<em>Acer okamotoanum</em> protects SH-SY5Y neuronal cells against hydrogen peroxide-induced oxidative stress

Introduction

Oxidative stress produced by excessive reactive oxygen species (ROS) is thought to be an important cause of various neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease (Finkel and Holbrook, 2000; Gorman et al., 1996). ROS, including superoxide anions, hydroxyl radicals and hydrogen peroxide (H₂O₂), are generated in normal conditions, but imbalances, due to over production and accumulation of ROS, attack biological components such as proteins, lipids, and nucleic acids (Finkel and Holbrook, 2000). These effects may contribute to the pathology caused by mitochondrial dysfunction, inflammation, and apoptosis (Perticone et al., 1997; Somayajulu et al., 2005). ROS production activates pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), to release inflammatory cytokines including interleukin (IL)-1β, IL-6, and IL-8 (Barnes and Karin, 1997). In addition, ROS-induced oxidative stress elevates the expression of pro-apoptotic proteins such as cleaved caspase-3, cleaved caspase-9 and Bcl-2-associated X protein (Bax), decreasing the expressions of anti-apoptotic proteins such as B cell lymphoma 2 (Bcl-2) and B-cell lymphoma extra-large (Bcl-xl), resulting in neuronal cell death (Thayyullathil et al., 2008). Protection against oxidative stress by modulating inflammation and apoptosis is thought to be an important therapeutic strategy for neurodegenerative diseases.

Numerous studies have been attempted to establish neuroprotective effects of antioxidant from natural sources such as curcumin, green tea and Ginkgo biloba (Koo et al., 2004; Mandel et al., 2008; Shi et al., 2010). In addition, antioxidants from natural sources have attracted much attention due to their low toxicity and minimal side effects compared with various synthetic antioxidants. Acer species have been used in traditional medicine against neuralgia, rheumatism, bruises, hepatic disorders, eye diseases, hemostasis, abnormal urination, constipation, and gastroenteric disorders for many years in East Asia and North America (Bae, 2000; Bi et al., 2016). In addition, various pharmacological studies have shown antioxidant, anti-cancer, anti-inflammatory and neuro-protective effects (Bi et al., 2016). Acer okamotoanum (A. okamotoanum) is a maple species found on the Korean island of Ulleungdo (Moon and Kwon, 2004). Several studies reported anti-oxidant, anti-cancer, immune improvement, and skin whitening activity of extracts from A. okamotoanum (Jeong et al., 2009; Jin et al., 2008; Qadir et al., 2007). Especially, A. okamotoanum demonstrated in vitro antioxidant activities via nitrite scavenging, superoxide dismutase-like activity, and glutathione S-transferase activity. In addition, active components from A. okamotoanum, such as flavonol glycosides and phenolic compounds, reported anti-oxidant and anti-cancer activity, as well as inhibition of HIV-1 integrase (Jin et al., 2006; Jin et al., 2007; Kim et al., 1998; Lee et al., 2018). In addition, we previously demonstrated that an ethyl acetate (EtOAc) fraction from A. okamotoanum improves cognition and memory function in Alzheimer’s disease mouse model (Choi et al., 2017b). However, the neuro-protective activity and mechanism of A. okamotoanum in neuronal cells has not been investigated. Therefore, this study was designed to examine the protective effects and mechanisms of the EtOAc fraction from A. okamotoanum against H₂O₂-induced oxidative stress in SH-SY5Y neuronal cells.

Materials and methods

Reagents

Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, and trypsin EDTA solution were purchased from Welgene (Daegu, Korea). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Bio Basic (Toronto, Canada) and dimethyl sulfoxide (DMSO) was purchased from Bio Pure (Ontario, Canada). Dichlorofluorescein diacetate (DCF-DA) was purchased from Sigma (St. Louis, MO, USA) and H₂O₂ was purchased from Junsei (Tokyo, Japan). RIPA buffer was purchased from Elpics Biotech (Daejeon, Korea), protease inhibitor cocktail from Calbiochem (Cambridge, MA, USA), and polyvinylidene fluoride (PVDF) membranes from Millipore (Bedford, MA, USA). Sample buffer and enhanced chemiluminescence (ECL) substrate solution were purchased from Bio-Rad (CA, USA). Primary antibodies such as iNOS, COX-2, IκBα, Bax, and Bcl-2 were obtained from Santa Cruz (CA, USA). Phospho-nuclear factor-kappaB (p-NF-κB), phosho IκBα (p-IκBα), β-Actin, caspase-3, -9, poly ADP ribose polymerase (PARP), and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Sample preparation

Acer okamotoanum samples were collected from Ulleung-do, Republic of Korea. A voucher specimen has been deposited at the Department of Integrative Plant Science, Chung-Ang University, Anseong, Republic of Korea (Voucher No. LEE 2014-04). Dried aerial parts of A. okamotoanum were extracted five times in methanol (MeOH), and then dried using a rotary evaporator. The MeOH extract was fractionated with EtOAc and used in our study.

Cell culture

SH-SY5Y cells were obtained from the American Type Culture Collection (OH, USA). The cells were grown in DMEM containing FBS (10% v/v) and penicillin–streptomycin (1% v/v) and maintained at 37 °C in a humidified atmosphere of 5% CO₂. The SH-SY5Y cells were seeded at a density of 5 × 10 ^{cells/mL in 96-well plate, and then incubated for 24 h. Afterward, the test wells were treated with EtOAc fraction of} A. okamotoanum at various concentrations (1, 5, 10, 25 and 50 µg/mL). After 2 h incubation at 37 °C, cells were treated with 300 µM H₂O₂ for 24 h.

Cell viability

Cell viability was determined using a MTT colorimetric assay (Mosmann, 1983). Cell culture media was replaced with medium containing MTT (5 mg/mL) in the wells. After 4 h of incubation, formazan crystals were dissolved in 200 µL of DMSO and the absorbance of each well was then read at 540 nm using a microplate reader (Thermo Fisher Scientific, Vantaa, Finland).

Reactive oxygen species (ROS) scavenging activity

Intracellular ROS production was measured using DCF-DA fluorescence assay (Wang and Zhu, 2003). After the culture supernatant had been removed, the cells were treated with the medium containing DCF-DA (80 µM). After 30 min of incubation at 37 °C, the fluorescence intensity was measured at an excitation − 485 nm and an emission − 535 nm using a fluorescence spectrophotometer (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany). Representative fluorescence images were obtained using fluorescent microscopy (Olympus BX50, Tokyo, Japan).

Lactate dehydrogenase (LDH) release activity

LDH release activity was measured using an LDH cytotoxicity detection kit (Clontech laboratories Inc., CA, USA), according to the manufacturer’s protocol. Briefly, 100 µL of culture supernatant from each well were allowed to react with 100 µL of the LDH reaction solution, and then incubated at room temperature for 30 min. The optical densities of the samples were measured at 490 nm using a microplate reader (Racher et al., 1990).

Western blot analysis

The cells were harvested and washed with PBS. After centrifugation, cells were lysed in 100 µL of RIPA buffer containing a protease inhibitor cocktail. The lysate was incubated on ice for 30 min and centrifuged at 12,000 rpm for 30 min at 4 °C. After the supernatant was collected, protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, CA, USA), and added to an equivalence volume of sample buffer. Equal amounts of protein (30 µg) in each sample were electrophoresed using 10% or 13% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in PBS-T for 1 h at room temperature, and then incubated overnight with primary antibodies at 4 °C. After washing the membrane with PBS-T, it was incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. The membrane was activated with ECL substrate solution, according the manufacturer’s instructions and visualized with the Davinch-chemi™ Chemiluminescence Imaging System (Core Bio, Seoul, Korea).

Statistics analysis

Results are presented as the mean ± standard deviation (SD). The statistical significance of data among the group differences was tested using one-way analysis of variance (ANOVA), followed by a Duncan’s multiple range test and Student’s t-test. Statistical significance was considered for P values < 0.05.

Results and discussion

Recently, many researchers have shown that oxidative stress is the cause of neurodegenerative diseases, and natural antioxidants have attracted much attention for use in the therapeutic strategy for neurodegenerative diseases. To search for natural antioxidants, ROS-induced in vitro cellular models are commonly used (González-Sarrías et al., 2016; Zhou et al., 2014). H₂O₂ is widely known as one of the most important toxic inducers of oxidative stress, which produces highly reactive hydroxyl radicals, resulting in cellular injury (Tabner et al., 2001). In addition, accumulation of H₂O₂ has been observed in neurodegenerative disease with inflammation, necrosis, and apoptosis in the brain (Finkel and Holbrook, 2000; Li et al., 2003). Human neuroblastoma SH-SY5Y cells are widely used as a cellular model for research on H₂O₂-induced oxidative stress (Nirmaladevi et al., 2014; Shin et al., 2007).

Natural plants contain various bioactive compounds. Especially, potential antioxidant activities are dependent on hydroxyl groups of hydrophilic bioactive compounds (Miller and Rice-Evans, 1997). Therefore, to extract a large quantity of hydrophilic bioactive compounds, we used MeOH and EtOAc. In addition, we previously reported that the EtOAc fraction from A. okamotoanum has higher in vitro free radical scavenging activities against DPPH and O2-and higher total phenolic and flavonoid contents, compared with n-BuOH, methylene chloride and n-hexane fractions, and the crude MeOH extract (Choi et al., 2017a). Furthermore, the EtOAc fraction of A. okamotoanum led to a significant protective effect in oxidative stress-induced C6 glial cells among other extracts and fractions (Choi et al., 2017a). The most active compounds from the EtOAc fraction of A. okamotoanum leaves were flavonol glycosides such as quercetin-3-O-β-D-galactopyranoside and quercetin-3-O-α-L-arabinopyranoside (Choi et al., 2017b). Kim et al. reported that many flavonoid glycosides containing galloyl isolated from the EtOAc fraction of A. okamotoanum leaves strongly inhibit HIV-1 integrase. Furthermore, two coumarins, cleomiscosin A and C, isolated from the EtOAc fraction from leaves and twigs of A. okamotoanum, led to strong anti-oxidant activities (Jin et al., 2007). In addition, we also isolated and identified active components such as afzelin, quercitrin, and isoquercitrin from the EtOAc fraction of A. okamotoanum (Lee et al., 2018). On the basis of these findings, the EtOAc fraction was used.

In this study, we demonstrated the protective activity of the EtOAc fraction from A. okamotoanum against H₂O₂-induced inflammation and apoptosis in SH-SY5Y cells via regulation of inflammatory and apoptotic gene expression. As shown in Fig. 1(A), cytotoxicity in SH-SY5Y cells was not observed up to the EtOAc fraction concentration of 25 µg/mL. At 50 µg/mL, cell viability was decreased to 81.31% of control cells. Therefore, we tested the EtOAc fraction from A. okamotoanum at concentrations up to 25 µg/mL in SH-SY5Y cells exposed to H₂O₂, and measured cell viability using the MTT assay. As illustrated in Fig. 1(B), the H₂O₂-treated control group showed significantly decreased cell viability of 55.31% compared with the non-treated normal group of 100.00%. However, treatment with the EtOAc fraction from A. okamotoanum showed dose-dependent increase in cell viability compared with H₂O₂-treated SH-SY5Y cells. In particular, the 10 and 25 µg/mL concentrations of the EtOAc fraction from A. okamotoanum increased cell viability from 55.31 to 95.77 and 94.43%, respectively. Li et al., (2003) demonstrated that SH-SY5Y cell viability was decreased by treatment with H₂O₂ (Li et al., 2003). Our results showed that H₂O₂ treatment led to a loss of cell viability in SH-SY5Y cells. However, cell viability was dose-dependently elevated by the treatment of various concentrations of EtOAc fraction from A. okamotoanum.

Fig. 1

Effects of the EtOAc fraction from A. okamotoanum on SH-SY5Y cell viability. (A) SH-SY5Y cells were seeded and cultured for 24 h. Cells were treated with the various concentrations (1, 5, 10, 25, 50 µg/mL) of the EtOAc fraction from A. okamotoanum for 24 h. Values are expressed as the mean ± SD. *P < 0.05 compared to the normal group by Student’s t-test. (B) SH-SY5Y cells were seeded and cultured for 24 h. Cells were treated with various concentrations (1, 5, 10, 25 µg/mL) of EtOAc fraction from A. okamotoanum, and then with 300 µM H₂O₂ for 24 h. Values are expressed as the mean ± SD. ^{Means with different letters indicate significant differences (}P < 0.05) by Duncan’s multiple range test. Normal group indicates untreated cells, whereas control group indicates the cells exposed to H₂O₂ alone

To evaluate the inhibition of ROS production associated with the EtOAc fraction from A. okamotoanum in H₂O₂-exposed SH-SY5Y cells, the DCF-DA assay was performed. As shown in Fig. 2, the H₂O₂-treated control group showed significantly increased ROS production. Figure 2(B) showed that ROS production was steadily increased in the control group by H₂O₂ exposure in a time-dependent manner during 60 min, indicating that H₂O₂ led to oxidative stress. Figure 2(C) showed the amount of ROS production at 60 min. However, the EtOAc fraction of A. okamotoanum, at the concentrations of 5, 10 and 25 µg/mL, produced dose-dependent declines in H₂O₂-induced accumulation of ROS, averaging 85.83, 68.33, and 64.70%, respectively. We also showed that H₂O₂ could evoke ROS-induced cytotoxicity in SH-SY5Y cells. Previous research found that SH-SY5Y cells increase ROS production mediated by H₂O₂ (Garcimartín et al., 2014; Park et al., 2015). However, the presence of the EtOAc fraction from A. okamotoanum alleviated H₂O₂ neurotoxicity by a reduction of ROS.

Fig. 2

Effects of the EtOAc fraction from A. okamotoanum on ROS levels in SH-SY5Y cells treated with H₂O₂. SH-SY5Y cells were seeded and cultured for 24 h. Cells were treated with various concentrations (1, 5, 10, 25 µg/mL) of the EtOAc fraction from A. okamotoanum, and then with 1 mM H₂O₂ for 24 h. ROS production was determined using the DCF-DA assay followed by fluorescence spectrophotometry (excitation—485 nm; emission—535 nm). (A) The representative fluorescence images under observation by fluorescent microscopy (×100 magnification). (B) Time course of change in intensity of ROS fluorescence within 60 min. (C) The intensity of ROS fluorescence at 60 min. Values are mean ± SD. ^{Means with different letters indicate significant differences (}P < 0.05) by Duncan’s multiple range test. Normal group indicates untreated cells, whereas control group indicates the cells exposed to H₂O₂ alone

Figure 3 shows the protective effects of EtOAc fraction from A. okamotoanum against toxicity in the LDH assay. LDH release in the H₂O₂-treated control group was significantly elevated to 100.00%, compared with the non-treated normal group with release of 76.13%. However, treatment with the EtOAc fraction from A. okamotoanum in H₂O₂-induced SH-SY5Y cells significantly decreased H₂O₂-induced LDH release. LDH is an intracellular enzyme present in cells. Increased LDH in culture medium indicates damage to cellular membranes and is a marker for cell death (Hansen et al., 1989). Consistent with another study (Garcimartín et al., 2014), our data demonstrated that LDH leakage into the culture medium was increased in H₂O₂-induced SH-SY5Y cells. Treatment with the EtOAc fraction from A. okamotoanum significantly reduced LDH in the medium of SH-SY5Y cells exposed to H₂O₂. Therefore, we confirmed the protective effects of the EtOAc fraction from A. okamotoanum in H₂O₂-exposed SH-SY5Y cells, through measured concentration-dependent increases in cell viability, decline in ROS, and decreased LDH release.

Fig. 3

Effects of the EtOAc fraction from A. okamotoanum on LDH release in SH-SY5Y cells treated with H₂O₂. SH-SY5Y cells were treated with various concentrations (1, 5, 10, 25 µg/mL) of the EtOAc fraction from A. okamotoanum, and then with 300 µM H₂O₂ for 24 h. LDH release was determined using an LDH assay. Values are expressed as the mean ± SD. ^{Means with different letters indicate significant differences (}P < 0.05) by Duncan’s multiple range test. Normal group indicates untreated cells, whereas control group indicates the cells exposed to H₂O₂ alone

To further evaluate the protective molecular mechanisms of the EtOAc fraction from A. okamotoanum, we measured inflammation-related protein expressions such as iNOS, COX-2, p-IκBα, and p-NF-κB. As shown in Fig. 4, the H₂O₂-treated control group showed significantly up-regulated expressions of iNOS, COX-2, p-NF-κB, and an increase in the ratio of p-IκBα/IκBα. However, treatment with the EtOAc fraction from A. okamotoanum in H₂O₂-induced SH-SY5Y cells showed significant and dose-dependent decreased expression of iNOS, COX-2, p-NF-κB, and a decrease in the ratio of p-IκBα/IκBα, compared with the control group. The NF-κB pathway is widely known as an important pro-inflammatory mediator and plays a role in neurodegenerative diseases (Amor et al., 2010; Tilstra et al., 2014). Under normal conditions, NF-κB is present in the cytosol bound to IκB inhibitors (NF-κB/IκB complex). However, when cells were stimulated by oxidative stress, activated IκB-α activates the translocation of NF-κB from the cytosol to the nuclei (Amor et al., 2010). Activated NF-κB can induce over-expression of inflammatory mediators, including iNOS and COX-2 (Jung et al., 2009). The over expression of COX-2 and iNOS leads to production of various inflammatory cytokines that are associated with degenerative diseases (Minghetti, 2004). Therefore, we observed expressions of iNOS, COX-2, IκBα, p-IκBα, and p-NF-κB in H₂O₂-induced SH-SY5Y cells. Our study demonstrated that the EtOAc fraction from A. okamotoanum significantly attenuated expressions of iNOS and COX-2 via the NF-κB pathway. Especially, gene expression levels of iNOS and COX-2 in H₂O₂-induced SH-SY5Y cells were reduced in a concentration-dependent manner by treatment with the EtOAc fraction from A. okamotoanum.

Fig. 4

Effects of the EtOAc fraction from A. okamotoanum on iNOS, COX-2, p-IκBα, IκBα, and p-NFκB protein expressions in H₂O₂-induced SH-SY5Y cells. SH-SY5Y cells were treated with concentrations (1, 5, 10, 25 µg/mL) of the EtOAc fraction from A. okamotoanum, and then with 300 µM H₂O₂ for 24 h. Protein expressions were measured using western blotting. Values are expressed as the mean ± SD. ^{Means with different letters indicate significant differences (}P < 0.05) by Duncan’s multiple range test. β-actin was used as a loading control. Normal group indicates untreated cells, whereas control group indicates the cells exposed to H₂O₂ alone

To identify the protective mechanisms of the EtOAc fraction from A. okamotoanum against H₂O₂-induced apoptosis, we investigated protein expression of pro- and anti- apoptotic genes using western blotting. As shown in Fig. 5, the expressions of cleaved caspase-9, caspase-3, and PARP were increased in the H₂O₂-exposed control group compared with the untreated normal group. However, in the groups treated with the EtOAc fraction from A. okamotoanum, dose-dependent down-regulation of the over-expression of cleaved caspase-9, caspase-3, and PARP was observed. In addition, Fig. 6 illustrates the expression of the pro-apoptotic gene, Bax, and the anti-apoptotic gene, Bcl-2. The H₂O₂-treated control group showed an increased ratio of Bax to Bcl-2 via enhancement of the expression of Bax and decreasing the expression of Bcl-2, compared with the normal group. However, the EtOAc fraction from A. okamotoanum significantly decreased the ratio of Bax to Bcl-2. Oxidative stress-induced neuronal cell death is associated with the mitochondria-related apoptotic pathway in H₂O₂-induced SH-SY5Y cells, and is an important mechanism in neurodegenerative diseases (Hu et al., 2015). The overproduction of ROS leads to increased permeability of the mitochondrial membrane, thus resulting in release of cytochrome C. Released cytochrome C activates pro-apoptosis factors, such as caspase-9, caspase-3 and PARP, eventually inducing cell death (Hu et al., 2015; Wang et al., 1998). Our results suggest up-regulation of proteolytic cleavage of caspase-9, caspase-3 and PARP following treatment with H₂O₂. However, the EtOAc fraction from A. okamotoanum protects neuronal cells against H₂O₂ by down-regulation of caspase-9, caspase-3, and PARP. In addition, mitochondrial membrane injury by oxidative stress leads to neuronal apoptosis by modulating expression of the Bcl-2 gene family (Pastorino et al., 1998; Zhang et al., 2007). Similar to our results, previous research showed that H₂O₂-treated SH-SY5Y cells increased pro-apoptotic gene expression such as Bax, and lowered anti-apoptotic gene such as Bcl-2 (Zhang et al., 2007). The present results indicate that the EtOAc fraction from A. okamotoanum could not only significantly reduce Bax, but also increase Bcl-2. Therefore, the EtOAc fraction from A. okamotoanum protects oxidative stress by regulation of inflammation and apoptosis in SH-SY5Y cells.

Fig. 5

Effects of the EtOAc fraction from A. okamotoanum on caspase-9, caspase-3 and PARP protein expressions in H₂O₂-induced SH-SY5Y cells. Cells were treated with concentrations (1, 5, 10, 25 µg/mL) of the EtOAc fraction from A. okamotoanum, and then with 300 µM H₂O₂ for 24 h. Protein expressions were measured using western blotting. Values are expressed as the mean ± SD. ^{Means with different letters indicate significant differences (}P < 0.05) by Duncan’s multiple range test. β-actin was used as a loading control. Normal group indicates untreated cells, whereas control group indicates the cells exposed to H₂O₂ alone

Fig. 6

Effects of the EtOAc fraction from A. okamotoanum on Bax and Bcl-2 protein expressions in H₂O₂-induced SH-SY5Y cells. Cells were treated with concentrations (1, 5, 10, 25 µg/mL) of the EtOAc fraction from A. okamotoanum, and then with 300 µM H₂O₂ for 24 h. Protein expressions were measured using western blotting. Values are expressed as the mean ± SD. ^{Means with different letters indicate significant differences (}P < 0.05) by Duncan’s multiple range test. β-actin was used as a loading control. Normal group indicates untreated cells, whereas control group indicates the cells exposed to H₂O₂ alone

In conclusion, this study demonstrated that the EtOAc fraction from A. okamotoanum prevents H₂O₂-induced oxidative stress in SH-SY5Y cells. The treatments with the EtOAc fraction elevated cell viability by decreasing ROS production and LDH release. In addition, the EtOAc fraction from A. okamotoanum protects SH-SY5Y cells from oxidative stress-induced inflammation and apoptosis by modulating the NF-κB pathway, inhibiting caspase activity and regulating the Bax/Bcl-2 ratio. The results suggest that A. okamotoanum may possess neuroprotective potential, but further study is necessary to elucidate the pharmacological effects in neurodegenerative diseases.