Protective effect and mechanism of procyanidin B2 against hypoxic injury of cardiomyocytes

Abstract

Background: Cardiomyocyte ischemia and hypoxia are important causes of oxidative stress damage and cardiomyocyte apoptosis in coronary heart disease (CHD). Epidemiological investigation has shown that eating more plant-based foods, such as vegetables and fruits, may significantly decrease the risk of CHD. As natural antioxidants, botanicals have fewer toxic side effects than chemical drugs and have great potential for development. Procyanidin B2 (PB2) is composed of flavan-3-ol and epicatechin and has been reported to have antioxidant and anti-inflammatory effects. However, whether PB2 exerts protective effects on hypoxic cardiomyocytes has remained unclear. This study aimed to explore the protective effect of PB2 against cardiomyocyte hypoxia and to provide new treatment strategies and ideas for myocardial ischemia and hypoxia in CHD.

Methods and results: A hypoxic cardiomyocyte model was constructed, and a CCK-8 assay proved that PB2 had a protective effect on cardiomyocytes in a hypoxic environment. DCFH fluorescence staining, DHE staining, and BODIPY lipid oxidation assessment revealed that PB2 reduced the oxidative stress levels of cardiomyocytes under hypoxic conditions. TUNEL staining, Annexin V/PI fluorescence flow cytometry, and Western blot analysis of the expression of the apoptosis marker protein cleaved caspase-3 confirmed that PB2 reduced cardiomyocyte apoptosis under hypoxic conditions. JC-1 staining indicated that PB2 reduced the mitochondrial membrane potential of cardiomyocytes under hypoxia. In addition, transcriptomic analysis proved that the expression of 158 genes in cardiomyocytes was significantly changed after PB2 was added during hypoxia, of which 53 genes were upregulated and 105 genes were downregulated. GO enrichment analysis demonstrated that the activity of cytokines, extracellular matrix proteins and other molecules was changed significantly in the biological process category. KEGG enrichment analysis showed that the IL-17 signaling pathway and JAK-STAT signaling pathway underwent significant changes. We also performed metabolomic analysis and found that the levels of 51 metabolites were significantly changed after the addition of PB2 to cardiomyocytes during hypoxia. Among them, 39 metabolites exhibited increased levels, while 12 metabolites exhibited decreased levels. KEGG enrichment analysis showed that cysteine and methionine metabolism, arginine and proline metabolism and other metabolic pathways underwent remarkable changes.

Conclusion: This study proves that PB2 can reduce the oxidative stress and apoptosis of cardiomyocytes during hypoxia to play a protective role. Transcriptomic and metabolomic analyses preliminarily revealed signaling pathways and metabolic pathways that are related to its protective mechanism. These findings lay a foundation for further research on the role of PB2 in the treatment of CHD and provide new ideas and new perspectives for research on PB2 in the treatment of other diseases.

Keywords: Apoptosis; Cardiomyocyte hypoxia; Coronary heart disease; Oxidative stress; Procyanidin B2.

Fig. 1

PB2 increases cardiomyocyte viability and reduces oxidative stress levels under hypoxic conditions. (A) Normoxic control group; hypoxic control group; and 20, 40, and 60 μM PB2 treatment groups under hypoxic conditions. CCK-8 assays were used to measure cell viability. (B) Representative images of DCFH fluorescence staining from flow cytometry and quantification of the flow cytometry results. (C) A fluorescence microscope was used to obtain representative images of DHE fluorescence staining and to perform quantitative analysis of fluorescence results. (D) Quantitative analysis of cellular lipid peroxidation using BODIPYTM581/591 C11. *P < 0.05 compared with normoxic control; #P < 0.05 compared with hypoxic control.

Fig. 2

PB2 reduces cardiomyocyte apoptosis and MMP under hypoxic conditions. (A) Annexin V/PI flow cytometry results for apoptosis and representative images. (B) A fluorescence microscope was used to obtain representative images of TUNEL fluorescence staining and to perform quantitative analysis of the fluorescence results. Scale bar, 60 μm. (C) Representative images and relative expression levels of apoptosis marker proteins from Western blotting. (D) Representative graph of MMP measured by JC-1 flow cytometry and statistical analysis of the flow cytometry results. *P < 0.05 compared with normoxic control; #P < 0.05 compared with hypoxic control.

Fig. 3

Transcriptomic analysis of the protective effect of PB2 on cardiomyocytes under hypoxia. (A) Volcano plot of differentially expressed genes. (B) Differentially expressed gene clustering heatmap. (C) GO enrichment analysis histogram. Note: The abscissa is the term in GO level 2, and the ordinate is the -log 10 (P) enrichment value for each term. (D) GO enrichment analysis bubble chart. (E) KEGG enrichment analysis bubble chart.

Fig. 4

Metabolomic analysis of the protective effect of PB2 on cardiomyocytes under hypoxia. (A) OPLS-DA S-plot. (B) Volcano plot of differential metabolites. (C) Differential metabolite clustering heatmap. (D) Differential metabolite KEGG enrichment bubble plot.