Activation of endothelial nitric oxide synthase by peptides isolated from skin gelatin of skate (Okamejei kenojei) protects against vasoconstriction in cardiovascular diseases

Abstract

Cardiovascular diseases, principally myocardial infarction, stroke and atherosclerosis, are a significant public health problem worldwide. Attempts to prevent cardiovascular diseases often imply modifications and improvement of major risk factors such as diabetes, hypertension and obesity. By modulating and improving physiological functions, bioactive peptides as components of functional foods or nutraceuticals with certain health claims may provide new therapeutic applications for the prevention or treatment of chronic diseases. Skin gelatin of skate (Okamejei kenojei) was hydrolyzed using Alcalase, flavourzyme, Neutrase and protamex. Among them, the Alcalase hydrolysate exhibited the highest angiotensin-I converting enzyme (ACE) inhibitory activity among them. Then, Alcalase hydrolysate was further hydrolyzed with protease and separated by a 1 kDa molecular weight cut-off membrane. The below 1 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. Two peptides responsible for ACE inhibitory activity were identified to be MVGSAPGVL (829 Da) (SP1) and LGPLGHQ (720 Da) (SP2) by time of flight-mass spectrometry/mass spectrometry (Q-TOF MS/MS) analysis. The IC50 values against ACE activity were 3.09 μM (SP1) and 4.22 μM (SP2). The purified peptides are safe materials in terms of cellular toxicity and thereby could be used as safe inhibitors of ACE without any cytotoxicity to human umbilical vein endothelial cells (EA.hy926 cells) and as effective antioxidants having no effect on the cell proliferation. The antioxidative potential of the peptides were characterized by analyzing the intracellular reactive oxygen species (ROS) scavenging effect of the peptides in EA.hy926 cells. Moreover, the peptides are capable of upregulating the expression of cellular antioxidative enzymes (superoxide dismutase and glutathione) and thereby enhanced the intracellular antioxidant mechanisms. The purified peptides augmented the production of nitric oxide (NO) by activating endothelial nitric oxide synthase (eNOS) expression in EA.hy926 cells. The peptides significantly suppressed angiotensin II-induced release of pro-inflammatory products such as prostaglandin E2 (PGE2), and cytokines; tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-8 in EA.hy926 cells. Furthermore, the peptides down-regulated the expression levels of cyclooxygenase-2 (COX-2), TNF-α, IL-1β, IL-6, IL-8 and endothelin-1 in EA.hy926 cells. Molecular signaling pathway studies showed that the peptides inhibited the nuclear factor kappa B (NF-κB) p65 and p50 subunits in EA.hy926 cells. Moreover, the peptides suppressed the phosphorylation of mitogen activated protein kinase (MAPK) pathway molecules; c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (ERK) and p38 in EA.hy926 cells. Antihypertensive effect in spontaneously hypertensive rats (SHRs) revealed that oral administration of protease hydrolysate below 1 kDa (PH1) can decrease systolic blood pressure significantly. PH1 inhibits vasoconstriction via peroxisome proliferator-activated receptor-gamma (PPAR-) expression, activation and phosphorylation of eNOS in rat lung tissues. Moreover, the level of endothelin-1, RhoA, α-smooth muscle actin, cleaved caspase 3 and MAPK expression were decreased by PH1 in rat lung tissues. The expression levels of inflammatory cytokines were also attenuated by PH1 in rat lung tissues. Vascularity, muscularization and cellular proliferation in rat lung tissues were detected by immunohistochemical staining. These results suggested that the cardioprotective mechanism of hydrolysate and peptides isolated from skin gelatin of skate (O. kenojei) may be partly due to improvement of endothelial function associated with eNOS and oxidative stress pathway. In conclusion, the purified hydrolysate and peptides would be beneficial ingredients for nutraceuticals and pharmaceuticals against cardiovascular diseases.