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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.46 No.1 pp.15-22
DOI : https://doi.org/10.11620/IJOB.2021.46.1.15

Alpha-lipoic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced cell injury by inhibiting autophagy and apoptosis

Kyeong-Rok Kang1, Jae-Sung Kim1, Tae-Hyeon Kim1, Jeong-Yeon Seo2, HyangI Lim1, Jong-Hyun Park1, Kwang Yeol Yang1, Sun-Kyoung Yu1, Heung-Joong Kim1, Chun Sung Kim1, Hong Sung Chun2, Lee3, Park3, Kim1*
1The Institute of Dental Science, Chosun University, Gwangju 61452, Republic of Korea
2Department of Integrative Biological Sciences & BK21 FOUR Educational Research Group for Age-associated Disorder Control Technology, Chosun University, Gwangju 61452, Republic of Korea
3Laboratory for the Study of Regenerative Dental Medicine, Department of Oral Histology-Developmental Biology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 08826, Republic of Korea
*Correspondence to: Do Kyung Kim, E-mail: kdk@chosun.ac.kr
February 24, 2021 March 10, 2021 March 10, 2021

Abstract


Alpha-lipoic acid (ALA) is a naturally occurring antioxidant and has been previously used to treat diabetes and cardiovascular disease. However, the autophagy effects of ALA against oxidative stress-induced dopaminergic neuronal cell injury remain unclear. The aim of this study was to investigate the role of ALA in autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cell line. We examined SH-SY5Y phenotypes using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (cell viability/proliferation), 4′,6-diamidino-2-phenylindole dihydrochloride nuclear staining, Live/Dead cell assay, cellular reactive oxygen species (ROS) assay, immunoblotting, and immunocytochemistry. Our data showed ALA attenuated hydrogen peroxide (H2O2)-induced ROS generation and cell death. ALA effectively suppressed Bax up-regulation and Bcl-2 and BclxL down-regulation. Furthermore, ALA increased the expression of the antioxidant enzyme, heme oxygenase-1. Moreover, the expression of Beclin-1 and LC-3 autophagy biomarkers was decreased by ALA in our cell model. Combined, these data suggest ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.


Alpha-lipoic acid , Autophagy , Hydrogen peroxide , Neuronal cell

초록

    © The Korean Academy of Oral Biology. All rights reserved.

    This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Introduction

    Parkinson’s disease is a common neurodegenerative disease that occurs in about 1 in 100 elderly people 65 years of age or older [1]. It is characterized by the selective loss of substantia nigra dopaminergic neurons [1]. The most widely accepted mechanism of dopaminergic cell death in Parkinson’s disease is the vicious cycle of oxidative stress [1,2]. Hydrogen peroxide (H2O2) is a key reactive oxygen species (ROS) and can induce cell injury in a variety of cell types [3,4]. Recently, naturally occurring antioxidants are receiving great attention as they are recognized as safe and functional compounds for treating neurodegenerative diseases including Parkinson’s disease [1].

    Alpha-lipoic acid (ALA, Fig. 1), a naturally occurring antioxi- dant, has been used to treat diabetes and cardiovascular disease [5-7]. In particular, ALA is easily absorbed into the bloodstream and can easily cross through the blood-brain barrier, so it has been suggested that it can protect the central nervous system [8,9].

    Autophagy, a different type of self-destructive process from apoptosis, is a major cellular pathway essential for cell survival [10,11]. This process removes cellular waste products, degenerative proteins and degraded cell organelles [12,13]. The proteins and cell organelles to be removed are sequestered into a vesicle called an autophagosome made of a double membrane, and this vesicle is bound to the lysosome to form an autophagolysosome [12,13]. These are broken down by lysosomal enzymes, and the broken down substances are used for the energy needed to survive the cell or for the creation of new organelles [12,13]. In other words, autophagy is one of the recycling systems in the cell [12,13]. Although the cytoprotective role of autophagy has been demonstrated in various experimental models, autophagy has also been reported to induce or participate in cell injury in certain circumstances [12- 15].

    Therefore, the purpose of this study is to investigate the role of ALA on autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cells, and to reveal the protective effect of ALA on dopamine neurons. Ultimately, we would like to present the efficacy of ALA as a treatment for degenerative brain diseases including Parkinson’s disease. The SH-SY5Y cells are widely used to study the dopaminergic etiology due to represent a typical dopaminergic markers, such as tyrosine hydroxylase and dopamine transporters [1,4,16]. So that, the SH-SY5Y cells used in this study could be suitable model cells to study the role of ALA on H2O2-mediated dopaminergic cell injury. Our results showed that ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.

    Materials and Methods

    1. Materials

    ALA (Fig. 1), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), H2O2, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Sigma (St. Louis, MO, USA). The Live/Dead cell viability assay kit was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Antibodies against Bcl-2, Bcl-xL, Bax, heme oxygenase-1 (HO-1), Actin, and Beclin-1 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The antibody against LC-3 was obtained from MBL Inc. (Woburn, MA, USA).

    2. Cell culture and cell treatments

    The human dopaminergic nuronal cell line, SH-SY5Y, was cultured in Dulbecco’s Modifed Eagls’s Medium/Nutrient Mixture F-12 (DMEM/F12, Thermo Scientific, Rockfrod, IL, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and penicillin (100 U/mL)-streptomycin (100 μg/mL) at 37℃ in 5% CO2. When indicated, ALA was added 30 minutes prior to the treatment of H2O2. To prevent the direct interaction between ALA and H2O2 in the culture medium, at the end of the ALA pretreatment, the medium was changed to fresh low-serum DMEM/F12 medium.

    3. Cytotoxicity assay

    SH-SY5Y cells were seeded at a concentration of 1 × 104 cells per well in 48-well plates. After 24 hours growth, the cells were treated with ALA or H2O2 at various concentrations for 24 hours. The cell viability test was evaluated using the MTT assay. At least 3 separate experiments were performed on each concentration combination. The supernatant was subsequently removed, and MTT crystals were dissolved in 200 μL/well dimethyl sulfoxide. Thereafter, optical density was measured at 570 nm using a spectrometer.

    4. Cell Live/Dead assay

    SH-SY5Y cell survival was measured using green calcein AM and ethidium homodimer-1, which stain live and dead cells, respectively. To evaluate cell survival, SH-SY5Y cells were plated on chamber slides, stimulated with H2O2 for 24 hours with or without pretreatment with ALA for 30 minutes, and then stained with green calcein AM and ethidium homodimer- 1 according to the manufacturer’s protocol. Cells were then examined and imaged using fluorescence microscopy (Eclipse TE200; Nikon Instruments, Melville, NY, USA).

    5. DAPI staining

    SH-SY5Y cells were cultured in chamber slides at a seeding density of 1 × 104 cells per well for 24 hours, and then treated with H2O2 for 24 hours with or without pretreatment with ALA for 30 minutes. The treated SH-SY5Y cells were fixed with 4% paraformaldehyde (in phosphate buffered saline, PBS) for 15 minutes at room temperature and washed with PBS. The fixed cells were stained with DAPI (300 nM) for 15 minutes at room temperature in dark, washed with PBS and examined under fluorescent microscopy (Eclipse TE2000).

    6. Measurement of intracellular ROS

    SH-SY5Y cells were cultured in chamber slides at a seeding density of 1 × 104 cells per well for 24 hours, and then treated with H2O2 for 24 hours with or without pretreatment with ALA for 30 minutes. And then loaded with 20 μM H2DCFDA for 30 minutes at 37℃. Then cells were washed twice with phenol red-free DMEM/F12 and were incubated with H2O2 for 30 minutes. Cells were then examined and imaged using fluorescence microscopy (Eclipse TE200).

    7. Immunoblotting

    The SH-SY5Y cells were treated with H2O2 for 24 hours with or without pretreatment with ALA for 30 minutes. Immunoblotting was done according to the previously described method with minor modifications [17]. The anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-HO-1, anti-Beclin-1, anti-LC-3, or anti-β-actin antibody was used as the primary antibody.

    8. Haematoxylin and eosin staining

    SH-SY5Y cells were cultured in chamber slides at a seeding density of 1 × 104 cells per well for 24 hours, and then treated with H2O2 for 24 hours with or without pretreatment with ALA for 30 minutes. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed with PBS. Haematoxylin and eosin staining were performed to evaluate the morphological alterations in SH-SY5Y cells. Cells were observed and imaged using Leica DM 750 microscope (Leica Microsystems, Heerbrugg, Switzerland).

    9. Immunocytochemistry

    SH-SY5Y cells were cultured in chamber slides at a seeding density of 1 × 104 cells per well for 24 hours, and then treated with H2O2 for 24 hours with or without pretreatment with ALA for 30 minutes. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed with PBS. Immunocytochemistry was performed using Vetastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Fixed cell were incubated with Beclin-1 antibody for 1 hour and incubated with peroxidase-conjugated goat antimouse antibody for 1 hour. Cells were observed and imaged using Leica DM 750 microscope (Leica Microsystems).

    10. Statistical analysis

    All experiments were performed at least 3 times. The results were presented as mean ± standard deviation. The statistical significance was analyzed by using Student’s t-test for two groups and one way analysis of variance for multi-group comparisons. All statistical analyses were performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). p-values ＜ 0.05 were considered statistically significant.

    Results

    1. ALA protects SH-SY5Y cells against H2O2-induced cytotoxicity

    In this study, the effect of ALA on H2O2-induced SH-SY5Y cell viability loss was assessed by MTT assay and Live/Dead assay. Treatment with 12.5–500 μM H2O2 decreased the viability of SH-SY5Y cells compared with the control in a dosedependent manner (Fig. 2A). H2O2 (250 μM) induced approximately 35% cell loss after 24 hours treatment (Fig. 2A). SHSY5Y cells were treated with 12.5–200 μM ALA for 24 hours, and an MTT assay was performed to verify if cell viability was altered. As shown in Fig. 2B, the viability of SH-SY5Y cells treated with 12.5, 25, 50, 100, and 200 μM ALA were 102 ± 4%, 108 ± 5%, 105 ± 3%, 104 ± 5%, and 96 ± 2%, respectively. These data indicate that ALA did not affect the cell viability.

    To investigate whether ALA could protect against H2O2-induced dopaminergic cell death, SH-SY5Y cells were pretreated with 25 and 50 μM ALA for 30 minutes, followed by 250 μM H2O2 treatment for 24 hours. As shown in Fig. 3A, H2O2- induced cell loss was attenuated by ALA treatment. To verify the survival of SH-SY5Y cells treated with ALA, cell Live/Dead assay using green calcein AM and ethidium homodimer-1 was performed. Cell Live/Dead assay showed an increase in the living population of SH-SY5Y cells incubated with ALA as compared with H2O2 treated cells (Fig. 3B upper panel). These results indicated that ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury.

    2. ALA inhibited H2O2-induced apoptosis and ROS production in SH-SY5Y cells

    The nuclear condensation changes were assessed using DAPI staining. A significant proportion of SH-SY5Y cells with condensed nuclei increased upon exposure to H2O2, which are the characteristics of apoptosis (Fig. 3B lower panel). The number of SH-SY5Y cells with condensed nuclei decreased by pretreatment with ALA in a dose-dependent manner.

    To identify the changes of intracellular ROS in SH-SY5Y cells during the H2O2-induced cell death and ALA mediated protection, we measured ROS production in SH-SY5Y cells using fluorescent dye H2DCFDA. As shown in Fig. 4, the levels of fluorescence H2DCFDA increased after 30 minutes treatment with H2O2. However, ALA decreased H2O2-induced ROS production in a ALA dose dependent-manner (Fig. 4).

    3. ALA protects H2O2-induced cell death through down-regulating oxidative stress

    Immunoblotting was performed to verify if the expressions of anti-apoptotic Bcl-2, Bcl-xL and pro-apoptotic Bax are modulated by ALA. After treatment with 250 μM H2O2 for 24 hours, the expression of Bcl-2 and Bcl-xL was markedly decreased but the expression of Bax was increased (Fig. 5A). However, pretreatment with ALA decreased the expression of Bax and increased the expression level of Bcl-2 and Bcl-xL in SH-SY5Y cells (Fig. 5A). To confirm the oxidative stress in SHSY5Y cells during the H2O2-induced cell death and ALA mediated protection, the alteration of biomarker associated with inducible enzyme with antioxidant was investigated. As shown in Fig. 5B, ALA significantly increased the expression of HO-1, biomarker associated with antioxidant. These data indicate that ALA protects H2O2-induced dopaminergic cell death through down-regulating apoptosis and oxidative stress.

    4. ALA inhibits autophagy in H2O2-induced SH-SY5Y cell injury

    To determine whether the autophagic processes is involved in H2O2-induced cell death, immunoblotting and immunocytochemistry were performed. As shown in Fig. 6A, after treatment with 250 μM H2O2 for 24 hours, the expressions of Beclin-1 and LC-3, the specific biomarkers associated with autophagy, were markedly increased. However, pretreatment with ALA decreased the expression levels of Beclin-1 and LC-3 in SH-SY5Y cells (Fig. 6A). Futhermore, the immunoreactivity of Beclin-1 decreased by 25 and 50 μM pretreatment with ALA in SH-SY5Y cells, as indicated by the results of immunocytochemistry (Fig. 6B). These results indicate that ALA inhibits autophagy in H2O2-induced SH-SY5Y cells.

    Discussion

    ALA, which is a potent antioxidant is easily absorbed into the bloodstream and can without difficulty cross through the bloodbrain barrier, so it has been advised that it can be protect the central nervous system [8,9]. SH-SY5Y cells are widely used to in the study dopaminergic pathogenesis [1,4,16]. This is because these cell line represent several typical dopaminergic pathological markers, such as tyrosine hydroxylase and dopamine transporter [1,4,16]. In this study, the protective effect of ALA in H2O2-induced cell death was examined.

    MTT assay and cell Live/Dead assay showed that ALA did not affect the cell viability and survival of SH-SY5Y cells. These data suggested that ALA has been nontoxicity to SH-SY5Y cells. In addition, we found that ALA could regulate H2O2- induced cell death in SH-SY5Y human dopaminergic neuronal cells (Figs. 2 and 3).

    It is well accepted that ROS may attack intracellular biological macromolecules, including nuclear acids, proteins and lipids, dysfunction of mitochondria, and activate signaling pathways leading to apoptosis [18,19]. In this study, our results showed the apoptotic activity, pro-apoptotic Bax up-regulation and anti- apoptotic Bcl-2 and Bcl-xL down-regulations when treated with H2O2 in SH-SY5Y cells (Figs. 3 and 5). Interestingly, ALA pretreatment reduced the formation of nuclear condensation and decreased the levels of Bax protein expression, but increase the levels of Bcl-2 and Bcl-xL protein expressions in SH-SY5Y cells (Figs. 3 and 5). Furthermore, our data showed that ALA exerted its protective effects through ROS reduction (Fig. 4). These data suggest that ALA protects H2O2-induced cell injury through down-regulating apoptosis and oxidative stress.

    Several studies reported that HO-1 has neuroprotective effects against oxidative stress-induced neuronal damage [20- 22]. Although HO-1 protein levels are normally low in neurons, HO-1 can be highly up-regulated in cerebral ischemia [23] and during the formation of neurofibrillary tangles in Alzheimer’ s disease [24] and markedly accumulated in neuronal Lewy bodies of Parkinson’s patients [25]. Heme and other oxidative stress stimuli enhance the HO-1 expression in practically all tissues and cells including neurons [25]. Similar to other previous studies [20-25], in this study, the pretreatment with ALA increased HO-1 expression in SH-SY5Y cells (Fig. 5). These data indicate that ALA may increase cell resistance to oxidative stress and delay the dopaminergic cell injury.

    Autophagy is an important physiological process that degrades intracellular components, such as cellular waste products, degenerative proteins and damaged cellular organelles [12,13]. Beclin-1 and LC-3 are common biomarkers of autophagy [14]. Autophagy may be a mechanism for cell survival but may also induce cell death [12]. At the early stage of cell stress, autophagy is induced and then cytoprotective effect [12]. When cell stress is too severe at late stage, excessive autophagy may trigger cell injury and death [12]. Hence, to verify whether autophagic processes is involved in H2O2- induced cell injury, the alteration of biomarkers associated with autophagy was investigated. In our study, the expressions of Beclin-1 and LC-3 were decreased by ALA pretreatment in SH-SY5Y cells. These findings indicate that ALA attenuated the autophagic processes in H2O2-induced dopaminergic cell injury (Fig. 6). On the other hand, the mechanisms of cell protective effects induced by ALA in dopaminergic neuronal cells were not fully understood [26]. Therefore, further studies are needed to investigate the precise cellular and molecular mechanisms of cell apoptosis induced by ALA.

    In conclusion, these results suggest that ALA protects SHSY5Y human dopaminergic neuronal cells against H2O2- induced cell injury by inhibiting autophagy and apoptosis. Subsequent studies will allow us to elucidate that ALA may be a promising candidate for the treatment and prevention of treating neurodegenerative diseases including Parkinson’s disease.

    Acknowledgements

    This research was supported by HysensBio Co., Ltd funded (2020).

    Figure

    IJOB-46-1-15_F1.gif

    Chemical structure of alpha-lipoic acid.

    IJOB-46-1-15_F2.gif

    Effects of hydrogen peroxide (H2O2) and alpha-lipoic acid (ALA) on cell cytotoxicity in SH-SY5Y human dopaminergic neuronal cells. The SH-SY5Y cells were treated with various concentrations of (A) H2O2 and (B) ALA for 24 hours. The cell viabilities were measured using an 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide assays. The percentage of cell viability was calculated as a ratio of A570nms of material treated cells and untreated control cells. Each data point represents the mean ± standard deviation of three experiments.

    *p < 0.05 vs. control, **p < 0.01 vs. control, and ***p < 0.001 vs. control (the control cells measured in the absence of H2O2 and ALA).

    IJOB-46-1-15_F3.gif

    Cell death induced by hydrogen peroxide (H2O2) and protective effect of alpha-lipoic acid (ALA) in SH-SY5Y cells. (A) Indicated concentrations of ALA were added to the SH-SY5Y cells for 30 minutes before the addition of H2O2 (250 μM). Cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide assay. Each data point represents the mean ± standard deviation of three experiments. (B) Cell Live/Dead assay (upper panel) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (lower panel) were performed to investigate the cell survival and chromatin condensation, respectively. The scale bar presented in each image is 100 μm.

    **p < 0.01 vs. the cells measured in the presence of H2O2 only.

    IJOB-46-1-15_F4.gif

    Effects of alpha-lipoic acid (ALA) on hydrogen peroxide (H2O2)-induced reactive oxygen species production in SH-SY5Y cells. Cells were cultured in chamber slides at a seeding density of 1 × 104 cells per well for 24 hours, and then treated with H2O2 for 24 hours with or without pretreatment with ALA for 30 minutes. And then loaded with 20 μM 2′,7′-Dichlorodihydrofluorescein diacetate for 30 minutes at 37℃. Images were observed by fluorescence microscopy (Eclipse TE2000; Nikon Instruments, Melville, NY, USA). The scale bar presented in each image is 100 μm.

    IJOB-46-1-15_F5.gif

    Effects of alpha-lipoic acid (ALA) on hydrogen peroxide (H2O2)-induced oxidative stress in SH-SY5Y cells. (A) Expressions of anti-apoptotic factors Bcl-2, Bcl-xL, and pro-apoptotic factor Bax in SH-SY5Y cells. (B) Expression of heme oxygenase-1 (HO-1) in SH-SY5Y cells. SH-SY5Y cells were treated with H2O2 (250 μM) for 24 hours. ALA was added to the media for 30 minutes before the addition of H2O2. The cell lysate was prepared and analyzed by immunoblotting as described in “Materials and Methods”.

    IJOB-46-1-15_F6.gif

    Effect of alpha-lipoic acid (ALA) on autophagic processes in hydrogen peroxide (H2O2)-induced cell injury. (A) Immunoblotting was performed to verify the expression of Beclin-1 and LC-3, the specific biomarkers associated with autophagy. (B) Immunocytochemistry was performed to verify the expression of Beclin-1. SH-SY5Y cells were treated with H2O2 (250 μM) for 24 hours. ALA was added to the media for 30 minutes before the addition of H2O2. Cells were observed and imaged using Leica DM 750 microscope (Leica Microsystems, Heerbrugg, Switzerland).

    H&E, haematoxylin and eosin.

    Table

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