In Vitro Vascular-Protective Effects of a Tilapia By-Product Oligopeptide on Angiotensin II-Induced Hypertensive Endothelial Injury in HUVEC by Nrf2/NF-κB Pathways

Figures

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  Figure 1. (a) Structure of Leu-Ser-Gly-Tyr-Gly-Pro (LSGYGP). Effect of LSGYGP on the viability of human umbilical vein endothelial cells (HUVEC). (b) and protective effect of LSGYGP on the viability of Ang II-treated HUVEC. (c) Cells were exposed to varying concentrations (10, 20, 50, and 100 μM) of LSGYGP and the cell viability was assessed by MTT assay. # p < 0.001, compared with blank group (untreated cells); * p < 0.05, ** p < 0.01, compared with control group (Ang II-treated cells).

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  Figure 2. (a) Representative 3-amino,4-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM DA) fluorescent images and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent images of LSGYGP in HUVEC. Mean optical density analysis of cellular DAF-FM DA staining (green fluorescence; an indicator of production of NO) and mean optical density analysis of cellular DCFH-DA staining (green fluorescence; an indicator of production of ROS). (b) Mean optical density analysis of fluorescent images. # p < 0.001, compared with blank group (untreated cells); *** p < 0.001, compared with control group (Ang II-treated cells).

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  Figure 3. Effects of LSGYGP on the expression of superoxide dismutase (SOD), gamma-glutamyltransferase (GGT), glutathione (GSH), and nuclear erythroid 2-related factor 2 (Nrf2) pathway in HUVEC (a,b). Protein expression was by western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was as control. # p < 0.001, compared with blank group (untreated cells); * p < 0.05, *** p < 0.001, compared with control group (Ang II-treated cells).

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  Figure 4. (a) HUVEC were exposed to different concentrations (10, 50, and 100 μM) of LSGYGP with 1 μM Ang II to stimulated mitogen-activated protein kinases (MAPK) phosphorylation in cells. (b) Equal amounts of protein were loaded in each lane. # indicate significantly compared with untreated cells; # p < 0.001, compared with blank group (untreated cells); * p < 0.05, *** p < 0.001, compared with control group (Ang II-treated cells).

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  Figure 5. Effects of LSGYGP on the expression of iNOS, COX-2, NF-κB p65, and IκBα in HUVEC (a,b). Protein expression was by western blot, GAPDH, NF-κB p65, and IκBα were as control, respectively. (c) The effect of LSGYGP on translocation of NF-κB p65 in HUVEC. Cells were pre-treated with LSGYGP (10 and 100 μM) and subsequently treated with Ang II (1 μM) for 24 h. Nucleus was stained with DAPI and NF-κB p65 was immunostained with p65 antibody. (d,e) LSGYGP suppressed the NF-κB activity inside the nucleus of NF-κB in Ang II-stimulated HUVEC. Electrophoretic mobility shift assay (EMSA) was performed to determine the NF-κB activity in nuclear reaction by using DNA probe specific to NF-κB p65. # p < 0.001, compared with blank group (untreated cells); ** p < 0.01, *** p < 0.001, compared with control group (Ang II-treated cells).

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  Figure 6. (a) The expressions of Bax, Bcl-2, procaspase-3, caspase-3 (p20), and phosphorylation of Akt in HUVEC. GAPDH was used as an internal control. (b) The ratios of Bcl-2/Bax and cleaved-caspase-3/procaspase-3 were calculated. # p < 0.001, compared with blank group (untreated cells); ** p < 0.01, *** p < 0.001, compared with control group (Ang II-treated cells).

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  Figure 7. (a) Comet assay of HUVEC: (A) cells without treatment (the blank group); (B) cells exposed to 1 μM Ang II (the control group); (C, D, E, and F) cells pretreated with LSGYGP (10, 20, 50, and 100 µM, respectively) prior to treatment with 1 μM Ang II, all both followed by staining with DAPI. Images were obtained using an inverted fluorescence microscope with blue fluorescence (magnification: 10×). (b) Tail lengths of the comets were analyzed. # p < 0.001, compared with blank group (untreated cells); *** p < 0.001, compared with control group (Ang II-treated cells).

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  Figure 8. General and local overview poses of the best interaction poses after automated docking of peptide-NF-κB p65 active site (a), amino acids of LSGYGP involved in hydrogen bonds are represented by thin sticks; (b) Bi-dimensional (2D) diagrams of predicted interactions between ligand and NF-κB p65 amino acid residues. LSGYGP is draw by gray lines, and hydrogen bonds of them shown with green dashed lines.

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  Figure 9. Summary diagram of signaling pathway of this study.