Indobufen alleviates apoptosis by the PI3K/Akt/eNOS pathway in myocardial ischemia‒reperfusion (I/R) injury

Figures

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  Fig. 1. The chemical structure of Indobufen.

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  Fig. 2. Indobufen alleviated myocardial ischemia injury in I/R rats. (A)TTC staining of representative sections, the white-colored areas represent the infarct areas and the red-colored areas represent non-infarcted areas. (B) Quantification of rat heart infarct size in different groups. Lane1: I/R group, lane2: clopidogrel (7.5 mg/kg) group, lane3: indobufen (20 mg/kg) group, lane4: indobufen (10 mg/kg) group, lane5: aspirin (10 mg/kg), lane6: aspirin (5 mg/kg) group. (C) Quantification of pathological indicators in H&E staining: hyperemia, hemorrhage, necrosis and degeneration. The detailed scoring criterion of pathological indicators was mentioned above. Lane1: sham group, lane2: I/R group, lane3: clopidogrel (7.5 mg/kg) group, lane4: indobufen (20 mg/kg) group, lane5: indobufen (10 mg/kg) group, lane6: aspirin (10 mg/kg), lane7: aspirin (5 mg/kg) group. (D) Histological analysis showed cardioprotection of Indubufen in I/R hearts (400×). Scale bar is 25 μm. (E) The arrow points to the observation site. All data are shown as mean ± S.E.M, n = 7-8. *P < 0.05, **P < 0.01 vs. I/R group; #P < 0.05, ##P < 0.01 vs. Sham.

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  Fig. 3. Indobufen improved left ventricular function in I/R rats. (A) Representative images of each group of echocardiograms. (B) Comparison of EF (%) in each group. (C) comparison of FS (%) in each group. All data are shown as mean ± S.E.M, n = 5. (B, C) Lane1: sham group, lane2: I/R group, lane3: clopidogrel (7.5 mg/kg) group, lane4: indobufen (20 mg/kg) group, lane5: indobufen (10 mg/kg) group, lane6: aspirin (10 mg/kg), lane7: aspirin (5 mg/kg) group. *P < 0.05, **P < 0.01 vs. I/R group; #P < 0.05, ##P < 0.01 vs. Sham.

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  Fig. 4. Indobufen alleviated oxidative stress injury in I/R rats. (A) Indobufen suppressed the up-regulation of CK level of I/R group. (B) Indobufen decreased LDH activity of I/R group. (C) Indobufen reduced MDA level of I/R group. (D) Indobufen up-regulated SOD level of I/R group. (A–D) Lane1: sham group, lane2: I/R group, lane3: clopidogrel (7.5 mg/kg) group, lane4: indobufen (20 mg/kg) group, lane5: indobufen (10 mg/kg) group, lane6: aspirin (10 mg/kg), lane7: aspirin (5 mg/kg) group. Data are shown as mean ± S.E.M, n = 8. *P < 0.05, **P < 0.01 vs. I/R group; #P < 0.05, ##P < 0.01 vs. Sham.

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  Fig. 5. Indobufen significantly inhibited cardiomyocyte apoptosis. (A) Suppression of cardiomyocyte apoptosis by Indobufen pretreatment (×400). Scale bar is 25 μm. Brown staining of the nucleus is indicative of apoptosis. The arrow points to the observation site (B) Percentage of apoptotic cells, n = 5. (C) Caspase-3 protein level was determined using Western blot analysis. (D) Quantification of Caspase-3 protein expression, n = 3. (E) Bax and BCL-2 protein levels were determined using Western blot analysis. (F) Quantification of Bax and BCL-2 proteins expression, n = 3. Each column represents the mean ± S.E.M. (B, D, F) Lane1: sham group, lane2: I/R group, lane3: clopidogrel (7.5 mg/kg) group, lane4: indobufen (20 mg/kg) group, lane5: indobufen (10 mg/kg) group, lane6: aspirin (10 mg/kg), lane7: aspirin (5 mg/kg) group. *P < 0.05, **P < 0.01 vs. I/R group; #P < 0.05, ##P < 0.01 vs. Sham. Original blots/gels are presented in Supplementary.

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  Fig. 6. Indobufen significantly elevated plasma levels of NO and eNOS and cardiac mRNA levels of eNOS, and down-regulated plasma ROS levels. (A) Indobufen elevated the level of eNOS protein in plasma (B) Indobufen up-regulated the level of eNOS mRNA in heart. (C) Indobufen increased NO production in rat plasma. (D) Indobufen reduced ROS generation in rat plasma. Lane1: sham group, lane2: I/R group, lane3: clopidogrel (7.5 mg/kg) group, lane4: indobufen (20 mg/kg) group, lane5: indobufen (10 mg/kg) group, lane6: aspirin (10 mg/kg), lane7: aspirin (5 mg/kg) group. All data are shown as mean ± S.E.M, n = 5–8. *P < 0.05, **P < 0.01 vs. I/R group; #P < 0.05, ##P < 0.01 vs. Sham.

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  Fig. 7. Effects of Indobufen on the expression of p-Akt and p-eNOS in cardiac tissue. Representative photomicrographs of immunohistochemical staining showed the effects of Indobufen on the expression of (A) p-Akt and (B) p-eNOS immunoreactivity in heart sections. All the photographs were taken at 400×. Scale bar = 25 μm. (C) The positive staining of p-Akt (%area). (D) The positive staining of p-eNOS (% area). (E) p-Akt/Akt, p-eNOS/eNOS protein levels were determined using western blot analysis. (F) Quantification of p-Akt/Akt proteins expression. (G) Quantification of p-eNOS/eNOS proteins expression. Lane1: sham group, lane2: I/R group, lane3: clopidogrel (7.5 mg/kg) group, lane4: indobufen (20 mg/kg) group, lane5: indobufen (10 mg/kg) group, lane6: aspirin (10 mg/kg), lane7: aspirin (5 mg/kg) group. Each column represents the mean ± S.E.M, n = 5. *P < 0.05, **P < 0.01 vs. I/R group; #P < 0.05, ##P < 0.01 vs. Sham. Original blots/gels are presented in Supplementary.

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  Fig. 8. Indobufen alleviated OGD/R-induced cytotoxicity in cultured H9c2 cells. (A) Microscope images showing representative cell morphology of H9c2 cells after pretreatment for 24 h and exposed to OGD/R (×200 magnification, scale bar = 100 μm). Drug concentration determination: (B) Indobufen (C) Aspirin. ((D) Cell viability. Lane1: control group, lane2: OGD/R group, lane3: aspirin (50 µM) group, lane4: indobufen (250 µM) group, lane5: indobufen (125 µM) group, lane6: indobufen (250 µM) + LY294002 (20 µM). Data are shown as mean ± S.E.M, n = 5. *P < 0.05, **P < 0.01 vs. OGD/R; ##P < 0.01 vs. Control.

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  Fig. 9. Indobufen alleviated oxidative stress injury after OGD/R injury in H9c2 cells. (A) Indobufen suppressed the up-regulation of CK level of OGD/R group. (B) Indobufen decreased LDH activity of MI/R group. (C) Indobufen reduced MDA level of OGD/R group. (D) Indobufen up-regulated SOD level of OGD/R group. Data are shown as mean ± S.E.M, n = 8. Lane1: control group, lane2: OGD/R group, lane3: aspirin (50 µM) group, lane4: indobufen (250 µM) group, lane5: indobufen (125 µM) group, lane6: indobufen (250 µM) + LY294002 (20 µM). *P < 0.05, **P < 0.01 vs. OGD/R; #P < 0.05, ##P < 0.01 vs. Control.

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  Fig. 10. Indobufen significantly inhibited H9c2 cells apoptosis. (A) Suppression of cardiomyocyte apoptosis by Indobufen pretreatment (×20). Scale bar is 100 μm. green staining of the nucleus is indicative of apoptosis, blue staining of the nucleus is indicative of nucleus. (B) Percentage of apoptotic cells. (C) Caspase-3 protein level was determined using Western blot analysis. (D) Quantification of Caspase-3 protein expression. (E) Bax and BCL-2 protein levels were determined using Western blot analysis. (F) Quantification of Bax and BCL-2 proteins expression. All data are shown as mean ± S.E.M, n = 3. (B, D, F) Lane1: control group, lane2: OGD/R group, lane3: aspirin (50 µM) group, lane4: indobufen (250 µM) group, lane5: indobufen (125 µM) group, lane6: indobufen (250 µM) + LY294002 (20 µM). *P < 0.05, **P < 0.01 vs. OGD/R; #P < 0.05, ##P < 0.01 vs. Control. Original blots/gels are presented in Supplementary.

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  Fig. 11. Effects of Indobufen on the expression of P-Akt and P-eNOS in H9c2 cells. Representative photomicrographs of immune-fluorescence staining showed the effects of Indobufen on the expression of (A) P-Akt and (C) P-eNOS immunoreactivity in H9c2 cells. All the photographs were taken at 20×. Scale bar = 100 μm. (B) The positive cells of P-Akt ratio (%). (D) The positive cells of P-eNOS ratio (%). (E) P-Akt/Akt, P-eNOS/eNOS protein levels were determined using Western blot analysis. (F) Quantification of P-Akt/Akt proteins expression. (G) Quantification of P-eNOS/eNOS proteins expression. Each column represents the mean ± S.E.M, n = 3. (B, D–G) Lane1: control group, lane2: OGD/R group, lane3: aspirin (50 µM) group, lane4: indobufen (250 µM) group, lane5: indobufen (125 µM) group, lane6: indobufen (250 µM) + LY294002 (20 µM). *P < 0.05, **P < 0.01 vs. OGD/R; #P < 0.05, ##P < 0.01 vs. Control. Original blots/gels are presented in Supplementary.

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  Fig. 12. Schematic diagram of Indobufen against I/R injury.