Apigenin-7-O-β-d-(6″-p-coumaroyl)-glucopyranoside reduces myocardial ischaemia/reperfusion injury in an experimental model via regulating the inflammation response

Figures

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  Figure 1.. Chemical structure of apigenin-7-O-β-d-(6″-p-coumaroyl)-glucopyranoside.

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  Figure 2.. Area at risk and infarct size in different groups. (A) The representative photomicrographs of heart sections stained with Evans blue and TTC for different treatment groups are shown above the bar graph. (B) Treatment with APG (50 and 100 mg/kg) markedly reduced the infarct size caused by MI/RI. (C) AAR/LV was similar among groups. Data are expressed as mean ± S.D. (n = 6). Significance was determined by ANOVA followed by Tukey’s test. **p < 0.01 vs. MI/RI + vehicle. AAR: area at risk; LV: left ventricle; IS: infarct size.

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  Figure 3.. Representative light microscopic images of rat myocardial histopathological morphology (H&E, ×400). (A) Sham-operated group, the myocardial cell membrane showed clear integrity and no inflammatory cell infiltration. MI/RI + vehicle group, the myocardial tissue showed widespread cell structure disorder, large amount of infiltrated neutrophil granulocytes, interstitial oedema and myofibrillar fracture. MI/RI + APG (25 mg/kg) group, rats treated with APG (25 mg/kg) showed similar injury extended as vehicle group. MI/RI + APG (50 mg/kg) group, rats treated with APG (50 mg/kg) showed mild degree of necrosis and less infiltration of inflammatory cells. MI/RI + APG (100 mg/kg) group, rats treated with APG (100 mg/kg) showed nearly normal myofibrillar structure with few neutrophil granulocytes’ infiltration. (B) Effects of APG on myocardial histopathological scores. Values are expressed as the mean ± S.D. (n = 6). Significance was determined by ANOVA followed by Tukey’s test. *p < 0.05 vs. MI/RI + vehicle group.

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  Figure 4.. Effects of APG on cell viability, LDH release, cell morphology and apoptosis in neonatal rat cardiomyocytes subjected to SI/RI-induced injury. (A) Effects of APG on the viability of cardiomyocytes subjected to SI/RI-induced injury, measured by MTT assay. (B) Effects of APG on the release of LDH in the culture medium, measured by an ELISA kit. (C) Cells were subjected to SI/RI with or without different concentrations of APG. Cells were observed under an inverted phase contrast microscope. Treatment with APG resulted in less shrinkage compared with the SI/RI group. (D) Representative images of TUNEL staining were obtained. Significance was determined by ANOVA followed by Tukey’s test. Values represent as mean ± S.D. (n = 3). #p < 0.05 vs. control group; *p < 0.05 vs. SI/RI group.

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  Figure 5.. Effects of APG on translocation of NF-κB p65 and expression levels of phospho-IκB-α and ICAM-1 in MI/RI rats. (A) Representative blots of NF-κB p65 expression in cytosolic and nuclear fractions, and expression levels of phospho-IκB-α and ICAM-1 in rat myocardium. Nuclear extracts (NE) of NF-κB were normalized to PCNA (nuclear marker). Cytosolic extracts (CE) of NF-κB, phospho-IκB-α and ICAM-1 were normalized to β-actin (cytoplasmic marker). (B) Expression levels of NF-κB (NE), NF-κB (CE), phospho-IκB-α and ICAM-1 are shown as fold-change in sham-operated group. Significance was determined by ANOVA followed by Tukey’s test. Data are presented as mean ± S.D. (n = 3). #p < 0.05 vs. Sham group, *p < 0.05 vs. MI/RI + vehicle group.

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  Figure 6.. Effects of APG on NF-κB p65 translocation and expression level of phospho-IκB-α in SI/RI-stimulated rat cardiomyocytes. (A) Representative western blots of NF-κB p65 expression in cytosolic and nuclear fractions, and expression level of phospho-IκB-α in neonatal rat cardiomyocytes. Nuclear extracts (NE) of NF-κB were normalized to PCNA (nuclear marker). Cytosolic extracts (CE) of NF-κB, phospho-IκB-α and ICAM-1 were normalized to β-actin (cytoplasmic marker). (B) Expression levels NF-κB (NE), NF-κB (CE) and phospho-IκB-α are shown as fold change in the control group. Significance was determined by ANOVA followed by Tukey’s test. Data are presented as mean ± S.D. (n = 3). #p < 0.05 vs. control group, *p < 0.05 vs. SI/RI group.

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  Figure 7.. Effects of suppression or overexpression of NF-κB on the protective role of APG. (A) Representative western blots showed the expression levels of NF-κB (CE) and NF-κB (NE) when the cells were transfected with siNF-κB and pcDNA3.1(+)-NF-κB. (B) Expression levels of NF-κB (NE) and NF-κB (CE) are shown as fold change in the control group. (C) The release of LDH in the culture medium was measured by ELISA method when the cells were transfected. (D) Representative images of TUNEL staining were obtained when the cells were transfected. Significance was determined by ANOVA followed by Tukey’s test. Data are presented as mean ± S.D. (n = 3). *p < 0.05 vs. NF-κB group.