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Introduction

Parkinson's disease (PD) is an age-related neurodegenerative disorder that is characterised by hypokinesia (1). An oxidative stress insult is one of the principal causes that is associated with the profound loss of dopamine (DA)-producing neurons in the substantia nigra pars compacta (2,3). Even though the mechanisms underlying neuronal damage remain unclear, apoptosis or oxidative stress, as crucial contributors to the pathogenesis of PD, have been reported (4–8). Oxidative stress-induced cell lesions are usually induced by hydrogen peroxide (H2O2), hydroxyl radicals and superoxide, which are known as reactive oxygen species (ROS) and are generated through metabolic processes in cells. The oxidative stress in vitro model has been proposed by using H2O2 as an inducer when it is added to the cell culture medium (9).

The Bcl-2 family, as regulatory proteins, are vital apoptosis-related factors in H2O2-induced oxidative stress in vitro models. Among this family, Bcl-2 is an anti-apoptotic protein and forms a heterodimer with Bax, an apoptotic activator, to control the fate of cells (10,11). The ratio of Bax to Bcl-2 protein is a common indicator with which to determine whether a cell is alive or is dead via apoptosis. In addition, the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun kinases (JNKs) and p38 kinases have been reported to participate in the regulation of apoptosis. ERK1/2, p38 and JNK are activated when a cell is injured by oxidative stress or a pro-inflammatory environment (12,13). Abnormal levels of phosphorylated JNK, p38 and ERK1/2 in the brains of patients with Alzheimer's disease (AD) are associated with oxidative stress (14). Consequently, the illumination of the biochemical processes surrounding H2O2-mediated neuronal apoptosis may aid in the understanding of the pathogenesis of neurodegenerative diseases and in discovering new drug targets for the treatment of diseases.

α-synuclein, a protein highly expressed in the human brain, is localised in the inner membrane of the mitochondria. α-synuclein not only dose-dependently inhibits complex I activity of the mitochondrial respiratory chain, but also aggregates to form insoluble fibrils in PD characterised by Lewy bodies (15). Tyrosine hydroxylase (TH) catalyses the synthesis of catecholamines in the rate-limiting step. Alterations in TH activity may be involved in PD. TH may help to produce H2O2 and other ROS in pathological conditions. Nevertheless, TH is also a possible target for the damaging alterations induced by ROS or may be a target for radical-mediated injury (16). It has been proposed that the abnormal expression of TH induced by oxidative damage leads to a reduction in DA levels, which is associated with the degeneration of dopaminergic neurons in PD (17). Therefore, α-synuclein and TH may be novel drug targets.

In recent years, natural substances extracted from plants have attracted increasing attention due to their unique biological activities, such as neuroprotective potential that can protect cells from oxidative damage. A number of Chinese herbal effects have been evaluated and have been shown to exert beneficial effects in various models related to PD (18,19), suggesting that herbs, as drug candidates, have a bright future in the treatment of PD. Astragaloside IV (AS-IV), an ingredient extracted from Astra galus membranaceus, is frequently used as a food additive and in herbal medicine. AS-IV is included in some efficacious medicinal prescriptions and as a supplement in various health foods (20). AS-IV has been shown to exert anti-hypertensive (21), positive inotropic (22), anti-inflammatory (23) and anti-infarction effects (24). Despite the use of Astragalus membranaceus as a traditional therapy for degenerative diseases in China, few scientific studies investigating the antioxidant mechanism of AS-IV in neurons have been reported to date, at least to the best of our knowledge. Moreover, further research is required in order to fully examine the effects of the antioxidant activity of AS-IV.

Thus, the aims of the present study were to evaluate the neuroprotective effects of AS-IV in vitro using SH-SY5Y cells exposed to H2O2 and to discover novel targets of AS-IV. Our findings demonstrate that AS-IV protects the cells from oxidative damage by downregulating the Bax/Bcl-2 ratio. The effects of AS-IV were also mediated via the downregulation of the expression of α-synuclein and the increase in TH expression via the p38 signalling pathway. To the very best of our knowledge, this is a fundamental new discovery of the mechanisms through which AS-IV protects neuronal cells from damage.

Materials and methods

Chemicals and reagents

AS-IV (Fig. 1), with a purity >98%, was obtained from the Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China). Bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), vitamin C (Vit C) and H2O2 were all purchased from Sigma-Aldrich (St. Louis, MO, USA). The Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from KeyGen Biotech Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium/F12 (DMEM/F12) were both purchased from Gibco (Grand Island, NY, USA). Anti-α-synuclein antibody (#ab138501) was purchased from Epitomics (Burlingame, CA, USA). Anti-β-actin (#3700), anti-Bcl-2 (#15071) and anti-Bax (#5023) monoclonal antibodies were all purchased from Cell Signalling Technology, Inc. (Beverly, MA, USA). Anti-p38 mitogen-activated protein kinase (MAPK; sc-7972), anti-p-p38 (sc-17852-R), anti-p-JNK (sc-293136), anti-ERK1/2 (sc-514302), anti-p-ERK1/2 (sc-16981-R) and anti-TH (sc-7847) mono clonal antibodies were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All of the other reagents were of analytical grade.

Figure 1 Chemical structure of astragaloside IV (AS-IV). Molecular formula, C41H68O14; molecular weight, 784.9702 g/mol.

Cell culture

The SH-SY5Y cell line (#CRL-2266, ATCC, Rockville, MD, USA) was cultured in DMEM supplemented with penicillin (final concentration, 100 U/ml), streptomycin (final concentration, 0.1 mg/ml) and 10% (v/v) FBS in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The SH-SY5Y cells were plated in 6-well plates for assays and 96-well plates (for MTT assay) at a density of approximately 1×104 cells/well. After 24 h, the cells were treated with various concentrations of AS-IV (50-200 µmol/l containing 0.1% DMSO) for 24 h and were then exposed to the same fresh medium containing 300 µmol/l H2O2 for 4 h.

Determination of cell viability

By means of MTT assay, we first assessed the drug treatment toxicity. The cells (8,000 cells/well) were plated in 96-well microplates and grown in DMEM for 24 h and exposed to various concentrations of H2O2 (100, 200, 300 and 400 µmol/l).

The effects of various concentrations of AS-IV on cell viability were analysed following treatment for 24 h. The medium was then replaced by 200 µl of fresh medium plus 10 µl of the MTT solution (1 mg/ml). The microplates were incubated at 37°C in 5% CO2 for 4 h. The precipitated formazan was dissolved in 100 µl DMSO. The optical density of the samples was measured at 492 nm. The untreated SH-SY5Y cells were used as controls. For pre-treatment with vitamin C (Vit C), the SH-SY5Y cells were treated with 200 mg/l Vit C (A8100; Solarbio, Beijing, China) for 1 h and then exposed to H2O2 for 4 h.

Cell morphological observation

Following exposure to 300 µmol/l H2O2, the SH-SY5Y cells were observed under a phase-contrast microscope (TE2000-U; Nikon, Tokyo, Japan). Moreover, the SH-SY5Y cells were observed under a microscope following exposure to H2O2 and DAPI staining.

Detection of apoptotic cells by flow cytometry

The cell apoptotic rate following exposure to H2O2 was assayed by flow cytometry using the Annexin V-FITC/propidium iodide (PI) double-labelling method. The SH-SY5Y cells (1×105 cells/ml) were seeded in 60-mm dishes and treated with AS-IV and H2O2 (300 µmol/l). The cells were trypsinised and collected by centrifugation at 800 rpm for 5 min. An Annexin V-FITC apoptosis detection kit was used to double-stain the cells according to the manufacturer's instructions. The samples were analysed using a FACSVantage SE flow cytometer (BD Biosciences, San Jose, CA, USA).

Measurement of intracellular ROS levels

The SH-SY5Y cells (4×104 cells/ml) were seeded in 6-well plates for 48 h. The cells were treated with AS-IV for 24 h prior to exposure to H2O2 (300 µmol/l) for 4 h. Following stimulation, the cells were incubated with 10 µmol/l DCFH-DA, which was oxidised to the highly fluorescent compound, DCF, at 37°C for 30 min and, the cells were then washed 3 times with phosphate-buffered saline (PBS). The fluorescence intensity, expressed as the intracellular ROS, was measured with a FACSVantage SE flow cytometer.

Immunofluorescence staining

The immunofluorescence staining technique was performed as follows: briefly, the cells, on coverslips, were fixed with a 4% paraformaldehyde solution for 20 min and were permeabilised with 0.5% Triton X-100 in PBS for 15 min, and the background was blocked with 5% BSA in PBS for 1 h before staining with primary and secondary antibodies. Primary antibodies to α-synuclein (1:100; #ab138501; rabbit anti-α-synuclein monoclonal antibody; Epitomics) and TH (1:100; sc-7847; goat anti-TH monoclonal antibody; Santa Cruz Biotechnology, Inc.) were diluted by 0.5% Triton X-100 and incubated with the cells for 90 min at 37°C. A secondary FITC-conjugated goat-anti-rabbit IgG (1:100; sc-2012; Santa Cruz Biotechnology, Inc.) and a TRITC-conjugated mouse-anti-goat IgG (sc-516243) were diluted in 0.5% Triton X-100 and were incubated with the cells for 60 min at room temperature. The cells were then stained with DAPI for 3 min. The slides were rinsed with PBS after each step. The slides were mounted with glycerol and stored for detection.

Western blot analysis

The SH-SY5Y cells (2×106 cells/well) were seeded and treated with 10 µmol/l SB203580 (S8307; Sigma-Aldrich) for 1 h or various concentrations of AS-IV for 24 h prior to exposure to H2O2 (300 µmol/l) for 4 h. The treated cells were collected and were resuspended in a lysis buffer (RIPA buffer with protease inhibitor cocktail) at 4°C for 2 h. The lysate was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was then collected. The Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a UV spectrophotometer were used to equalize the protein loading. Equal amounts of protein (40 µg) were subjected to 12% SDS-PAGE and were transferred onto polyvinylidene difluoride membranes. Blocking buffer [5% (v/v) non-fat dry-milk in TBS containing 0.1% Tween-20 (TBST), pH 7.5] was used to treat the membranes for 1 h at room temperature. The membranes were then incubated with primary antibodies to Bcl-2 (1:1,000), Bax (1:1,000), α-synuclein (1:1,000), TH (1:1,000), p38 (1:1,000), p-p38 (1:1,000), ERK1/2 (1:1,000), p-ERK1/2 (1:1,000), p-JNK (1:1,000) and β-actin (1:1,000) overnight at 4°C and were then incubated with HRP-conjugated rabbit or mouse IgG secondary antibodies (1:1,000) for 1 h. The secondary antibodies HRP-conjugated goat-anti-rabbit IgG (sc-2004), HRP-conjugated goat-anti-mouse IgG (sc-2005) and HRP-conjugated mouse anti-goat IgG (sc-2354) were obtained from Santa Cruz Biotechnology, Inc. The proteins were detected by an enhanced chemiluminescence (ECL) solution (Amersham Biosciences, Buckinghamshire, UK), and densitometric analysis was performed with the use of a PDI ImageWare System (Bio-Rad Laboratories, Inc.).

Statistical analysis

The results were analysed using a one-way analysis of variance (one-way ANOVA) followed by the Tukey's test to examine the effects of the different drug concentrations. The data are expressed as the means ± SEM. A Dunnett's test and an ANOVA followed by a Bonferroni correction were performed to determine the statistical significance. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

Protective effects of AS-IV against H2O2-induced SH-SY5Y cell damage

We initially examined the viability of the SH-SY5Y cells, which was examined by MTT assay, after incubating the cells for 2, 4 and 6 h with H2O2 in the absence or presence of AS-IV for 24 h. As shown in Fig. 2A, exposure to H2O2 induced a gradual reduction in cell viability in a time- and dose-dependent manner. The cell viability was approximately 57% of the control value at 4 h following H2O2 exposure (300 µmol/l). Therefore, for all the subsequent experiments, a concentration of H2O2 at 300 µmol/l was used for the cell damage model.

Figure 2 Effect of astragaloside IV (AS-IV) on the viability of cells exposed to hydrogen peroxide (H2O2) in a time- and concentration-dependent manner. The viability of the cells was determined by MTT assay after incubating the cells for 2, 4 and 6 h with H2O2 for 24 h. (A) Cell viability at 4 h after H2O2 exposure (300 µmol/l). (B) Cell viability group in the absence or (C) presence of H2O2 exposure. (D) DAPI staining showing nuclear fragmentation and nuclear DNA condensation following exposure to 300 µmol/l H2O2, AS-IV and vitamin C (Vit C) at 200 mg/l. Medium with 0.1% DMSO was used as a standard to normalise the results. The data are the means ± SEM (n=3). ##P<0.01 vs. control (untreated cells); **P<0.05 vs. H2O2 (300 µmol/l).

As shown in Fig. 2B, cell viability was not affected by AS-IV at a concentration of up to 200 µmol/l. After the SH-SY5Y cells were treated with AS-IV (50–200 µmol/l) for 24 h and then exposed to H2O2 (300 µmol/l) for 4 h, cell viability was increased with the increasing concentrations of AS-IV compared with the control group. As shown in Fig. 2C, cell viability following exposure to H2O2 for only 4 h was 59% of the control value. However, treatment with AS-IV (50, 100 and 200 µmol/l) for 24 h prior to exposure to H2O2 increased cell viability to 70, 71 and 75%, respectively, which was similar to the activity of Vit C, a drug that was used as a positive control. Evidently, AS-IV was effective in protecting the SH-SY5Y cells against H2O2-induced injury.

We also assessed the cell morphological changes using a phase-contrast microscope. Exposure to 300 µmol/l H2O2 for 4 h clearly induced the aggregation and shrinkage of cell bodies and reduced the number of SH-SY5Y cells. However, treatment with AS-IV (200 µmol/l) prior to H2O2 exposure significantly prevented the morphological manifestations of cell damage (Fig. 2D, upper panel). DAPI staining also revealed that nuclear fragmentation and nuclear DNA condensation occurred following exposure to 300 µmol/l H2O2; however, pre-treatment with AS-IV inhibited these apoptotic features, exerting effects similar to those of Vit C at 200 mg/l (Fig. 2D, bottom panel). These results suggested that AS-IV exerted an anti-apoptotic effect in SH-SY5Y cells exposed to H2O2.

AS-IV inhibits the H2O2-induced apoptosis of SH-SY5Y cells

The inhibitory effects of AS-IV on the apoptosis of SH-SY5Y cells induced by H2O2 were assessed by flow cytometry. As shown in Fig. 3A, the percentage of apoptotic cells increased to 33.2% following exposure to 300 µmol/l H2O2 for 4 h. However, treatment with AS-IV (50, 100 and 200 mg/l) for 24 h prior to H2O2 exposure prevented apoptosis in a concentration-dependent manner, and the rate of apoptosis decreased to 28.9, 22.6 and 14.8%, respectively. AS-IV at 200 mg/l exerted similar effects to those of Vit C. Moreover, incubation with AS-IV alone for 24 h had no effects on the cell apoptotic ratio of the SH-SY5Y cells (data not shown).

Figure 3 Astragaloside IV (AS-IV) inhibits hydrogen peroxide (H2O2)-induced apoptosis and attenuates reactive oxygen species (ROS) production stimulated by H2O2 (300 µmol/l). (A) The results of flow cytometry show that pre-treatment with AS-IV (50–200 mg/l) for 24 h prior to H2O2 exposure decreased apoptosis and (B) suppressed the production of DCFH fluorescence (for ROS production) in concentration-dependent manner. The medium was used as a standard to normalise the results. The data are shown as the means ± SEM (n=3). **P<0.01 vs. control (untreated cells); #P<0.05 vs. H2O2 (300 µmol/l); ##P<0.01 vs. H2O2 (300 µmol/l).

AS-IV inhibits ROS production in SH-SY5Y cells induced by H2O2

Intracellular ROS plays a crucial role in oxidative stress-induced cell damage; thus, the effects of AS-IV on H2O2-induced ROS production in SH-SY5Y cells were assessed by flow cytometry. As shown in Fig. 3B, the cells with fluorescence induced by 300 µmol/l H2O2 for 4 h displayed a 6.5-fold increase greater than that of the control group (P<0.05). By contrast, tre atment with AS-IV for 24 h prior to exposure to H2O2 suppressed the production of DCFH fluorescence (for ROS production) in a concentration-dependent manner. The inhibitory effects of AS-IV at 200 µmol/l were significant (53.8% compared to the H2O2-exposed group not treated with AS-IV (P<0.05) and were similar to the effects of Vit C at 200 mg/l.

AS-IV decreases the H2O2-induced increase in the Bax/Bcl-2 ratio in SH-SY5Y cells

The levels of Bax and Bcl-2 were determined by western blot analysis as Bcl-2 family members are involved in apoptosis. As shown in Fig. 4A, compared with the control, Bax protein expression was upregulated in the H2O2-exposed group, while AS-IV treatment downregulated Bax expression. The levels of Bcl-2 decreased in the H2O2 group and increased following AS-IV pre-treatment. The Bax/Bcl-2 ratio wass increased 4.2-fold of the normal control group upon exposure to H2O2, whereas this increase was attenuated in the cells pre-treated with 50–200 µmol/l AS-IV.

Figure 4 Astragaloside IV (AS-IV) decreases the hydrogen peroxide (H2O2) -induced increase in the Bax/Bcl-2 ratio, decreases the expression of α-synuclein (α-syn) and increases the expression of tyrosine hydroxylase (TH) in SH-SY5Y cells. The expression of Bax and Bcl-2 was determined by western blot analysis. (A) Bax/Bcl-2 ratio following exposure to H2O2 and pre-treatment with 50–200 µmol/l of AS-IV. (B) Western blot nalysis and (C) a double immunofluorescence assay showing the expression of α-syn and TH in SH-SY5Y cells exposed to H2O2. The data are the means ± SEM (n=3). **P<0.01 vs. control (untreated cells); ##P<0.05 vs. H2O2 (300 µmol/l).

AS-IV decreases the expression of α-synuclein and increases the expression of TH in SH-SY5Y cells

α-synuclein is a major component of Lewy bodies that plays an important role in H2O2-induced apoptosis in neurons (25). Thus, to determine whether the observed neuroprotective effects of AS-IV were associated with the expression of α-synuclein, we performed western blot analysis using a monoclonal rabbit antibody against α-synuclein. As shown in Fig. 4B, when the cells were exposed to H2O2, the expression of α-synuclein significantly increased 2.25-fold compared with the control group. By contrast, pre-treatment with AS-IV (50, 100 and 200 µmol/l) markedly decreased the expression of α-synuclein by 22.2, 27.1 and 42.2%, respectively, compared with the H2O2-exposed cells. To further confirm this effect, we performed a double immunofluorescence assay using a monoclonal rabbit antibody against α-synuclein. As shown in Fig. 4C, the results were consistent with those of western blot analysis. These results suggest that AS-IV inhibited the overexpression of α-synuclein induced by H2O2. Consequently, we hypothesized that the neuroprotective effects of AS-IV against oxidative stress-induced damage are mediated by the decrease in the expression of α-synuclein.

Previous studies have shown that α-synuclein, implicated in the pathogenesis of PD, is involved in the regulation of DA metabolism, possibly by downregulating the expression of TH (26). TH catalyses the rate-limiting step in the biosynthesis of the catecholamines, DA, norepinephrine and epinephrine (27). Therefore, TH expression was also evaluated by double immunofluorescence assay. As shown in Fig. 4C, pre-treatment with AS-IV prevented the H2O2-induced downregulation of TH levels in the SH-SY5Y cells. The results indicated that AS-IV treatment inhibited H2O2-induced α-synuclein upregulation.

AS-IV attenuates the H2O2-induced phosphorylation of p38 in SH-SY5Y cells

To clarify the mechanisms underlying the neuroprotective effects of AS-IV, we evaluated the expression of p-p38, p-JNK and p-ERK to determine whether the protective effects of AS-IV are mediated via the MAPK signalling pathway. Our results revealed that the levels of phosphorylated p38 increased (Fig. 5A); however, no effect was observed on the levels of phosphorylated ERKl/2 (Fig. 5B) and phosphorylated JNK (Fig. 5C) in the H2O2-exposed cells. By contrast, AS-IV (200 µmol/l) pre-treatment markedly decreased the levels of phosphorylated p38 compared with those in the untreated cells exposed to H2O2 (Fig. 5A).

Figure 5 Astragaloside IV (AS-IV) decreases the hydrogen peroxide (H2O2)-induced phosphorylation of p38, but has no effect on the phosphorylation of c-Jun kinase (JNK) or extracellular signal-regulated kinase (ERK). Western blot analysis showing the levels of (A) phosphorylated p38, (B) phosphorylated ERK1/2 and (C) phosphorylated JNK in the H2O2-exposed groups. (A) Effects of AS-IV (200 µmol/l) pre-treatment on the levels of phosphorylated p38 compared with those of the H2O2 group. The data are the means ± SEM (n=3). **P<0.01 vs. control (untreated cells); #P<0.05 vs. H2O2 (300 µmol/l); ##P<0.01 vs. H2O2 (300 µmol/l).

To elucidate whether H2O2 stimulates α-synuclein expression via the p38 MAPK signalling pathway, a chemical inhibitor targeting p38 kinase (SB203580) was used. The SH-SY5Y cells were pre-incubated with SB203580 (10 µmol/l) for 1 h and were then exposed to H2O2 (300 µmol/l) for 4 h, and the levels of α-synuclein were determined. As shown in Fig. 6A, AS-IV (200 µmol/l) pre-treatment markedly decreased the expression of α-synuclein 0.73-fold compared with the untreated cells exposed to H2O2.

Figure 6 Chemical inhibitor targeting p38 kinase (SB203580) inhibits the expression of α-synuclein (α-syn) and increases the expression of tyrosine hydroxylase (TH) in SH-SY5Y cells. (A) Incubation with SB203580 in the SH-SY5Y cells decreased the expression of α-syn compared with the hydrogen peroxide (H2O2) group as shown by western blot analysis. (B) Results of the double immunofluorescence assay in SH-SY5Y cells. The data are the means ± SEM (n=3). **P<0.01 vs. control (untreated cells); #P<0.05 vs. H2O2 (300 µmol/l).

Of note, when the H2O2-exposed SH-SY5Y cells were incubated with SB203580 alone, the expression of α-synuclein was attenuated compared with the H2O2 group, and incubation of the cells with SB203580, H2O2 and AS-IV (200 µmol/l) together also decreased the expression of α-synuclein. We found identical results in the double immunofluorescence analysis, as shown in Fig. 6B. Thus, these results indicated that AS-IV protected the SH-SY5Y cells from oxidative stress-induced damage and that these effects were mediated via the decrease in the expression of α-synuclein through the p38 MAPK signalling pathway.

Discussion

PD is a neurodegenerative disorder resulting from the gradual and progressive loss of dopaminergic neurons in the substantia nigra. Accumulating evidence suggests that a pathological mechanism for this is that oxidative stress is implicated in different neurodegenerative diseases, such as PD and AD (28–32). H2O2, as an oxidative stress inducer, is widely used in vitro models (4,33). The human neuroblastoma cell line, SH-SY5Y, is extensively used as a cell model for researching neuronal cell death induced by H2O2 (34,35). In this study, H2O2-induced oxidative stress in SH-SY5Y cells was used to examine H2O2-induced neurotoxicity and the effects of AS-IV.

Although the exact mechanisms of oxidative stress are not completely clear, the use of antioxidant agents as a method of neuroprotection may be a potential treatment strategy for neurode generative diseases (36). AS-IV, as a major active constituent of Astragalus membranaceus, exerts multipotent effects under pathophysiological conditions. As previously demonstrated, AS-IV not only protected primary DA neurons from 6-OHDA-induced neurotoxicity and neurodegeneration, but also promoted dopaminergic neurite outgrowth (37). In our study, we demonstrated that AS-IV exerted protective effects against the H2O2-induced loss of cell viability. Similarly, the results of DAPI staining suggested that AS-IV prevented H2O2-induced morphological changes associated with apoptosis in the SH-SY5Y cells.

It is well known that ROS can injure the cardinal cellular components, such as proteins, DNA and lipids, resulting in subsequent cell death by necrosis or apoptosis. To provide further evidence, we examined the effects of AS-IV on cells by flow cytometry. When the SH-SY5Y cells were pre-treated with AS-IV for 24 h and were then cultured with 300 µmol/l H2O2 for 4 h, the percentage of apoptotic cells decreased and the production of ROS was also reduced compared with the cells exposed to H2O2 alone. Therefore, the protective effects of AS-IV may contribute to the antioxidant activity. However, the balance and maintenance of the intracellular redox state is largely dependent on the existance of glutathione, particularly the ratio of glutathione (GSH) to oxidized glutathione (GSSG). An increased GSSG-to-GSH ratio is an important indicator of oxidative stress. Some studies have analysed the status of GSH in SH-SY5Y cells induced by H2O2 (38–40). In this study, we wished to determine the mechanisms involved in the protective effects of AS-IV against oxidative stress. We focused on α-synuclein, a very important factor, and thus the levels and the status of GSH in the SH-SY5Y cells were not analysed in our study. α-synuclein is a 14-kDa soluble, intrinsically unfolded protein that is expressed in all neurons (41,42). The overexpression of α-synuclein may be associated with the selective dege neration and toxicity of dopaminergic neurons (43). TH, as a key enzyme, plays a critical role in the differentiation and survival of dopaminergic neurons. In this study, we found that AS-IV inhibited the overexpression of α-synuclein induced by H2O2 and increased the levels of TH. Our results provide new evidence that AS-IV protects cells from H2O2-induced injury and is associated with the expression of α-synuclein and TH. Cell survival depends mostly on the balance between the anti- and pro-apoptotic proteins of the Bcl-2 family in the early phases of the apoptotic cascade. Any changes in the balance between the anti- and pro-apoptotic proteins will affect cell death. Bax (pro-apoptotic group) and Bcl-2 (anti-apoptotic group) play a vital role in the apoptotic pathway. The ratio of Bax/Bcl-2 is a better predictor of apoptosis than the absolute expression of either Bcl-2 or Bax alone (44). Our results revealed that the expression of these Bcl-2 family members was influenced profoundly by H2O2. Treatment with AS-IV upregulated the expression of anti-apoptotic Bcl-2 and reduced the expression of pro-apoptotic Bax. Thereby, AS-IV decreased the H2O2-induced increase in the Bax/Bcl-2 ratio in SH-SY5Y cells.

ROS play a key role as a second messenger in signal transduction cascades and regulate signalling pathways. The intracellular redox state may lead to the activation of MAPKs (45). H2O2, as an oxidative stress inducer, may activate ERK and p38 proteins in cells, which may be closely related to MAPK signalling (46,47). In our experiments, treatment with AS-IV notably inhibited the H2O2-induced increase in phosphorylated p38 levels. We further found that the protective effects of AS-IV were associated with the expression of α-synuclein and TH through the p38 MAPK signalling pathway using chemical inhibitors targeting p38 kinase (SB203580).

In conclusion, the results of this study demonstrated that AS-IV decreased H2O2-induced cell damage, prevented cell morphologic changes, and decreased ROS production and the apoptotic rate. Furthermore, the neuroprotective effects of AS-IV against the H2O2-induced apoptosis of SH-SY5Y cells were associated with the downregulation of the Bax/Bcl-2 ratio, decreased levels of α-synuclein and increased levels of TH via the p38 MAPK signalling pathway. Our study may provide a novel therapeutic strategy for PD, although further research into the neuroprotective mechanisms of AS-IV is warranted.

Acknowledgments

We are grateful for the financial support from the National Nature Science Foundation of China (no. 81370403), Chongqing Foundation and Advanced Research Project (no. CSTC2015jcyjBX0053), and Chongqing Medical University Scientific Research Cultivating Fund (no. 201414).

References