Cardioprotective Effect of Danhong Injection against Myocardial Infarction in Rats Is Critically Contributed by MicroRNAs

Abstract

Background: Danhong injection (DHI) has been mainly used for the treatment of myocardial infarction, atherosclerosis, and coronary heart disease in clinical practice. Our previous studies have shown that DHI improves ventricular remodeling and preserves cardiac function in rats with myocardial infarction (MI). In this study, we focused on the potential mechanism of DHI in protecting cardiac function in MI rats.

Methods: Sprague-Dawley rats were subjected to ligation of the left anterior descending coronary artery (LAD) to prepare a myocardial infarction (MI) model. After 14 day DHI intervention, cardiac function was measured by echocardiography and myocardial fibrosis was assessed by Masson staining. Differentiated miRNAs were screened using rat immunopathology miScript miRNA PCR arrays, and their results were verified by RT-PCR, immunofluorescence, and immunoblotting.

Results: DHI treatment significantly reduced infarct size and improved cardiac function and hemodynamics in MI rats by echocardiography and morphology. miRNA PCR array results showed that DHI reversed 25 miRNAs known to be associated with inflammation and apoptosis. Moreover, the expression of inflammatory factors TNF-α, IL-1β, and IL-6 was significantly reduced in the treated DHI group. Mechanistically, DHI downregulated the inflammatory transcription factor NF-κB (as reflected by inhibition of NF-κB p65 nuclear translocation and phosphorylation of the IκBα).

Conclusions: DHI is effective in mitigating inflammation associated with MI by preventing NF-κB nuclear translocation and regulating miRNAs, thereby improving cardiac function in myocardial infarction rats.

Figure 1

Effects of DHI on cardiac functionality and hemodynamics index. Quantitative assessment of dilation and systolic function based on LVEF (LV ejection fraction) (a), LVIDs (LV end-systolic dimensions) (b), LVAWs (LV end-systolic anterior walls) (c), LVFS (LV fractional shortening) (d), LVVols (LV systolic volumes) (e), Tei value ((IVCT + ICRT)/MVET) (f), isovolumic contraction time plus isovolumic relaxation time (IVCT + IVRT), mitral valve ejection time (MVET), and LVSP (LV systolic pressure) (g), LVEDP (LV end-diastolic pressure) (h), +dp/dt max (LV maximum upstroke velocity) (i), −dp/dt max (LV maximum descent velocity) (j), HR (heart rate) (k), and SW (stroke work) (l). Representative echocardiographic images (M mode) in different groups (m). From left to right: sham group, model group, DHI group, valsartan group. All values are means ± SD (n = 8 or n = 6). ^∗p < 0.05 and ^∗∗p < 0.01 versus sham group; ^#p < 0.05 and ^##p < 0.01 versus model group.

Figure 2

Left ventricle stained with H&E and Masson. (a) Representative photomicrographs of H&E-stained myocardium (400×). (b) Representative photomicrographs of Masson-stained myocardium (400×). From left to right: sham group, model group, DHI group, and valsartan group. (c) Proportion of the infarct ratio represented in bar graph; the cavity on the right represents left ventricular; red represents cardiomyocytes, and blue represents collagen fiber. All values are expressed in means ± SD, (n = 5). ^∗p < 0.05 versus the sham group; ^#p < 0.05 versus the model group.

Figure 3

Regulation of DHI on miRNA through the immunopathology miScript miRNA PCR array analysis. (a) Scatter diagram of differentially expressed miRNA of the DHI group upon model group. 2-fold regulation was selected, with DHI as group 1, and MI model was selected as the control group. Red indicates upregulated genes. miRNA above the oblique line is upregulated and below the line is downregulated (n = 3). (b) Unsupervised hierarchical clustering analysis on the differentially expressed miRNAs (>2-fold change) in the model group vs DHI group. Note: (1) red represents upregulation of miRNA; green represents downregulation of miRNA. (2) (b) model group and (c) DHI group. (c) miRNA-GO-network was generated according to the relationship of significant functions and miRNAs. A high enrichment degree between the differentially expressed miRNAs and the functions in the network. Enrichment degree means the contribution of a miRNA to the surrounding GOs or the opposite. The blue circle represented GOs, the red square represented miRNAs, and their relationship was represented by lines art. (d) The miRNA-KEGG signal pathway analysis of the differentially expressed miRNA related to the signal pathway. The black bar represents the signal pathway regulated by overexpressed and underexpressed miRNAs. The vertical axis is the pathway category, and the horizontal axis is the −log p value of each pathway. (e) Using differentially expressed miRNA and prediction target gene to construct the miRNA-mRNA network. The blue circles represent miRNAs, the red circles represent mRNA, and lines art represent the relationship between miRNA and mRNA.

Figure 4

Effects of DHI on mRNA expression in myocardial tissue following MI. The relative levels of cardiac TNF-α, IL-1β, Bcl-2, and caspase-3 mRNA were assessed by RT-PCR. Results were normalized to GAPDH, and all values are expressed in means ± SD (n = 4). ^∗p < 0.05 and ^∗∗p < 0.01 compared with the sham group; ^#p < 0.05 and ^##p < 0.01 compared with the model group.

Figure 5

Inhibitory effect of DHI on inflammatory cytokines. (a–c) Effects of DHI on serum TNF-α, IL-1β, and IL-6 levels recorded at the end of 14 days after surgery; all values are expressed in means ± SD (n = 8). ^∗p < 0.05 and ^∗∗p < 0.01 compared with the sham group; ^#p < 0.05 and ^##p < 0.01 compared with the model group.

Figure 6

Effect of DHI on the activation of the NF-κB pathway. (a) DHI inhibits phosphorylation of NF-κB and IκB-α in cardiomyocytes cytoplasm from infarct marginal zone. (b) DHI prevents the nucleation of NF-κB in myocardial tissue from the infarct marginal zone. The level of p-NFκB and p-IκB-α in the cytoplasm was corrected by NF-κB and IκB-α, respectively. The expression of NF-κB in the nucleus was corrected by Lamin B. Data are expressed as mean ± SD (n = 3). ^∗∗p < 0.01 compared with the sham group; ^##p < 0.01 compared with the model group.