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Spent hens are egg-laying hens reaching the end of their egg-laying cycles, being a major byproduct of the egg industry. Recent studies have been focusing on finding new value-added uses for spent hens. We have previously identified four bioactive peptides from spent hen muscle proteins, including three angiotensin-converting enzyme (ACE) inhibitory (ACEi) peptides (VRP, LKY, and VRY), and one ACE2 upregulating (ACE2u) peptide (VVHPKESF (V-F)). The current study aims to explore the antioxidant effects of these bioactive peptides from spent hen muscle proteins, as well as the underlying mechanisms underlying their antioxidant actions.
Peptides, including VRP, LKY, VRY, and V-F (purity > 98%) were synthesized by Genscript Corp (Piscataway, NJ, USA). Triton-X-100, dithiothreitol (DTT), and Ang II were obtained from Sigma (St Louis, MO, USA). The cytotoxicity of peptides against VSMCs and ECs followed an alamarBlue assay. After reaching 80% of confluency on a 96-well plate, cells were treated with 100 μM of peptides for 24 h in growth media, after which the media were replaced with 200 μL of 10% alamarBlue solution (in growth medium, v/v) for 4 h avoiding light. Then, 150 μL of the solution was transferred into an opaque 96-well plate for fluorescence signal detection, with an emission wavelength at 590 nm and excitation wavelength at 560 nm. A control without any treatment was added. Both cells were grown in DMEM containing 10% FBS and 1% penicillin–streptomycin at 37 °C in with 5% CO2 and 100% humidity; ECs were supplemented with NEAAs (1%). The growth media were changed every three days for both cells. Superoxide detection, lipid peroxidation assay, and western blotting were performed.
In the current study, we further assessed their antioxidant and cytoprotective activities in two vascular cell lines—vascular smooth muscle A7r5 cells (VSMCs) and endothelial EA.hy926 cells (ECs)— upon stimulation by tumor necrosis factor alpha (TNFα) and angiotensin (Ang) II, respectively. The results from our study revealed that all four peptides attenuated oxidative stress in both cells. None of the investigated peptides altered the expression of TNFα receptors in ECs; however, VRY and V-F downregulated Ang II type 1 receptor (AT1R), while V-F upregulated the Mas receptor (MasR) in VSMCs. Further, we found that the antioxidant effects of VRP, LKY, and VRY were likely through acting as direct radical scavengers, while that of V-F was at least partially ascribed to increased endogenous antioxidant enzymes (GPx4 and SOD2) in both cells. Besides, as an ACE2u peptide, V-F exerted antioxidant effect in a MasR- dependent manner, indicating a possible involvement of the upregulated ACE2-MasR axis underlying its antioxidant action.
The antioxidant effects of four previously identified, spent hen-derived peptides, including three ACEi peptides (VRP, LKY, and VRY) and one ACE2u peptide (V-F), were studied in two vascular cells, ECs and VSMCs, in vitro. All the four peptides reduced oxidative stress in both ECs and VSMCs, initiated by TNFα or Ang II stimulation, respectively. None of these peptides altered the expression of TNFα receptors (TNF-R1 and TNF-R2) in ECs, while VRY and V-F downregulated AT1R and meanwhile V-F upregulated MasR in VSMCs. In addition, we found that V-F exerted its antioxidant effect partially dependent on MasR in VSMCs, which might involve upregulating ACE2 and counteracting the ROS produced from the binding of Ang II with AT1R. Further analysis on the expression of endogenous antioxidant enzymes demonstrated that only V-F upregulated GPx4 and SOD2. The antioxidant activities of VRP, LKY, VRY, and V-F in vascular cells indicated their multifunctional properties, in addition to their ACEi or ACE2u activity, which supports their potential use as functional food ingredients against hypertension.