Neuroprotective Effects of Probucol against Rotenone-Induced Toxicity via Suppression of Reactive Oxygen Species Production in SH-SY5Y Cells

Abstract

Probucol is a hyperlipidemic drug with antioxidant properties. It has been reported to prevent mitochondrial dysfunction, reduce oxidative stress, and suppress neurotoxicity in neurodegenerative disease models, including Parkinson’s disease models. However, the molecular mechanisms underlying the neuroprotective effects of probucol have been not examined yet. Thus, in this study, we investigated whether probucol can alleviate the effects of a mitochondrial complex I inhibitor, rotenone, on a human neuroblastoma cell line (SH-SY5Y). We evaluated the cell viability and cytotoxicity and apoptosis rates of SH-SY5Y cells treated with rotenone and probucol or edaravone, a known free-radical scavenger. Subsequently, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels in the cells were evaluated to determine the effects of probucol on mitochondrial function. We found that rotenone caused cytotoxicity, cell apoptosis, and mitochondrial dysfunction, enhanced ROS generation, and impaired MMP. However, probucol could inhibit this rotenone-induced decrease in cell viability, MMP loss, intracellular ROS generation, and apoptosis. These results suggest that probucol exerts neuroprotective effects via MMP stabilization and the inhibition of ROS generation. Additionally, this effect of probucol was equal to or greater than and more persistent than that of edaravone. Thus, we believe probucol may be a promising drug for the treatment of neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases.

INTRODUCTION

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease¹⁾ with an increasing prevalence.²⁾ PD is characterized by the loss of dopaminergic neurons in the substantia nigra, which impairs an individual’s ability to regulate involuntary movements, leading to movement disorders. The main medications used for treating PD are dopaminergic drugs and L-3,4-dihydroxyphenylalanine (L-DOPA); however, their long-term application often causes side effects. Therefore, it is imperative to develop a therapeutic drug that can provide lasting or permanent improvement.^3,4) However, the molecular mechanisms underlying PD pathogenesis, which can facilitate the development of new therapeutic drugs and treatment approaches, have not been elucidated yet.

Mitochondrial complex I deficiency has been observed in the brain of patients with PD,⁵⁾ indicating that mitochondrial dysfunction plays a key role in PD development. Mitochondrial dysfunction is associated with both genetic and environmental factors of PD.⁶⁾ Impairment of various mitochondrial functions, such as mitochondrial dynamics and the electron transport chain, increases the production of reactive oxygen species (ROS). Thus, mitochondrial dysfunction and increased ROS generation can lead to the degeneration of dopaminergic neurons underlying the pathogenesis and progression of PD.^7,8)

Rotenone is a natural organic compound and an active ingredient in pesticides and insecticides.⁹⁾ The most potent effect of rotenone is inhibition of mitochondrial complex I,¹⁰⁾ which induces cell death by promoting ROS generation.¹¹⁾ It also reduces antioxidant enzyme activity, increasing oxidative stress and inducing cell death.^12,13) Hence, rotenone-treated cells and animals are widely used as models to investigate the mechanisms of PD.^14–16)

Probucol is a hyperlipidemic drug commonly used to lower cholesterol levels and mitigate secondary cardiovascular events^17,18) and is known to possess antioxidant properties. Recent studies have reported the effects of probucol on oxidative stress. Probucol can enhance antioxidant enzymes and inhibit caspase-3 activation in myocarditis,¹⁹⁾ as well as suppress high glucose-induced ROS generation and apoptosis.²⁰⁾

Probucol has been shown to prevent oxidative stress and excitotoxicity, such as ROS generation and lipid peroxidation, in an in vitro Huntington’s disease model.²¹⁾ In Alzheimer’s disease models, probucol has been demonstrated to reduce hippocampal oxidative stress and cognitive decline in vivo²²⁾ and inhibit ROS generation and restore impaired mitochondrial dynamics in vitro.²³⁾ Probucol reduces mitochondrial complex I inhibition and ROS generation and protects brain cells from neurotoxicity caused by manganese poisoning with PD-like symptoms.²⁴⁾ In addition, probucol can inhibit the decrease in antioxidant enzymes induced by 6-hydroxydopamine and alleviate lipid peroxidation and hyperlocomotion, which are signs of neurotoxicity, in mice.²⁵⁾ Probucol has been reported to inhibit dopaminergic neurodegeneration induced by bacterial metabolites of Streptomyces venezuelae with ROS generation and mitochondrial complex I impairment in Caenorhabditis elegans.²⁶⁾ It has also been shown to reduce rotenone-induced brain cell death that causes mitochondrial membrane potential (MMP) reduction in zebrafish.²⁷⁾ These reports indicate that probucol prevents mitochondrial dysfunction and protects cells against neurotoxicity; however, the mechanism underlying this protective effect is not entirely clear.

Therefore, in the present study, we investigated the neuroprotective effects of probucol on rotenone-induced toxicity, mitochondrial function, and ROS generation in a human neuroblastoma cell line (SH-SY5Y) to determine its mechanism of action.

MATERIALS AND METHODS

Cell Culture and Compound Treatment

SH-SY5Y cells (provided by ATCC, Manassas, VA, U.S.A.) were cultured in Eagle’s minimal essential medium (EMEM; FUJIFILM Wako, Osaka, Japan)/nutrient mixture F12-Ham (Ham’s F-12; FUJIFILM Wako) supplemented with 10% fetal bovine serum (FBS; Hyclone, New Zealand) and 1 mM sodium pyruvate (FUJIFILM Wako) and cultured at 37 °C in air containing 5% CO₂. After growth to 90% confluence, cells were seeded and cultured for 24 h for cell attachment before treatment. Probucol (a gift from Otsuka Pharmaceutical), edaravone (Sigma-Aldrich, St. Louis, MO, U.S.A.), and rotenone (Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan). The solutions were prepared under sterile conditions. Probucol and edaravone were administered 1 h prior to the addition of rotenone. Edaravone is an approved medication for the treatment of amyotrophic lateral sclerosis (ALS) in some regions. It is known to have antioxidant properties acting as a free radical scavenger.^28,29) Thus, it was used in the present study for comparison with probucol. Groups of cells cultured in medium containing only DMSO were used as vehicle controls, and the DMSO concentration in each group was standardized at 0.1%.

Measurement of Cell Viability

Cell viability was measured using a Cell Counting Kit-8 (CCK-8/WST-8; Dojindo, Kumamoto, Japan), according to the manufacturer’s protocol. Briefly, to maintain a consistent cell density during cell viability measurements, SH-SY5Y cells were seeded in 96-well plates at densities of 2 × 10⁴, 1 × 10⁴, and 5 × 10³ cells/well. Rotenone was added with or without probucol or edaravone, and the cells were cultured for 24, 48, and 72 h in descending order of the number of seeded cells. CCK-8 reagent was added at 10 µL/well and incubated for 90–120 min under 5% CO₂ at 37 °C, and the absorbance was measured at 405 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific, Waltham, MA, U.S.A.). The cell viability of each group was expressed relative to the cell viability of the control group at 100%. Based on the WST-8 assay results, an effective dose of probucol against rotenone toxicity was administered in subsequent experiments.

Measurement of Cytotoxicity Rate

The cytotoxicity rate was determined by measuring the activity of lactate dehydrogenase (LDH) released into the medium using a Cytotoxicity LDH Assay Kit-WST (Dojindo). SH-SY5Y cells were seeded in 96-well plates at 2 × 10⁴, 1 × 10⁴, and 5 × 10³ cells/well. Rotenone was added with or without probucol or edaravone and cultured for 24, 48, and 72 h. Briefly, the lysis buffer included in the kit was added to the high-control wells of each group at 20 µL/well, according to the manufacturer’s protocol, and the cells were cultured for 30 min at 37 °C and 5% CO₂. Subsequently, 100 µL of the supernatant was transferred from all wells to a 96-well plate for measurement, followed by the addition of 100 µL of working solution. The reaction mixture was then incubated in the dark at approximately 25 °C for 30 min. Then, 50 µL of stop solution was added to all wells, and absorbance was measured at 490 nm using a microplate reader. Cytotoxicity was evaluated based on the high-control LDH activity set as 100%.

Detection of Apoptosis

SH-SY5Y cells were seeded at 4 × 10⁵ and 3 × 10⁵ cells in 6 cm dishes. After 48 and 72 h of probucol, edaravone, and rotenone addition, apoptosis was examined using the MEBCYTO-Apoptosis Kit (MBL Life Sciences, Tokyo, Japan), according to the manufacturer’s instructions. Briefly, cells were first treated with trypsin (Nacalai Tesque) and then washed with phosphate-buffered saline (PBS; TaKaRa-Bio, Shiga, Japan). Next, the cells were suspended in 85 µL of binding buffer and treated with 10 µL of FITC-labeled annexin-V and 5 µL of propidium iodide (PI). Finally, the cells were suspended in 400 µL of binding buffer, and 10000 cells from each group were analyzed using a flow cytometer (BD FACSCalibur HGTM; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan).

Western Blotting

SH-SY5Y cells were seeded in a 10 cm dish at 2 × 10⁶, 1.5 × 10⁶, and 1 × 10⁶ cells. Rotenone was added in the presence or absence of probucol or edaravone and cultured for 24, 48, and 72 h. Cells were washed with PBS and lysed with a cell lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Nonidet P-40, 0.1% Triton, and 50 mM NaF) containing a protease inhibitor cocktail (Nacalai Tesque). The cells were then kept on ice for 20 min and centrifuged for 15 min at 13200 rpm at 4 °C. The protein concentration of the supernatant was quantified using the BCA Protein Assay Kit (FUJIFILM Wako). The concentration of each group was equalized, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for separating the proteins according to their molecular weight. The separated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Clear Blot Membrane-Plus; ATTO, Tokyo, Japan) using horizontal blotting (ATTO). After blocking the membrane with Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% skim milk (Nacalai Tesque), primary antibodies diluted in TBS-T with 2% skim milk were added, and the membranes were incubated at 4 °C for 24 h. Anti-caspase-3 (1 : 1000; MBL Life Sciences), anti-caspase-9 (1 : 1000; Cell Signaling Technology, MA, U.S.A.), and anti-actin monoclonal antibodies (1 : 30000; Merck Millipore, MA, U.S.A.) were used as primary antibodies. After washing with TBS-T, secondary antibodies diluted in TBS-T with 2% skim milk were added and reacted for 1 h at room temperature. Horseradish peroxidase (HRP)-conjugated goat anti-rat immunoglobulin G (IgG) (1 : 1000; Proteintech, IL, U.S.A.), and anti-mouse IgG (1 : 30000; Cell Signaling Technology) were used as secondary antibodies. Proteins on the membrane were detected by the chemiluminescence method using ECL™ Prime Western blotting Detection Reagent (Cytiva, Buckinghamshire, U.K.) or Immobilon™ Western Chemiluminescent HRP substrate (Merck Millipore) and analyzed using ImageJ (National Institutes of Health, MD, U.S.A.).

Measurement of MMP

MMP was measured using an MT-1 MitoMP Detection Kit (Dojindo), according to the manufacturer’s instructions. Because MT-1 aggregates and emits red fluorescence when mitochondria are healthy and membrane potential is maintained, the loss of MMP can be assessed by a decrease in fluorescence levels. Cell nuclei were detected using PureBlu Hoechst 33342 (Bio-Rad Laboratories, Tokyo, Japan). SH-SY5Y cells were seeded in 96-well plates at 2 × 10⁴ and 1 × 10⁴ cells/well. The cells were then incubated with MT-1 dye working solution containing Hoechst for 30 min at 37 °C and 5% CO₂. Probucol or edaravone was added to the medium, and the cells were incubated for 1 h, followed by the addition of rotenone for 1, 24, and 48 h. Cells were washed with Hanks’ balanced salt solution without phenol red (HBSS (−); FUJIFILM Wako) and exchanged into an imaging buffer solution. Subsequently, they were detected on a ZOE™ fluorescence cell imager (Bio-Rad) and analyzed using Image J. The fluorescence intensity per 100 cells was calculated relative to the control group, whose intensity was considered 100%, and the MMP of each group was calculated.

Measurement of ROS

Intracellular ROS production was measured using the ROS Assay Kit Photo-oxidation Resistant DCFH-DA (Dojindo), according to the manufacturer’s protocol. The non-fluorescent probe 2,7-diacetyldichlorofluorescein (DCFH-DA) is taken up by the cells and oxidized by intracellular ROS to form fluorescent dichlorofluorescein. Briefly, SH-SY5Y cells were seeded in 96-well black plates at a density of 1 × 10⁴ cells/well. Probucol or edaravone was added to the medium, and the cells were incubated for 1 h, followed by the addition of rotenone for 2, 24, and 48 h. Thereafter, they were treated with DCFH-DA working solution containing Hoechst. After incubation for 30 min at 37 °C with 5% CO₂, the cells were washed and HBSS (−) was added. Subsequently, the cells were detected using a fluorescent cell imager and analyzed using ImageJ. The amount of intracellular ROS produced by each group was calculated using the fluorescence intensity per 100 cells in the control group as 1.

Statistical Analyses

The data are expressed as mean ± standard error of the mean (S.E.M.). Statistical analyses were performed using the GraphPad Prism 6 software (GraphPad Software, San Diego, CA, U.S.A.) with one-way ANOVA (non-parametric), followed by Tukey’s multiple comparison test. The significance level was set at p < 0.05.

RESULTS

Probucol Alleviates Rotenone-Induced Cell Death

We first confirmed the toxicity of rotenone, an inhibitor of mitochondrial complex I, to SH-SY5Y cells. After 48 h of treatment, we observed that rotenone exhibited cytotoxicity in a concentration-dependent manner from 10 to 1000 nM (Fig. 1A). Rotenone treatment at 250 nM reduced cell viability by approximately 50%. Thus, this concentration was used to examine the cytoprotective effects of probucol against rotenone. Probucol was simultaneously compared with edaravone, a known antioxidant compound. Probucol did not affect cell viability within the investigated concentration range (Fig. 1B); in contrast, edaravone significantly increased cell viability at 50 µM (Fig. 1C).

Fig. 1. Probucol Inhibits Rotenone-Induced Cell Viability Loss

Graphs show cell viability measured by the WST-8 assay. SH-SY5Y (1 × 10⁴ cells/well, 96-well plate) were treated with (A) rotenone (10–1000 nM), (B) probucol (1–50 µM), and (C) edaravone (1–50 µM) for 48 h. (D) Probucol (1–50 µM) or (E) edaravone (1–50 µM) was added 1 h before adding rotenone (250 nM) (n = 4, mean ± S.E.M.). Vehicle control (0.1% DMSO) cell viability was 100%. Significance was compared with the control in (A), (B), and (C) and rotenone alone in (D) and (E). The p-values are shown in the figure.

Probucol alleviated rotenone-induced decrease in cell viability in a concentration-dependent manner (Fig. 1D); edaravone exhibited similar protective effects (Fig. 1D). The protective effects of probucol at 5 and 10 µM were comparable to those of edaravone at 5 and 10 µM, respectively (Fig. 1E), Thus, 10 µM was used in subsequent experiments.

The effects of probucol and edaravone on cell viability and cytotoxicity were examined 24, 48, and 72 h after rotenone treatment (Fig. 2). No difference in cytotoxicity or cell viability was observed after 24 h of treatment; however, rotenone significantly increased cytotoxicity and decreased cell viability after 48 and 72 h of treatment. Probucol significantly inhibited the effects of rotenone, and its reversal of cytotoxicity was greater than that of edaravone.

Fig. 2. Probucol Reduces Rotenone-Induced Cytotoxicity

Graphs represent (A) the cytotoxicity rate measured by LDH assay and (B) cell viability measured by WST-8 assay. SH-SY5Y cells (2 × 10⁴, 1 × 10⁴, and 5 × 10³ cells/well, 96-well plate) were treated with 10 µM probucol (PB) or 10 µM edaravone (Ed), treated with 250 nM rotenone (Rot) 1 h later, and cultured for 24, 48, and 72 h (n = 4, mean ± S.E.M.). The significance was compared with rotenone only. The p-values are shown in the figure.

Probucol Reduces Rotenone-Induced Apoptosis

Next, we examined the effects of probucol on apoptosis. Apoptosis was detected 48 and 72 h after rotenone treatment (Fig. 3). Probucol significantly inhibited rotenone-induced apoptosis, with an effect equivalent to that of edaravone after 48 h. However, the effect of probucol after 72 h was significantly stronger than that of edaravone.

Fig. 3. Probucol Inhibits Rotenone-Induced Apoptosis

SH-SY5Y cells (4 × 10⁵ and 3 × 10⁵ cells, 6 cm dish) were treated with 10 µM probucol (PB) or 10 µM edaravone (Ed). After 1 h, 250 nM rotenone (Rot) was added, and the plates were incubated for 48 and 72 h. (A) Apoptosis was detected using flow cytometry 72 h after rotenone addition. Apoptosis was considered to have occurred in both the upper and lower right regions of the figure. (B) Percentage of apoptotic cells detected by flow cytometry (n = 5, mean ± S.E.M.). Significance was compared with Rot only; The p-values are shown in the figure.

Furthermore, the expression levels of caspase-3 were analyzed using Western blotting (Fig. 4). The expression of the active form of cleaved caspase-3 was increased, whereas there was no difference in the expression levels of non-activated pro-caspase-3. Probucol inhibited rotenone-induced caspase-3 activation more strongly than edaravone at 48 and 72 h (Fig. 4B).

Fig. 4. Probucol Inhibits Rotenone-Induced Caspase-3 and Caspase-9 Expression

SH-SY5Y (2 × 10⁶, 1.5 × 10⁶, and 1 × 10⁶ cells, 10 cm dish) were treated with 10 µM probucol (PB) or 10 µM edaravone (Ed), followed by 250 nM rotenone (Rot) addition. After 24, 48, and 72 h, the cells were collected and analyzed using Western blotting. The amount of protein per sample was 20 µg. Actin was used as a loading control. (A) Western blot results of caspase-3 48 h after the addition of rotenone. (B) Expression levels of cleaved caspase-3, detected by Western blotting, are expressed as a ratio to actin (n = 9, mean ± S.E.M.). (C) Results of caspase-9 detection by Western blotting 48 h after rotenone addition. (D) Expression levels of cleaved caspase-9, detected by Western blotting, are expressed as a ratio to actin (n = 3, mean ± S.E.M.). Significance was compared with Rot only. The p-values are shown in the figure.

We also investigated the activation of caspase-9, which functions upstream of capsase-3 in the mitochondrial apoptotic pathway. Although it was not significant, probucol and edaravone slightly inhibited rotenone-induced apoptosis at 48 h (Fig. 4D).

Probucol Inhibits Rotenone-Induced MMP and ROS Production

Rotenone inhibits mitochondrial complex I and is cytotoxic. Therefore, we measured MMP, an index of mitochondrial function, to investigate the effect of probucol on the action of rotenone in the mitochondria. MMP levels were significantly decreased at 1 and 24 h after rotenone treatment. However, probucol treatment significantly suppressed this effect (Fig. 5). The effect of probucol on MMP decline was the same as that of edaravone at 1 h, but probucol was less potent than edaravone at 24 h.

Fig. 5. Probucol Inhibits Rotenone-Induced Imbalance of Mitochondrial Membrane Potential (MMP)

SH-SY5Y (2 × 10⁴ and 1 × 10⁴ cells/well, 96-well plate) were treated with 10 µM probucol (PB) or 10 µM edaravone (Ed), after 1 h of 250 nM rotenone (Rot) addition. The MMP was detected after 1, 24, and 48 h of incubation. The image shows the results obtained 1 h after rotenone addition. (A) Results were obtained using a fluorescent cell imager 1 h after the addition of rotenone. MMP (upper: red), cell nucleus (middle: blue), merged (lower); scale bars, 100 µm. (B) Fluorescence intensity of MT-1 per 100 cells (n = 3, 4, mean ± S.E.M.). The fluorescence intensity of the control (0.1% DMSO) was set at 100%. Significance was compared with Rot only. The p-values are shown in the figure.

Because mitochondrial dysfunction increases ROS production, we investigated the effect of probucol on rotenone-induced ROS production (Fig. 6). Rotenone significantly increased intracellular ROS levels 2 and 24 h after addition. However, probucol significantly suppressed this rotenone-induced ROS production. Additionally, this effect of probucol was stronger than that of edaravone at 24 h.

Fig. 6. Probucol Inhibits Rotenone-Induced Oxidative Stress

SH-SY5Y (2 × 10⁴ and 1 × 10⁴ cells/well, 96-well plate) were treated with 10 µM probucol (PB) or 10 µM edaravone (Ed), after 1 h of 250 nM rotenone (Rot) addition. ROS was detected after 2, 24, and 48 h of incubation. (A) Results were obtained using a fluorescent cell imager 24 h after rotenone addition. ROS (upper: green), cell nucleus (middle: blue), merged (lower); scale bars, 100 µm. (B) Fluorescence intensity of ROS per 100 cells (n = 4, 6, mean ± S.E.M.). The fluorescence intensity of the control (0.1% DMSO) was set at 1. The p-values are shown in the figure.

DISCUSSION

Probucol, a hyperlipidemic drug, is known to have antioxidant properties.^30,31) Its antioxidant effects have been examined using various disease models, including PD. Previous studies have suggested that the antioxidant properties of probucol exert a protective effect in PD models,^25–27) preventing mitochondrial dysfunction^26,27) and oxidative stress.^25,26) However, the mechanisms behind the neuroprotective effects of probucol in PD are unclear, as there is a lack of comprehensive reports on the effects of probucol on mitochondrial dysfunction, oxidative stress, and neuronal death in PD models. Therefore, the present study attempted to investigate the effects of probucol on mitochondrial function and ROS production using a PD model.

We found that probucol inhibited the rotenone-induced decrease in cell viability and increase in cytotoxicity and apoptosis (Figs. 1–4). Damage to the mitochondrial electron transport chain alters MMP, ROS production, and ATP synthesis, resulting in abnormal mitochondrial morphology, intracellular calcium homeostasis, and intracellular signaling, ultimately leading to reduced cell viability.³²⁾ Therefore, we next examined MMP and ROS levels to investigate the effects of probucol on mitochondrial function (Figs. 5, 6). Probucol inhibited the rotenone-induced decrease in MMP and increase in intracellular ROS production. Our results suggested that probucol suppresses the cytotoxicity of rotenone through its protective effects on the mitochondria and inhibitory effects on ROS production.

Edaravone has been approved as a neuroprotective agent to treat ALS; it has the ability to scavenge free radicals.²⁸⁾ Edaravone has been shown to inhibit apoptosis and exhibit neuroprotective effects in PD models in vivo and in vitro.^33,34) In the present study, the potent antioxidant effects of probucol were compared with those of edaravone. Comparing the effects of probucol and edaravone on rotenone-induced events, their effects on decreased cell viability were comparable; however, the alleviating effect of probucol on increased cytotoxicity rate and apoptosis was significantly stronger than that of edaravone (Figs. 2, 3). The WST-8 assay measures intracellular dehydrogenase activity and cannot distinguish between cell cycle arrest and cell death,³⁵⁾ whereas the LDH assay can measure dead cells with damaged cell membranes,³⁶⁾ and apoptosis assays can detect cells with apoptotic features. The results of these assays suggested that probucol may be more protective than edaravone against rotenone-induced cell death.

The alleviating effect of probucol on ROS production was stronger than that of edaravone, whereas edaravone had a stronger effect on MMP than probucol (Fig. 5). Mitochondrial dysfunction in PD may be due to a vicious cycle of mitochondrial dynamics, electron transport chain impairment, mitophagy deficiencies, increased ROS production, or a combination of these factors.⁸⁾ The mechanism of probucol’s antioxidant action involves the direct activation of the intracellular antioxidant enzyme glutathione peroxidase³⁷⁾ and the activation of the Keap1/Nrf2/ARE antioxidant signaling pathway.^38,39) In contrast, edaravone donates its electrons to free radicals and is ultimately transformed into compounds with different structures.²⁸⁾ We speculate that the differences in the effects of probucol and edaravone on MMP are due to differences in their mechanisms of action. However, further investigations are required to confirm this hypothesis.

Taken together, our results suggest that the inhibition of mitochondrial complex I by rotenone leads to a decrease in MMP and an increase in ROS, which subsequently promotes caspase-3 activation and induces apoptosis. Probucol has a suppressive effect on all these symptoms, and the effects of probucol are greater than those of edaravone.

This study has some limitations. Because this study was conducted using cultured cells, further in vivo research is required to validate our results. Additionally, the ROS and MMP results do not explicitly indicate that probucol acts on mitochondria. To confirm the effects of probucol on mitochondria, it is necessary use methods that involves directly injecting probucol into the mitochondria, such as a mitochondria-specific drug delivery system.⁴⁰⁾ Furthermore, recent reports have indicated that probucol enhances mitophagy⁴¹⁾; thus, further research from multiple perspectives is required to investigate the effects of probucol on mitochondrial dysfunction.

CONCLUSION

We confirmed that probucol exerted a protective effect against rotenone-induced toxicity in SH-SY5Y cells by suppressing oxidative stress and mitochondrial dysfunction. This effect was equal to or greater than and more persistent than that of edaravone, a free-radical scavenger. Thus, probucol may be a novel therapeutic agent for treating diseases such as PD and Alzheimer’s disease, which are associated with oxidative stress and mitochondrial dysfunction.

Acknowledgments

The authors would like to express their sincere gratitude to Otsuka Pharmaceutical Co., Ltd., for providing probucol for use in this study. We would also like to thank all the research students in the laboratory for helping us complete the experiments.

Conflict of Interest

M. Miyake is a former employee of Otsuka Pharmaceutical Co., Ltd.; T. Takeo received probucol from Otsuka Pharmaceutical Co., Ltd.; the other authors have no conflict of interest.

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