Alginate oligosaccharide alleviates myocardial reperfusion injury by inhibiting nitrative and oxidative stress and endoplasmic reticulum stress-mediated apoptosis

Figures & data

Figure 1 Schematic representation of the molecular structure of AOS prepared by enzymatic degradation.

Abbreviation: AOS, alginate oligosaccharide.

Figure 2 Effect of AOS pretreatment on myocardial infarct size 24 hours after I/R injury as measured by Evans blue-TTC double staining.

Notes: (A) Representative pictures of TTC staining in the four groups. Red color = AAR; white color = IS. (B–D) Graphs showing the percentage of AAR/LV, IS/AAR, and IS/LV. All data are shown as mean ± SD (n=8–10 independent samples per group). *P<0.05 vs SHAM; ^#P<0.05 vs I/R. A1: AOS (200 mg/kg/d, 7 days); A2: AOS (400 mg/kg/d, 7 days).
Abbreviations: AOS, alginate oligosaccharide; I/R, ischemia/reperfusion; TTC, triphenyltetrazolium chloride; AAR, area at risk; IS, infarct size; LV, left ventricle size.

Figure 3 Effect of AOS pretreatment on cardiac function 24 hours after I/R injury.

Notes: (A) Representative echocardiographic images in the four groups; (B) EF; (C) FS; (D) LVESP; (E) LVEDP; (F) dp/dt_max; (G) dp/dt_min. All data are shown as mean ± SD (n=6 independent animals per group). *P<0.05 vs SHAM; ^#P<0.05 vs I/R. A1: AOS (200 mg/kg/d, 7 days); A2: AOS (400 mg/kg/d, 7 days).
Abbreviations: AOS, alginate oligosaccharide; I/R, ischemia/reperfusion; EF, left ventricular ejection fraction; FS, left ventricular fractional shortening; LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure; dp/dt_max, maximal slope of systolic pressure increment; dp/dt_min, maximal slope of diastolic pressure decrement.

Figure 4 Effect of AOS pretreatment on plasma cTnI level 24 hours after I/R injury as measured by ELISA.

Notes: All data are shown as mean ± SD (n=6 independent samples per group). *P<0.05 vs SHAM; ^#P<0.05 vs I/R. A1: AOS (200 mg/kg/d, 7 days); A2: AOS (400 mg/kg/d, 7 days).
Abbreviations: AOS, alginate oligosaccharide; cTnI, concentration of cardiac troponin-I; I/R, ischemia/reperfusion.

Figure 5 Effect of AOS pretreatment on myocardial apoptosis 3 hours after I/R injury as determined by TUNEL staining.

Notes: (A) Representative photomicrographs of TUNEL staining at 400× magnification in the four groups. Apoptotic cardiomyocyte nuclei appear brown, and normal nuclei appear blue. (B) The graph shows the percentage of TUNEL-positive cells. All data are shown as mean ± SD (n=6 independent samples per group).*P<0.05 vs SHAM; ^#P<0.05 vs I/R. A1: AOS (200 mg/kg/d, 7 days); A2: AOS (400 mg/kg/d, 7 days).
Abbreviations: AOS, alginate oligosaccharide; I/R, ischemia/reperfusion; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 6 Effect of AOS pretreatment on the level of nitrative/oxidative stress 3 hours after I/R injury.

Notes: (A) Representative photomicrographs of 3-nitrotyrosine at 400× magnification detected by immunohistochemistry. (B) Representative Western blot and quantitative analysis of iNOS. (C) Representative images of in situ dihydroethidium staining at 400× magnification acquired by confocal microscopy. (D) Myocardial superoxide generation detected by lucigenin-enhanced chemiluminescence (E) Representative Western blot and quantitative analysis of NOX2. (F) Representative Western blot and quantitative analysis of 4-HNE. All data are shown as mean ± SD (n=6 independent samples per group). *P<0.05 vs SHAM; ^#P<0.05 vs I/R. A1: AOS (200 mg/kg/d, 7 days); A2: AOS (400 mg/kg/d, 7 days).
Abbreviations: AOS, alginate oligosaccharide; I/R, ischemia/reperfusion; iNOS, inducible nitric oxide synthase, NOX2, NADPH oxidase2; 4-HNE, 4-hydroxynonenal.

Figure 7 Effect of AOS pretreatment on the expression of Bax and Bcl-2 3 hours after I/R injury as detected by Western blot analysis.

Notes: (A and B) Quantitative analysis of Bax and Bcl-2 protein expression. (C) Representative Western blots of Bax, Bcl-2, and GAPDH. All data are shown as mean ± SD (n=6 independent samples per group). *P<0.05 vs SHAM; ^#P<0.05 vs I/R. A1: AOS (200 mg/kg/d, 7 days); A2: AOS (400 mg/kg/d, 7 days).
Abbreviations: AOS, alginate oligosaccharide; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2; I/R, ischemia/reperfusion.

Figure 8 Effect of AOS pretreatment on the expression of ER stress-related proteins 3 hours after I/R injury detected by Western blot.

Notes: (A–C) Quantitative analyses of CHOP, GRP78, and caspase-12 protein expression. (D) Representative Western blots of CHOP, GRP78, caspase-12, and GAPDH. All data are shown as mean ± SD (n=6 independent samples per group). *P<0.05 vs SHAM; ^#P<0.05 vs I/R. A1: AOS (200 mg/kg/d, 7 days); A2: AOS (400 mg/kg/d, 7 days).
Abbreviations: AOS, alginate oligosaccharide; ER, endoplasmic reticulum; I/R, ischemia/reperfusion; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78.