Shexiang Tongxin Dropping Pill Protects Against Chronic Heart Failure in Mice via Inhibiting the ERK/MAPK and TGF-β Signaling Pathways

Abstract

Background: Chronic heart failure (CHF) is a major public health problem with high mortality and morbidity worldwide. Shexiang Tongxin Dropping Pill (STDP) is a widely used traditional Chinese medicine preparation for coronary heart disease and growing evidence proves that STDP exerts beneficial effects on CHF in the clinic. However, the molecular mechanism of the therapeutic effects of STDP on CHF remains largely unknown. Objective: This study aimed to elucidate the mechanism of action of STDP against CHF by integrating network pharmacology analysis and whole-transcriptome sequencing. Methods: First, the mouse model of CHF was established by the transverse aortic constriction (TAC) surgery, and the efficacy of STDP against CHF was evaluated by assessing the alterations in cardiac function, myocardial fibrosis, and cardiomyocyte hypertrophy with echocardiography, Masson's trichrome staining, and wheat germ agglutinin staining. Next, a CHF disease network was constructed by integrating cardiovascular disease-related genes and the transcriptome sequencing data, which was used to explore the underlying mechanism of action of STDP. Then, the key targets involved in the effects of STDP on CHF were determined by network analysis algorithms, and pathway enrichment analysis was performed to these key genes. Finally, important targets in critical pathway were verified in vivo. Results: STDP administration obviously improved cardiac function, relieved cardiomyocyte hypertrophy, and ameliorated myocardial fibrosis in CHF mice. Moreover, STDP significantly reversed the imbalanced genes that belong to the disease network of CHF in mice with TAC, and the number of genes with the reverse effect was 395. Pathway analysis of the crucial genes with recovery efficiency revealed that pathways related to fibrosis and energy metabolism were highly enriched, while TGF-β pathway and ERK/MAPK pathway were predicted to be significantly affected. Consistently, validation experiments confirmed that inhibiting ERK/MAPK and TGF-β signaling pathways via reduction of the phosphorylation level of Smad3 and ERK1/2 is the important mechanism of STDP against CHF. Conclusion: Our data demonstrated that STDP can recover the imbalanced CHF network disturbed by the modeling of TAC through the multi-target and multi-pathway manner in mice, and the mechanisms are mainly related to inhibition of ERK/MAPK and TGF-β signaling pathways.

Keywords: ERK/MAPK signaling pathway; Shexiang Tongxin dripping pill; TGF-β signaling pathway; chronic heart failure; network pharmacology; whole-transcriptome sequencing.

FIGURE 1

STDP relieves TAC-induced cardiac dysfunction in CHF mice. (A) Representative echocardiograms after 6 weeks of STDP treatment (86 mg/kg/d). (B) The cardiac function indexes of mice were quantified by the Vevo 1100 software. ^## p < 0.01 versus the Sham group; ^** p < 0.01 versus the Model group; n = 7.

FIGURE 2

STDP reduces cardiomyocyte hypertrophy in CHF mice. (A) Representative images of mouse left ventricular sections stained with WGA and photographed with fluorescence microscope (magnification, ×400). Bars = 50 μm. (B) Statistics of cardiomyocyte cross sectional area in mice. ^## p < 0.01 versus the Sham group; ^** p < 0.01 versus the Model group; n = 3.

FIGURE 3

STDP attenuates myocardial fibrosis in CHF mice. After 6 weeks of STDP treatment, representative images of mouse left ventricular sections stained with Masson’s trichrome and photographed with fluorescence microscope (magnification, ×400). Bars = 50 µm. n = 3. Collagen fibers were stained blue and myocytes were stained red, reflecting the degree of myocardial fibrosis.

FIGURE 4

STDP recovers imbalanced CHF network disturbed by TAC modeling. (A) CHF disease network in the Sham group. Each node represents a gene, and each edge represents the relationship between two genes based on the protein-protein interaction. The size of node represents NTRA rank, and the circles in the center of the network indicate the crucial pathways. (B) TAC modeling disrupted CHF network. The red color of the genes represents log2 Fold Change (Model/Sham) > 0, implying upregulated expression after TAC modeling; whereas blue indicate log2 Fold Change (Model/Sham) < 0, meaning downregulated gene expression by TAC. The gradation of the color indicates the degree of gene upregulation and downregulation. (C) The recovery regulation of STDP on CHF network. The purple color of the gene indicates that the recovery efficiency EoR >0. It means that STDP treatment shows a recovery effect on the gene. The black color indicates no effect. The gradation of the color represents the efficiency of recovery regulation.

FIGURE 5

STDP improves TAC-induced CHF in mice mainly by energy metabolism and fibrotic signaling pathway. (A) The top 25 classical pathways significantly enriched after STDP treatment by IPA. (B) A network showing interactions among the gene clusters with significant enrichment in molecular functions and biological processes using Metascape.

FIGURE 6

STDP protects mice against CHF via downregulating ERK/MAPK and TGF-β pathways. (A) The gene expression levels of Tgfb3, Tgfbr1, Mybphl1, and Bmp10 detected by transcriptome sequencing and qRT-PCR. The internal reference was Actb, n = 3. (B) The protein expression levels of p-ERK1/2, ERK1/2, p-Smad3, and Smad3 in heart tissues detected by immunoblot analysis. GAPDH was used as the internal standard. (C) The quantitative results calculated by Image Lab software. ^## p < 0.01 versus the Sham group; ^* p < 0.05 and ^** p < 0.01 versus the Model group; n = 3.