**1. Introduction**

The imbalance between the antioxidant defense mechanism and reactive oxygen species (ROS) generation in a physiological system leads to oxidative stress and associated disease consequences. Regulated ROS generation is critical for the activation of protective signaling pathways, but when in excess amount it induces oxidative stress. Oxidative stress induces depolarization of the mitochondrial membrane. When the mitochondrial membrane potential is reduced, a series of signaling proteins is activated, which leads to the activation of several stress-responsive genes, such as p53, Bax, Bcl-2, and caspase-3 [1]. This results in enhanced reactive oxygen species generation, severe cell damage, and apoptosisinduced cell death [2,3]. These risk factors can induce endothelial dysfunction (ED) through a variety of processes [4,5]. The endothelium, particularly the terminal arteries, is damaged by too much ROS, which disrupts the intracellular reduction-oxidation balance. Hence, ED is considered as an early indicator in the progression of cardiovascular disease (CVD) [6,7]. Since oxidative stress is defined as a possible cause of cardiovascular disease, treatment with antioxidants is a good strategy to prevent CVD-causing endothelial vein damage.

Recently, marine-derived food proteins have attracted much attention because of their wide range of bioactivity. Seafood consumption is thought to lower the risk of various

**Citation:** Suryaningtyas, I.T.; Ahn, C.-B.; Je, J.-Y. Cytoprotective Peptides from Blue Mussel Protein Hydrolysates: Identification and Mechanism Investigation in Human Umbilical Vein Endothelial Cells Injury. *Mar. Drugs* **2021**, *19*, 609. https://doi.org/10.3390/md19110609

Academic Editors: Donatella Degl'Innocenti and Marzia Vasarri

Received: 9 October 2021 Accepted: 26 October 2021 Published: 27 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

diseases, as peptides derived from marine-food protein have an anti-inflammatory, antihypertensive, antidiabetic, anticancer, antioxidant, and anti-obesity potential [8–14]. Some identified antioxidant peptides or protein hydrolysates are important subjects of interest, due to their specific properties as therapeutic agents to protect the body from diseases related to oxidative damage [8,15,16]. Blue mussel (*Mytilus edulis*) is one of the prominent protein-rich marine food sources that can be converted into bioactive peptides (BAPs) through enzymatic hydrolysis to optimize their health benefits, such as anti-inflammation, antioxidant, and anti-obesity [17–19]. Fermented blue mussel sauce, a popular Asian-style culinary condiment, has also been associated with CVD risk by producing BAPs with antihypertensive activity [20,21]. An effective strategy to prevent CVD is to protect the venous endothelial cells, which are damaged by oxidative stress. In a previous study, α-chymotrypsin-assisted protein hydrolysate of blue mussel showed cytoprotective activity in protecting HUVECs from damage induced by H2O2-mediated oxidative stress [1]. However, there is insufficient information in previous reports about specific BAPs that play an important role in endothelial cell protection and certain mechanisms associated with ROS-mediated CVD that need to be understood. The purification of peptides is one of the procedures that needs to be carried out, in order to further expand the use of this compound as a pharmaceutical raw material or functional food source in the future. Therefore, the purpose of this study was to evaluate the capacity of purified peptides derived from blue mussel to protect HUVEC from oxidative stress caused by H2O2 exposure, as well as to understand the protective mechanism of these peptides.

#### **2. Results**

#### *2.1. Purification and Identification of Cytoprotective Peptides*

Cytoprotective peptides were purified from α-chymotrypsin-assisted protein hydrolysates of blue mussel by a cytoprotective activity-guided purification process. First, separation was performed by Sephadex G-25 gel filtration and four fractions (F1~4) were collected. After evaluating cytoprotective activity, the F3 fraction that showed the highest protective effect on HUVEC against H2O2-induced oxidative cell damage was selected and further purified by HPLC equipped with a C18 column (Figure 1). Six fractions were obtained by HPLC separation. After determination of cytoprotective activity, fractions H3 and H4 showed similar HUVEC protective activity and were analyzed by LC-MS/MS to identify the peptide sequence. Finally, two peptides of EPTF (calculated MW, 493 Da) and FTVN (calculated MW, 480 Da) were identified in H4 fraction (Figure 2). No peptides were identified in the H3 fraction. To evaluate more about their potential to protect HUVECs, these two peptides were chemically synthesized to further investigate their cytoprotective activity and underlying mechanism.

**Figure 1.** Peptide purification from α-chymotrypsin-assisted blue mussel hydrolysates. (**A**) Gel filtration chromatogram, (**B**) cytoprotective activity of gel filtration fraction, (**C**) HPLC chromatogram, and (**D**) cytoprotective activity of HPLC fraction. Detailed separation conditions are described in Section 2.1. Cells were treated with fraction for 2 h followed by the addition of 600 μM H2O2 and further incubation for 24 h.

**Figure 2.** Identification of cytoprotective peptides from α-chymotrypsin-assisted protein hydrolysates of blue mussel by LC-MS/MS.
