*2.5. Anti-Apoptotic Activity against H2O2-Induced HUVECs Damage*

A high dose exposure of powerful oxidants such H2O2 causes severe damage to macromolecules, which leads to cell death through apoptosis and/or necrosis mechanisms [24]. The cytoprotective activity of EPTF, FTVN, as well as their combination was evaluated by measuring the anti-apoptotic activity. Annexin V-FITC/PI and Hoechst 33342 staining was performed after the peptide treatments.

The results revealed that 94.1% of HUVECs were located in the lower left quadrant in the non-treated cells (control), which decreased to 60.8% in the H2O2 group. There was a high percentage of necrotic cell (28.5%) compared to apoptotic cell (10.72%) in the presence of H2O2 treatment alone. Pretreatment of HUVECs with EPTF, FTVN, and their combination significantly reduced the total percentage of dead cells compared to the H2O2 treatment group (Figure 5A). This indicates that our model treatment leads to apoptosis first, followed by necrosis. However, peptide treatment decreased predominantly the necrosis rate (Figure 5B). Figure 6 confirmed that peptide treatment modulated the protein expression related to apoptosis, indicating that cell survival by peptide treatment is attributed to downregulation of apoptotic protein expression.

**Figure 5.** The effect of EPTF, FTVN, and combination in inhibiting HUVEC apoptosis. (**A**) Quadrant dot analysis showing live–dead cells, and (**B**) apoptotic and necrotic ratio using flow cytometer analysis using Annexin V-FITC/PI staining assay. (**C**) Morphological changes under fluorescence microscope with Hoechst 33342 staining assay (white arrows showed apoptosis occurrence). HUVECs were pretreated with 0.1 mg/mL of peptides for 2 h before being challenged with 600 μM H2O2 for 24 h. All treatment was carried out in triplicate.

**Figure 6.** Western blot analysis of (**A**) released mitochondrial cytochrome C (Cyt-C) found in cytosol, and (**B**) Bax, Bcl-2, and activated caspase-3 (procaspase-3/cleaved caspase-3) expression. As protein loading control, Cox IV and β-actin were used. Expression level of (**C**) released Cyt C within mitochondria and cytoplasm, (**D**) ratio of Bax/Bcl-2 expression, and (**E**) cleaved caspase-3/β-actin for caspase-3 activation. HUVECs were incubated with 0.1 mg/mL of peptides for 2 h before being challenged with 600 μM H2O2 for 24 h. All treatment was carried out in triplicate. The data are provided as means ± SD (*n* = 3). Different letters show significance difference at *p* < 0.05.

Observation of nuclear morphology with a fluorescence microscope shows that H2O2 treatment leads to morphological changes, especially nuclear shrinkage, segregation, and chromatin condensation (Figure 5C). HUVECs in the peptides-pretreated group were comparable to those in the non-treated cells (control), suggesting that EPTF, FTVN, and their combination protect the HUVECs from apoptotic cell death.

It is known that disruption of mitochondrial membrane integrity by H2O2 insult leads to the release of cytochrome C (Cyt C), which in turn causes apoptosis in cells [25]. Western blot analysis was performed to investigate the effect of EPTF, FTVN, and their combination on the released of Cyt C into the cytoplasm. As shown in Figure 6A, mitochondrial Cyt C was strongly detected in the non-treated cells, while cytosolic Cyt C was weakly detected. On the other hand, H2O2 treatment resulted in cytoplasmic release of Cyt C from mitochondria into the cytoplasm, and Cyt C was strongly detected in the cytoplasm, indicating activation of apoptosis mediated by the intrinsic pathway. However, the release of the Cyt C from mitochondria in the cytoplasm by H2O2 treatment was significantly reduced after pretreatment with EPTF, FTVN, and their combination (Figure 6A,C), indicating suppression of the intrinsic pathway by H2O2 exposure.

The expression ratio of Bcl-2 and Bax plays a crucial role in the apoptosis process by regulating mitochondrial membrane permeability, which is associated with the disruption of mitochondrial membrane integrity [26]. In this study, H2O2 exposure resulted in an increase in Bax expression but a decrease in Bcl-2 expression (Figure 6B). In the cells pretreated with EPTF, FTVN, and their combination, the Bax and Bcl-2 expression was reversed and the Bax/Bcl-2 ratio was also significantly reduced compared to the cells with H2O2 treatment (Figure 6B,D). Finally, this study examined the activation of caspase-3, which is known to be the execution caspase in apoptosis. High expression of procaspase-3 was detected in the untreated cells, whereas the cleaved caspase-3, the active form of

caspase-3, was negligible (Figure 6B,E). On the other hand, procaspase-3 was converted to cleaved caspase-3 in the presence of H2O2 treatment, but this process was abolished by pretreatment with EPTF, FTVN, and their combination, suggesting that the anti-apoptotic effect of the peptides came from suppression of the caspase-3 pathway.

#### **3. Discussion**

Recent studies have shown that BAPs derived from marine dietary proteins by enzymatic hydrolysis have versatile health benefits, as they have antioxidant, antihypertensive, and antidiabetic effects. To date, many BAPs have been isolated and identified from marine dietary protein hydrolysates [27]. In addition, previous studies have identified specific BAPs in blue mussel protein hydrolysates that have been attributed antioxidant, antithrombotic, antihypertensive, osteogenic, and anti-osteoporotic effects [19,20,28–31]. Blue mussel protein hydrolysate produced by α-chymotrypsin has been previously identified as a potential cytoprotective agent [1]. However, there is limited information on specific BAPs with cytoprotective effects of blue mussel protein hydrolysates in alleviating HUVEC damage caused by oxidative stress. In this study, two cytoprotective peptides were isolated and identified as EPTF and FTVN, and their molecular mechanism underlying the cytoprotective activity was investigated.

The idea of using antioxidants with cryoprotective affects to treat CVD is based on the evidence that the excess amount of ROS generates oxidative stress, which then leads to endothelial cell damage and induces apoptosis [31]. Endothelial dysfunction (ED) a physiological condition that occurs in the early development of atherosclerosis, is often characterized by cell death, including the mechanism of apoptosis [32]. H2O2 is one of the most understood ROS, serving as a second messenger in a variety of critical cellular signaling pathways, but when it presents in high concentrations it has toxic consequences that can lead to cellular dysfunction or even cell death. Therefore, to evaluate the cytoprotective effects of EPTF and FTVN, an H2O2-mediated HUVEC injury model was used. Since these two peptides were identified in the same fraction (H4), we investigated the cytoprotective effect of each peptide as well as their combination (synergic effect). It was found that pretreatment with EPTF, FTVN and both combinations reversed the cell death induced by H2O2 treatment. Moreover, increased ROS generation by H2O2 treatment was remarkedly quenched by pretreatment of EPTF, FTVN, and their combination. There was no significant difference in the cytoprotective effect between the peptides and their combination. Each peptide had a potent cytoprotective effect on its own. This indicates that the contribution of each peptide to cytoprotective is similar.

To uncover the mechanism underlying the cytoprotective activity, the effect of EPTF, FTVN, and their combination in the cellular antioxidant defense system was investigated. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a regulator of species lifespan that regulates the expression of genes coding for detoxifying proteins, antioxidants, and antiinflammatories [33]. In this study, H2O2 plays a role as an exogenous stimulus that induces the oxidative damage and triggers the activation of Nrf2. In this condition, the Nrf2- Keap1-Cullin3 complex was disrupted, which then allowed the translocation of Nrf2 from cytoplasm to nucleus [34]. From Figure 4E, we can see that Nrf2 was accumulated in nucleus as an indication of cytoprotective activity. One of the genes regulated through Nrf2 is HO-1, which is recognized as a cytoprotective enzyme through catalyzing heme protein into ferritin, biliverdin, and carbon monoxide [35]. In our study, H2O2 stimulated the production of HO-1 but only with insignificant concentration; this result is similar to another study using H2O2 as the stress model [36,37]. This might be due to the very high concentrations of exogenous stimuli that exceed the cell capacity to perform cytoprotective mechanisms, which also results in cellular damage. To eliminate damaged cells, the apoptosis process occurs. We demonstrated that pretreatment of EPTF, FTVN, and their combination upregulated HO-1 expression through Nrf2 activation in H2O2-mediated HUVEC injury.

Induction of HO-1 by peptide treatment may have influenced cell survival and the decrease in intracellular ROS generation. Mitochondria is a natural source of cellular ROS; their membranes contain certain key compounds involved in antioxidant responses and in stimulating apoptotic pathways [38]. When oxidative stress surpasses a cell's ability to balance it, mitochondrial dysfunction occurs, which then leads mitochondria to generate more ROS. Furthermore, oxidative stress promotes nuclear damage and triggers the cascade of apoptotic cell death. Apoptosis is a programmed cell death that is governed by two major pathways including the intrinsic mitochondria pathway and the extrinsic death receptor pathway [39]. Since H2O2 is known to induce apoptosis through the intrinsic mitochondria pathway, in this study the role of EPTF, FTVN, and their combination was investigated in the intrinsic mitochondria pathway [40]. A key event of this pathway is mitochondria Cyt C released into the cytoplasm through the relative ratio of Bax and Bcl-2 proteins expression, i.e., a high Bax/Bcl-2 ratio increased cell death probability through an intrinsic mitochondria pathway-mediated apoptosis [41]. The released Cyt C then binds to apoptotic protease-activating factor-1 and forms an apoptosome with procaspase-9. This activates caspase-3, an important trigger of apoptosis. In this study, H2O2 exposure increased Bax expression while decreasing Bcl-2 expression in HUVEC and showed a high ratio of Bax/Bcl-2, indicating the increase of mitochondrial membrane permeability. However, its value was decreased in HUVEC that received pretreatment with EPTF, FTVN, and their combination. In addition, our data clearly showed H2O2 mediated mitochondria dysfunction, which is proved by the accumulation of Cyt C in the cytoplasm, while pretreatment of EPTF, FTVN, and their combination inhibited this event in the cytoplasm.

Finally, H2O2-mediated activation of caspase-3 in HUVEC was inhibited by cascade activation via alteration of the Bax-Bcl-2 ratio and release of Cyt C by pretreatment of EPTF, FTVN, and their combination. This suggests that the intrinsic mitochondrial pathway is involved in the cytoprotective effect induced by EPTF, FTVN and their combination in H2O2-mediated HUVEC damage.

Similar results of BAPs from various food proteins, including edible seahorse, rice bran, and *Mytilus coruscus*, have been reported in H2O2-mediated HUVEC injury where these BAPs showed the cytoprotective effect through modulation of the intrinsic mitochondria pathway [24,42,43]. These findings suggest that BAPs may be useful for ameliorating oxidative stress-mediated ED and may be helpful for treating CVD.
