Amino Acid Composition, Antioxidant, and Cytoprotective Effect of Blue Mussel (Mytilus edulis) Hydrolysate through the Inhibition of Caspase-3 Activation in Oxidative Stress-Mediated Endothelial Cell Injury

Figures

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  Figure 1. Antioxidant abilities of blue mussel hydrolysates with various proteolytic enzymes determined by (A) DPPH scavenging assay, (B) ORAC assay, (C) ABTS+ radical assay, and (D) TBARS assay. a–d Different letters on the bar charts indicated significant differences (p < 0.05). * p < 0.05 vs. control.

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  Figure 2. Effect of BMCH on oxidative stress-mediated HUVEC injury. (A) Cell viability and (B) the protective effect of BMCH in H2O2-mediated HUVEC injury, as determined by MTT assay, and (C) live-dead cell assay by calcein-AM/PI double staining. Cells were pretreated with BMCH for 30 min followed by exposure of H2O2 (600 μM) and incubation for 24 h. * p < 0.05 and ** p < 0.01 vs. H2O2 only treatment.

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  Figure 3. (A) Determination of intracellular ROS production by DCFH-DA staining under a fluorescence microscope and (B) quantitative intracellular DCF fluorescence intensity in H2O2-treated HUVEC. Cells were pretreated with BMCH for 30 min, followed by exposure of H2O2 (600 μM) and incubation for 24 h. * p < 0.05 and ** p < 0.01 vs. H2O2 only treatment.

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  Figure 4. Quantification of intracellular GSH levels under (A) normal and (B) oxidative stress condition and determination of antioxidant enzyme levels (C) SOD, (D) CAT, and (E) GPx activities. Cells were treated with BMCH for 24 h for measurement of GSH level under normal condition. Cells were pretreated with BMCH for 30 min followed by exposure of H2O2 (600 μM) and incubation for 24 h for measurement of GSH under oxidative stress condition and antioxidant enzyme activities. Differences were considered statistical significance at # p < 0.05 and ## p < 0.01 compared with the control, and * p < 0.05 and ** p < 0.01 compared with the cells treated with H2O2 only treatment.

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  Figure 5. Effects of BMCH on apoptosis in H2O2-treated HUVEC injury. (A) Cellular morphology and (B) nuclear morphology by H33342 staining under a fluorescence microscope. (C) FACS analysis using Annexin V-FITC apoptosis kit and (D) quantification of apoptotic and necrotic cells by FACS analysis. * p < 0.05 and ** p < 0.01 vs. H2O2 only treatment.

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  Figure 6. Effect of BMCH on mRNA expressions by RT-qPCR. (A) p53, (B) bax/bcl-2 ratio, and (C) caspase-3. Cells were pretreated with BMCH for 30 min followed by exposure of H2O2 (600 μM) and incubation for 24 h. * p < 0.05 and ** p < 0.01 vs. H2O2 only treatment.

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  Figure 7. (A) Representative western blotting of procaspase-3 and cleaved caspase-3, and (B) the relative expression level of cleaved caspase-3 protein level in H2O2-mediated HUVEC injury. Cells were pretreated with BMCH for 30 min followed by exposure of H2O2 (600 μM) and incubation for 24 h. * p < 0.05 and ** p < 0.01 vs. H2O2 only treatment.