The effect and mechanism of dl-3-n-butylphthalide on angiogenesis in a rat model of chronic myocardial ischemia

Abstract

Objective: To assess the effect of dl-3-n-butylphthalide (NBP) on angiogenesis and its underlying mechanism in a rat model of chronic myocardial ischemia (CMI).

Methods: Forty Sprague-Dawley rats were randomly divided into four groups: model, low-dose NBP (L-NBP), middle-dose NBP (M-NBP), or high-dose NBP (H-NBP) (n=10/group). All groups received intraperitoneal injections of isoprinosine hydrochloride daily for 14 days. Additionally, the L-NBP, M-NBP, and H-NBP groups received NBP at 3, 6, and 12 mg per kg body weight, respectively, by intraperitoneal injection. An additional 10 rats (control group) received 0.9% sodium chloride via intraperitoneal injection for 14 consecutive days. Echocardiography was used for the measurement of heart function. Immunohistochemical staining for factor VIII-related antigen and microvascular density determination were performed. The protein and mRNA expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in CMI areas were measured by western blot and RT-PCR, respectively.

Results: Electrocardiograms showed that NBP improved cardiac function by regulating left ventricular end-diastolic and end-systolic diameters, ejection fraction, and fractional shortening. Compared with the control and model groups, the L-NBP, M-NBP, and H-NBP groups showed increased mRNA and protein expression of VEGFA and HIF-1α in myocardial tissue. The mRNA and protein expression of VEGFA and HIF-α in the H-NBP group were the highest.

Conclusion: NBP treatment promotes VEGF and HIF-1α protein expression during myocardial ischemia, which may represent useful biomarkers for coronary collateral establishment and offer potential targets for therapeutic induction of angiogenesis in patients with CMI.

Keywords: Dl-3-n-butylphthalide; angiogenesis; chronic myocardial ischemia; hypoxia-inducible factor 1-α; vascular endothelial growth factor.

Figure 1

Motion-mode echocardiography images of rats among the control group (A), model group (B), L-NBP group (C), M-NBP group (D), and H-NBP group (E). Compared with the model group, the L-NBP, M-NBP, and H-NBP groups showed significantly improved heart function. The H-NBP group recovered heart function to the level of the control group.

Figure 2

The effect of NBP on factor VIII-related antigen expression in capillary endothelial cells of rats with CMI. Immunohistochemical staining in the control group (A), model group (B), L-NBP group (C), M-NBP group (D), and H-NBP group (E). All images are 400× in magnification.

Figure 3

The effect of NBP on HIF-1α and VEGFA expression in rat ischemic myocardium. Photographs show representative western blots by group for HIF-1α and VEGFA. Graphs show the gray levels of bands. Quantitative data were normalized to β-actin. A. Protein bands for HIF-1α by western blot. B. Protein expression of HIF-1α. C. Protein bands for VEGFA by western blot. D. Protein expression of VEGFA. Values are the means ± SD (n=10 per group). #Compared with the control group, P<0.05; *Compared with the L-NBP group, P<0.05.

Figure 4

Hif1a and Vegfa mRNA expression levels in rat ischemic myocardium. Data are the means ± SD (n=10 per group). A. mRNA expression of Hif1a. B. mRNA expression of Vegfa. #Compared with the control (CO) group, P<0.05; *Compared with the L-NBP group, P<0.05.