Protective effect of the combinations of glycyrrhizic, ferulic and cinnamic acid pretreatment on myocardial ischemia-reperfusion injury in rats

Abstract

The aim of this study was to find an effective drug cocktail pretreatment to protect myocardial tissue of the heart from ischemia-reperfusion (I/R) injury. The mechanisms underlying the effects of the drug cocktail were subsequently explored in order to expand the application of Dang-gui-si-ni-tang (DGSN), a Traditional Chinese Medicine. The active components of DGSN in the serum following oral administration were investigated using high-performance liquid chromatography. The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) levels were then analyzed to show the effect of the active components in the treatment of myocardial I/R injury. An L16 (4⁴) orthogonal experiment was utilized to determine the most effective cocktail mix and the mechanism underlying the effect of this mix on myocardial I/R injury was investigated. It was observed that FCG, a mixture of glycyrrhizic (50 mg/kg), cinnamic (200 mg/kg) and ferulic (300 mg/kg) acid, was the optimal drug cocktail present in DGSN. This was absorbed into the blood following oral administration and was shown to decrease MDA levels and increase the activity of SOD. In conclusion, the findings suggest that FCG, a combination of active ingredients in the DGSN decoction, can be absorbed into the blood and protect the myocardium from I/R injury.

Keywords: cinnamic acid; ferulic acid; glycyrrhizic acid; ischemia-reperfusion cocktail.

Figure 1

High-performance liquid chromatography chromatograms of (A) Dang-gui-ni-tang decoction extracted by n-butanol; (B) the standard sample; (C) the serum sample; and (D) the blank serum. The peaks marked as ➀ ➁ ➂ and ➃ are peoniflorin and ferulic, cinnamic and glycyrrhizic acids, respectively. The detection wavelength was 275 nm.

Figure 2

Effects of FCG on (A) myocardial AAR and (B) IS. (C) Images of the myocardial AAR and infarct area in the sham, I/R and PPC+I/R groups (left, center and right, respectively). The area of infarct is white, the myocardial AAR is deep red and the nonischemic area is blue. Data are presented as the mean ± standard deviation; n=6. ^**P<0.01 vs. I/R; ^###P<0.001 vs. sham, n=6. Sham, sham-operated animal without ligation; I/R, 30 min ischemia followed by 2 h reperfusion; PPC+I/R, administration of FCG for 5 days prior to the induction of myocardial ischemia. FCG, ferulic acid (300 mg/kg), cinnamic acid (200 mg/kg) and glycyrrhizic acid (50 mg/kg); AAR, area at risk; IS, infarct size.

Figure 3

Morphology of rat myocardium (hematoxylin and eosin staining; magnification, ×200). Sham, sham-operated animal without ligation; I/R, 30 min ischemia followed by 2 h reperfusion; PPC+I/R, administration of FCG for 5 days prior to the induction of myocardial ischemia. The arrow indicates muscle fiber disarranging and clear hydropic degeneration. FCG, ferulic acid (300 mg/kg), cinnamic acid (200 mg/kg) and glycyrrhizic acid (50 mg/kg).

Figure 4

Electron microscopic examination of rat myocardium (magnification, ×15,000). Sham, sham-operated animal without ligation; I/R, 30 min ischemia followed by 2 h reperfusion; PPC+I/R, administration of FCG for 5 days prior to the induction of myocardial ischemia. Thin arrows indicate apoptotic bodies, Δ indicates swelling mitochondria, large arrows indicate chromatin condensation and aggregation. FCG, ferulic acid (300 mg/kg), cinnamic acid (200 mg/kg) and glycyrrhizic acid (50 mg/kg).

Figure 5

Effect of FCG pretreatment on cytoplasmic and nuclear NF-κBp65 expression in different groups. Data are presented as the mean ± standard deviation; n=6. ^*P<0.05 vs. I/R; ^##P<0.001 and ^#P<0.05 vs. sham. Sham, sham-operated animal without ligation; I/R, 30 min ischemia followed by 2 h reperfusion; PPC+I/R, administration of FCG for 5 days prior to the induction of myocardial ischemia. FCG, ferulic acid (300 mg/kg), cinnamic acid (200 mg/kg) and glycyrrhizic acid (50 mg/kg); NF-κB, nuclear factor-κB.

Figure 6

Effect of FCG pretreatment on cytoplasmic I-κBα and phospho-I-κBα expression in different groups. Data are presented as the mean ± standard deviation; n=6. ^*P<0.05 vs. I/R; ^##P<0.001 and ^#P<0.05 vs. sham. Sham, sham-operated animal without ligation; I/R, 30 min ischemia followed by 2 h reperfusion; PPC+I/R, administration of FCG for 5 days prior to the induction of myocardial ischemia. FCG, ferulic acid (300 mg/kg), cinnamic acid (200 mg/kg) and glycyrrhizic acid (50 mg/kg); I-κBα, inhibitory-κBα; phospho-, phosphorylated-.

Figure 7

Effect of FCG pretreatment on PPARα protein expression in different groups. Data are presented as the mean ± standard deviation; n=6. ^**P<0.05 vs. I/R; ^#P<0.05 vs. sham. Sham, sham-operated animal without ligation; I/R, 30 min ischemia followed by 2 h reperfusion; PPC+I/R, administration of FCG for 5 days prior to the induction of myocardial ischemia. FCG, ferulic acid (300 mg/kg), cinnamic acid (200 mg/kg) and glycyrrhizic acid (50 mg/kg); PPARα, peroxisome proliferator-activated receptor α.

Figure 8

Effect of FCG pretreatment on phospho-Akt protein expression in different groups. Data are presented as the mean ± standard deviation; n=6. ^**P<0.05 vs. I/R. Sham, sham-operated animal without ligation; I/R, 30 min ischemia followed by 2 h reperfusion; PPC+I/R, administration of FCG for 5 days prior to the induction of myocardial ischemia. FCG, ferulic acid (300 mg/kg), cinnamic acid (200 mg/kg) and glycyrrhizic acid (50 mg/kg); phospho-, phosphorylated-.