Fucoxanthin protects neonatal rat cardiomyocytes and attenuates high glucose-mediated oxidative stress via the AMPK pathway

Figures

• 

  Figure 1. Fucoxanthin increases the cell viability in Iry neonatal rat cardiomyocytes treated with high glucose. A – The cell viability decreased significantly when the cells were exposed to high glucose environment (30 mM/l). B – The cell viability decreased significantly when the incubation time increased to 24 h in 30 mM/l glucose, 30 mM/l mannitol was adopted as an osmotic control. C – Addition of fucoxanthin (40 and 80 µM/l) increased the cell viability in high glucose treated cardiomyocytes. D – Fucoxanthin (40 and 80 µM/l) decreased the release of LDH in the incubating medium of cardiomyocytes exposed to high glucose. *P < 0.05 compared to control, #p < 0.05 compared to high glucose treated rats (n = 10)

• 

  Figure 2. Fucoxanthin halted the generation of reactive oxygen species in Iry cardiomyocytes treated with high glucose. DHE and fluorescent probe were used for measuring intracellular levels of reactive oxygen species in cardiomyocytes. A – Cardiomyocytes staining with DHE showing representative fluorescence images (400×, scale bar: 20 µm). B – Intensity of red fluorescence (quantitative analysis). C – HPLC analysis for dihydroethidium fluorescence, fucoxanthin 40 µM. *P < 0.05 compared to control, #p < 0.05 compared to high glucose treated group (n = 10)

• 

  Figure 3. Fucoxanthin blocked NADPH oxidase subunits in Iry neonatal rat cardiomyocytes treated with high glucose. A – Fucoxanthin decreased the activity of Rac1 in high glucose treated cardiomyocytes. B – Fucoxanthin suppressed the levels of NADPH oxidase subunits gp91phox, p47phox, p67phox, whereas the levels of p22phox remained unaltered in cardiomyocytes treated with high glucose. Fucoxanthin 40 µM. C – Quantitative results showing expression of GTP-Rac1 against total Rac1. D – Quantitative results showing expression of NADPH oxidase subunits gp91phox, p47phox and p67phox and p22phox. *P < 0.05 compared to control, #p < 0.05 compared to high glucose media treated group (n = 6)

• 

  Figure 4. Fucoxanthin suppressed the levels of MDA, elevated the activity of SOD, CAT, and GSH-px, and maintained the content of GSH in Iry neonatal rat cardiomyocytes treated with high glucose media. A – Biochemical estimation of markers of oxidative stress, MDA, B – SOD, C – catalase, D – GSH-px respectively. E – The content of GSH was measured by biochemical analysis. Fucoxanthin: 40 µM. *P < 0.05 compared to control, #p < 0.05 compared to high glucose media treated group (n = 10)

• 

  Figure 5. Fucoxanthin blocked high glucose-mediated apoptosis in Iry neonatal rat cardiomyocytes treated with high glucose. A – TUNEL staining of apoptosis induced cardiomyocytes (400×, scale bar: 20 µm). B – Apoptosis rate % TUNEL staining of cardiomyocytes. C – PI and Annexin V staining for apoptotic cells by flow cytometer. Fucoxanthin 40 µM. *P < 0.05 compared to control, #p < 0.05 compared to high glucose media treated group (n = 6)

• 

  Figure 6. Fucoxanthin modulated the expression levels of Bax and Bcl-2 in Iry neonatal rat cardiomyocytes treated with high glucose. A – Immunohistochemical staining of Bcl-2 and Bax in cardiomyocytes in control, high glucose and FXN treated cells (400×, scale bar: 20 µm). B – Immunostaining of Bax and Bcl-2 in control, high glucose and FXN treated cells. C – Western blot analysis for expression of Bcl-2 and Bax in cardiomyocytes. Fucoxanthin 40 µM. *P < 0.05 compared to control, #p < 0.05 compared to high glucose media treated group (n = 6)

• 

  Figure 7. Effect of fucoxanthin on expression levels of AMPK and Akt/GSK-3β in Iry neonatal rat cardiomyocytes treated with high glucose. A – Expression of p-Akt and GSK-3β proteins by western blot analysis. B – p-AMPK analysis by western blot study. DDH (10 µM) could abolish the elevated levels of pAMPK levels by fucoxanthin in cardiac cells treated with high glucose. Fucoxanthin 40 µM. *P < 0.05 compared to control, #p < 0.05 compared to high glucose media treated group, &p < 0.05 compared to high glucose + FXN treated group of cells (n = 6)

• 

  Figure 8. Blockade of AMPK abolished the effect of fucoxanthin on the activity of NADPH oxidase and expression its subunit in Iry neonatal rat cardiomyocytes treated with high glucose. A – Fucoxanthin suppressed the activity of Rac1, which was blocked by DDH. B – Fucoxanthin along with DDH reversed the expression levels of NADPH oxidase subunits p67phox, p47phox, and gp91phox. C – DDH reversed the inhibition of NADPH activity mediated by fucoxanthin. Fucoxanthin 40 µM. *P < 0.05 compared to control, #p < 0.05 compared to high glucose media treated group, &p< 0.05 compared to high glucose + FXN treated group of cells (n = 6)

• 

  Figure 9. Inhibition of AMPK aggravated apoptosis in Iry neonatal rat cardiomyocytes treated with high glucose. A – DDH blocked the effect of fucoxanthin on expression of Bcl-2 and Bax. B – Image showing apoptosis in myocytes by TUNEL staining. Quantitative analysis of cardiomyocytes subjected to TUNEL staining. Fucoxanthin 40 µM. *P < 0.05 compared to control, #p < 0.05 compared to high glucose media treated group, &p < 0.05 compared to high glucose + FXN treated group of cells (n = 6)