3,4-Dihydroxybenzalacetone Inhibits the Propagation of Hydrogen Peroxide-Induced Oxidative Effect via Secretory Components from SH-SY5Y Cells

Abstract

The polyphenol derivative 3,4-dihydroxybenzalacetone (DBL) is the primary antioxidative component of the medicinal folk mushroom Chaga (Inonotus obliquus (persoon) Pilat). In this study, we investigated whether the antioxidative effect of DBL could propagate to recipient cells via secreted components, including extracellular vesicles (EVs), after pre-exposing SH-SY5Y human neuroblastoma cells to DBL. First, we prepared EV-enriched fractions via sucrose density gradient ultracentrifugation using conditioned medium from SH-SY5Y cells exposed to 100 µM hydrogen peroxide (H₂O₂) for 24 h, with and without 1 h of 5 µM DBL pre-treatment. CD63 immuno-dot blot analysis demonstrated that fractions with density of 1.06–1.09 g/cm³ had CD63-like immuno-reactivities. Furthermore, the 2,2-diphenyl-1-picrylhydrazyl assay revealed that the radical scavenging activity of fraction 11 (density of 1.06 g/cm³), prepared after 24-h H₂O₂ treatment, was significantly increased compared to that in the control group (no H₂O₂ treatment). Notably, 1 h of 5 µM DBL pre-treatment or 5 min of heat treatment (100 °C) diminished this effect, although concentrating the fraction by 100 kDa ultrafiltration enhanced it. Overall, the effect was not specific to the recipient cell types. In addition, the uptake of fluorescent Paul Karl Horan-labeled EVs in concentrated fraction 11 was detected in all treatment groups, particularly in the H₂O₂-treated group. The results suggest that cell-to-cell communication via bioactive substances, such as EVs, in conditioned SH-SY5Y cell medium, propagates the H₂O₂-induced radical scavenging effect, whereas pre-conditioning with DBL inhibits it.

INTRODUCTION

Chaga (Inonotus obliquus (persoon) Pilat) is a fungus particularly abundant in Russia and Northern Europe.¹⁾ It has traditionally been used as an alternative folk medicine with many beneficial effects, including antioxidative, anti-inflammatory, neuroprotective, and anti-tumor effects.²⁾ Oxidative stress is highly associated with the pathogenesis of oxidative stress-related diseases, such as cancer, and neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease (PD).³⁾ Therefore, the strong antioxidant properties of Chaga have been extensively studied against oxidative stress-related diseases. One of Chaga’s primary antioxidative components is 3,4-dihydroxybenzalacetone (DBL), a low catechol-containing phenylbutanoid.^4,5) DBL acts as a potent reactive oxygen species scavenger in hydrogen peroxide (H₂O₂)-treated PC12 cells⁶⁾ and inhibits amyloid β aggregation.⁷⁾ Furthermore, DBL activates the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione pathway via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling, preventing inflammation in acute lung injuries in mice.⁸⁾ Moreover, it has a neuroprotective effect against the PD-related neurotoxin 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells.⁹⁾ DBL activates protein unfolding and the oxidative stress response, which can pre-condition the endoplasmic reticulum (ER) for stress and autophagy against 6-OHDA-induced neurotoxicity in SH-SY5Y cells.¹⁰⁾ Further, it suppresses cancer cell proliferation by inhibiting cellular topoisomerase II¹¹⁾ and inhibits the inflammatory effect of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation, thereby leading to enhanced apoptosis and inhibited cellular invasion.^12,13)

Extracellular vesicles (EVs) are secretary vesicles with diameter of 50–150 nm and comprising lipid bilayers released from various cell sources. They stably circulate in body fluids, including blood, breastmilk, urine, and saliva¹⁴⁾ and participate in cell-to-cell communication by the horizontal transfer of components, such as internalized proteins, messenger RNAs, and micro RNAs.¹⁵⁾ Recent attempts have been made to use nanosized EVs as carriers for therapeutic drugs and bioactive molecules.¹⁶⁾ Polyphenols are prime candidates for natural compounds loading on EVs, owing to their potent antioxidant properties and cell-death preventive effects.¹⁷⁾ For example, a mouse model study had demonstrated that curcumin-loaded exosomes effectively cross the blood–brain barrier, improving PD pathology.¹⁸⁾ However, little is known about the effects of cellular exposure to DBL on EV physiology.

This study aimed to understand the effects of DBL on EV secretion and contents in H₂O₂-treated SH-SY5Y cells and to assess whether the antioxidant effects after DBL exposure propagate to recipient cells.

MATERIALS AND METHODS

Materials

Bis-Tris and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Dojindo Laboratories Co., Ltd. (Kumamoto, Japan). PKH67 Green Fluorescent and PKH24 Red Fluorescent Cell Linker Mini Kits (MINI67 and MINI24) for General Cell Membrane Labeling were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Dulbecco’s Modified Eagle Medium (DMEM)/F12, Opti-MEM I Reduced-Serum Medium, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). The 96-well plates were from Corning/Falcon (Corning, NY, U.S.A.). The 1,2-diphenyl-2-picrylhydrazyl (DPPH) reagent was purchased from Cayman Chemical Company (Ann Arbor, MI, U.S.A.). The Premix WST-1 Cell Proliferation Assay System was purchased from TaKaRa Bio Inc. (Kusatsu, Shiga, Japan). DBL (purity >95%), H₂O₂, lactate dehydrogenase (LDH)-Cytotoxic Test Wako (No. 299-50601), Silver Stain MS Kit (No. 299-58901), mouse anti-CD63 immunoglobulin G (IgG) monoclonal antibody 3-13 (1.1 mg/mL, 1 : 1000 dilution for Western blotting (WB) and dot-blot analysis), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IgG monoclonal antibody 5A12 (1.0 mg/mL, 1 : 1000 dilution for WB) were purchased from FUJIFILM-Wako Pure Chemical Corporation (Osaka, Japan). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1.0 mg/mL, 1 : 4000 dilution for WB) was obtained from MP Biomedicals (Irvine, CA, U.S.A.). The Clarity™ Western ECL substrate, nitrocellulose membrane, and polyvinylidene fluoride membrane were obtained from Bio-Rad Laboratories (Hercules, CA, U.S.A.). Finally, the 0.22 µm Millex^®-GV Filter Unit and Amicon^® Ultra-4 Centrifugal Filter Unit (MWCO 10 kDa) were purchased from Merck Millipore Ltd. (Burlington, MA, U.S.A.), and the Vivacon^® 500 Centrifugal Unit (MWCO 100 kDa) was obtained from Sartorius Stedim Biotech (Aubagne Cedex, France). Coomassie brilliant blue (CBB) (one step CBB staining solution) was purchased from Bio Craft Co. (Tokyo, Japan). The 9-cm culture dish was purchased from AS ONE CORPORATION (Osaka, Japan). The CELVIEW™ Cell culture dishes were obtained from Greinor Japan (Tokyo, Japan).

Cell Cultures

SH-SY5Y human neuroblastoma cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan), and the PC12 rat pheochromocytoma cell line was obtained from the RIKEN BioResource Research Center, Japan. The C6BU-1 rat glioma cell line was gifted by Professor N. Miki (Medical School of Osaka University, Japan).

All cells were cultured at 37 °C in a humidified 5% CO₂ incubator. The SH-SY5Y cells were maintained in DMEM/F12 supplemented with 15% FBS. The PC12 and C6BU-1 cells were grown in DMEM containing 10% FBS. The cells were cultured in 100 µL/well of culture medium in 96-well plates until >90% confluency was achieved for the cell-based assays.

EV Preparation via Sucrose Density Gradient Ultracentrifugation

First, 5 mL of the culture medium was recovered per SH-SY5Y condition group (described below) 24 h after incubating with Opti-MEM I at 37 °C in a humidified 5% CO₂ atmosphere. The SH-SY5Y condition groups were as follows: the groups without H₂O₂ exposure after DMSO and DBL pretreatments were described as “Control” and “DBL,” respectively. The groups with H₂O₂ exposure after DMSO and DBL pretreatments were denoted as “H₂O₂” or “DBL + H₂O_2,” respectively. The culture medium was filtered through a 0.22 µm Millex^®-GV Filter Unit. The filtered supernatant was concentrated and then replaced with 25-mM Bis-Tris buffer (pH 6.5) using an Amicon^® Ultra-4 Centrifugal Filter Unit (MWCO 10 kDa) by centrifuging at 4700 × g for 30 min at 4 °C. The supernatant was loaded onto a 12 mL of 10–60% sucrose density gradient prepared by stepwise stacking of 2 mL of 60, 50, 40, 30, 20, and 10% sucrose solutions (prepared in Bis-Tris buffer). Ultracentrifugation was performed with an Optima™ L-100 XP (Beckman Coulter, Brea, CA, U.S.A.) using a SW41 rotor at 75600 × g for 5 h at 4 °C. The samples were fractionated into approximately 1 mL portions.

Dot-Blot, WB, CBB, and Silver Staining Analyses

Dot-blot analysis was performed using a 96-well Bio-Dot microfiltration apparatus (Bio-Rad Laboratories). Briefly, 100 µL/well of the fractionated sample prepared via sucrose density gradient ultracentrifugation was blotted onto a nitrocellulose membrane (Bio-Rad Laboratories) under vacuum. The blot was blocked with 10% dried milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 and then incubated with specific antibodies for the target proteins at 4 °C overnight. The Clarity™ Western ECL substrate was used to detect chemiluminescent target signals after incubation with the HRP-conjugated goat anti-mouse IgG secondary antibody using a ChemiDoc XRS Plus apparatus (Bio-Rad Laboratories). In addition, WB analysis was performed as previously described.¹⁹⁾ Silver staining analysis was conducted according to the attached manufacture’s protocol.

For dot-blot CBB staining to analyze protein concentration and distribution, nitrocellulose membranes (Bio-Rad Laboratories) blotted on 100 µL/well of the fractionated sample were incubated with one step CBB staining solution (Bio Craft) for 2 h at room temperature. The stained blots were washed three times with 70% ethanol and finally with Milli Q water. Images of CBB-stained nitrocellulose membranes were scanned using a COREFIDO MC362w (Oki Electric Industry Co., Ltd., Tokyo, Japan).

For semiquantitative analysis of EV content in all four groups, both CD63 signal intensity of WB and protein signal intensity of dot-blot CBB staining were analyzed using open-source image processing software Image J2 (Version 2.90, http://imagej.net/Contributors). The amount of EV estimated by CD63 signal was normalized to the total protein amount estimated via dot-blot CBB staining, and the relative EV content among the four groups was compared and analyzed.

Cell-Based Assays

LDH and WST-1 assays were performed following the manufacturer’s protocol. The DPPH radical scavenging assay was performed as previously described.²⁰⁾ All assays used DBL dissolved in methanol. Data were acquired in duplicate and presented as a percentage of the control. Briefly, SH-SY5Y, C6BU-1, or PC12 cells were plated at approximately 3.0 × 10⁴ cells/well in the 96-well plate and cultured in 15% FBS-DMEM/F12 (for SH-SY5Y) or 10% FBS-DMEM (for C6BU-1 or PC12) at 37 °C under 5% CO₂ until >95% confluency was achieved. For the DPPH assays, cells were exposed for 24 h to a mixture of 99 µL/well of Opti-MEM and 1 µL/well of supernatant concentrated 10-fold with 100 kDa ultrafiltration (Vivacon^® 500 Centrifugal Unit, MWCO 100 kDa) of fraction 10 or 11 prepared from SH-SY5Y medium via sucrose density ultracentrifugation. A mixture of 10 µL/well of fraction 10 or 11 with 90 µL/well of Opti-MEM was used in the no treatment and heat (100 °C and 5 min) groups. After incubation at 37 °C under 5% CO₂, 50 µL/well of culture medium was transferred into each well of a new 96-well plate and mixed with 50 µL/well of 0.2 mM DPPH dissolved in MES (pH 6.1) buffer. The mixture was incubated at room temperature for 30 min in the dark, and absorbance was then measured at 570 nm using the BioRad Model 680 Plate Reader. To examine the neuroprotective effect of DBL by WST-1 assays, SH-SY5Y cells were pre-incubated with Opti-MEM I containing 5 µM DBL for 2 h. The medium was replaced by 100 µL/well of Opti-MEM I containing 200 µM H₂O₂ for 48 h. Furthermore, to evaluate cell toxicity and cell viability for H₂O₂ by LDH assay and WST-1 assays, respectively. SH-SY5Y cells were exposed to 100 µL/well of Opti-MEM I containing various H₂O₂ concentrations for 24 h for the LDH assays and 48 h for the WST-1 assays at 37 °C under 5% CO₂. For the LDH assays, the mixture of 50 µL/well of culture medium and 50 µL/well of the LDH assay reagent supplied with the kit was incubated at room temperature for 30 min in the dark. For the WST-1 assays, 5 µL/well of the WST-1 assay reagent included in the kit was directly added to each well of the 96-well plate after removal of 50 µL/well of culture medium. The mixture was incubated at room temperature for 60 min in the dark, and absorbance was then measured at 450 nm using the BioRad Model 680 Plate Reader.

Fluorescent Labeling of EVs with PKH Dye

EVs were fluorescently labeled with PKH67 Green Fluorescent and PKH24 Red Fluorescent Cell Linker Mini Kits, following the manufacturer’s protocol. In brief, samples were prepared for PKH-labeling by concentrating 400 µL of each fraction 10-fold with a Vivacon^® 500 Centrifugal Unit. After completing the labeling procedure, the PKH-labeled samples were washed thrice with Bis-Tris buffer using a Vivacon^® 500 Centrifugal Unit to remove free PKH dye. Subsequently, the samples were used for the uptake assay as following. EVs in the fractions of the control group, prepared via sucrose density gradient ultracentrifugation from SH-SY5Y medium, were labeled with PKH 67 Green Fluorescent dye. EVs in the fractions of the other groups (H₂O₂, DBL+ H₂O₂, and DBL) were labeled with PKH 24 Red Fluorescent dye. In the cellular uptake assay, cells were cultured in CELVIEW™ Cell culture dishes with 500 µL/well of the culture medium. Subsequently, 5 µL/well of a mixture of equal volumes of PKH 67-labeled and PKH 24-labeled samples was added, and the cells were incubated at 37 °C for 24 h in 5% CO_2. After washing the cells three times with PBS, they were fixed with 4% paraformaldehyde. The incorporation of PKH-labeled EVs into cells was observed using a BZ-X710 fluoresce microscope (KEYENCE, Osaka, Japan).

Statistical Analyses

All data are presented as means ± standard errors of the mean. A one-way ANOVA was performed to analyze group differences using KaleidaGraph ver. 5.0 software (Synergy Software, Reading, PA, U.S.A.). A p-value less than 0.05 was considered statistically significant.

RESULTS

Effect of DBL on H₂O₂-Induced Oxidative Stress in SH-SY5Y Cells

One of Chaga’s primary antioxidant compounds is DBL, a catechol-containing phenylbutanoid derivative (Fig. 1C). Thus, we examined the cytotoxicity and cell viability after exposure to various H₂O₂ concentrations to evaluate the physiological response of SH-SY5Y cells to H₂O₂-induced oxidative stress.

Fig. 1. Hydrogen Peroxide (H₂O₂)-Induced Oxidative Stress Responses of SH-SY5Y Cells

(A) Dose-dependent effect of H₂O₂ for 24 h on SH-SY5Y cell cytotoxicity by lactate dehydrogenase assay. Data are represented as the mean ± standard error of the mean (S.E.M; n = 3). (B) Dose-dependent effects of H₂O₂ on SH-SY5Y cell viability. Cell viability was evaluated by WST-1 assay after exposure to various H₂O₂ concentrations for 48 h. Data are represented as the mean ± S.E.M. (n = 3). (C) Chemical structure of 3,4-dihydroxybenzalacetone (DBL). (D) SH-SY5Y cell morphology 2 h after exposure to dimethyl sulfoxide (DMSO) or 5 µM DBL, with or without 100 µM H₂O₂, for 24 h. Representative images of each condition are shown in (a–d). Scale bar, 200 µm. (E) Effect of 48 h exposure to 200 µM H₂O₂ on the viability of SH-SY5Y cells pre-treated with various DBL concentrations for 2 h (Ea). Effect of 48 h DBL exposure on viability of SH-SY5Y cells (Eb). Data are represented as the mean ± S.E.M., n = 6, p < 0.001; one-way ANOVA, ** p < 0.01 vs. control group.

Cytotoxicity was remarkably increased in an H₂O₂ dose-dependent manner; 50% effective dose (ED₅₀) was 43.6 ± 4.7 µM, based on LDH activity, 24 h after H₂O₂ exposure (Fig. 1A). Cell viability significantly decreased in an H₂O₂ dose-dependent manner; IC₅₀ was 224.3 ± 5.0 µM (Fig. 1B). The SH-SY5Y cell morphology did not differ across the various conditions (Fig. 1D). Pre-incubation with 5 or 10 µM DBL for 2 h significantly affected the antioxidative effect of DBL on SH-SY5Y cells exposed to 200 µM H₂O₂ for 48 h (Fig. 1Ea). However, 10 µM DBL had a cytotoxic effect, affecting cell viability (Fig. 1Eb). Therefore, we selected a 24 h treatment with 100 µM H₂O₂ after pre-exposure to 5 µM DBL for 1 h to evaluate the physiological properties of EVs.

EV Fractionation via Sucrose Density Gradient Ultracentrifugation

We collected cell culture medium from various experimental conditions and prepared sub-fractionated samples via sucrose density gradient ultracentrifugation to investigate whether the oxidative effect of H₂O₂ or antioxidative effect of DBL could propagate by secretory components from SH-SY5Y cells (Fig. 2A). We evaluated the total protein amount, fractional density, and CD63-like immunoreactivity (a representative EV marker) for each sub-fractioned sample. Semiquantitative CBB staining revealed that fractions 8 to 12 had detectable protein levels, which corresponded to fractional densities between 1.05 and 1.09 g/cm³ (Fig. 2B). CD63-like immunoreactivity was observed in fractions 10 and 11 per group and was significantly enhanced by H₂O₂ treatment (Fig. 2C). Overall, fractions 10 and 11 were considered to primarily contain EVs.

Fig. 2. Fractionation of SH-SY5Y Extracellular Vesicles via Sucrose Gradient Ultracentrifugation

(A) Schematic of SH-SY5Y-conditioned medium fractionation. (B) Protein evaluation per fraction by Coomassie brilliant blue staining. A standard series of bovine serum albumin samples were blotted as controls to semi-quantitatively assess the protein amount per fraction. (C) Immuno-dot blot analysis of the extracellular vesicles per fraction using CD63.

Propagation of the Oxidative Effect by EVs

We used fractions 10 and 11 to examine the physiological properties of SH-SY5Y cells by performing DPPH and LDH assays. The DPPH assay exhibited a significant increase in radical scavenging activity in fraction 11, which was treated with H₂O₂ for 24 h, compared to the baseline activity levels of the control group. However, the effect was blocked by pre-treatment with 5 µM DBL for 1 h (Fig. 3A, left panel). The radical scavenging activity did not change in fraction 10, which was prepared after each treatment. The LDH assay also demonstrated that fractions 10 and 11 were not cytotoxic, regardless of the treatment (data not shown). Moreover, the radical scavenging activity of fraction 11 completely diminished after a 100 °C heat treatment for 5 min (Fig. 3A, middle panel) but was detected in the supernatant after concentrating fraction 11 by 100 kDa ultrafiltration (Fig. 3A, right panel). PKH-labeled EV uptake was observed in the supernatant of concentrated fraction 11 and was the most apparent in the H₂O₂-treated group (Fig. 3B). CD63-immunoreactivity was detected in all the concentrated samples via WB. However, GAPDH-immunoreactivity was only detected in the H₂O₂-treated groups (Fig. 3C). In addition, the EV content of all groups in fraction 11 was semi-quantitatively analyzed using data obtained from CD63 WB analysis and dot-blot CBB staining analysis. The results showed a trend toward low EV amounts in the DBL alone group; however, no statistically significant difference was observed between the groups (Fig. 3C). Comparative analysis of the protein components of concentrated samples, via sodium dodecyl-sulfate polyacrylamide gel electrophoresis, indicated that some proteins specifically changed in a H₂O₂ or DBL (Fig. 3D, black and white arrowheads, respectively).

Fig. 3. Propagation and Inhibition of the Hydrogen Peroxide-Induced Oxidative Effect

(A) Radical scavenging activity of fraction 10 or 11 prepared from SH-SY5Y cells. Samples directly taken from fraction 10 or 11 of each group were pre-treated with or without heat (100 °C for 5 min). Data are presented as the mean ± S.E.M., n = 8, p < 0.01; one-way ANOVA, * p < 0.05 vs. control group. (B) Uptake activity of PKH-labeled extracellular vesicles (EVs) of fraction 11 in each group. PKH-labeled EVs were co-incubated with SH-SY5Y cells for 24 h. Scale bar, 50 µm. (C) Detection of EV marker in fraction 11 by Western blotting after concentrating the samples by 100 kDa ultrafiltration. Semiquantitative analysis of the amount of EVs in all groups in fraction 11 was performed using data from CD63 Western blotting analysis and dot-blot CBB staining analysis. No statistically significant difference was observed among all groups. Data are presented as the mean ± S.E.M., n = 5, one-way ANOVA, p = 0.50. (D) Comparative analysis of the protein components of concentrated fraction 11 samples via sodium dodecyl-sulfate polyacrylamide gel electrophoresis, visualized by silver staining. Black and white arrowheads indicate proteins whose EV content levels appeared to change in a H₂O₂ and DBL, respectively.

Propagation of Radical Scavenging Activity to Various Cell Types

We conducted DPPH and LDH assays using C6BU-1 glioma cells and undifferentiated PC12 cells to determine whether the oxidative effects of H₂O₂ treatment in fraction 11 propagated to glial cells and other types of neuronal cells. DPPH assay demonstrated that the radical scavenging activity significantly increased in the concentrated fraction 11 supernatant after H₂O₂ treatment compared to the baseline activity levels of control group in both cell types; however, pre-treatment with 5 µM DBL for 1 h blocked this effect (Fig. 4A). The LDH assay demonstrated that the concentrated fractions did not induce cytotoxicity in either cell type (Fig. 4B). To examine the differences in the recognition ability of control and H₂O₂-induced EVs on the same cell surface, control and H₂O₂-induced EVs were stained in different colors with PKH 67 Green and PKH 24 Red Fluorescent dyes, respectively. The SH-SY5Y EVs transferred to the C6BU-1 and PC12 recipient cells (Fig. 4C), and some SH-SY5Y EVs induced by H₂O₂ treatment recognized different cell surface sites from the ones control EVs recognized (Figs. 4Cd, Ce, Cm, Cn).

Fig. 4. Cell-to-Cell Communication Ability of SH-SY5Y EVs to Various Cell Types

(A) Radical scavenging activity of SH-SY5Y fraction 11 (MWCO 100 kDa) against C6BU-1 and PC12 cells by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Data are presented as the mean ± S.E.M., n = 10, p < 0.001; one-way ANOVA, ** p < 0.01 vs. control group. (B) Cytotoxicity of SH-SY5Y fraction 11 (MWCO 100 kDa) against C6BU-1 and PC12 cells by lactate dehydrogenase assay. Data are represented as the mean ± S.E.M. (n = 10 for C6BU-1 cells, n = 8 for PC12 cells). p < 0.001; one-way ANOVA, * p < 0.05 vs. control group. (C) Uptake activity of PKH-labeled EVs in fraction 11 from SH-SY5Y cells to C6BU-1 glioma cells and undifferentiated PC12 cells. The PKH67-labeled control EVs or PKH24-labeled hydrogen peroxide-treated EVs were co-incubated with the cells for 24 h. Negative control (N.C.) samples were prepared by the same PKH-labeling procedure using Bis-Tris buffer instead of fraction 11. Scale bar, 50 µm.

DISCUSSION

In this study, we showed that the radical scavenging activity of SH-SY5Y cells treated with 100 µM H₂O₂ for 24 h propagated to various cell types, including C6BU-1, PC12, and SH-SY5Y cells via bioactive substances, likely EVs, in the culture medium. This effect was abolished by 1 h pre-treatment with 5 µM DBL, suggesting that the EVs released from SH-SY5Y host cells may be altered by DBL pre-treatment.

Previous reports had demonstrated that DBL affects cell viability differently across different cell types.^6,10) Therefore, to accurately assess the response of DBL to H₂O₂-induced oxidative stress, we quantitatively evaluated the levels of DBL cytotoxicity and H₂O₂ oxidative stress in SH-SY5Y cells (Figs. 1A, B, E). A previous study had reported that exposing SH-SY5Y cells to more than 10 µM DBL for 8 h induces antioxidant protein and phase II detoxification protein expression along with ER stress.¹⁰⁾ Similarly, we observed cytotoxicity after pre-exposing the cells to 10 µM DBL for 48 h; however, exposure to 5 µM DBL was not cytotoxic (Fig. 1Eb). The degree of H₂O₂-induced oxidative stress in our study was comparable to that in a previous report,²¹⁾ evidenced by similar IC₅₀ values after H₂O₂ exposure (Fig. 1B). Moreover, the ED₅₀ value from LDH assay after 24 h of H₂O₂ exposure and cell morphology after 24 h of 100 µM H₂O₂ or 5 µM DBL treatment (Fig. 1D) strongly supported that 24 h exposure to 100 µM H₂O₂ is cytotoxic to SH-SY5Y cells while having only little effect on mitochondrial function.

Bioactive substances in the conditioned medium from SH-SY5Y cells treated with 100 µM H₂O₂ for 24 h propagated the radical scavenging activity of fraction 11 (Figs. 3A, 4A). Heat treatment (100 °C for 5 min) eliminated this activity, whereas the concentration of the fraction did not change it (Fig. 3A). Moreover, CD63-immunoreactivity was clearly detected in fraction 11 (Fig. 2C), as was the uptake of fluorescent PKH-labeled EVs (Figs. 3B, 4C). To prevent contamination due to EVs from the FBS in the SH-SY5Y cell culture medium, we washed the host SH-SY5Y cells five times with PBS during the conditioned medium exchange. Furthermore, in the purification step via sucrose gradient ultracentrifugation, we used samples from which most low molecular weight factors were removed by concentrating and washing with 10 kDa ultrafiltration. Therefore, EVs secreted by the SH-SY5Y cells were most likely the bioactive components in fraction 11. However, we could not completely exclude the possibility of the bioactive components with high molecular weights being temperature sensitive.

Notably, the scavenging activity and cytotoxicity of fraction 10 did not differ across the treatment groups, and the PKH-labeled EV uptake was low in recipient SH-SY5Y cells in all treatment groups (Fig. 3A). However, the intensities of CD63-like immunoreactivities in fraction 10 of the H₂O₂-treated groups were stronger than those in fraction 11 (Fig. 2C). EV diversity might explain this discrepancy. Rapid technological innovations have recently enabled the analysis of the composition of EVs with high sensitivity and precision, and combined proteomics and multiple analyses, such as lipidomes, have shown that differently sized EVs released from one cell type have different compositions, indicating EV heterogeneity.^14,22) Notably, cell surface recognition patterns of the control and H₂O₂-induced EVs differed (Figs. 4Cd, Cm). In addition, silver staining analysis demonstrated that at least some protein components differed between the control and H₂O₂-induced EVs (Fig. 3D). Thus, the EVs of fraction 11 could have different physiological properties than those of fraction 10.

GAPDH is overexpressed and can accumulate in the nucleus during apoptosis in various cell types, suggesting that GAPDH may be an intracellular sensor of oxidative stress during early apoptosis.²³⁾ Therefore, the increase in GAPDH expression in the H₂O₂-treated group observed in our experiment is not a surprising phenomenon. Cells release diverse types of EVs, such as exosomes, microvesicles, and apoptotic bodies.²⁴⁾ Microvesicles and apoptotic bodies are derived from the plasma membrane and contain intracellular components, such as GAPDH. Therefore, under the H₂O₂-induced oxidative stress condition, the release of apoptotic bodies increased, suggesting that fraction 11 contained more apoptotic bodies in addition to exosomes and microvesicles. Furthermore, it was impossible to separate these three EVs (exosome, apoptotic bodies, and microvesicles) in the fraction 11 via the ultracentrifugation recovery method commonly used today. Thus, in Fig. 3C, we speculate that both CD63 and GAPDH are clearly detected under the H₂O₂- and DBL + H₂O₂-treatment conditions and not under the other conditions.

In this study, DBL pre-treatment inhibited the radical scavenging activity of fraction 11 obtained from H₂O₂-exposed SH-SY5Y cells. DBL pre-conditioned the ER to stress. The homeostatic phenomenon induced an adaptive response to weak stress levels without cytotoxicity by inducing antioxidant genes and autophagy.¹⁰⁾ We used 5 µM DBL, which was not cytotoxic to SH-SY5Y cells. Therefore, the decreased radical scavenging activity of secreted EVs in response to H₂O₂-induced oxidative stress after DBL pre-treatment could reflect antioxidant genes and autophagy induction during DBL pre-treatment. In addition, DBL induces the expression of antioxidant genes including heme oxygenase-1 (HO-1) in SH-SY5Y cells.¹⁰⁾ HO-1 is a downstream factor of E74-like factor 2 and Nrf2 signaling to cross talk between UPR and oxidative stress response in SH-SY5Y cells.²⁵⁾ Therefore, DBL pre-treatment contributes to the production of EVs containing antioxidant molecules, such as HO-1, that can prevent the radical scavenging activity of secreted EVs in response to H₂O₂-induced oxidative stress. However, the EV components that inhibit the radical scavenging effect are currently unknown and would require further investigation.

The uptake assay demonstrated that H₂O₂ treatment significantly increased the amount of fluorescent PKH-labeled EVs taken up by target cells; conversely, DBL pre-treatment suppressed the uptake in various cell types (Figs. 3B, 4C). However, the amount of EV in each of the four groups in fraction 11 showed no statistically significant difference although there was a trend toward low EVs in the DBL alone-treated group (Fig. 3C), suggesting that DBL pre-treatment had no effect on the amount of EVs released from H₂O₂-stimulated host cells. These results demonstrated the possibility that there are differences in the ability of EVs to be taken up into the recipient cells.

Curcumin, a polyphenol with chemical structure similar to that of DBL, stimulates exosome release to remove cholesterol and reduce lipid concentration accumulated in the endolysosomal compartment.^26,27) However, it is unclear whether there are differences in the physiological effects and secretion mechanisms between EVs stimulated by DBL and curcumin. As exosomes pass through the blood-brain barrier,²⁸⁾ clarifying the differences and similarities between both EVs may help to understand the mechanism of action of polyphenols in improving cognitive function, a typical physiological effect of polyphenols.

We found that bioactive substances follow cell-to-cell transmission to propagate radical scavenging activity in the 1.06 g/cm³ fraction obtained via sucrose gradient ultracentrifugation using a conditioned medium from H₂O₂-treated SH-SY5Y cells. Conversely, the activity was inhibited by 1 h pre-treatment with 5 µM DBL. Furthermore, the active substances were heat-sensitive, had high molecular weights, and were present in the EV-enriched fraction, suggesting that the substances were EVs. However, the current study could not completely rule out the possibility of factors other than EVs being responsible for this activity. Therefore, further studies would be required, by comparative analysis of factors, such as miRNAs contained in EVs, to establish that the activity was indeed due to EVs.

In conclusion, the radical scavenging activity of SH-SY5Y cells was induced by 24 h of treatment with 100 µM H₂O₂ and was inhibited after pre-conditioning with DBL. The response was propagated by cell-to-cell communication through bioactive substances, likely EVs. The results suggested that DBL might modulate the physiological effects of target recipient cells by regulating the properties of EVs released from host cells.

Acknowledgments

This work was supported by the National Institute of Advanced Industrial Science and Technology (AIST) and University of Tsukuba (Grant: FY2016 AWASEWAZA awarded to M.J. Ikemoto and H. Shigemori).

Conflict of Interest

The authors declare no conflict of interest.

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