Methanol Extract of Cassia mimosoides var. nomame Attenuates Myocardial Injury by Inhibition of Apoptosis in a Rat Model of Ischemia-Reperfusion

Abstract

Interruption of blood flow through coronary arteries and its subsequent restoration triggers the generation of a burst of reactive oxygen species (ROS), leading to myocardial cell death. In this study, we determined whether a methanol extract of Cassia mimosoides var. nomame Makino could prevent myocardial ischemia-reperfusion injury. When radical scavenging activity of the extract was measured in vitro using its α,α-diphenyl-β-picrylhydrazyl (DPPH) radical quenching ability, the extract showed an activity slightly lower than that of ascorbic acid. Three days after oral administration of the extract (400 mg/kg/day) to rats, myocardial ischemia/reperfusion injury was generated by 30 min of ligation of the left anterior descending coronary artery (LAD), followed by 3 hr reperfusion. Compared with the vehicle-treated group, administration of the extract significantly reduced infarct size (IS) (ratio of infarct area to area at risk) in the extract-treated group by 28.3%. Reduction in the cellular injury was mediated by attenuation of Bax/Bcl-2 ratio by 33.3%, inhibition of caspase-3 activation from procas-pase-3 by 40%, and subsequent reduction in the number of apoptotic cells by 66.3%. These results suggest that the extract attenuates myocardial injury in a rat model of ischemia-reperfusion by scavenging ROS, including free radicals, and consequently blocking apoptotic cascades. Therefore, intake of Cassia mimosoides var. nomame Makino might be beneficial for preventing ischemic myocardial injury.

Keywords: Cassia mimosoides var. nomame Makino; apoptosis; heart; ischemia-reperfusion; methanol extract; reactive oxygen species.

Fig. 1

Assessment of DPPH radical scavenging activity of methanol extract. Absorbance at 540 nm was measured after various concentrations of the extract and ascorbic acid were reacted with DPPH. Electron donating ability (EDA) of the methanol extract of Cassia mimosoides var. nomame Makino and ascorbic acid was calculated using the following equation: EDA (%)=(1–extract or ascorbic acid absorbance/blank absorbance)×100 in which extract, ascorbic acid, and blank absorbance represent absorbance of solutions containing the extract, ascorbic acid, and nothing (negative control), respectively.

Fig. 2

Effects of the extract on infarct size in rats. Following ischemia-reperfusion, Evans blue was injected through jugular vein, from which area at risk (AAR) was defined as the area not infiltrated with Evans blue. Then, the myocardium was excised into 4 pieces about 3 mm thick, and the slices were stained with TTC, from which infarct area (IA) was defined as the area not stained with TTC. Infarct size (IS) [IA (% AAR)] was expressed as a percentage of IA to AAR, and the area at risk [AAR (% LV)] was expressed as a percentage of AAR to the left ventricle mass. The numbers of rats used in vehicle-, and extract-treated groups were 5 and 6, respectively. Each column represents the means±SE. ^*p<0.05 vs. vehicle-treated control group.

Fig. 3

Effects of the extract on apoptosis. A. Photomicrographs of myocardial tissue sections showing TUNEL staining (400 ×): (a),(c) vehicle-treated control group; (b),(d) extract-treated group; (a),(b) and (c),(d) were taken from border zone (BZ) and infarct area (IA), respectively. B. Quantitative analysis of TUNEL-positive cells. The ratio (apoptotic cell number/total cell number) in AAR was assessed by TUNEL staining in two separate regions: IA and BZ where AAR represents the sum of IA and BZ. The numbers of rats used in vehicle- and extract-treated groups were 5 and 6, respectively. ^*p<0.05 vs. vehicle-treated control group.

Fig. 4

Effect of the extract on the activation of procaspase-3 to cleaved caspase-3. A. Photomicrographs of myocardial tissue sections showing cleaved caspase-3 expression (200×): (a) vehicle-treated control group; (b) extract-treated group; Photomicrographs were taken from AAR randomly. B. Quantitative analysis of cleaved caspase-3 protein was performed with arbitrary units. The numbers of rats used in vehicle- and extract-treated groups were 5 and 6, respectively. ^*p<0.05 vs. vehicle-treated control group.

Fig. 5

Effect of the extract on the expression of Bcl-2 and Bax. A. Photomicrographs of myocardial tissue sections showing Bcl-2 and Bax expression (200×): (a),(d) sham-operated group; (b),(e) vehicle-treated control group; (c),(f) extract-treated group; Photomicrographs were taken from area at risk (AAR) randomly. (a~c) and (d~f) represent Bcl-2 and Bax, respectively. B. Quantitative assessment of Bcl-2/Bax ratio, with the value for the sham group arbitrarily set at 1.0. The numbers of rats used in sham, vehicle- and extract-treated groups were 3, 5, and 6, respectively. Each column represents the means±SE. ^**p<0.01 vs. vehicle-treated group. ^##p<0.01 vs. sham group.