Nutr Res Pract.  2019 Jun;13(3):205-213. 10.4162/nrp.2019.13.3.205.

Supplementation with psyllium seed husk reduces myocardial damage in a rat model of ischemia/reperfusion

• Lim SH
• Lee J

• KMID: 2453284
• DOI: http://doi.org/10.4162/nrp.2019.13.3.205

Abstract

BACKGROUND/OBJECTIVES
Myocardial infarction (MI) is caused by extensive myocardial damage attributed to the occlusion of coronary arteries. Our previous study in a rat model of ischemia/reperfusion (I/R) demonstrated that administration of arabinoxylan (AX), comprising arabinose and xylose, protects against myocardial injury. In this study, we undertook to investigate whether psyllium seed husk (PSH), a safe dietary fiber containing a high level of AX (> 50%), also imparts protection against myocardial injury in the same rat model.
MATERIALS/METHODS
Rats were fed diets supplemented with PSH (1, 10, or 100 mg/kg/d) for 3 d. The rats were then subjected to 30 min ischemia through ligation of the left anterior descending coronary artery, followed by 3 h reperfusion through release of the ligation. The hearts were harvested and cut into four slices. To assess infarct size (IS), an index representing heart damage, the slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC). To elucidate underlying mechanisms, Western blotting was performed for the slices.
RESULTS
Supplementation with 10 or 100 mg/kg/d of PSH significantly reduces the IS. PSH supplementation (100 mg/kg/d) tends to reduce caspase-3 generation and increase BCL-2/BAX ratio. PSH supplementation also upregulates the expression of nuclear factor erythroid 2-related factor 2 (NRF2), and its target genes including antioxidant enzymes such as glutathione S-transferase mu 2 (GSTM2) and superoxide dismutase 2 (SOD2). PSH supplementation upregulates some sirtuins (NAD+-dependent deacetylases) including SIRT5 (a mitochondrial sirtuin) and SIRT6 and SIRT7 (nuclear sirtuins). Finally, PSH supplementation upregulates the expression of protein kinase A (PKA), and increases phosphorylated cAMP response element-binding protein (CREB) (pCREB), a target protein of PKA.
CONCLUSIONS
The results from this study indicate that PSH consumption reduces myocardial I/R injury in rats by inhibiting the apoptotic cascades through modulation of gene expression of several genes located upstream of apoptosis. Therefore, we believe that PSH can be developed as a functional food that would be beneficial in the prevention of MI.

Keyword

Infarction; apoptosis; sirtuin; functional food

MeSH Terms

Animals
Apoptosis
Arabinose
Blotting, Western
Caspase 3
Coronary Vessels
Cyclic AMP Response Element-Binding Protein
Cyclic AMP-Dependent Protein Kinases
Diet
Dietary Fiber
Functional Food
Gene Expression
Glutathione Transferase
Heart
Infarction
Ischemia
Ligation
Models, Animal*
Myocardial Infarction
Psyllium*
Rats*
Reperfusion
Sirtuins
Superoxide Dismutase
Xylose
Arabinose
Caspase 3
Cyclic AMP Response Element-Binding Protein
Cyclic AMP-Dependent Protein Kinases
Glutathione Transferase
Psyllium
Sirtuins
Superoxide Dismutase
Xylose

Figure

• 

  Fig. 1 Summary of development of myocardial infarction (Modified from [4]). In the preocclusion steps, LDL enters the intima due to endothelial dysfunction. LDL absorbed is oxidized to oxLDL, and oxLDL is engulfed by the macrophages. The macrophages are then transformed to foam cells which subsequently proliferate, resulting in the formation of atherosclerotic plaques and narrowing of the arteries. In the post-occlusion steps, abrupt rupture of the plaques leads to clot formation in the lesion. This event can occlude the artery and subsequently result in myocardial ischemia. As a result, ATP generation is greatly reduced due to interruption of oxidative phosphorylation, and myocardial cells die through apoptosis and necrosis. As regions of cell death become extensive, myocardial infarction ensues. PSH, psyllium seed husk; LDL, low-density lipoprotein; oxLDL, oxidized LDL.

• 

  Fig. 2 Effect of PSH supplementation on infarct size. Rats underwent 30 min ischemia through ligation of LAD, followed by 3 h reperfusion through release of the ligation. (A) Evans blue dye was infused into the heart after LAD was re-ligated. The heart was harvested and cut into four slices. The slices were stained with TTC. AAR, IA, and BZA were determined as the area without infiltration of Evans blue dye, the area without TTC stain, and the area equivalent to (AAR-IA), respectively. (B) IS, the ratio of IA to AAR, and RS, the ratio of AAR to LVA, are presented. In the PSH-treated groups, PSH (1, 10, or 100 mg/kg/d) supplements were fed for 3 days prior to LAD ligation. In the control group, no PSH was administered prior to LAD ligation. The number of rats used in the control and PSH-treated (1, 10, or 100 mg/kg/d) groups were 6 per group. Values are expressed as means ± SEM. *P < 0.05, when compared to control group. PSH, psyllium seed husk; LAD, left anterior descending coronary artery; TTC, 2,3,5-triphenyltetrazolium chloride; AAR, area at risk; IA, infarct area; BZA, border zone area; IS, infarct size; RS, risk size; LVA; left ventricular area.

• 

  Fig. 3 Effect of PSH supplementation on the formation of caspase-3 (CASP3) and expression of BCL-2 and BAX. (A) Western blots of CASP3 (cleaved caspase-3 generated from procaspase-3), BCL-2, and BAX in the AAR. Protein levels were measured by Western blotting for the sham, control, and PSH-treated (100 mg/kg/d) groups. ERK1 was used as the loading control. (B) Quantitative analysis of CASP3 (cleaved caspase-3), BCL-2, and BAX. C-CASP3 (CASP3)/ERK1 and BCL-2/BAX ratios are presented. The ratios were calculated by setting the control group value (0 mg/kg/d of PSH) at 1. The number of rats used in the sham, control and PSH-treated (100 mg/kg/d) groups were 6 per group. Values are expressed as means ± SEM. CASP3, caspase-3; C-CASP3, cleaved caspase-3; PSH, psyllium seed husk; AAR, area at risk.

• 

  Fig. 4 Effect of PSH supplementation on the expression of NRF2, GSTM2, and SOD2. Western blots of NRF2, GSTM2, and SOD2 in the AAR are presented. (A) Protein levels were measured by Western blotting for the sham, control, and PSH-treated (100 mg/kg/d) groups. ERK1 was used as a loading control. (B) Quantitative analysis of NRF2, GSTM2, and SOD2: NRF2/ERK1, GSTM2/ERK1, and SOD2/ERK1 ratios are presented. The ratios were calculated by setting the control group value (0 mg/kg/d of PSH) at 1. The number of rats used in the sham, control and PSH-treated (100 mg/kg/d) groups were 6 per group. Values are expressed as means ± SEM. ***P < 0.001, and *P < 0.05 vs. control group. PSH, psyllium seed husk; NRF2, nuclear factor erythroid 2-related factor 2; GSTM2, glutathione S-transferase mu 2; SOD2, superoxide dismutase 2; AAR, area at risk.

• 

  Fig. 5 Effect of PSH supplementation on the expression of SIRT (1–7). Western blots of SIRT (1–7) in the AAR are presented. (A) Protein levels were measured by Western blotting for the sham, control, and PSH-treated (100 mg/kg/d) groups. ERK1 was used as the loading control. (B) Quantitative analysis of SIRT (1–7): SIRT (1–7)/ERK1 ratios are presented. The ratios were calculated by setting the control group value (0 mg/kg/d of PSH) at 1. The number of rats used in the sham, control and PSH-treated (100 mg/kg/d) groups were 6 per group. Values are expressed as means ± SEM. **P < 0.01 and *P < 0.05 vs. control group. PSH, psyllium seed husk; SIRT, sirtuins; AAR, area at risk.

• 

  Fig. 6 Effect of PSH supplementation on the expression of PKAβ, pCREB, and CREB. Western blots of PKAβ, pCREB, and CREB in the AAR are presented. (A) Protein levels were measured by Western blotting for the sham, control, and PSH-treated (100 mg/kg/d) groups. ERK1 was used as the loading control. (B) Quantitative analysis of PKAβ/ERK1, pCREB/ERK1, and CREB/ERK1 ratios are presented. The ratios were calculated by setting the control group value (0 mg/kg/d of PSH) at 1. The number of rats used in the sham, control and PSH-treated (100 mg/kg/d) groups were 6 per group. Values are expressed as means ± SEM. **P < 0.01 and *P < 0.05 vs. control group. PSH, psyllium seed husk; PKAβ, protein kinase Aβ; pCREB, phosphorylated cAMP response element-binding protein; AAR, area at risk.

• 

  Fig. 7 A proposed, underlying mechanism for the myocardial protection through PSH supplementation. PSH supplementation protects against myocardial I/R injury through upregulation of PKA expression, CREB phosphorylation, sirtuin expression, NRF2 expression, expression of antioxidant enzymes such as GSTM2 and SOD2, and subsequent reduction of ROS toxicity, followed by reduction of apoptosis through increase of BCL-2/BAX ratio and subsequent reduction of CASP3 generation. PKA, protein kinase A; pCREB, phosphorylated cAMP response element-binding protein; SIRT5, 6, 7, sirtuins 5, 6, 7; NRF2, nuclear factor erythroid 2-related factor 2; GSTM2, glutathione S-transferase mu 2; SOD2, superoxide dismutase 2; CASP3, caspase-3; MI, myocardial infarction; PSH, psyllium seed husk; I/R, ischemia/reperfusion; ROS, reactive oxygen species.

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