Sailuotong Prevents Hydrogen Peroxide (H2O2)-Induced Injury in EA.hy926 Cells

Sailuotong (SLT) is a standardised three-herb formulation consisting of Panax ginseng, Ginkgo biloba, and Crocus sativus designed for the management of vascular dementia. While the latest clinical trials have demonstrated beneficial effects of SLT in vascular dementia, the underlying cellular mechanisms have not been fully explored. The aim of this study was to assess the ability and mechanisms of SLT to act against hydrogen peroxide (H2O2)-induced oxidative damage in cultured human vascular endothelial cells (EAhy926). SLT (1–50 µg/mL) significantly suppressed the H2O2-induced cell death and abolished the H2O2-induced reactive oxygen species (ROS) generation in a concentration-dependent manner. Similarly, H2O2 (0.5 mM; 24 h) caused a ~2-fold increase in lactate dehydrogenase (LDH) release from the EA.hy926 cells which were significantly suppressed by SLT (1–50 µg/mL) in a concentration-dependent manner. Incubation of SLT (50 µg/mL) increased superoxide dismutase (SOD) activity and suppressed the H2O2-enhanced Bax/Bcl-2 ratio and cleaved caspase-3 expression. In conclusion, our results suggest that SLT protects EA.hy916 cells against H2O2-mediated injury via direct reduction of intracellular ROS generation and an increase in SOD activity. These protective effects are closely associated with the inhibition of the apoptotic death cascade via the suppression of caspase-3 activation and reduction of Bax/Bcl-2 ratio, thereby indicating a potential mechanism of action for the clinical effects observed.


Introduction
Cerebrovascular disease (CVD), such as stroke and vascular dementia, is a leading cause of morbidity and mortality, imposing a huge financial burden on the health care system worldwide. Progression of CVD is mediated via a numbers of factors, such as aging, hypertension, inflammation, and atherosclerosis, over a prolonged period [1,2]. It is now well established that many of these factors are closely associated with a chronic increase of oxidative stress, which can lead to vascular endothelial damage [3,4]. Oxidative stress is caused by excessive generation of reactive oxygen species (ROS),

Effects of SLT on LDH Leakage and SOD Activity in H2O2 Treated EA.hy926 Cells
Lactate dehydrogenase (LDH) is one of the major representative indicators of cell injury. Therefore, the protective effect of SLT on H2O2-treated EA.hy926 cells was confirmed using LDH assay. As shown in Figure 2A, H2O2 (0.5 mM; 24 h) markedly increased LDH leakage from the  ( EA.hy926 cells (p < 0.05, n = 3), while SLT reduced this H2O2-mediated LDH leakage in a concentration-dependent manner (p < 0.05 at 50 µg/mL compared to H2O2 alone; n = 3).
To further examine the protective effects of SLT, we measured SOD activity in H2O2-treated EA.hy926 cells. SOD activity was significantly reduced by H2O2 (p < 0.05 compared to control, n = 3). This significant reduction of SOD activity was partly reversed by SLT at 50 µg/mL ( Figure 2B).

Effect of SLT on the Intracellular ROS Generation in H2O2 Treated EA.hy926 Cells
In order to elucidate whether the protective effect of SLT is mediated by a reduction of intracellular oxidative stress, intracellular ROS generation was determined by 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), ROS specific dye. As shown in Figure 3, H2O2 markedly increased (~2-fold) intracellular ROS generation in EA.hy926 cells (p < 0.001 compared to control, n = 3) and SLT (1-50 µg/mL) suppressed this H2O2-induced ROS generation in a concentration-dependent manner (p < 0.001 at 50 µg/mL compared to H2O2 alone; n = 3). Interestingly, the effect of SLT at 50 µg/mL in suppressing H2O2-induced ROS generation is comparable to gallic acid (10 µg/mL), a known potent anti-oxidant [29].  To further examine the protective effects of SLT, we measured SOD activity in H 2 O 2 -treated EA.hy926 cells. SOD activity was significantly reduced by H 2 O 2 (p < 0.05 compared to control, n = 3). This significant reduction of SOD activity was partly reversed by SLT at 50 µg/mL ( Figure 2B).
To further examine the protective effects of SLT, we measured SOD activity in H2O2-treated EA.hy926 cells. SOD activity was significantly reduced by H2O2 (p < 0.05 compared to control, n = 3). This significant reduction of SOD activity was partly reversed by SLT at 50 µg/mL ( Figure 2B).

Effect of SLT on the Intracellular ROS Generation in H2O2 Treated EA.hy926 Cells
In order to elucidate whether the protective effect of SLT is mediated by a reduction of intracellular oxidative stress, intracellular ROS generation was determined by 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), ROS specific dye. As shown in Figure 3, H2O2 markedly increased (~2-fold) intracellular ROS generation in EA.hy926 cells (p < 0.001 compared to control, n = 3) and SLT (1-50 µg/mL) suppressed this H2O2-induced ROS generation in a concentration-dependent manner (p < 0.001 at 50 µg/mL compared to H2O2 alone; n = 3). Interestingly, the effect of SLT at 50 µg/mL in suppressing H2O2-induced ROS generation is comparable to gallic acid (10 µg/mL), a known potent anti-oxidant [29].

Effect of SLT on Protein Expression Level of Bax, Bcl-2, and Cleaved Caspase-3 in H 2 O 2 Treated EA.hy926 Cells
The protein expressions of Bax (pro-apoptotic factor) and Bcl-2 (anti-apoptotic factor) in H 2 O 2 treated EA.hy926 cells were evaluated using Western blotting with the result expressed as Bax/Bcl-2 ratio. As shown in Figure 4A, H 2 O 2 treatment markedly increased the Bax/Bcl-2 ratio (p < 0.05 compared to control, n = 3), while SLT at 50 µg/mL reduced this H 2 O 2 -mediated effect significantly (p < 0.05 compared to H 2 O 2 alone; n = 3). The effect of SLT on cleaved caspase-3 protein expression was also determined using Western blotting. It was found that H 2 O 2 caused a significant increase of cleaved capsase-3 expression (n = 3) in EA.hy926 cells, and this effect was completely reversed by SLT at 50 µg/mL (n = 3) ( Figure 4B).

Effect of SLT on Protein Expression Level of Bax, Bcl-2, and Cleaved Caspase-3 in H2O2 Treated EA.hy926 Cells
The protein expressions of Bax (pro-apoptotic factor) and Bcl-2 (anti-apoptotic factor) in H2O2 treated EA.hy926 cells were evaluated using Western blotting with the result expressed as Bax/Bcl-2 ratio. As shown in Figure 4A, H2O2 treatment markedly increased the Bax/Bcl-2 ratio (p < 0.05 compared to control, n = 3), while SLT at 50 µg/mL reduced this H2O2-mediated effect significantly (p < 0.05 compared to H2O2 alone; n = 3). The effect of SLT on cleaved caspase-3 protein expression was also determined using Western blotting. It was found that H2O2 caused a significant increase of cleaved capsase-3 expression (n = 3) in EA.hy926 cells, and this effect was completely reversed by SLT at 50 µg/mL (n = 3) ( Figure 4B).

Discussion
It is well established that endothelial dysfunction caused by elevated cerebrovascular oxidative stress is one of the major mechanisms of CVDs [3,6,30]. Therefore, interventions that can protect endothelial cells from ROS-induced damage would be beneficial for CVDs. In the present study, we demonstrated that SLT could protect EA.hy926 cells from oxidative stress and cell damage caused by H 2 O 2 . Furthermore, our results suggest that this effect is possibly mediated by a reduction of the Bax/Bcl-2 ratio and an increase of SOD activity in the EA.hy926 cells.
ROS, such as superoxide anions and hydroxyl radicals, are generated during normal cellular metabolism [31]. Under normal physiological conditions, vascular endothelial cells are in continuous contact with steady-state levels of oxidative metabolites. This constant level of ROS is tightly regulated by a number of anti-oxidative enzymes, such as SOD and glutathione peroxidase [32,33]. However, when this anti-oxidative mechanism is disrupted, excessive ROS will be generated, leading to endothelium dysfunction, which contributes to the development and progression of a number of cerebral and vascular diseases, such as atherosclerosis, stroke, and hypertension [6, 34,35]. H 2 O 2 is one of the ROS that have been shown to play a major role in vascular and endothelial dysfunction [3,10,36]. Numerous in vitro studies have demonstrated that high levels of H 2 O 2 can cause significant injury and reduce endothelial cell viability [37][38][39]. In line with this, our results showed that H 2 O 2 (0.5 mM, 24 h) significantly reduced viability and increased LDH leakage in the EA.hy926 cells. Interestingly, pre-incubation of the cells with SLT one hour prior to the addition of H 2 O 2 significantly suppressed the H 2 O 2 -mediated cell death and LDH leakage, demonstrating the anti-apoptotic property of SLT.
A recent clinical study demonstrated that SLT can improve cognitive function and memory in people with vascular dementia [18]. Animal studies have suggested these clinically beneficial effects are possibly associated with increases in cerebral blood flow and reductions in platelet aggregation rate and whole blood viscosity [21]. However, the underlying cellular mechanisms of SLT in protecting endothelial cell from ROS-mediated injury had not been explored previously. H 2 O 2 has been shown to induce oxidative stress via an increase in the generation of intracellular ROS in endothelial cells [40]. Our results showed that SLT suppressed the H 2 O 2 -induced intracellular ROS generation in a concentration-dependent manner. More importantly, SLT at 50 µg/mL produced a similar effect to our positive control, gallic acid (a known potent anti-oxidant) [29]. In addition, SOD is a major enzyme that protects against oxidative stress damage in endothelial cells [33,41]. In the present study, SLT at 50 µg/mL partly reversed the H 2 O 2 -suppressed SOD activity in the EA.hy926 cells. These results highlight the potent anti-oxidant properties of SLT through reduction in intracellular ROS generation and modulation of anti-oxidative enzyme activity.
A number of apoptosis-related proteins, including Bax, Bcl-2, and caspase-3, are required for cellular survival regulation [42]. In this study, we examined if SLT can suppress the apoptotic signalling pathway transduction triggered by H 2 O 2 . Our results showed that H 2 O 2 increased the Bax/Bcl-2 ratio; this H 2 O 2 -induced effect was inhibited by pre-treatment of SLT. It has been demonstrated that intracellular ROS can increase cytosolic caspases activity via activation of Bax and dissociation of cytochrome C from the inner mitochondrial membrane [43]. Several studies have suggested that Bax/Bcl-2 plays a role in determining cell apoptosis process [44,45]. H 2 O 2 has been shown to downregulate Bcl-2 and upregulate Bax (i.e., increased Bax/Bcl-2 ratio), leading to caspase-3 cleavage, and eventually apoptosis [46]. Caspases-3 is one of the most important enzymes responsible for the cleaving of many cellular substrates during apoptosis [47]. In line with this, we demonstrated that the anti-apoptotic effect of SLT appears to be associated with the inhibition of the apoptotic death cascade via suppression of caspase-3 activation and a reduction in the Bax/Bcl-2 ratio in EA.hy926 cells.
Endothelial dysfunctions are closely associated with vascular dementia and other neurological disorders [9,30]. For instance, changes in brain vascular endothelial cell morphology can reduce the blood-brain barrier permeability, leading to cognitive decline and dementia [48]. Moreover, an increase in ROS generation and oxidative stress in vasculature has been suggested as one of the central pathologies of both vascular dementia and Alzheimer's disease [49]. Given that current therapies for these diseases are limited [50,51], the development of new therapies/interventions is urgently needed. In this regard, SLT has recently been demonstrated to improve cognitive function and memory in people with vascular dementia. Despite the relatively small sample size in this study, the findings have highlighted the therapeutic potential of SLT in improving cognitive function in people with dementia [18]. It is important to point out that, although some preclinical pharmacokinetic, toxicity, and pharmacodynamics studies of SLT and its individual components have been conducted in several animal models of cerebral disorders [20][21][22]27,52], the cellular and molecular effects of SLT in endothelial cells have not been fully explored. The results of this study demonstrate the effects and underlying signalling mechanisms of SLT against oxidative stress in EA.hy926 cells, providing new insights and molecular evidence to previous in vivo and clinical observations.
The present study has several limitations. Multiple sources of ROS have been suggested to contribute to endothelial cell damage [53,54]. For example, Xie et al. demonstrated the significant role of mitochondrial-derived ROS in age-related cardiovascular diseases [55]. Our current experiments have only examined the global cellular ROS generation in response to exogenous H 2 O 2 ; more detailed studies, such as direct measurement of mitochondrial ROS, are required to clarify the cellular target and anti-oxidative property of SLT in endothelial cell. Although our results show that the anti-apoptotic property of SLT was associated with reduction in caspase-3 activation and Bax/Bcl-2 ratio, the effects of SLT on other cellular signalling pathways should also be explored using additional experimental models (e.g., heavy metal-induced [56] and hypoxia-induced [57] oxidative stress models). Additionally, contributions of individual components of SLT to the observed effects were not determined. 3-(4,5-di-methylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) was purchased from Astral Scientific (Caringbah, Australia). The superoxide dismutase (SOD) activity assay kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). CytoTox non-radioactive cytotoxicity assay was obtained from Promega Corporation (Madison, WI, USA). The cellular ROS/Superoxide detection assay kit was purchased from Abcam (Cambridge, UK). Anti-Bax, anti-Bcl-2, anti-cleaved caspase-3, and anti-beta actin antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). All other reagents and chemicals were of chemical analytical grade.

EA.hy 926 Cell Culture
The permanent human endothelial cell line EA.hy926 was originally derived from a human umbilical vein obtained from ATCC (Manassas, VA, USA). In this study, cells were grown in DMEM/F12 (1:1 Mix) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO 2 at 37 • C. During cell culture, the medium was changed every three days until the cells reached 90% confluence. To assess the effects of SLT on EA.hy926 cells, the cells were treated with increasing concentrations of SLT (0.1, 1, 10, 50 µg/mL) for 1 h followed by H 2 O 2 (0.5 mM) or vehicle for 24 h unless stated otherwise.

Measurement of Cell Viability
Cell viability was determined using MTT assay. In brief, cells were seeded in 96-well plates at a density of 1.0 × 10 5 cells/well and allowed to attach for 24 h. After incubation with the above-mentioned treatments, the culture supernatant was removed, then the cells were incubated with MTT (5 mg/mL) in DMEM/F12 medium at 37 • C for 4 h. After MMT incubation, the culture medium with dye was replaced with 150 µL DMSO and was agitated in a plate shaker for 5 min. Next, the optical density (O.D.) of each well was measured at 560 nm using a Microplate Reader (BMG Labtech, Ortenberg, Germany). Cell viability was expressed as a percentage relative to control.

Measurement of Intracellular ROS Level
Intracellular ROS level was evaluated using the cellular ROS/superoxide detection assay kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. In brief, cells were seeded in 96-well plates at a density of 1.0 × 10 5 cells/well and allowed to attach for 24 h. After incubation with the above-mentioned treatments, the culture supernatant was removed and the cells were washed with 100 µL/well of 1× assay buffer. The ROS specific stain, DCFH-DA, was added to the cells and allowed to incubate in the dark for 60 min. After the incubation, intracellular ROS level was determined using a fluorescence microplate reader (Ex = 488 nm, Em = 520 nm) (BMG Labtech, Ortenberg, Germany).

Measurement of Lactate Dehydrogenase (LDH) Leakage
Lactate dehydrogenase (LDH) release was evaluated using the non-radioactive assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates at a density of 1.0 × 10 5 cells/well and allowed to attach for 24 h. After incubation with the above-mentioned treatments, 50 µL of supernatant per well was transferred to a 96-well flat clear bottom plate. An equal amount of CytoTox reagent was added to each well and allowed to incubate for 30 min at room temperature. A stop solution was added to terminate the reaction at the end of the incubation period. LDH level was measured at 490 nm using a microplate reader (BMG Labtech, Ortenberg, Germany).

Measurement of Intracellular SOD Activity
Cells were seeded in six-well plates at a density of 1.0 × 10 6 cells/well and allowed to attach for 24 h. The cells were treated with vehicle, H 2 O 2 (0.5 mM; 24 h) alone or H 2 O 2 (0.5 mM; 24 h) with SLT (50 µg/mL; 1 h prior addition of H 2 O 2 ). After the treatment, the cells were lysed by Freeze Thaw method three times. The activity of SOD was determined using a commercially available kit, according to the manufacturer's instructions.

Western Blotting
Cells were seeded in six-well plates at a density of 1.0 × 10 6 cells/well and allowed to attach for 24 h. The cells were treated with vehicle, H 2 O 2 (0.5 mM; 24 h) alone or H 2 O 2 (0.5 mM; 24 h) with SLT (50 µg/mL; 1 h prior addition of H 2 O 2 ). After the treatment, the cells were homogenized and lysed in a Radioimmunoprecipitation assay RIPA buffer (Thermo Scientific, Waltham, MA, USA) in the presence of protease inhibitors (Roche Applied Science, Penzberg, Germany) to obtain protein extracts. Protein concentrations were determined using the bovine serum albumin (BSA) protein assay kit (Pierce, Waltham, MA, USA). Samples (25 µg of protein per lane) were loaded onto a mini-PROTEAN TGXTM precast electrophoresis gel (BioRad, Hercules, CA, USA). After electrophoresis (110 V, 90 min), the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes using iBlot 2 gel transfer system (Thermofisher, Waltham, MA, USA). Non-specific sites were blocked with 5% non-fat dry milk in Phosphate Buffered Saline Tween-20 (PBSt) for 60 min, and the blots were then incubated with anti-Bax, 1:1000 (Santa Cruz), anti-Bcl-2, 1:1000 (Santa Cruz), anti-cleaved caspase-3, 1:1000 (Santa Cruz, Dallas, TX, USA), and anti-beta actin, 1:10,000 (Santa Cruz) in PBSt overnight at 4 • C. Anti-rabbit horseradish peroxidase (HRP) conjugated immunoglobulin G (IgG), 1:1000 (DakoCytomation, Glostrup, Denmark) in PBSt (60 min, room temperature) was used to detect the binding of its correspondent antibody. β-actin was used to verify equal loading of protein in each lane. The protein expression was detected with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Waltham, MA, USA) and quantified by Quantity One (version 4.6.7) software (BioRad).

Statistical Analysis
Data were presented as mean ± SEM of n experiments. Statistical comparisons were performed using t-test or one-way analysis of variance (ANOVA), where appropriate. Differences were considered to be statistically significant at p < 0.05. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Conclusions
In conclusion, our results revealed that SLT can inhibit H 2 O 2 -induced endothelial cell injury via the direct reduction of intracellular ROS generation and increase of SOD activity. These protective effects are closely associated with the inhibition of the apoptotic death cascade through suppression of caspase-3 activation and reduction of Bax/Bcl-2 ratio. Our data suggest that SLT possesses potent anti-oxidative and anti-apoptotic activities, which at least partially contribute to its cognitive enhancing effects observed in the clinical study. Further studies are required to investigate the effects and mechanisms of SLT between different cell types within the neurovascular unit and the interaction/synergistic effects between individual components of SLT