Protective Effect of Pycnogenol® in Human Neuroblastoma SH-SY5Y Cells Following Acrolein Induced Cytotoxicity

Abstract

Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer’s disease (AD). Considerable attention has focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol® (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione (GSH) levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH-oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein induced cytotoxicity, protein damages, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress related neurodegenerative diseases such as AD.

Introduction

Intracellular oxidative stress, represented by ROS production/lipid peroxidation, is a naturally occurring physiological process that may be required in response to some cytotoxic agent as part of downstream signaling pathway [1-3]. It is well known that excessive oxidative stress can be toxic, causing membrane damage and activating pathways of cell death through protein oxidation, lipid peroxidation, and DNA cleavage processes [4-7]. Oxidative stress is associated with neurodegenerative diseases, such as Alzheimer’s disease (AD) [8, 9] and Parkinson’s disease (PD) [10-12], which depletes the antioxidant system and increases lipid peroxidation products [13-15].

Naturally occurring flavonoids possess free radical scavenging properties and neuroprotection from oxidative injury by their ability to modulate intracellular signals [16]. These flavonoids are found in fruits, vegetables and plant-derived beverages, and may have important roles as dietary components via cytoprotective actions in many organs [17, 18]. Flavonoids can act as a vasodilator [19], anti-carcinogenic, anti-inflammatory, antibacterial, immune-stimulating anti-allergic, and antiviral compounds [20]. Pycnogenol® (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), has a high capacity to scavenge free radicals and promote cellular health. Major constituents of PYC are polyphenols, specifically, monomeric and oligomeric units of catechin, epicathechin, and taxifolin. PYC has been shown to prevent neurotoxicity and apoptotic cell death in the presence of oxidative stressors [11, 21]. Concomitantly, PYC protects against lipid peroxidation, and pro-oxidants such as H₂O₂, HO⁻ NO⁻, O₂⁻, and peroxynitrite (ONOO⁻) [22-24]. The beneficial effects of PYC have been demonstrated previously and include inhibition of caspase-3 activation [25, 26].

Antioxidant therapy has been shown to be beneficial in neurological disorders including both AD and PD [4, 11, 13, 14, 21]. In the present study, using human neuroblastoma (SH-SY5Y) cell line, we investigated the effect of PYC, a potent antioxidant and reactive oxygen species (ROS) scavenger, on acrolein induced cytotoxicity and ROS generation. Acrolein (2-propen-1-al), the most reactive of the strongest electrophile among the unsaturated aldehydes, is a toxic compound that can be endogenously produced as a product of lipid peroxidation and from polyamine metabolism [27]. Acrolein can induce oxidative stress [28-30], react with proteins, phospholipids[28, 31], and DNA, forming stable Michael adducts [32]. This compound plays an important role in the development of oxidative damage [32] and, consequently, in the pathogenesis of selected diseases, such as AD [33]. The mechanisms by which acrolein causes oxidative damage and neurotoxicity are not completely defined, but accumulating evidence indicates that this alkenal primarily binds and depletes cellular nucleophiles, such as reduced glutathione (GSH), lipoic acid and thioredoxin [27]. Acrolein preferentially attacks free thiol (SH) groups of cysteine residues, γ-amino groups of lysine residues and histidine residues, resulting in an acrolein-amino acid adduct and introducing a carbonyl group to proteins [34] and impairs the function of selected proteins [29]. In the present study, we examined intracellular ROS production, antioxidants/oxidants level, and cell viability in SH-SY5Y neuroblastoma cells treated with both acrolein and pycnogenol.

Materials and Methods

Materials

PYC (gift from Horphag Research Ltd., Le Sen, France), acrolein (Fisher Scientific Inc. USA), dimethylsulfoxide (DMSO), 2,7-dichlorofluoresceindiacetate (DCFH-DA), 3-(3,4-dimehylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless stated otherwise. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) were purchased from Gibicol-BRL. Hybond-polyvinylidene difluoride (PVDF) membrane was purchased from BioRad.

Cell culture and cell treatment

SH-SY5Y cells obtained from ATCC (American Type Culture Collection, Manassas, VA) were maintained in DMEM and kept at 37 °C in a humidified 5% CO₂ incubator, supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and cultured in 48-well plates as well as in 100 mm culture plates. SH-SY5Y cells were exposed to different concentrations of acrolein (1, 5, 10 and 25 μM) freshly prepared in DMEM media each day before use. These concentrations were equal to 0.35, 1.75, 3.5 and 8.75 nmol/mg protein, respectively, with calculation based on the average protein concentration of the cells. The in vivo acrolein concentrations in AD brain have been reported to be 2.5 nmol/mg in amygdala and 5.0 nmol/mg in the parahippocampal gyrus [33]. The levels of acrolein used in this study are relevant to the pathological conditions observed in AD. SH-SY5Y cells were pre-incubated with different concentrations of PYC (25, 50 and 100ug/ml) for 1 h before acrolein was added. Assays for cell viability, GSH system, protein oxidation, lipid peroxidation, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, and ROS were performed 24 h after treatments.

Estimation of Cell Viability

SH-SY5Y cells cultures were grown in 48-well plates. In order to assay cell viability, mitochondrial function was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay as described by Zhao and colleagues [35]. Briefly, PYC treated/untreated SH-SY5Y cells were assessed 24 h after exposure to acrolein. Photomicrographs of each culture were taken by phase contrast microscopy prior to the MTT assay. A stock solution of MTT (prepared in identical media) was added to each well with a final concentration of 1.0 mg/ml, and incubated for 1 h. The dark blue formazan crystals formed in intact cells were extracted with 200 ml of dimethyl sulfoxide (DMSO), and absorbance at 595 nm was measured with a microplate reader (BIO-TEK). Results were expressed as the percent of MTT reduction, assuming that the absorbance of control cells was 100%.

Total ROS assay

Generation of ROS was assessed by DCFH-DA, an oxidation-sensitive fluorescent probe as previously described [35]. Intracellular H₂O₂ or low-molecular-weight peroxides can oxidize DCFH-DA to the highly fluorescent compound dichlorofluorscein (DCF). DCFH-DA was chosen because acrolein itself does not interfere with the fluorescence of DCFH-DA [36]. Twenty four hours after treatment with PYC and acrolein, cells were incubated with 10 μM DCFH-DA at 37 °C for 30 min, and then washed twice with PBS. The fluorescence intensity of DCF was measured in a microplate-reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

Estimation of Superoxide (O₂^·−) Production

O₂^·− production in SH-SY5Y cell was quantified with lucigenin-enhanced chemiluminescence, as previously described [37], with some modifications. SH-SY5Y cells were collected in 2.0 ml tubes and homogenized with an ultrasonic cell disruptor (Microson Farmingdale, NY). The sample volume was increased by the addition of 900 μL HEPES buffer pH 7.4 (NaCl, 119 mM; HEPES, 20 mM; KCl, 4.6 mM; MgSO₄, 1 mM; Na₂HPO₄, 0.15 mM; KH₂PO₄, 0.4 mM; NaHCO₃, 1 mM; CaCl₂, 1.2 mM and glucose 5.5 mM), protease inhibitors; pepstatin A, leupeptin, aprotinin, and phenylmethylsulfonyl fluoride (PMSF), L-NAME (1 mmol/L), triethylenetetramine (1.0 mmol/L), SDS (100 μmol/L), and 10 μL lucigenin (1800 μmol/L) was added to produce a final concentration of 20 μmol/L. Samples were aliquoted in 96-well plates, allowed to dark adapt at room temperature, and the luminescence recorded. Blanks were subtracted from cell homogenate-added wells and chemiluminescence data after blank subtraction are reported as percent counts/mg protein. O₂^·− production was also examined in the presence of diphenyliodonium and quinacrine (100 μmol/L and 1.0 mmol/L, respectively to inhibit O₂^·− producing via NADPH-oxidase system), oxypurinol (100 μmol/L), rotenone (100 μmol/L), or indomethacin (10 μmol/L) to assess the effects of xanthine oxidase, mitochondrial respiration, and cyclo-oxygenase activity on the production of O₂^·−.

Estimation of NADPH-oxidase Activity

NADPH-oxidase activity was measured according to the method described earlier [38]. Briefly, SH-SY5Y cells were homogenized by freeze and thawing in 2.0 ml 0.1M, PBS (pH 7.4) containing HEPES, 10 mM; EDTA, 2.0 mM; EGTA, 2.0 mM; MgSO₄ 0.6 mM; KCl, 4.6 mM; and protease inhibitors; pepstatin A, leupeptin, aprotinin, and phenylmethylsulfonyl fluoride (PMSF). The 20 μL of homogenate was added to 900 μL HEPES–buffer (described above) containing L-NAME (1 mmol/L), triethylenetetramine (1.0 mmol/L), SDS (100 μmol/L), 20 μmol/L lucigenin, and the samples were aliquoted in 96-well plate in the dark at room temperature. Plates were placed in the luminometer (Synergy HT, BIO-TEK), allowance was made for basal luminescence, and enzyme activity was initiated by the addition of NADPH (0.2 mmol/L), and readings taken for 15 min. Blanks were subtracted from cell homogenate-added wells and chemiluminescence data after blank subtraction are reported as percent counts/min/mg protein. Samples were also assayed in the presence of diphenyliodonium and quinacrine (100 μmol/L and 1.0 mmol/L, respectively) to inhibit the NADPH oxidases producing O₂^·−.

Estimation of GSH

Determination of GSH was performed as described previously [4]. The reaction mixture contained 0.1 M sodium phosphate buffer (pH 8.0), 5.0 mM EDTA, 20 μl o-phthaldehyde (1.0 mg/ml), and 20 μl of above mentioned sample. After incubation for 15 min at room temperature, fluorescence at emission 420 nm and excitation at 350 nm was recorded.

Estimation of GSSG

The estimation of GSSG was performed as described previously [4]. The samples were incubated first with 0.04 M N-ethyleimide (NEM) for 30 min to interact with GSH present in the sample. The reaction mixture contained 0.1 N NaOH, 5.0 mM EDTA, 20 μl o-phthaldehyde (1.0 mg/ml), and 20 μl of above mentioned sample. After incubation for 15 min at room temperature, fluorescence at emission 420 nm and excitation at 350 nm was recorded.

Estimation of Protein Carbonyls

Protein carbonyls are an index of protein oxidation and were determined as described previously [4]. Briefly, the cell extract (5mg of protein) was derivatized with 10 mM 2,4-dinitrophenylhydrazine in the presence of 5ml of 12% SDS for 20 min at room temperature. The samples were neutralized with 7.5ml of the neutralization solution (2 M Tris in 30% glycerol). Derivatized protein samples were blotted onto nitrocellulose membranes with a slot-blot apparatus (250 ng/slot). The membrane was then washed with wash buffer [10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20], blocked by incubation in the presence of 5% BSA, followed by incubation with rabbit polyclonal anti-DNPH antibody as primary antibody for 1 h. The membranes were washed with buffer and further incubated with alkaline phosphatase (ALP)-conjugated goat anti-rabbit antibody as secondary antibody for 1 h. Blots were developed using Sigma Fast tablets (BCIP/NBT) and quantified using Scion Image (PC version of Macintosh compatible NIH Image) software.

Estimation of 3-nitrotyrosine (3-NT)

The content of 3-NT was determined by incubating the above sample with Laemmli sample buffer (0.125 M Trizma base, pH 6.8, 4% SDS, 20% glycerol) for 20 min. Then 250 ng of protein were blotted onto the nitrocellulose paper using a slot-blot apparatus and immunochemical method as described above for protein carbonyls. The mouse anti-nitrotyrosine antibody was used as primary antibody and ALP- conjugated anti-mouse secondary antibody was used for detection. Densitometric analysis of bands in images of the blots was used to calculate levels of 3-NT.

Estimation of 4-HNE conjugated proteins

Levels of HNE were quantified by slot-blot analysis as described previously [4]. Anti-HNE antibody raised in rabbit was used as the primary antibody. Controls in which the primary antibody was reacted with free HNE resulted in faint, non-specific binding of the antibody (data not shown). However, since both acrolein and acrolein + PYC-treated samples used the same antibody, background correction was identical in both samples.

Western-blots

NADPH-oxidase subunits- gp91^Phox, p67^Phox, p47^Phox, p40^Phox, and p22^Phox were studied by Western-blot as described previous [4], with some modifications. The cells were lysed using an ultrasonic cell disruptor (Microson Farmingdale, NY) on ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 10% glycerol including above mentioned protease inhibitors) and centrifuged at 1000 × g for 10 min. Cytosolic and membrane fractions were prepared with standard methods [39]. Protein (50μg) was loaded with the appropriate marker (Bio-Rad) on a gradient gel (4-20% Tris-HCl), followed by a transfer to a polyvinylidene fluoride membrane using a semi-dry transfer system (Bio-Rad) in transfer buffer (25 mM Tris, 150 mM glycine, 20% MeOH) 2 h at 15 volt. The membrane was blocked with 5% milk or bovine serum albumin (BSA) in Tris/saline buffer-Tween (TBST). Primary antibodies for gp91^Phox, p67^Phox, p47^Phox, p40^Phox, and p22^Phox, including beta actin, Na+/K+-ATPase and GAPDH were added at a concentration of 1:1,000 and incubated overnight at 4°C. The blot was then washed thee times in TBST and incubated for 1 h with alkaline phosphatase conjugated secondary antibodies in a 1:8000 dilution. The membrane was washed three times in TBST for 5 min and developed in Sigma Fast tablets (BCIP/NBT substrate). Blots were dried, scanned with Adobe Photoshop, and quantified with Scion Image (PC version of Macintosh-compatible NIH Image).

Protein Estimation

Total protein concentrations were measured using the Pierce BCA method (Sigma, St. Louis, MO).

Statistical Analysis

Dose-dependent differences in oxidative/nitrosative stress markers, cell viabilities, and differences in NADPH oxidase activity are reported as mean ± SD. Possible differences between group means were evaluated with a one-way ANOVA coupled with a Student Newman-Keuls post-hoc test (StatView 5.0, SAS Institute). For significant differences, alpha was set at 0.05.

Results

Effect of PYC on Acrolein-Induced Cytotoxicity

Exposure of SH-SY5Y cell cultures to acrolein for 24 h reduced cell viability in a dose dependent fashion [F (15, 80) = 27.00, p < 0.0001] (Fig. 1 & 2). PYC alone has no affect on cell (thus cells can tolerate relatively high doses up to 200 μM). When acrolein is applied to cells pretreated with PYC for 1 h, the amount of cell death is significantly reduced suggesting that PYC plays a protective role. Regardless of whether the cells are pretreated with 50 and 100 μg/ml of PYC, the level of protection is the same, suggesting that more than one mechanism is involved in acrolein-related cell death. Pretreatment of cells with PYC at either low doses (5, 10 and 25 μg/ml), or high doses (150 and 200 μg/ml), did not provide significant (p > 0.05) protection (data not shown). PYC attenuated acrolein-induced cell loss at both 50 and 100μg/ml. Since 100μg/ml of PYC failed to show protection greater than 50μg/ml dose, following 1 or 25μM acrolein treatment, the high dose of PYC was not pursued further.

Fig. 1.

PYC prevented loss of SH-SY5Y cell viability. The viability was measured by the reduction of MTT. Acrolein caused loss of cell viability compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h protected SH-SY5Y cells from acrolein cytotoxicity compared with group acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

Fig. 2.

Phase contrast microphotographs of SH-SY5Y cells treated with acrolein with and without PYC. Acrolein treatment resulted in significant cell death with some membrane blobbing, but no other morphological changes in surviving SH-SY5Y cells. PYC (50 and 100μg/ml) significantly has reduced cell death, even at high acrolein levels. (A) Control; (B) treated with acrolein 1 μM; (C) acrolein 5 μM; (D) acrolein 10 μM; (E) acrolein 25 μM; (F) treated with PYC 50 μg/ml; (G) acrolein 1 μM + PYC 50 μg/ml; (H) acrolein 5 μM + PYC 50 μg/ml; (I) acrolein 10 μM + PYC 50 μg/ml; (J) acrolein 25 μM + PYC 50 μg/ml; (K) treated with PYC 100 μg/ml; (L) acrolein 1 μM + PYC 100 μg/ml; (M) acrolein 5 μM + PYC 100 μg/ml; (N) acrolein 10 μM + PYC 100 μg/ml; (O) acrolein 25 μM + PYC 100 μg/ml. Microphotographs were taken at 40 X and calibration bar was 10μ.

Effect of PYC on Acrolein-Induced ROS

As shown in Fig. 3, exposure of SH-SY5Y cell cultures to acrolein significantly increased ROS levels is a dose dependent fashion [F (13, 70) = 69.00, p < 0.0001]. 1 μM acrolein treatment did not significantly increase ROS levels and PYC alone has no affect on ROS production. Pretreating cells with PYC significantly reduced acrolein-induced ROS levels. A 1 h pretreatment of PYC at either 50 and 100μg/ml and subsequent acrolein exposure significantly decreased ROS levels compared to corresponding acrolein alone treatment. At the higher concentrations of acrolein (10 and 25 μM), while PYC did offer significant reductions in ROS, values were still significantly elevated compared to control. The highest concentration of PYC (100μg/ml) did not provide significantly greater antioxidant activity than 50μg/ml.

Fig. 3.

(A) PYC reduces ROS production in SH-SY5Y cells. ROS level was measured by the oxidation of DCFH-DA. Acrolein treatment increased ROS production in SH-SY5Y cells compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h reduced ROS production by acrolein treatment in SH-SY5Y cell compared the culture treated with acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only. PYC depletes O₂^·− production in SH-SY5Y cells. (B) O₂^·− level and (C) O₂^·− producing enzyme NADPH-oxidase activity was increased with acrolein treatment in SH-SY5Y cells compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h reduced O₂^·− production by acrolein treatment in cell cultures compared with acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p <0.05 and ^##p < 0.001 versus acrolein only.

Effect of PYC on Acrolein-Induced O₂^·− Production

Exposure of SH-SY5Y cell cultures to acrolein significantly [F (13, 70) = 27.00, p < 0.0001] increased O₂^·− production [Fig. 4(A)] in a dose dependent fashion. 1 μM acrolein treatment did not significantly increase O₂^·− levels. Pre-exposure with PYC for 1 h at either 50 or 100μg/ml followed by treatment with acrolein significantly decreased O₂^·− levels compared to corresponding acrolein treatment alone. The higher dose of PYC (100μg/ml) was not significantly more effective than 50μg/ml.

Fig. 4.

Level of GSH system was alleviated with PYC treatment in SH-SY5Y cells. Acrolein caused loss of GSH (A) increased GSSG (B) and discrepancy of GSH/GSSG (C) in SH-SY5Y cells compared with control. 1 h pretreatment with PYC (50 and 100 μg/ml) and incubation for 24 h protected cells from acrolein induced adverse effect on GSH system compared with group acrolein only. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

NADPH-oxidase Activity

Exposure of SY5Y cell cultures to acrolein significantly increased NADPH-oxidase activity [F (13, 70) = 19.80, p < 0.0001] [Fig. 4(B)] in a dose dependent fashion. 1 μM acrolein treatment or PYC alone had no significant effect on NADPH-oxidase activation. Pretreatment of SH-SY5Y cells with PYC 50 or 100μg/ml and subsequently treated with acrolein showed significant depletion in NADPH-oxidase activity. At 25 μM acrolein and treatment with PYC 50μg/ml, NADPH-oxidase activity was significantly reduced as compared to acrolein alone.

Effect of PYC on Acrolein-Induced GSH Depletion

The level of internal antioxidant tri-peptide molecule GSH was measured in SH-SY5Y cell cultures [Fig. 5(A)]. Analysis of GSH levels demonstrated a significant [F (13, 70) = 17.00, p < 0.0001] dose dependent decrease with acrolein. Acrolein treated cells when pretreated with either 50 or 100μg/ml of PYC significantly attenuated GSH declines compared to corresponding acrolein alone treatment. Pre-treatment with PYC at 100μg/ml was not effective in maintaining GSH levels when cells were subjected to 10μM of acrolein. Higher concentrations of PYC (100μg/ml) were not significantly more effective than 50μg/ml.

Fig. 5.

PYC treatment protects SH-SY5Y cells from protein and lipid damages. Acrolein increased protein oxidation (protein carbonyl formation A & a), lipid peroxidation (4-HNE formation B & b), and protein nitration (3-NT formation C & c) in SH-SY5Y cells compared from acrolein induced protein damages compared with group acrolein only. Blot represents one sample of each group and each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

Effect of PYC on Acrolein-Induced GSSG Elevation

Analysis of GSSG levels in SY5Y cell cultures demonstrated a significant dose dependent increase with acrolein treatment [F (13, 70) = 45.07, p < 0.0001] [Fig. 5(B)]. Pretreatment with PYC (50 μg/ml) and subsequent acrolein exposure significantly decreased GSSG levels as compared to acrolein alone. PYC alone has no affect on GSSG whether cells are treated at 25, 50 or 100 μg/ml. SH-SY5Y cells pretreated with 100 μg/ml PYC and subsequently exposed to 10μM acrolein, failed to significantly lower GSSG levels and were significantly higher than all other pretreatment groups.

Effect of PYC on Acrolein-Induced Changes in GSH/GSSG ratio

As shown in Fig. 5(C), exposure of SH-SY5Y cell cultures to acrolein for 24 h significantly reduced the GSH/GSSG ratio [F (13, 70) = 51.27, p < 0.0001]. Pretreatment with PYC for 1 h significantly attenuated acrolein-induced declines in the GSH/GSSG ratio. Pre-treatment with 50 μg/ml of PYC increased GSH/GSSG ratio in all acrolein exposure groups, although levels did not reach control values. Although pre-treatment with100 μg/ml of PYC alone resulted in a decline, it was capable of offsetting a decline normally observed with 5 μM acrolein treatment alone. At 10 μM acrolein exposure, pre-treatment of 100 μg/ml PYC failed to significantly elevate the GSH/GSSG ratio.

Effect of PYC on Acrolein-Induced Protein Carbonylation

Fig. 6(A) shows the levels of protein carbonyls in SH-SY5Y cell cultures treated with acrolein. The levels of protein carbonyls were significantly increased [F (13, 70) = 23.00, p < 0.0001] with acrolein in a dose dependent fashion. Pretreatment with either 50 or 100μg/ml of PYC significantly reduced protein carbonyl levels compared to acrolein treatment alone, although the levels were significantly higher compared to control. Higher concentration of PYC (100μg/ml) was not more effective than 50μg/ml.

Fig. 6.

NADPH-oxidase subunit proteins in SH-SY5Y cells were significantly affected with acrolein exposure. Cells were processed for immunobloting followed by Western-blot. The levls of p67^Phox (B), p47^Phox (C), and p40^Phox (D) demonstrated increase in membrane and p67^Phox decreased in cytosol (G). The effect of acrolein exposure was attenuated with PYC (50 μg/ml ). The gp91^Phox (A) and p22^Phox (E) failed to demonstrate significant changes. Na⁺/K⁺-ATPase (F) and GAPDH (G) show equal proteins.

Effect of PYC on Acrolein-Induced lipid peroxidation (4–HNE bound proteins)

The levels of 4-HNE bound proteins were significantly increased [F (13, 70) = 21.00, p < 0.0001] in SH-SY5Y cell cultures exposed to acrolein as shown in Fig. 6(B). The lower concentration of acrolein (1 μM) treatment had no significant effect on 4-HNE formation. Pretreatment with either 50 or 100 μg/ml PYC significantly decreased 4-HNE levels. Even at the highest treatment of acrolein (25 μM), PYC 50 μg/ml demonstrated a significant reduction in 4HNE levels. There were no significant differences between pretreatment of PYC at 50 and 100 μg/ml.

Effect of PYC on Acrolein-Induced Protein Nitration (3-NT, Formation)

Acrolein exposure significantly increased the levels of 3-NT in the SH-SY5Y cell culture [F (13, 70) = 17.70, p < 0.0001] as shown in Fig. 6(C). The levels of 3-NT were only affected by the highest doses of acrolein (10 μM, and 25 μM). Pretreatment with PYC significantly decreased acrolein induced 3-NT formation. Increasing the concentration of PYC from 50 to 100 μg/ml did not have a significant difference.

Effect of PYC on Acrolein-induced NADPH-oxidase activation

Western-blot analysis shows acrolein-induced changes in NADPH-oxidase complex proteins (gp91^Phox, p67^Phox, p47^Phox, p40^Phox, and p22^Phox) in SH-SY5Y cells (Fig. 7) and PYC significantly reduce active complex formation.

Fig. 7.

Cytosolic protein subunits of NADPH-oxidase p67^Phox (A & B), levels in the SH-SY5Y cells increased in membrane and declined in cytosol with acrolein exposure were significantly attenuated by PYC (50 μg/ml). In same fashion p47^Phox (C), and p407^Phox (D) also show increase with acrolein exposre and significantly decreased with PYC treatment. Each bar represents the group mean ± SD of six cultures/group. *p < 0.05 and **p < 0.001 versus control, ^#p < 0.05 and ^##p < 0.001 versus acrolein only.

The cytosolic component of NADPH-oxidase p67^Phox, p47^Phox, and p40^Phox, showed a significant dose-dependent increase [F (7, 40) = 15.43, p > 0.0001] in membrane (Fig. 8A,C & D) with acrolein treatment. Like p67^Phox and p47^Phox, p40^Phox also significantly decreased [F (7, 40) = 7.955, p > 0.0001] in the membrane with PYC treatment. In reverse fashion, p67^Phox subunit significantly declines [F (7, 40) = 17.44, p > 0.0001] in the cytosol (Fig. 8B), indicate a shift of the subunit to the membrane, which is attenuated by PYC significantly (Fig. 7D).

Fig. 8.

Membrane protein subunits of NADPH-oxidase gp91^Phox (A) and p22^Phox (B) show non significant elevation with acrolien exposure. PYC attenuates the levels of both subunits in membrane.

A major membrane subunits of NADPH-oxidase, gp91^Phox and p22^Phox, showed no significant change [F (7, 40) = 1.3429, p < 0.2559] with acrolein treatment (Fig. 9A & B).

Fig. 9.

Schematic presentation of proposed mechanism of protective effects of pycnogenal (PYC) on acrolein-induced cytotoxicity in SH-SY5Y cells. The thiol (-SH) – reactive property of acrolein activates NADPH-oxidase complex, leading to the production of superoxide (O₂^−.). Normally superoxide dismutase (SOD) converts O₂^−. into hydrogen peroxide (H₂O₂). O₂^−. activates nitric oxide synthase (iNOS) leading to the production of nitric oxide (NO) and the formation of peroxynitrites (ONOO⁻), which ultimately causes oxidative/nitrosative stress. Increased oxidative/nitrosative stress causes mitochondrial dysfunction, lipid peroxidation, protein and DNA damage, which can result in cell death. Lipid peroxidation further produces toxic aldehyde products including elevated acrolein that further aggravates degenerative process. ONOO⁻ is known to activate poly(ADP-ribrosyl) polymerase (PARP) leading to ATP depletion and cell death. PYC prevents assembling of NADPH-oxidase subunits (both in cytosol and the membrane), and has protective effects on the beginning of acrolein-induced deleterious cascade. PYC prevents O₂^−. production from NADPH-oxidase activation and NO from iNOS. PYC scavenges O₂^−., NO, ONOO⁻, H₂O₂ and hydroxyl radicals (OH^.) to prevent oxidative/nitosative stress, and elevates internal antioxidant glutathione (GSH) defenses that can enhance cell survival.

Discussion

The present set of in vitro experiments characterized the effects of Pycnogenol® on a variety of oxidative stress markers. PYC, a patented combination of bioflavonoids, scavenges oxidants and thus promotes cellular health. PYC is a mixture of polyphenols, that decrease toxic byproducts of lipid peroxidation such as H₂O₂, HO^−., O₂^−. and O₂⁻ [22, 23]. Previous studies have reported increased SH-SY5Y cell survival with PYC [21, 25, 26]. The present study demonstrates that PYC can protect SH-SY5Y cells from cytotoxicity induced by acrolein in part by (1) reducing ROS production; (2) remediating suppressed NADPH-oxidase, and (3) enhancing the GSH system [25, 26]. It is been suggested that PYC may not only suppress the generation of ROS, but also attenuate apoptotic neuronal death in Aβ-induced animal models of AD, possibly by decreasing free radical generation [11]. Data from this present study shows that treatment with acrolein results in a significant increase of ROS level in a dose-dependent manner. This is consistent with previous descriptions of acrolein-induced oxidative stress [33, 40, 41].

GSH protects cultured neurons against oxidative damage resulting from acrolein, and 4-HNE [33]. GSH can also protect the brain from damage by peroxynitrite, hydroxyl free radicals, or reactive alkenals [42]. These findings suggest that these changes, are a common feature of various types of oxidative and nitrosative stress, which may be associated with the protein damages, modification of redox regulation and cellular function [43]. There is evidence that oxidative stress, including free radicals, plays a key role in AD and PD [8, 9, 44]. Brain membrane lipids are rich in polyunsaturated fatty acids, which are especially sensitive to free radical-induced lipid peroxidation [45, 46].

Several studies have reported that increased endogenous antioxidant levels, resulting from dietary or pharmacological intake of antioxidants precursors or substrates, can protect GSH from oxidative depletion and consequently protect the brain against oxidative stress [47-49]. PYC has been used as a nutritional supplement to be taken orally, and studies have indicated that this phytochemical is indeed being absorbed and metabolized by humans [50, 51]. Other studies indicate that PYC pretreatment is associated with the protection of cells undergoing oxidative stress associated death, with potential applications in neurodegenerative diseases [24].

Acrolein is the strongest electrophilic aldehyde in nature [29] that occurs as a ubiquitous pollutant in the environment. It is also formed in vivo from cellular lipid peroxidation [52, 53], and reacts with various biomolecules including proteins, DNA, and phospholipids, with the potential to disrupt their function especially in neurodegeneration [29, 33, 40, 41, 54]. The mechanisms of how acrolein inflicts tissue damage are not fully established, but data suggests that it can induce oxidative stress [54, 55] (Fig. 3). It has been reported that the acrolein-induced damaging molecules strongly react with GSH, reducing levels and subsequently induces the production of ROS [40, 41, 56, 57]. Depletion in the GSH defense favors an increase in apoptosis and a decrease in cell survival [36, 55, 58, 59] (Fig. 4). In the presence of acrolein, NADPH-oxidase can also be an important source of ROS over-production and protein modification, leading to neuronal oxidative stress. It is known that the thiol (-SH)-reactive property of acrolein are responsible for the activation of NADPH-oxidase complex and the inducible nitric oxide synthase (iNOS) [60]. In the present set of experiments, acrolein significantly increased ROS production, O₂^−. levels and activation of O₂^−. producing enzyme NADPH-oxidase (Fig. 6 & 7). Such events have been linked to apoptotic cell death via caspase-3 activation, DNA fragmentation, and poly (ADP-ribose) polymerase (PARP) cleavage [11]. PYC suppressed both ROS and NADPH oxidase activation (assembling of cytosolic subunits (p67^Phox, p47^Phox and p40^Phox) to the membrane subunits (gp91^Phox and p22^Phox)), while also attenuating GSH depletion, protein damages, and acrolein-induced cell death.

The results shown in this in vitro study demonstrate that PYC probably acts as an antioxidant at the maximal dose 50 μg/ml. We demonstrated that PYC protected SH-SY5Y cells from acrolein-induced oxidative stress by decreasing the percentage of ROS production and as well as reducing NADPH-oxidase activation. In addition, we also found that PYC attenuated the reduction in GSH levels, by the decline in oxidative/nitrosative burden, which was a normal consequence of acrolein exposure. But the elevation in GSH was not due to its synthesis; since PYC treatment was not effective even at 200 μg/ml. Lower concentrations, were not enough to remove acrolein induced oxidative stress, while high concentrations of PYC, being a xenobiotic for the system, might itself be creating a burden, that would favor oxidative stress in the presence of other oxidants. This may explain why, at higher concentrations (100μg/ml) PYC did not provide sufficient antioxidant defenses. Data showing PYC protects SH-SY5Y cells from acrolein cytotoxicity, suggests that the mechanism of acrolein neurotoxicity involves oxidative stress, and that the neuroprotective effects of PYC are associated with its antioxidant properties. We also observed that acrolein may have destructive effects through mechanisms other than oxidative stress, since pretreatment with PYC never resulted in values equivalent to that of controls. PYC could be a key component for the development of therapeutics for neurodegenerations including AD, but case in its effective concentrations and timings for treatment must be observed.

Abbreviations used

DCFH-DA

2,7-dichlorofluoresceindiacetate

DMSO

dimethylsulfoxide

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

4-HNE

4-hydroxynonenal

L-NAME

N_ω-Nitro-L-arginine methyl ester hydrochloride

MTT

3-(3,4-dimehylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

NEM

N-ethyleimide

NADPH

reduced nicotinamide adenine dinucleotide phosphate

3-NT

3-nitrotyrosine

PBS

phosphate-buffered saline

PMSF

phenylmethylsulfonyl fluoride

PMS

post-mitochondrial supernatant

PVDF

polyvinylidene difluoride

PC

protein carbonyl

PYC

Pycnogenol®

RNS

reactive nitrogen species

ROS

reactive oxygen species