Dl-3-n-butylphthalide protects the heart against ischemic injury and H9c2 cardiomyoblasts against oxidative stress: involvement of mitochondrial function and biogenesis

Abstract

Background: Myocardial infarction (MI) is an acute and fatal condition that threatens human health. Dl-3-n-butylphthalide (NBP) has been used for the treatment of acute ischemic stroke. Mitochondria may play a protective role in MI injury. However, there are few reports on the cardioprotective effect of NBP or the potential mitochondrial mechanism for the NBP-induced protection against cardiac ischemia injury. We investigated the therapeutic effects of NBP in an in vivo MI model and an in vitro oxidative stress model, as well as the potential mitochondrial mechanism.

Methods: This study comprised two different experiments. The aim of experiment 1 was to determine the protective effects of NBP on MI and the underlying mechanisms in vivo. In part 1, myocardial infarct size was measured by staining with 2,3,5-triphenyltetrazoliumchloride (TTC). Myocardial enzymes and mitochondrial enzymes were assayed. The aim of experiment 2 was to investigate the role of NBP in H₂O₂-induced myocardial ischemic injury in H9c2 cells and to determine the potential mechanism. In part 2, H9c2 cell viability was evaluated. ROS levels, mitochondrial morphology, and mitochondrial membrane potential of H9c2 cells were measured. ATP levels were evaluated using an assay kit; mitochondrial DNA (mtDNA), the expressions of NRF-1 and TFAM, and mitochondrial biogenesis factors were determined.

Results: NBP treatment significantly reduced the infarct ratio, as observed by TTC staining, decreased serum myocardial enzymes in MI, and restored heart mitochondrial enzymes (isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and a-ketoglutarate dehydrogenase (a-KGDH) activities after MI. Moreover, in in vitro studies, NBP significantly increased the viability of H9c2 cells in a dose-dependent manner, reduced cell apoptosis, protected mitochondrial functions, elevated the cellular ATP levels, and promoted H₂O₂-induced mitochondrial biogenesis in H9c2 cardiomyoblasts.

Conclusion: Collectively, the results from both the in vivo and in vitro experiments suggested that NBP exerted a cardioprotective effect on cardiac ischemic injury via the regulation of mitochondrial function and biogenesis.

Keywords: Dl-3-n-butylphthalide; Mitochondrial biogenesis; Mitochondrial function; Myocardial infarction.

Fig. 1

Experiment design. In experiment 1 a, animals were divided into four groups: Sham, MI, 40 mg/kg NBP, and 80 mg/kg NBP groups. Rats in the NBP-treated groups were injected intraperitoneally with 40 mg/kg NBP and 80 mg/kg NBP at 30 min after MI. The infarct area, cardiac enzymes, and mitochondrial enzyme were measured 24 h after MI. In experiment 2 b, H9c2 cardiomyoblasts were divided into three groups: control, H₂O₂, and H₂O₂ + NBP 10 μM groups. The cells were pre-treated with 100 μM H₂O₂ for 2 h and subsequently treated with NBP for 24 h. Then, cell viability, apoptosis, mitochondrial function, and biogenesis were detected

Fig. 2

Infarct area and cardiac enzymes in the different groups in experiment 1. At 24 h after MI, the hearts of the rats were collected for the detection of the infarct area and cardiac enzymes. Data revealed that NBP significantly reduced the infarct area of rats following MI a, b. Moreover, the levels of cardiac enzymes were reduced after NBP treatment c-e. # P < 0.05 vs Sham group; *P < 0.05 vs MI group

Fig. 3

Effect of NBP on mitochondrial enzyme activities in MI. The activity is expressed as nM of NADH oxidized/h/mg protein for ICDH; nM of ferrocyanide formed/h/mg protein for a-KGDH; nM of succinate oxidized/min/mg protein for SDH; and nM of NADH oxidized/min/mg protein for MDH. Values represent the mean ± S.E.M. (n = 6). # P < 0.05 vs Sham group; *P < 0.05 vs MI group

Fig. 4

Effect of NBP on cell viability and apoptosis in H9c2 cardiomyoblasts exposed to H₂O₂. a The cell viability was determined by CCK-8 assay. Value were the mean ± S.E.M, n = 6, # P < 0.05 vs. Control group; * P < 0.05 vs H₂O₂ group. b The cell apoptosis was determined by Annexin V-FITC/PI staining. Depicted are the mean ± S.E.M, # P < 0.05 vs Control group; * P < 0.05 vs H₂O₂ group

Fig. 5

The influence of NBP on mitochondrial function in H9c2 cardiomyoblasts exposed to H₂O₂. a The generation of ROS in different groups (Control group; H₂O₂ group; H₂O₂ + NBP group). b Micrographs of mitochondrial morphology by MitoTracker Red staining in H9c2 (Control group; H₂O₂ group; H₂O₂ + NBP group). c Mitochondrial membrane potential (MMP) was determined by evaluation of JC-1 staining under an upright fluorescence microscope after use of the JC-1 assay kit. d Cells were exposed to H₂O₂ for 2 h and then MMP was determined in the absence or presence of NBP. Results were expressed as the mean ± S.E.M. # P < 0.05 vs Control group; * P < 0.05 vs H₂O₂ group

Fig. 6

NBP promotes mitochondrial biogenesis after H₂O₂ treatment. The mitochondrial ATP generation a and mitochondrial DNA (mtDNA) content b were measured in different groups. The expression of NRF-1, and TFAM at protein levels was measured by western blot c and d Data are shown as the mean ± S.E.M. # P < 0.05 vs Control group. * P < 0.05 vs H₂O₂ group