Danhong Injection Enhances the Therapeutic Efficacy of Mesenchymal Stem Cells in Myocardial Infarction by Promoting Angiogenesis

Abstract

Stem cell-based therapies have the potential to dramatically transform the treatment and prognosis of myocardial infarction (MI), and mesenchymal stem cells (MSCs) have been suggested as a promising cell population to ameliorate the heart remodeling in post-MI. However, poor implantation and survival in ischemic myocardium restrict its efficacy and application. In this study, we sought to use the unique mode of action of Chinese medicine to improve this situation. Surrounding the myocardial infarct area, we performed a multi-point MSC transplantation and administered in conjunction with Danhong injection, which is mainly used for the treatment of MI. Our results showed that the MSC survival rate and cardiac function were improved significantly through the small animal imaging system and echocardiography, respectively. Moreover, histological analysis showed that MSC combined with DHI intervention significantly reduced myocardial infarct size in myocardial infarcted mice and significantly increased MSC resident. To investigate the mechanism of DHI promoting MSC survival and cell migration, PCR and WB experiments were performed. Our results showed that DHI could promote the expression of CXC chemokine receptor 4 in MSC and enhance the expression of stromal cell-derived factor-1 in myocardium, and this effect can be inhibited by AMD3100 (an SDF1/CXCR4 antagonist). Additionally, MSC in combination with DHI interfered with MI in mice and this signifies that when combined, the duo could the expression of vascular endothelial growth factor (VEGF) in the marginal zone of infarction compared with when either MSC or DHI are used individually. Based on these results, we conclude that DHI enhances the residence of MSCs in cardiac tissue by modulating the SDF1/CXCR4 signaling pathway. These findings have important therapeutic implications for Chinese medicine-assisted cell-based therapy strategies.

Keywords: Danhong injection; SDF-1/CXCR4; mesenchymal stromal cells; myocardial infarction; stem cell transplantation.

FIGURE 1

MSC assessment. (A) Cell surface marker of UC-MSCs at P5 showing positive for CD73, CD90, and CD105 and negative for CD19, CD11b, CD34, and CD45. (B) Morphological images of UC-MSCs at P5 (×100).

FIGURE 2

Effects of MSC combined with DHI on cardiac function and hemodynamics in MI mice. LV ejection fraction (A) and fractional shortening (B) of mice in each group after intervention for 14 days. (C–F) indicated LV ejection fraction (EF), fractional shortening (FS), early and late diastolic mitral flow velocity ratio (E/A), and mitral valve diastolic velocity ratio (E′/A′) in each group after 28 days of intervention, respectively. (G,H) The LV maximum upstroke velocity (+dp/dt_max) and maximum descent velocity (–dp/dt_max) were examined by cardiac catheterization. (I) Representative echocardiographic images (M-mode) in different groups. All values are means ± SD (n = 8 or n = 6). ^#p < 0.05, ^##p < 0.01 versus model group; NS means no significant difference.

FIGURE 3

Pathological examination was performed by HE and Masson staining. (A,B) Representative photomicrographs of HE-stained and Masson-stained myocardium (200×). (C) Representative heart longitudinal panoramic view. (D) The infarct area ratio was quantified by midline method. (E,F) Representative percentage of heart and lung wet weight to body weight. All values are means ± SD (n = 4 or n = 8). ^#p < 0.05, ^##p < 0.01 versus model group; NS means no significant difference.

FIGURE 4

Molecular imaging of Dil-MSCs retention after transplantation. (A,B) Effect of DHI on MSC retention after 7 and 14 days of transplantation. (C,D) Representative fluorescence images of mice transplanted with MSC and MSC + DHI at 7 and 14 days were obtained by IVIS. (E) Representative myocardial photomicrographs of mice after transplantation of MSC and MSC + DHI for 28 days. All values are means ± SD (n = 8). ^##p < 0.01 versus model group.

FIGURE 5

DHI promotes the survival of MSC through the SDF-1/CXCR4 signaling pathway. Quantitative real-time PCR analysis of gene expression of SDF-1 (A), CXCR4 (B), and VEGF (C) in heart samples with various treatments. (D) The representative western blotting bands of SDF-1 and CXCR4 protein in different groups. (E) Semi-quantitative analysis of SDF-1 and CXCR4 protein were normalized against GAPDH expression. All values are means ± SD (n = 3). ^#p < 0.05, ^##p < 0.01 versus model group; NS means no significant difference.

FIGURE 6

The effect of DHI main component of Danshensu on MSC cells. (A) Effect of Danshensu on the expression of CXCR4 protein in MSCs. (B) Representative flow cytometric analysis of CXCR4-PE in MSCs. (C) MSC cell migration was assessed by transwell assay under different interventions. (D) The number of MSC migration statistics. All values are means ± SD (n = 3). ^#p < 0.05, ^##p < 0.01 versus control group; NS means no significant difference.