*3.2. Cytoprotective Effect of Phenolic Acids against Hydrogen Peroxide*

Excessive ROS levels lead to endothelial dysfunction and elevated blood pressure [22]. MDA, a marker of oxidative damage, can cause an abnormal physiological state in the body [23]. GSH, an active peptide with good antioxidant activity, can modulate oxidative balance and suppress oxidative damage [24]. In this study, we investigated the cytoprotective effects of phenolic acids on H2O2-induced oxidative stress in EA.hy 926 endothelial cells. Treatment with H2O2 (600 μM) decreased cell viability by 24.8%. However, treatments by caffeic, ferulic, gallic, and sinapic acid markedly increased the cell viability by 43.4, 43.6, 35.5, and 39.1%, respectively, compared to H2O2-induced cells (Figure 3A). To examine whether phenolic acids protect endothelial cells against oxidative damage, we measured ROS, GSH, and MDA levels (Figure 3B–D). Sinapic acid markedly reduced ROS generation by 44.1% compared to that in H2O2-treated cells. Caffeic acid, cinnamic

acid, coumaric acid, ferulic acid, gallic acid, sinapic acid, and syringic acid significantly enhanced the GSH levels. Our findings show that H2O2 treatment increased ROS levels and decreased intracellular GSH levels, whereas treatment with phenolic acids significantly reduced oxidative damage-induced ROS production and GSH depletion. In addition, we investigated the effect of phenolic acids on oxidative stress-induced lipid peroxidation in EA.hy 926 cells. Among the phenolic acids, sinapic acid showed the strongest inhibitory effect on lipid peroxidation. Lee and Lee (2021) reported that protocatechuic acid and gallic acid significantly decreased ROS levels, thereby regulating insulin resistance [25]. Caffeic acid and chlorogenic acid decreased blood pressure in hypertensive rats by increasing GSH and reducing MDA levels [16]. Taken together, these results suggest that sinapic acid plays a crucial role in the protection of endothelial cells by regulating ROS, MDA, and GSH levels.

**Figure 3.** Effect of selected phenolic acids (50 μM) on cell viability (**A**), the generation of reactive oxygen species (**B**), glutathione (**C**), and malondialdehyde (**D**) against hydrogen peroxide (600 μM) in EA.hy 926 cells. Quercetin (25 μM) was used as positive control. Each value was expressed as the mean <sup>±</sup> standard error (*<sup>n</sup>* = 3). Statistical significance was analyzed using the Tukey test. ### *<sup>p</sup>* < 0.001 versus nontreated cells. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 versus hydrogen-peroxide-treated cells.

#### *3.3. Effects of Sinapic Acid on the Expression of Phase II Enzymes and the Activation of Nrf2*

Based on our results, sinapic acid was selected for exploring the mechanism underlying the antihypertensive effect of phenolic acid. We measured the protein expression levels of HO-1, NQO-1, GCLC, and Nrf2. As shown in Figure 4, treatment with sinapic acid enhanced HO-1, NQO-1, and GCLC expression levels in a dose-dependent manner. In addition, sinapic acid significantly increased the nuclear translocation of Nrf2. The Nrf2 pathway is important for protection against various stressors [26]. Cytotoxicity caused by t-BHP-induced oxidative damage was recovered by caffeic acid via an increase in the expression of detoxifying enzymes, including HO-1 and GCLC [27]. Luo et al. (2018) reported that HO-1 ameliorates oxidative stress-induced endothelial aging by modulating eNOS activation [28]. Ginsenoside Rg3 upregulates the Nrf2 signaling pathway via Akt activation and improves endothelial dysfunction [29]. Moreover, sinapic acid reduces renal apoptosis, inflammation, and oxidative damage [30]. These results suggest that sinapic acid-mediated endothelial cell protection against oxidative damage may be associated with the antioxidative properties of sinapic acid.

**Figure 4.** Effect of sinapic acids on HO-1 (**A**), NQO-1 (**B**), and GCLC (**C**) protein expression and Nrf-2 expression levels in nucleus (**D**) and cytosol (**E**) against hydrogen peroxide (600 μM) in EA.hy 926 cells. Each value was expressed as the mean ± standard error (*n* = 3). Statistical significance was analyzed using the Tukey test. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 versus hydrogen-peroxide-treated cells.

#### *3.4. Effects of Phenolic Acids on Endothelial Dysfunction*

NO is essential for maintaining vascular function in the endothelium. Phosphorylation of eNOS can regulate NO production [31] and is essential for the improvement of CVD [32]. Akt mediates NO production via phosphorylation of eNOS, which promotes endothelial cell migration and angiogenesis [33]. A previous study reported that eNOS phosphorylation facilitates vasorelaxation via the PI3K/Akt signaling pathway in HU-VECs [34]. Therefore, phosphorylation of eNOS and Akt is important for the treatment of endothelial dysfunction. As shown in Figure 5, treatment with H2O2 (600 μM) significantly reduced the phosphorylation of eNOS and Akt. However, sinapic acid treatment at concentrations of 12.5, 25, and 50 μM enhanced the phosphorylation of eNOS by 14.1, 26.3, and 48%, respectively, compared to that in the H2O2-treated group. Sinapic acid increased Akt phosphorylation in a dose-dependent manner. Chen et al. (2020) reported that phenolic acids extracted from ginseng protect against vascular endothelial cell injury via the activation of the PI3K/Akt/eNOS pathway [35]. Yan et al. (2020) reported that gallic acid attenuated vascular dysfunction and hypertension in angiotensin II-induced C57BL/6J mice by suppressing eNOS degradation [36]. Taken together, our results showed that sinapic acid may be effective in the treatment of endothelial dysfunction via phosphorylation of eNOS and Akt.

**Figure 5.** Effect of sinapic acids on p-eNOS (**A**) and p-Akt (**B**) protein expression against hydrogen peroxide (600 μM) in EA.hy 926 cells. Each value was expressed as the mean ± standard error (*n* = 3). Statistical significance was analyzed using the Tukey test. # *p* < 0.05 and ## *p* < 0.01 versus nontreated cells. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 versus hydrogen-peroxide-treated cells.
