Cardioprotective effects of alpha-lipoic Acid on myocardial reperfusion injury: suppression of reactive oxygen species generation and activation of mitogen-activated protein kinase

Abstract

Background and objectives: Reactive oxygen species (ROS) and mitogen-activated protein (MAP) kinase play an important role in the development of myocardial reperfusion injury. In this study, we examined whether treatment with alpha-lipoic acid (ALA) before reperfusion could prevent myocardial reperfusion injury in vivo.

Materials and methods: Sprague-Dawley rats were subjected to a 45-minute left anterior descending coronary artery ligation followed by 45- or 10-minute reperfusion. ALA was administered 10 minutes prior to reperfusion. The infarct size ratio of the infarct area to the ischemic area at risk, was measured based on 10, 25, 50, and 100 mg/kg of ALA, with propidium iodide (PI) fluorescence. Apoptosis was evaluated by TdT-mediated dUDP nick end labeling (TUNEL) staining. The generation of intracellular ROS was evaluated using the fluorogenic probe, dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA). Western blot analysis was performed for MAP kinase (pERK 1/2 and pJNK 1/2) activity.

Results: The infarct size, according to ALA dose, was significantly suppressed 29.1% with ALA 25 mg/kg (p<0.0001), 41.5% with 50 mg/kg (p<0.05), and 41.4% with 100 mg/kg (p<0.05) compared to the controls (54.3%). However, the results were not significantly different with 47.2% of the ALA 10 mg/kg (p=0.192). A few apoptotic nucleoli were detected in the ALA 25 mg/kg group, but were frequently detected in the control group. The ROS generation was significantly suppressed (p<0.0001), the activity of pERK 1/2 was significantly increased (p<0.05) and the activity of pJNK 1/2 was significantly decreased (p<0.05) in the ALA 25 mg/kg group compared to the controls.

Conclusion: The results of this study suggested that adequate doses of ALA before reperfusion was effective for the prevention of myocardial reperfusion injury in vivo. This cardioprotective activity of ALA might be associated with an anti-apoptotic effect of ALA via suppression of ROS generation, increase of pERK 1/2 and decrease of pJNK 1/2 activity.

Keywords: Alpha-lipoic acid; Apoptosis; Mitogen-activated protein kinase; Myocardial reperfusion injury; Reactive oxygen species.

Fig. 1

This graph shows the infarct size (ratio of infarct to ischemic area) according to ALA dose at 45 minutes after reperfusion by propidium iodide fluorescence. The ALA at doses of 25 mg/kg (p<0.0001), 50 mg/kg (p<0.05), and 100 mg/kg (p<0.05) significantly reduced the infarct size compared to the control group, but not the ALA 10 mg/kg (p=0.192) group. The ALA 25 mg/kg group had the smallest infarct size. ALA: alpha-lipoic acid.

Fig. 2

Light microscopic findings of TUNEL staining in the infarct area at 10 minutes after reperfusion in the control (A) and ALA 25 mg/kg (B) groups. Apoptotic nucleoli (brown spot) were frequently detected in the control group (A). By contrast, only a few apoptotic nucleoli were detected in the ALA 25 mg/kg group (B). TUNEL: terminal deoxynucleotidyl transferase-mediated biotin-dUDP nick end labeling, ALA: alpha-lipoic acid.

Fig. 3

Confocal laser microscopic findings of fluorescence on intracellular ROS generation detected by CM-H₂DCFDA at 10 minutes after reperfusion in the control (A) and ALA 25 mg/kg (B) groups. The fluorescence expression (bright green color) was more frequently detected in the control group (A) than in the ALA 25 mg/kg group (B). The relative ROS generation and the relative level of CM-H₂DCFDA fluorescence by confocal microscopy, was significantly suppressed in the ALA 25 mg/kg group compared to the control group (C). ROS: reactive oxygen species, CM-H₂DCFDA: 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate, ALA: alpha-lipoic acid.

Fig. 4

The expression of mitogen-activated protein (MAP) kinase by Western blot analysis pERK 1/2 expression was increased more in the ALA 25 mg/kg group compared to controls. By contrast, pJNK 1/2 expression was decreased more in the ALA 25 mg/kg group than in controls. ALA: alpha-lipoic acid, ERK: extracellular signal-regulated kinase, JNK: c-Jun-NH2-terminal kinase.

Fig. 5

This graph shows the relative expression of pERK 1/2 (A) and pJNK 1/2 (B) in the infarct area by Western blot and densitometry analysis. The relative density, of the Western blot analysis by densitometry, was compared with the sham density. The relative density of pERK 1/2 was significantly increased (A), and for pJNK 1/2 it was significantly decreased (B) in the ALA 25 mg/kg group compared to controls. ^*p<0.05 compared with control. ALA: alpha-lipoic acid, ERK: extracellular signal-regulated kinase, JNK: c-Jun-NH2-terminal kinase.