Introduction

ETM

Experimental and Therapeutic Medicine

1792-0981 1792-1015

D.A. Spandidos

10.3892/etm.2017.4045

ETM-0-0-4045

Articles

α- and β-Naphthoflavone synergistically attenuate H₂O₂-induced neuron SH-SY5Y cell damage

Zhu

Yong

1 Bi

Fangfang

2 Li

Yanchun

1 Yin

Huiming

3 Deng

2 Pan

Haiquan

2 Li

Dongfang

2 Xiao

1Department of Neurology, The First Affiliated Hospital of Hunan University of Medicine, Huaihua, Hunan 418000, P.R. China 2Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China 3Department of Respiration, The First Affiliated Hospital of Hunan University of Medicine, Huaihua, Hunan 418000, P.R. China

Correspondence to: Professor Bo Xiao, Department of Neurology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, P.R. China, E-mail: boxiaoboxiao12@163.com

03 2017

13 01 2017

13 3 1143 1150 06112015 11112016

2017

Previous studies have demonstrated an association between neurological diseases and oxidative stress (OS). Naphthoflavone is a synthetic derivative of naturally occurring flavonoids that serves an important role in the treatment and prevention of OS-related diseases. The current study was designed to apply α- and β-Naphthoflavone individually and in combination to counteract the detrimental effects of OS on neurons in vitro. Neuronal SH-SY5Y cells were subjected to 20 µM H₂O₂, followed by exposure to 20 µM α-Naphthoflavone and/or 10 µM β-Naphthoflavone. Results indicated that α- and β-Naphthoflavone effectively antagonized the apoptosis-promoting effect of H₂O₂ on neuronal SH-SY5Y cells, and that β-Naphthoflavone significantly (P<0.05) reversed H₂O₂-inhibited cell viability. Notably, co-treatment of α- and β-Naphthoflavone reversed the H₂O₂-induced apoptosis rate elevation and cell viability reduction. Further analysis demonstrated that H₂O₂ inhibited the activities of antioxidant enzymes including catalase, superoxide dismutase and glutathione peroxidase, but this was reversed by the co-treatment with α- and β-Naphthoflavone and selectively enhanced by the treatment with α- or β-Naphthoflavone. H₂O₂-stimulated p38 mitogen-activated protein kinase activation was repressed following treatment with α- and/or β-Naphthoflavone, along with a decreased expression of the apoptosis-related factors and inhibited caspase-3 activation. In conclusion, co-treatment with α- and β-Naphthoflavone minimized H₂O₂-led neuron damage compared with treatment with α- or β-Naphthoflavone, suggesting a synergetic effect between α- and β-Naphthoflavone. This indicates that utilizing α- and β-Naphthoflavone together in the clinical setting may provide a novel therapeutic for neurological disease.

α-Naphthoflavone β-Naphthoflavone antioxidant enzyme activity oxidative stress p38 mitogen-activated protein kinase

Introduction

Oxidative stress (OS) is an important contributor to the damage induced by acute ischemic stroke and is a potent etiologic factor for chronic neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis and amyotrophic lateral sclerosis (1,2). The implication of OS in neurological diseases is associated with its effect on neurocytes; the structural and functional characteristics present in neurocytes include easily peroxidizable unsaturated fatty acids, high oxygen consumption rate and relative paucity of antioxidant enzymes, resulting in the vulnerability of neurocytes to OS (2). Furthermore, the incidence of OS and the opportunity of neurocytes exposed to OS are increased by exposure to environmental toxins (3), deficient antioxidants present in the diet source (4) and pathological conditions such as diabetes (5). An increase in OS is caused by excessive production of oxidative radical species (ROS), leading to the endogenous antioxidant defenses being overwhelmed (6). ROS exert a strong oxidative property and may result in cell membrane injury, mitochondrial dysfunction, protein misfolding and DNA destruction, finally leading neurocyte apoptosis (7).

OS-induced neurocyte apoptosis is a primary contributor to neurological disease with OS in the reoxygenation/reperfusion setting of stroke constituting a major driving force for neurocyte apoptosis (8–10). The immediate supplement of exogenous antioxidants has been demonstrated to decrease neuronal mortality and ease the burden of stroke in vivo and in vitro (8–10). By contrast, OS derived from microglial activation in PD induces neurocyte apoptosis and consequently assists the degeneration of dopaminergic neurons that is a marked pathological feature of PD (11,12). In AD, the incidence of neuronal apoptosis is notably elevated, which is associated with the induction of OS production through the disruption of ceramide metabolism and accumulation of amyloid β-peptides (13). Neurocytes are unable to be regenerated following apoptosis and the deficit of neurocytes may trigger typical characteristics of neurological diseases including memory loss, cognitive impairment and motor disturbance. Therefore, counteracting the detrimental effects of ROS, especially ROS-induced neuronal apoptosis, is an important approach in treating neurological diseases (10–13).

A number of botanical extracts, specifically those rich in flavonoids, confer neuroprotective effects via the inhibition of OS generation and inhibiting OS-mediated deleterious actions (14). Lin et al (15) demonstrated that the Wedelia chinensis extract effectively alleviates tertbutyl hydroperoxide-induced oxidative damage in neuronal PC12 cells and D-galactose-induced lipid peroxidation in the cerebral cortex of mice. Ratanasampil contains numerous Chinese herbs that have been demonstrated to have neuroprotective effects on OS-induced neuronal SH-SY5Y cell damage (16). In the present study, Naphthoflavone, a synthetic derivative of naturally occurring flavonoids, observed in Passiflora incarnata Linn (17), was assessed. Naphthoflavone has the potential to be a readily available and cheap therapeutic agent, if validated to be effective against neurological diseases. Naphthoflavone only contains two structural isomers, α-Naphthoflavone and β-Naphthoflavone, which is convenient for the investigation of their biological functions. By contrast, many botanical extracts, despite their neuroprotective effects have been identified to contain a number of different types of bioactive components (18). Therefore, the specific components of botanical extracts that are neuroprotective and their underlying mechanisms are typically poorly understood, resulting in restricted application of many botanical extracts in clinical settings. It has been demonstrated that Naphthoflavone upregulates the expression or activity of antioxidant-associated enzymes, including glutathione peroxidase (GPx), quinone oxidoreductase-1, glutathione transferase and heme oxygenase-1 (19–22), and represses ROS-producing enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (23). This suggests a potential effect exerted by Naphthoflavone in antioxidation. Currently, limited studies have assessed the role of Naphthoflavone in neurons that suffer from OS. Therefore, the present study aimed to administer α- and β-Naphthoflavone individually and combined to determine their capacity to counteract the detrimental effects of OS on neurons in vitro.

Materials and methods <sec> <title>Agents and cell culture

α- and β-Naphthoflavone were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The human neuron SH-SY5Y cells obtained from the American Type Culture Collection (Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Hyclone) and 100 U/ml streptomycin and penicillin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in a 5% CO₂ incubator at 37°C.

Cell viability assay

The cell viability was evaluated using a cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) using the following method. SH-SY5Y cells were seeded in 96-well plates at 1×10⁴ cells/well and cultured in DMEM without FBS at 37°C for 48 h. The cells were subjected to H₂O₂ at concentration of 0–320 µM for 24 h to evaluate the impairment. In an independent experiment, the cells underwent 20 µM H₂O₂ for 24 h followed by treatments with α- and β-Naphthoflavone, alone or in combination. SH-SY5Y cells that were subjected to 20 µM H₂O₂ for 24 h were subsequently treated with 0.5 µM SB203580 (Selleck Chemicals, Houston, Texas, USA), a p38MAPK-specific inhibitor, for 24 h. Finally, 10 µl CCK-8 solution was added to each well followed by an additional 5–8 h of incubation at 37°C. The optical density was then measured using a microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm.

Apoptosis rate analysis

The apoptosis rate was evaluated using the Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection kit I (cat. no. 550475; BD Biosciences, San Jose, CA, USA). A total of 5 µl Annexin V-FITC and 5 µl propidium iodide were added to SH-SY5Y cells (~2×10⁵) that were treated with Naphthoflavone and incubated at room temperature for 15 min in the dark. The extent of apoptosis was analyzed with a dual laser FACSCalibur flow cytometer (BD Biosciences) and estimated using the ModFit LT™ software version 2.0 (Verity Software House, Topsham, ME, USA).

Total antioxidant capacity (TAC) and malondialdehyde (MDA) measurement

The supernatant of the cells was lysed using a radioimmunoprecipitation assay (RIPA) buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) in order to measure the TAC using the azino-diethyl-benzthiazoline sulfate (ABTS) method (24). Total antioxidant capacity detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd. Incubation of ABTS with H₂O₂ and a peroxidase (metmyoglobin) produces a blue-green radical cation, ABTS⁺. Antioxidants in the sample suppress this color production proportionally to their concentration. Trolox (Nanjing KeyGen Biotech Co., Ltd.), a water-soluble vitamin E analogue was used to standardize the system. The results were expressed as µmol Trolox equivalent/protein concentration of plasma supernatant of lysed cells. In the MDA assay, 2 ml 0.6% thiobarbituric acid (Nanjing KeyGen Biotech Co., Ltd.) was added to 2 ml supernatant in a 10 ml tube. This tube was incubated in boiling water for 15 min and placed on ice to cool down prior to the optical density being measured at 532 nm via a microplate reader (ELx800; BioTek Instruments, Inc.). The results were expressed in nmol MDA/g protein.

Antioxidant enzyme activity assay

Catalase (CAT), superoxide dismutase (SOD) and GPx activity detection kits were all obtained from Beyotime Institute of Biotechnology (Haimen, China). In regard to CAT, cell homogenates were placed in a cuvette that had contained 250 mM hydrogen peroxide (H₂O₂) for 1–5 min. The remaining hydrogen peroxide was coupled with a chromogenic substrate (Beyotime Institute of Biotechnology) to generate a red product, N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinonemonoimine, which has a maximal absorption at 520 nm. Testing the remaining hydrogen peroxide enabled the quantity of H₂O₂ that reacted with CAT to be calculated and in turn determine the CAT activity. In the present study, SOD was present in the samples; this inhibited the process of superoxide transforming WST-8, a 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (Nanjing KeyGen Biotech Co., Ltd.), to a stable water-soluble WST-8 formazan. The latter may be evaluated by testing the optical density at 450 nm in order to determine SOD activity. The determination of GPx activity was based on the principle that NADPH continually diminishes in the cycle of GPx (6), transforming reduced glutathione to oxidized glutathione, which is reversed by glutathione reductase. Detecting reduced NADPH in absorbance at 340 nm may indirectly estimate the activity of GPx.

Western blot analysis

The cells were washed in PBS and lysed with RIPA buffer (50 mM Tris-buffer, 170 µg/ml leupeptin, 150 mM sodium chloride, 5 mM EDTA, 1 mM sodium orthovanadate, 1% NP-40, 81 µg/ml aprotinin, 0.5% deoxycholic acid, and 100 µg/ml phenylmethylsulfonyl fluoride, all these agents were purchased from Sigma-Aldrich; Merck Millipore, containing phosphatase inhibitors (okadaic acid; cat. no. ab120375; Abcam, Cambridge, UK). Protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A 10% SDS-PAGE gel loaded with ~20 µg protein from the cell lysis buffer was used to separate the proteins; these were then transferred onto polyvinylidene fluoride membranes (Merck Millipore) that were initially soaked in absolute methanol. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing Tween for 1 h. Proteins were probed with the following primary antibodies: anti-p38 mitogen-activated protein kinase (p38MAPK; 1:1,000, ab27986), anti-p38MAPK (phospho T180 + Y182; 1:800, ab195049), anti-cytochrome c (Cyt C; 1:200, ab53056), anti-caspase-3 (1:500, ab47131) purchased from Abcam. Anti-p38MAPK (phospho T322; 1:800, AP50174; Abgent, Inc., San Diego, CA, USA), anti-Bax (1:300, sc-493; Santa Cruz Biotechnology, Inc., La Jolla, CA, USA), and anti-GAPDH (1:5,000, ab16884; Abcam) at 37°C for 2 h. Following three washes, the membranes were incubated with anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibodies (1:20,000; cat. no. 611-903-002, Rockland Immunochemicals, Inc., Plottstown, PA, USA) at room temperature for 1 h. The blots on the membranes were scanned to quantify the optical density (Odyssey Image-forming System; LI-COR Biotechnology, Lincoln, NE, USA).

Statistical analysis

Data are presented as the mean ± standard error of the mean following three independent experiments. Data from these experiments were analyzed using SPSS software, version 12.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance with post hoc testing was used for multiple comparisons between each group. Significant differences were determined as P<0.05.

Results <sec> <title>Naphthoflavone attenuates H2O2-induced cell viability reduction and apoptosis elevation

H₂O₂ is recognized as a powerful oxidant and is commonly used to establish the OS model in a number of cell types. In the present study, the alteration of SH-SY5Y cell viability following cell exposure to H₂O₂ at concentration from 0–320 µM for 24 h was investigated. The cell viability was decreased by H₂O₂ in a dose-dependent manner, with the half cell viability loss at 20 µM H₂O₂ (Fig. 1A). Following the exposure to 20 µM H₂O₂ for 24 h, H₂O₂ was removed and cells were cultivated for an additional 24 h in the presence or absence of Naphthoflavone. The dose-response curves for the H₂O₂-pretreated cells cultivated with different doses of α- and β-Naphthoflavone are presented in Fig. 1B and C, respectively. The H₂O₂-inhibited cell viability increased in a dose-dependent manner by α-Naphthoflavone prior to the cell viability reaching a plateau at the dose of 20 µM α-Naphthoflavone (Fig. 1C). By contrast, the cell viability was increased with β-Naphthoflavone concentration raised from 0–10 µM, followed by a slight drop (Fig. 1C). Therefore, 20 µM α-Naphthoflavone and 10 µM β-Naphthoflavone, caused the maximal effect on cell viability recovery and were used individually and in combination in subsequent experiments of the present study to counteract 20 µM H₂O₂-induced detrimental effects. Results presented in Fig. 1D indicate that 20 µM H₂O₂ administered to the positive control (PC) cells induced a significant reduction of the cell viability compared to negative control (NC) cells without any treatment (P<0.05). This reduced cell viability was significantly increased by 10 µM β-Naphthoflavone (P<0.05 vs. PC), but not 20 µM α-Naphthoflavone (P=0.064; Fig. 1D). Furthermore, a significant increase in cell viability was observed following co-treatment with 20 µM α-Naphthoflavone and 10 µM β-Naphthoflavone compared with PC group (P<0.05). The co-treatment recovered the cell viability to a level close to that of the NC group.

As indicated by the apoptosis rate assay, H₂O₂ administration increased the apoptosis rate of SH-SY5Y cells in the PC group to 24.1%, ~5-fold increase compared with the NC group (P<0.01). Following this treatment, the addition of α- and β-Naphthoflavone, respectively, significantly decreased the apoptosis rate in comparison to the PC cells (P<0.05; Fig. 2A and B). Notably, co-treatment with α-Naphthoflavone and β-Naphthoflavone significantly reduced the apoptosis rate compared with the PC group, to 7.2% (P<0.01).

Naphthoflavone reverses H2O2-induced TAC decrease and MDA increase

To understand the H₂O₂-induced oxidative effect and potential antioxidant function of Naphthoflavone, the oxidative/antioxidative status of SH-SY5Y cells following different treatments was assessed via TAC and MDA assays. The TAC of SH-SY5Y cells (PC group) was significantly decreased following exposure to 20 µM H₂O₂ (P<0.01, Fig. 3A). Both α- and β-Naphthoflavone significantly reversed the H₂O₂-induced reduction in TAC compared with the PC group (P<0.05). Combined treatment with α- and β-Naphthoflavone had the most marked effect in on TAC, which significantly increased TAC in comparison to the PC group (P<0.05). MDA is regarded as a typical and sensitive biochemical marker following cell exposure to ROS and its accumulation reflects the extent of OS and indirectly that of cellular antioxidant capacity (25). In the current study, MDA in cells subjected to H₂O₂ (PC group) increased to ~2.5-fold higher compared with NC cells, reaching 13.24 µmol/mg protein (P<0.05, Fig. 3B). Post-treatment with α- and β-Naphthoflavone decreased MDA to 10.24 and 9.01 µmol/mg protein, respectively, which was a significant reduction compared with the PC group (P<0.05). Co-treatment with α- and β-Naphthoflavone reduced MDA to 6.74 µmol/mg, which was a significantly reduction compared with the PC (P<0.05).

Antioxidant enzyme activities are upregulated by Naphthoflavone

As presented in Fig. 3C-E, exposure to H₂O₂ decreased the activities of CAT, SOD and GPx by 82, 79 and 63%, compared with their respective NC values (P<0.01, P<0.01 and P<0.05, respectively). Post-treatment with α-naphthoflavone significantly increased SOD activity compared with the PC group (P<0.05; Fig. 3D), but only marginally restored the enzyme activities of CAT and GPx (Fig. 3C and E). Post-treatment with β-Naphthoflavone significantly increased CAT and SOD enzyme activities compared with the PC group (Fig. 3C and D; P<0.05). Combined treatment with α- and β-Naphthoflavone produced the most marked effect, which predominantly elevated SOD enzyme activity compared to PC (P<0.01; Fig. 1D), as well as increased CAT and GPx activities significantly compared with the PC group (Fig. 3C and E; P<0.05).

Naphthoflavone attenuates H2O2-induced damage via p38MAPK inhibition

Previous studies have demonstrated that H₂O₂ activates p38MAPK-mediated signaling to induce neuronal apoptosis (26). In the present study, SH-SY5Y cells exposed to H₂O₂ exhibited a 2.2-fold increase in p38MAPK phosphorylation at the sites of Thr180 and Tyr182 (P<0.05, Fig. 4A), although non-phosphorylated p38MAPK and phosphorylated p38MAPK at site of Thr322 were almost unaffected, as assessed by western blot analysis (Fig. 4A). By contrast, H₂O₂ administration markedly increased the expression of apoptosis-associated factors including Bax (P<0.01, Fig. 4B), Cyt C (P<0.01, Fig. 4C) and the ratio of cleaved to non-cleaved caspase-3 (P<0.01, Fig. 4D). Subsequent addition of a p38MAPK-specific inhibitor (SB203580) effectively reversed the elevation of p38MAPK phosphorylation on Thr180 and Tyr182 (P<0.05 vs. PC, Fig. 4A), and is associated with the reduction in the rate of apoptosis identified in Bax and Cyt C expression (P<0.05 vs. PC, Fig. 4B and C), the cleaved ratio of caspase-3 (P<0.01 vs. PC, Fig. 4D) and SH-SY5Y cells (P<0.05 vs. PC; Fig. 2). The data from the present study suggest that H₂O₂-induced p38MAPK activation facilitates neuron SH-SY5Y cell apoptosis, as previously considered (26). There is a limited data regarding the antioxidant role of SB203580; however, in the present study the inhibitor exhibited an antioxidant capacity by effectively reversing the H₂O₂-mediated increase of MDA (P<0.05; Fig. 3B) while H₂O₂-decreased CAT (P<0.05; Fig. 3C) and SOD activities (P<0.05; Fig. 3D).

Post-treatment with Naphthoflavones also repressed the p38MAPK phosphorylation at the Thr180 and Tyr182 sites and this inhibitory effect, in order of least to most marked, was exerted by α-Naphthoflavone (P<0.05 vs. PC), β-Naphthoflavone (P<0.05 vs. PC) and combined α- and β-Naphthoflavone (P<0.05 vs. PC; Fig. 4A). Additionally, α-Naphthoflavone decreased the expression of Bax and Cyt C and cleaved ratio of caspase-3 (P<0.05 vs. PC). These effects were also exhibited by β-Naphthoflavone, which was more effective than α-Naphthoflavone, specifically in the inhibition of Cyt C expression. By contrast, combined utilization of α- and β-Naphthoflavone minimized Bax and Cyt C expression and the ratio of cleaved caspase-3 (P<0.05, P<0.05 and P<0.01 vs. PC, respectively).

Discussion

Naphthoflavone has been documented to alleviate and confer protection against deleterious effects induced by OS. Previous studies have indicated that α-Napthoflavone inhibits OS and serves potential roles is estrogen-induced breast carcinogenesis and ultraviolet-led human skin aging (27,28). β-Naphthoflavone has been demonstrated to protect mice against aristolochic acid-I-induced acute kidney injury, and to attenuate hyperoxic lung injury in premature infants primarily via mitigating OS (29,30). In the present study, α- and β-Naphthoflavone were identified to effectively antagonize the apoptosis-promoting effect of H₂O₂ on neuronal SH-SY5Y cells. β-Naphthoflavone significantly reversed H₂O₂-inhibited the cell viability (P<0.05). Naphthoflavone was represented as a toxic substance in a previous study (31); however, the harmful effect of Naphthoflavone was not observed in the concentration range of α- o β-Naphthoflavone in the present study. Co-treatment with α- and β-Naphthoflavone abrogated the elevation of H₂O₂-induced apoptosis rate and cell viability reduction, which suggests a synergetic effect between α- and β-Naphthoflavone in these respects. This advocates the combined application of α- and β-Naphthoflavone in the clinical settings of neurological disease treatment.

OS causes structural and functional damage to neurons, H₂O₂-induced apoptosis mortality. As a frequent event in nervous system, it is therefore associated with a wide range of neurological diseases (1,2). OS is an important therapeutic target, and the supplement of exogenous antioxidants has been validated to be an effective approach to prevent and manage neurological diseases clinically (13–16). In the present study, neuronal SH-SY5Y cells underwent OS following exposure to H₂O₂, as indicated by TAC and MDA assays, and post-treatment with α-Naphthoflavone and/or β-Naphthoflavone effectively alleviated H₂O₂-induced OS.

A notable property of Naphthoflavone is an ablity to modulate antioxidant enzyme activity. It has been suggested that the antioxidant function exerted by natural flavones is completely dependent on their reducibility, derived from specific phenolic hydroxyl groups in their molecular structure (32). Evidence from Yang et al (6) indicates that natural flavones from grape seed extract elevate the activities of CAT, SOD and GPx, and mitigate the H₂O₂ insult to muscle cells, further suggesting that flavones serve an antioxidant role. It has been demonstrated previously that β-Naphthoflavone serves as a modulator of antioxidant enzyme activity (19). However, a previous study has indicated that antioxidant enzyme activity is unaffected following treatment with certain flavones (33). Thus, the present results indicate that the ability of antioxidant enzyme activity regulation is restricted in a number of flavones. In the current study, H₂O₂-inhibited CAT and SOD activities were promoted by β-Naphthoflavone. Furthermore, α-Naphthoflavone induced a compensatory increase in SOD activity. The activity of GPx was not significantly recovered by α- or β-Naphthoflavone, whereas it was effectively reversed by co-treatment with α- and β-Naphthoflavone. It has been demonstrated that antioxidant enzyme activity regulated by certain flavones potentially results in flavones stimulating or inhibiting aryl hydrocarbon receptors and subsequently regulating signaling mediated by nuclear factor erythroid 2-related factor, MAPK and peroxisome proliferator-activated receptors (6). This was partly supported by the results of the present study as SB203580 interfered with p38MAPK activation, resulting in the alteration of CAT and SOD enzyme activity, which may further explain why the addition of SB203580 may increase TAC and decrease MDA.

It has been indicated that OS-led neuronal apoptosis represents a major mechanism for the development and progression of neurological diseases (11–13) and OS-led neuronal apoptosis is at least in part dependent upon p38MAPK-mediated signaling. Park et al (26) demonstrated that the phosphorylation level of p38MAPK is markedly increased in SH-SY5Y cells following H₂O₂ stimulation, but those of extracellular signal regulated kinase and c-Jun N-terminal were not affected. Furthermore, increased p38MAPK phosphorylation is associated with an increase in the rate of SH-SY5Y cell apoptosis, which was eradicated following treatment with SB203580, a p38MAPK specific inhibitor (26). A similar outcome was observed in the present study and it was observed that OS-led neuronal apoptosis relies on p38MAPK phosphorylation at two sites (Thr180 and Tyr182), rather than at Thr322. α- and β-Naphthoflavone repressed p38MAPK phosphorylation at Thr180 and Tyr182, suggesting that α- and β-Naphthoflavone inhibition of H₂O₂-led SH-SY5Y cell apoptosis is partly dependent on p38MAPK signaling. However, it is unclear whether Naphthoflavone is inhibiting p38MAPK activation completely depends on repressing OS and its action on p38MAPK phosphorylation, or only partly depends. The latter suggests that Naphthoflavone may potentially inhibit p38MAPK activation through direct interference of its upstream signaling. To understand the mechanisms involved, future experiments to determine how dependent cell apoptosis in SH-SY5Y cells is on p38MAPK signaling are required.

Under OS conditions, numerous apoptosis-associated factors were upregulated or activated. Upregulated Bax and the released Cyt C from the mitochondria are tightly associated to mitochondrial dysfunction-mediated apoptosis (34). Conventional western blot assays are unable to examine proteins present in the mitochondria as the organelle is not lysed in this setting, and lysing mitochondria requires more specific conditions. However, a clear upregulation of Cyt C observed in the western blot analysis in the present study indicates that Cyt C was released from the mitochondria in large quantities. Caspase-3 activation is dependent on the cleavage of its inactive form, and is widely regarded as a key step in apoptosis execution (34). Notably, α- and β-Naphthoflavone individually and synergistically suppressed the expression of Bax and Cyt C and Caspase-3 activation, further confirming that Naphthoflavone may inhibit OS-led neuronal apoptosis.

The present results indicate that β-Naphthoflavone is more effective against OS-led neuron damage compared with α-Naphthoflavone. α-Naphthoflavone only induced a minor change to H₂O₂-inhibited SH-SY5Y cell viability, but it was markedly reversed by β-Naphthoflavone. Furthermore, the post-treatment with β-Naphthoflavone led to a reduced apoptosis rate compared with α-Naphthoflavone post-treatment. β-Naphthoflavone is more efficient at upregulating the activity of CAT, as well as inhibiting Cyt C released from the mitochondria compared with α-Naphthoflavone. Further differences between the mechanisms underlying α- and β-Naphthoflavone against OS-led neuron damage may potentially exist, as combined α- and β-Naphthoflavone had a more marked effect in this regard compared with α- or β-Naphthoflavone individually. Moreover, increasing doses did not continue to promote cell viability once the doses of α- and β-Naphthoflavone reached 20 and 10 µM, respectively. An improved understanding of the mechanisms by which α- and β-Naphthoflavone counteract OS-led neuron damage is necessary in future study. Furthermore, co-treatment with α- and β-Naphthoflavone abrogated H₂O₂-induced apoptosis rate elevation and cell viability reduction. This suggests a synergetic effect between α- and β-Naphthoflavone. Further analysis demonstrated that the activities of antioxidant enzymes including CAT, SOD and GPx, were inhibited by increased H₂O₂, but enhanced by α- and/or β-Naphthoflavone. H₂O₂-stimulated p38MAPK activation was repressed by α- and/or β-Naphthoflavone, along with a decreased expression of the apoptosis-related factors and inhibited caspase-3 activation.

In conclusion, co-treatment with α- and β-Naphthoflavone minimized H₂O₂-induced neuron damage compared with treatment with α- or β-Naphthoflavone individually. Therefore, it is advocated that utilizing α- and β-Naphthoflavone together in the clinical setting of neurological disease treatment may be beneficial.

Acknowledgements

The present study was supported by the Research Project of Health and Family Planning Commission of Hunan Province (grant no. B2016196).

References 1

Weaver

Liu

Does normobaric hyperoxia increase oxidative stress in acute ischemic stroke? A critical review of the literature

Med Gas Res5112015

10.1186/s13618-015-0032-4

26306184

Amara

Berbenni

Fragni

Leoni

Viggiani

Ippolito

Larocca

Rossano

Alberghina

Riccio

Colangelo

Neuroprotection by cocktails of dietary antioxidants under conditions of nerve growth factor deprivation

Oxid Med Cell Longev20152172582015

10.1155/2015/217258

26236423

Choong

Say

Neuroprotection of α-synuclein under acute and chronic rotenone and maneb treatment is abolished by its familial Parkinson's disease mutations A30P, A53T and E46K

Neurotoxicology328578632011

10.1016/j.neuro.2011.05.012

21658409

Tchantchou

Graves

Ortiz

Rogers

Shea

Dietary supplementation with apple juice concentrate alleviates the compensatory increase in glutathione synthase transcription and activity that accompanies dietary- and genetically-induced oxidative stress

J Nutr Health Aging84924962004

15543422

Villegas-Rivera

Román-Pintos

Cardona-Muñoz

Arias-Carvajal

Rodríguez-Carrizalez

Troyo-Sanromán

Pacheco-Moisés

Moreno-Ulloa

Miranda-Díaz

Effects of ezetimibe/simvastatin and rosuvastatin on oxidative stress in diabetic neuropathy: A randomized, double-blind, placebo-controlled clinical trial

Oxid Med Cell Longev20157562942015

10.1155/2015/756294

26290682

Yang

Zhu

Chen

Sun

Tan

Kang

Alteration of antioxidant enzymes and associated genes induced by grape seed extracts in the primary muscle cells of goats in vitro

PLoS One9e1076702014

10.1371/journal.pone.0107670

25238394

Soundararajan

Wishart

Rupasinghe

Arcellana-Panlilio

Nelson

Mayne

Robertson

Quercetin 3-glucoside protects neuroblastoma (SH-SY5Y) cells in vitro against oxidative damage by inducing sterol regulatory element-binding protein-2-mediated cholesterol biosynthesis

J Biol Chem283223122452008

10.1074/jbc.M703583200

18032389

Wang

Qin

Bai

Rong

Deng

Diallyl trisufide protects against oxygen glucose deprivation-induced apoptosis by scavenging free radicals via the PI3K/Akt-mediated Nrf2/HO-1 signaling pathway in B35 neural cells

Brain Res161438502015

10.1016/j.brainres.2015.04.014

25896937

Wang

Qiu

Shao

Chen

Tert-butylhydroquinone alleviates early brain injury and cognitive dysfunction after experimental subarachnoid hemorrhage: Role of Keap1/Nrf2/ARE pathway

PLoS One9e976852014

10.1371/journal.pone.0097685

24848277

Singh

Agrawal

Doré

Neuroprotective properties and mechanisms of resveratrol in vitro and in vivo experimental cerebral stroke models

ACS Chem Neurosci4115111622013

10.1021/cn400094w

23758534

Bournival

Plouffe

Renaud

Provencher

Martinoli

Quercetin and sesamin protect dopaminergic cells from MPP+-induced neuroinflammation in a microglial (N9)-neuronal (PC12) coculture system

Oxid Med Cell Longev20129219412012

10.1155/2012/921941

22919443

Mena

Casarejos

Solano

Rodríguez-Navarro

Gómez

Rodal

Medina

de Yebenes

NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice

FEBS Lett5831681742009

10.1016/j.febslet.2008.11.051

19084014

Jazvinšćak Jembrek

Hof

Šimić

Ceramides in Alzheimer's disease: Key mediators of neuronal apoptosis induced by oxidative stress and Aβ accumulation

Oxid Med Cell Longev20153467832015

10.1155/2015/346783

26090071

Leonardo

Doré

Dietary flavonoids are neuroprotective through Nrf2-coordinated induction of endogenous cytoprotective proteins

Nutr Neurosci142262362011

10.1179/1476830511Y.0000000013

22005287

Lin

Wang

Kuo

Lee

Tseng

Ethyl acetate extract of Wedelia chinensis inhibits tert-butyl hydroperoxide-induced damage in PC12 cells and D-galactose-induced neuronal cell loss in mice

BMC Complement Altern Med144912014

10.1186/1472-6882-14-491

25510435

Zhu

Meng

McGeer

Zhu

Nakanishi

The neuroprotective effects of ratanasampil on oxidative stress-mediated neuronal damage in human neuronal SH-SY5Y cells

Oxid Med Cell Longev20157923422015

10.1155/2015/792342

26064424

Dhawan

Drug/substance reversal effects of a novel tri-substituted benzoflavone moiety (BZF) isolated from Passiflora incarnata Linn.-a brief perspective

Addict Biol83793862003

10.1080/13556210310001646385

14690874

Yang

Kimatu

Fang

Pei

Identification of flavonoids from Flammulina velutipes and its neuroprotective effect on pheochromocytoma-12 cells

Food Chem2042742822016

10.1016/j.foodchem.2016.02.138

26988502

Nannelli

Rossignolo

Tolando

Rossato

Longo

Gervasi

Effect of beta-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig

Toxicology26569792009

10.1016/j.tox.2009.09.010

19786062

Morrissy

Strom

Purdom-Dickinson

Chen

NAD(P)H: Quinone oxidoreductase 1 is induced by progesterone in cardiomyocytes

Cardiovasc Toxicol121081142012

10.1007/s12012-011-9144-9

21947872

Higgins

Hayes

Mechanisms of induction of cytosolic and microsomal glutathione transferase (GST) genes by xenobiotics and pro-inflammatory agents

Drug Metab Rev43921372011

10.3109/03602532.2011.567391

21495793

Reed

Cawley

Backes

Inhibition of cytochrome P450 1A2-mediated metabolism and production of reactive oxygen species by heme oxygenase-1 in rat liver microsomes

Drug Metab Lett56162011

10.2174/187231211794455253

20942796

Hsu

Liou

Cheng

Peng

Lin

Chu

Luo

Huang

Jiang

Beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation

Pharmacol Res1021921992015

10.1016/j.phrs.2015.10.001

26453957

Milardovic

Kereković

Derrico

Rumenjak

A novel method for flow injection analysis of total antioxidant capacity using enzymatically produced ABTS*+ and biamperometric detector containing interdigitated electrode

Talanta712132202007

10.1016/j.talanta.2006.03.042

19071291

Tsikas

Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges

Anal BiochemOct242016(Epub ahead of print)

10.1016/j.ab.2016.10.021

Park

Lee

Park

Jeon

Cho

Neuroprotective effects of Liriope platyphylla extract against hydrogen peroxide-induced cytotoxicity in human neuroblastoma SH-SY5Y cells

BMC Complement Altern Med151712015

10.1186/s12906-015-0679-3

26054856

Liao

Chang

Lin

Tse

Cheng

Anti-ageing effects of alpha-naphthoflavone on normal and UVB-irradiated human skin fibroblasts

Exp Dermatol215465482012

10.1111/j.1600-0625.2012.01517.x

22716253

Mense

Singh

Remotti

Liu

Bhat

Vitamin C and alpha-naphthoflavone prevent estrogen-induced mammary tumors and decrease oxidative stress in female ACI rats

Carcinogenesis30120212082009

10.1093/carcin/bgp093

19406931

Xiao

Xue

Xin

Qian

Xie

Gong

Ren

Beta-Naphthoflavone protects mice from aristolochic acid-I-induced acute kidney injury in a CYP1A dependent mechanism

Acta Pharmacol Sin30155915652009

10.1038/aps.2009.156

19890363

Sinha

Muthiah

Jiang

Couroucli

Barrios

Moorthy

Attenuation of hyperoxic lung injury by the CYP1A inducer beta-naphthoflavone

Toxicol Sci872042122005

10.1093/toxsci/kfi226

15958656

Kober

Meyer-Alert

Grienitz

Hollert

Frohme

Intact cell mass spectrometry as a rapid and specific tool for the differentiation of toxic effects in cell-based ecotoxicological test systems

Anal Bioanal Chem407772177312015

10.1007/s00216-015-9161-9

26255296

Csepregi

Neugart

Schreiner

Hideg

Comparative evaluation of total antioxidant capacities of plant polyphenols

Molecules21pii.E2082016

10.3390/molecules21020208

26867192

Orozco

Wang

Keen

Chronic consumption of a flavanol- and procyanindin-rich diet is associated with reduced levels of 8-hydroxy-2′-deoxyguanosine in rat testes

J Nutr Biochem141041102003

10.1016/S0955-2863(02)00273-5

12667602

Burri

Kim

Fang

Chang

Perkins

Harris

Davis

Thompson

Bhalla

‘Loop’ domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity and apoptosis

Int J Radiat Oncol Biol Phys434234301999

10.1016/S0360-3016(98)00385-X

10030271

Figure 1.

Naphthoflavone reverses H₂O₂-inhibited neuron SH-SY5Y cell viability. (A) Neuron SH-SY5Y cell viability decreased by H₂O₂ in a dose-dependent manner, with half the cell viability loss at 20 µM H₂O₂. (B) H₂O₂-inhibited neuron SH-SY5Y cell viability increased in a dose-dependent manner with α-Naphthoflavone, until the viability reached the plateau at a dose of 20 µM α-Naphthoflavone. (C) H₂O₂-inhibited neuron SH-SY5Y cell viability increased with β-Naphthoflavone concentration, 0–10 µM, followed by a slight decrease. (D) Neuron SH-SY5Y cell viability following different treatments. α- and β-Naphthoflavone at 20 and 10 µM, respectively, were used individually and in combination following exposure of neuron SH-SY5Y cells to 20 µM H₂O₂. *P<0.05 vs. NC; ^#P<0.05 vs. PC (n=8). NC, negative control, cells without any treatment; PC, positive control, cells treated with 20 µM H₂O₂ only; α-, β-Naphthoflavone, co-treatment with 20 µM α-Naphthoflavone and 10 µM β-Naphthoflavone; SB203580, p38MAPK inhibitor; H₂O₂, hydrogen peroxide.

Figure 2.

Naphthoflavone decreases elevation of apoptosis in H₂O₂-induced elevation in neuron SH-SY5Y cells. α- and β-Naphthoflavone at a dose of 20 and 10 µM, respectively, were used individually and in combination following exposure of neuron SH-SY5Y cells to 20 µM H₂O₂. Apoptosis rate was (A) detected by flow cytometry and (B) quantified. *P<0.05, **P<0.05 vs. NC; ^#P<0.05, ^##P<0.01 vs. PC (n=4). NC, negative control, cells without any treatment; PC, positive control, cells treated with 20 µM H₂O₂ only; α-, β-Naphthoflavone, co-treatment with 20 µM α-Naphthoflavone and 10 µM β-Naphthoflavone; SB203580, p38MAPK inhibitor; H₂O₂, hydrogen peroxide.

Figure 3.

Oxidative or antioxidative status of neuron SH-SY5Y cells following different treatments. α- and β-Naphthoflavone at a dose of 20 and 10 µM, respectively, were used individually and in combination following exposure of neuron SH-SY5Y cells to 20 µM H₂O₂. The oxidative or antioxidative status of neuron SH-SY5Y cells following treatment with (A) TAC, (B) MDA, (C) CAT, (D) SOD and (E) GPx. *P<0.05, **P<0.05 vs. NC; ^#P<0.05, ^##P<0.01 vs. PC (n=4). NC, negative control, cells without any treatment; PC, positive control, cells treated with 20 µM H₂O₂ only; α-, β-Naphthoflavone, co-treatment with 20 µM α-Naphthoflavone and 10 µM β-Naphthoflavone; SB203580, p38MAPK inhibitor; TAC, total antioxidant capacity; MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase; GPx, glutathione peroxidase.

Figure 4.

Expression levels of proteins following different treatments. α- and β-Naphthoflavone at a dose of 20 and 10 µM, respectively, were used individually and in combination following exposure of neuron SH-SY5Y cells to 20 µM H₂O₂. (A) Western blot analysis indicates p38MAPK phosphorylation at Thr180 and Tyr182. The protein expression of (B) Bax (C) cytochrome c and (D) caspase-3 following different treatments. *P<0.05, **P<0.05 vs. NC; ^#P<0.05, ^##P<0.01 vs. PC (n=4,). NC, negative control, cells without any treatment; PC, positive control, cells treated with 20 µM H₂O₂ only; α-, β-Naphthoflavone, co-treatment with 20 µM α-Naphthoflavone and 10 µM β-Naphthoflavone; SB203580, p38MAPK inhibitor; p-p38, phosphorylated p38; H₂O_2, hydrogen peroxide.