-L, and NGR1-H groups).

FIGURE 3

Effects of NGR1 on serum biochemical indicators in LADCA-ligated rats. (A) LDH; (B) CK-MB; (C) α-HBDH; (D) IL-6; (E) NF-κB; (F) TNF-α; and (G) FFA. Values expressed as the mean ± SD. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001 vs. the MI group; ^# p < 0.05, ^## p < 0.01, and ^#### p < 0.001 vs. the sham group (n = 6 for sham, MI, Met, NGR1-L, and NGR1-H groups).

FIGURE 4

Network pharmacology of NGR1. (A) Reverse-docked targets of NGR1; (B) Functional annotation of reverse-docked targets. These reverse-docked targets were annotated by GO terms. The pathways were evaluated by a false-positive rate, ^* F < 0.05, ^** F < 0.01, and ^*** F < 0.001. (C) Functional target association network by String analysis. (D) Functional annotation of reverse-docked targets by KEGG terms.

FIGURE 5

Myocardial proteomics combined with network pharmacology revealed that NGR1 improved lipid metabolism in the ischemic myocardium. (A) Heatmap of differential proteins between the NGR1-H group and the MI group. (B) Functional annotation of differential proteins. (C) Lipid metabolism–associated network including reverse-docked targets and differential proteins. Spheres represent reverse-docked targets and diamonds represent differential proteins. (D) Functional annotation of proteins in the lipid metabolism–associated network. These proteins in the lipid metabolism–associated network were annotated by GO terms. The pathways were evaluated by a false-positive rate, ^* F < 0.05, ^** F < 0.01, and ^*** F < 0.001.

FIGURE 6

Validation of key differential protein expression in the lipid metabolism–associated network. (A) Heatmap of differential proteins in the lipid metabolism–associated network. (B) Expression of COX2, CPT2, ACADVL, and CD36 by Western blots. Values expressed as the mean ± SD. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001 vs. the MI group; ^# p < 0.05, ^## p < 0.01, and ^#### p < 0.001 vs. the sham group (n = 3 for sham, MI, NGR1-L, and NGR1-H groups).

FIGURE 7

Conventional MD simulation for evaluating the binding affinity of mTOR and AKT with or without NGR1. (A) Overview structure of the mTOR-AKT complex with NGR1 and a zoom-in view of NGR1 and the surrounding amino acids. (B) Backbone (alpha-C, C, N atoms) RMSDs are shown as a function of time for mTOR and AKT without NGR1 during 50-ns molecular dynamics simulation. (C) Backbone (alpha-C, C, and N atoms) RMSDs are shown as a function of time for mTOR and AKT with NGR1 during 50-ns molecular dynamics simulation. (D) RMSF of mTOR and AKT residues in 50-ns molecular dynamics simulation.