Nutr Res Pract.  2014 Aug;8(4):391-397.

Apple pectin, a dietary fiber, ameliorates myocardial injury by inhibiting apoptosis in a rat model of ischemia/reperfusion

• Lim SH
• Kim MY
• Lee J

Abstract

BACKGROUND
/OBJECTIVE: Myocardial cell death due to occlusion of the coronary arteries leads to myocardial infarction, a subset of coronary heart disease (CHD). Dietary fiber is known to be associated with a reduced risk of CHD, the underlying mechanisms of which were suggested to delay the onset of occlusion by ameliorating risk factors. In this study, we tested a hypothesis that a beneficial role of dietary fiber could arise from protection of myocardial cells against ischemic injury, manifested after occlusion of the arteries.
MATERIALS/METHODS
Three days after rats were fed apple pectin (AP) (with 10, 40, 100, and 400 mg/kg/day), myocardial ischemic injury was induced by 30 min-ligation of the left anterior descending coronary artery, followed by 3 hr-reperfusion. The area at risk and infarct area were evaluated using Evans blue dye and 2,3,5-triphenyltetrazolium chloride (TTC) staining, respectively. DNA nicks reflecting the extent of myocardial apoptosis were assessed by TUNEL assay. Levels of cleaved caspase-3, Bcl-2, and Bax were assessed by immunohistochemistry.
RESULTS
Supplementation of AP (with 100 and 400 mg/kg/day) resulted in significantly attenuated infarct size (IS) (ratio of infarct area to area at risk) by 21.9 and 22.4%, respectively, in the AP-treated group, compared with that in the control group. This attenuation in IS showed correlation with improvement in biomarkers involved in the apoptotic cascades: reduction of apoptotic cells, inhibition of conversion of procaspase-3 to caspase-3, and increase of Bcl-2/Bax ratio, a determinant of cell fate.
CONCLUSIONS
The findings indicate that supplementation of AP results in amelioration of myocardial infarction by inhibition of apoptosis. Thus, the current study suggests that intake of dietary fiber reduces the risk of CHD, not only by blocking steps leading to occlusion, but also by protecting against ischemic injury caused by occlusion of the arteries.

Keyword

Apple pectin; coronary heart disease; ischemia; apoptosis; dietary fiber

MeSH Terms

Animals
Apoptosis*
Arteries
Biomarkers
Caspase 3
Cell Death
Coronary Disease
Coronary Vessels
Dietary Fiber*
DNA Breaks, Single-Stranded
Evans Blue
Immunohistochemistry
In Situ Nick-End Labeling
Ischemia
Models, Animal*
Myocardial Infarction
Rats
Risk Factors
Caspase 3
Evans Blue

Figure

• 

  Fig. 1 Effect of apple pectin (AP) on infarct size in rats. Rats underwent 30 min-ischemia/3 hr-reperfusion, followed by injection of Evans blue through the jugular vein, from which area at risk (AAR) and non-ischemic region were defined as the area with Evans blue not infiltrated and the area with Evans blue infiltrated, respectively. (A) The myocardium was then excised into four pieces, approximately 3 mm thick, and two pieces (a-b and b-c) were used for quantification of infarct area. (B) The slices were stained with TTC, from which area at risk (AAR), infarct area (IA), border zone (BZ), and non-ischemic (Non-ischemic) heart tissue were defined: (a) Vehicle-treated control group; (b) Extract-treated group. C. Infarct size [IS(IA/AAR)] and risk size [(RS(AAR/LV)) were expressed as a percentage of IA to AAR and of AAR to the left ventricle (LV), respectively. AP (with 10, 40, 100, and 400 mg/kg/day) was fed for three days prior to occlusion. The numbers of rats used in the control and AP (with 10, 40, 100, and 400 mg/kg/day)-treated groups were 5, 8, 6, 5, and 7, respectively. *P < 0.05 vs. control group.

• 

  Fig. 2 Effect of apple pectin (AP) on apoptosis. (A) Photomicrographs of myocardial tissue sections showing TUNEL staining (400×): (a), (d) sham group; (b), (e) control group; (c), (f) AP-treated group (with 100 mg/kg/day); (a)~(c) and (d)~(f) were taken from the border zone (BZ) and infarct area (IA), respectively. (B) Quantitative analysis of TUNEL-positive cells. The ratio of apoptotic cells to the total cells is presented. The numbers of rats used in the sham, control, and AP-treated groups were 5, 5, and 5, respectively. *P < 0.05 vs. control group.

• 

  Fig. 3 Effect of apple pectin (AP) on activation of procaspase-3 to cleaved caspase-3. (A) Photomicrographs of myocardial tissue sections showing cleaved caspase-3 expression (200×): (a), sham group; (b), control group; (c), AP-treated group (with 100 mg/kg/day). Photomicrographs were randomly taken from area at risk (AAR). (B) Quantitative analysis of cleaved caspase-3 protein. Arbitrary units were used. The numbers of rats used in the sham, control, and AP-treated groups were 5, 5 and 5, respectively. *P < 0.05 vs. control group.

• 

  Fig. 4 Effect of apple pectin (AP) on expression of Bcl-2 and Bax. (A) Photomicrographs of myocardial tissue sections showing Bcl-2 and Bax expression (200×): (a), (d) sham group; (b), (e) control group; (c), (f) AP-treated group (with 100 mg/kg/day). Photomicrographs were randomly taken from BZ. (a)~(c) and (d)~(f) represent Bcl-2 and Bax, respectively. (B) Quantitative analysis of Bcl-2 and Bax. Bcl-2/Bax ratio was used. The numbers of rats used in the sham, control, and AP-treated groups were 5, 5, and 5, respectively. *P < 0.05 control vs. sham group, #P < 0.05 AP-treated vs. control group.

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