Glyceryl 1,3-Dipalmitate Produced from Lactobacillus paracasei

Aug 22, 2017 - Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Number 1, Section 4,. Roosevelt ...
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Glyceryl 1,3-Dipalmitate Produced from Lactobacillus paracasei subspecies. paracasei NTU 101 Inhibits Oxygen−Glucose Deprivation and Reperfusion-Induced Oxidative Stress via Upregulation of Peroxisome Proliferator-Activated Receptor γ in Neuronal SH-SY5Y Cells Meng-Chun Cheng and Tzu-Ming Pan* Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Number 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan ABSTRACT: Glyceryl 1,3-dipalmitate (GD) purified from Lactobacillus paracasei subsp. paracasei NTU 101-fermented products has been demonstrated to possess neuroprotective properties. We determined the effect of GD on oxygen−glucose deprivation and reperfusion (OGD/R)-induced SH-SY5Y neuroblastoma cell death. GD ameliorated OGD/R-induced apoptosis by elevating the protein expression of nuclear peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor erythroid 2-related factor 2 (Nrf2), thereby attenuating reactive oxygen species (ROS) generation. Pretreatment with GD reduced nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) expression from 1.54 ± 0.27 to 0.84 ± 0.46, thereby attenuating the induction of pro-inflammatory mediators, and increased the plasma membrane Ca2+ ATPase (PMCA) levels from 0.81 ± 0.02 to 1.08 ± 0.06, thus reducing the levels of cytosolic Ca2+; this also correlated with reduced cell death. We conclude that GD prevents SH-SY5Y cells from injury after OGD/R insult, possibly by modulating oxidative stress and inflammatory response. KEYWORDS: glyceryl 1,3-dipalmitate, Lactobacillus paracasei subsp. paracasei NTU 101, fermented products, oxygen−glucose deprivation and reperfusion, peroxisome proliferator-activated receptor γ

1. INTRODUCTION Cerebral ischemia interrupts blood flow in the brain and, thus, deprives brain cells of oxygen and nutrients required to meet their energy demands.1 Cerebral ischemia/reperfusion can induce neuronal injury and is associated with complicated biochemical and molecular events that hamper neurological functions. These events include excitotoxicity, apoptosis, oxidative stress, ionic imbalance, and inflammation.2 In vitro oxygen−glucose deprivation and reperfusion (OGD/R) experiments can mock the acute restriction of metabolites and oxygen supply that occurs following cerebral ischemia/reperfusion. Reperfusion after OGD disrupts the cell membrane permeability and finally results in neuronal cell death.3 OGD promotes reactive oxygen species (ROS) production, which can induce apoptosis. Several components of ROS that are generated after OGD/R insult play the key role in neuronal loss after cerebral ischemia.4 Among these are nitric oxide (NO) and the NO derivatives. The major source of potentially damaging NO in inflammatory cells is inducible nitric oxide synthase (iNOS).5 Increased expression levels and activation of peroxisome proliferator-activated receptor γ (PPARγ) can antagonize these harmful effects,6 indicating a neuroprotective role for PPARγ signaling in OGD/R. The mechanisms by which PPARγ is neuroprotective following ischemia include (1) inhibition of excitotoxicity, (2) repression of pro-inflammatory responses [such as inhibition of nuclear factor κ-lightchain-enhancer of activated B cells (NF-κB)], (3) upregulation of antioxidant enzymes, (4) activation of phagocytic activities, and (5) modification of neutrophil phenotypes.7 Finally, PPARγ © 2017 American Chemical Society

induces nuclear factor erythroid-2-related factor (Nrf2) expression. Nrf2 is a transcription factor that binds to antioxidant response elements (AREs) in antioxidant and detoxification genes (so-called “phase II” genes), including heme oxygenase-1 (HO-1). HO-1 confers protection by modulating intracellular redox states.8 Moreover, previous studies indicate that HO-1 can be upregulated through the Nrf2/ARE-mediated pathway and has an essential role in the protection of ischemia.9 Thus, the net effect of Nrf2 activity is the reduction in levels of prooxidative molecules, thereby removing one of the primary sources of NF-κB activation.10,11 Diglycerides (DGs) have a variety of uses in foods, cosmetic ingredients, and pharmaceuticals. It has both nutritional properties and dietary effects, many of which may be mediated by its 1,3-DG derivative.12 In Japan, certain cooking oils contain 70% 1,3-DG; these oils are classified as “health-specific foods” as a result of their clinical benefits. For example, Econa Healthy Cooking Oil (which was marketed by the ADM/Kao Group of Japan) is now available in Japan and the U.S.A.12,13 The prohealth benefits of 1,3-DG include its ability to raise β-oxidation, decrease body weight, suppress fat accumulation, and lower the triacylglycerol levels in the serum.14 Moreover, DG ameliorates the symptoms of illnesses, such as cardiovascular diseases, diabetes mellitus, and hypertension, thereby reducing the risk Received: Revised: Accepted: Published: 7926

June 12, 2017 August 18, 2017 August 22, 2017 August 22, 2017 DOI: 10.1021/acs.jafc.7b02728 J. Agric. Food Chem. 2017, 65, 7926−7933

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Journal of Agricultural and Food Chemistry of stroke.13 However, whether 1,3-DG itself can prevent strokerelated conditions has not yet been addressed. In the current study, we purified glyceryl 1,3-dipalmitate (GD) from Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101)-fermented products, and investigated its neuroprotective activity in the context of OGD/R-induced SH-SY5Y cell death.

2.2. GD Isolation and Purification. GD (Figure 1A) was isolated from NTU 101-fermented 25.0% skimmed milk sample supplemented with 5% filter-sterilized glucose (NTU 101-fermented products). Briefly, crude extracts of NTU 101-fermented products were concentrated under reduced pressure. The crude extract was subjected to silica gel column chromatography and eluted through several solvent gradients, including dichloromethane and methanol, hexane and acetone, and dichloromethane and ethyl acetate. Each fraction was concentrated and dehydrated before analysis by nuclear magnetic resonance spectra recorded on a Bruker Avance 400 spectrometer and liquid chromatography− electrospray ionization−mass spectrometry (LC−ESI−MS) system consisting of an ultraperformance liquid chromatography (UPLC) system (Ultimate 3000 RSLC, Dionex, Sunnyvale, CA, U.S.A.) and an electrospray ionization (ESI) source of a quadrupole time-of-flight (TOF) mass spectrometer (MS, maXis HUR-QToF system, Bruker Daltonics, Billerica, MA, U.S.A.). Preparation of GD (95.49% purity) was validated via LC−ESI−MS system.15 2.3. Cell Culture. The human neuroblastoma cell line SH-SY5Y was purchased from American Type Culture Collection (ATCC) and maintained in F12 and MEM (1:1, v/v) media, supplemented with 15% FBS, 10 mM sodium pyruvate, and 2 mM glutamine. Cells were incubated at 37 °C in 95% humidity and 5% CO2. Confluent cells were subcultured by diluting 1:3 in a 10 cm dish, and the culture medium was replaced every 3−4 days. To measure the cell viability, cells were seeded at a density of 2 × 104 cells/well of a 96-well plate. After incubation for 24 h, the cells were treated with GD (1 or 2 μM) for another 24 h, followed OGD/R insult. 2.4. OGD/R Experiments. OGD/R was performed as described previously.16 To initiate OGD, glucose-free F12/MEM (1:1, v/v)

2. MATERIALS AND METHODS 2.1. Chemicals. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), trypsin, and glucose were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Glutamine, sodium pyruvate, minimum essential medium (MEM), and Ham’s F12 (F12) (were purchased from Invitrogen Corp. (Carlsbad, CA, U.S.A.). Sodium dodecyl sulfate (SDS) was purchased from Merck (Darmstadt, Germany). Fetal bovine serum (FBS) was obtained from Invitrogen Life Technologies (Carlsbad, CA, U.S.A.). The Bio-Rad protein assay reagent was purchased from Bio-Rad Laboratories (Hercules, CA, U.S.A.).

Figure 1. GD inhibits OGD/R-induced SH-SY5Y cell death. (A) Chemical structure of GD, (B) SH-SY5Y cell viability following OGD treatment of various lengths (0−6 h), followed by 24 h of reperfusion, and (C) effect of GD pretreatment on SH-SY5Y viability after OGD/R. Data are the mean ± SD of experiments performed in triplicate. (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 compared to the OGD/R group, using Duncan’s multiple range test. GD, glyceryl 1,3-dipalmitate; con., concentration; and OGD/R, oxygen−glucose deprivation and reperfusion.

Figure 2. Effect of GD on OGD/R-induced ROS production. (A) Time course of the effect of reperfusion on ROS accumulation and (B) effect of GD pretreatment on ROS production, with the data obtained after 4 h of reperfusion. Intracellular ROS accumulation was measured using the fluorescent probe, DCFH-DA. Data are the mean ± SD of experiments performed in triplicate. (∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001 compared to the OGD/R group, using Duncan’s multiple range test. GD, glyceryl 1,3-dipalmitate; con., concentration; and OGD/R, oxygen−glucose deprivation and reperfusion. 7927

DOI: 10.1021/acs.jafc.7b02728 J. Agric. Food Chem. 2017, 65, 7926−7933

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Journal of Agricultural and Food Chemistry medium was bubbled with an anaerobic gas mixture (95% N2 and 5% CO2) for 30 min to remove any residual oxygen. After pretreatment with GD for 24 h, the sample medium was discarded and the cells were rinsed with glucose-free F12/MEM (1:1, v/v) and placed in fresh glucose-free F12/MEM. The medium in OGD groups was switched to glucose-free F12/MEM and then incubated for 6 h at 37 °C in 95% N2 and 5% CO2 for adaptation. OGD was terminated by adding glucose at a final concentration of 4.5 mg/L, followed by incubation under normal conditions for 24 h (reperfusion). In the control group, cells were incubated with growth medium under normal oxygen levels and changed to glucose-free F12/MEM medium at a final concentration of 4.5 mg/L, as described for reperfusion of the OGD group. 2.5. Determination of Cell Viability. Cell viability was quantified by the methyl thiazolyl tetrazolium (MTT) assay. After reperfusion, cells were incubated with MTT (0.5 mg/mL) in phosphate-buffered saline (PBS) for 4 h at 37 °C. Subsequently, the medium was removed and 200 μL of culture grade dimethyl sulfoxide (DMSO) was added to the culture wells to solubilize formazan. The absorbance was measured at 570 nm using a microplate reader (ELISA Universal Microplate Reader EXL 800, Bio-Tek Instruments, Inc., Winooski, VT, U.S.A.). Cell viability (percentage of the control) was calculated using the equation: percentage of the control = (absorbancetreatment/absorbancecontrol) × 100%. 2.6. Determination of ROS Generation. To monitor the net intracellular accumulation of ROS, the cell-permeable fluorescent probe DCFH-DA was used. Following treatment, cells were harvested with PBS and incubated with DCFH-DA (20 μM) in the dark for 30 min at 37 °C. The fluorescence level was measured by flow cytometry (BD FACSCanto, BD Biosciences, San Jose, CA, U.S.A.). ROS intensity was expressed as the relative value compared to that of the control. 2.7. Apoptosis Assays. Cell death was measured by the Annexin V−fluorescein isothiocyanate (AV−FITC) apoptosis detection kit (BD Pharmingen, San Jose, CA, U.S.A.). SH-SY5Y cells were centrifuged

to remove the medium, washed twice with PBS, and resuspended in 500 μL of binding buffer. Cells were counted to obtain a final density of 1 × 106 cells/mL. AV−FITC (5 μL) was added to the cells. Following incubation at 37 °C for 30 min under dark conditions, samples were washed with binding buffer. Propidium iodide (PI, 5 μL) was added to the mixtures, and stained cells were immediately analyzed on FACSCanto flow cytometry (BD FACSCanto). A total of 10 000 events were recorded for each sample. 2.8. Nuclear Protein Extraction. The nuclear protein extraction kit (BioVision, Mountain View, CA, U.S.A.) was used according to the instructions of the manufacturer. In brief, cytosol extraction buffer containing dithiothreitol and protease inhibitors was added (200 μL) to the cell pellet. After incubation, cell pellets were centrifuged (16000g for 5 min at 4 °C), and the supernatant was separated from the pellets (which contained nuclei) as the cytoplasmic extract. The pellets were resuspended in 100 μL of ice-cold nuclear protein extraction buffer, vortexed vigorously, and kept on ice for 10 min. This was repeated 4 times. After centrifugation (16000g at 4 °C) for 10 min, the supernatant (nuclear extract) was frozen and stored for future use. The sample protein concentration was determined by the Bio-Rad protein assay kit. 2.9. Western Immunoblotting. Cells were washed with ice-cold PBS twice, lysed by adding cold lysis buffer, and centrifuged (12000g for 10 min at 4 °C) to obtain the supernatant. Protein concentrations for each sample were determined using the Bio-Rad protein assay kit. A total of 20 μg of proteins was electrophoresed on 12% sodium dodecyl sulfate−polyacrylamide gel. Separated proteins were transferred onto a polyvinylidene difluoride membrane. The membrane was blocked for 1 h at room temperature with blocking buffer [0.05% Tween-20 and 5% (w/v) non-fat dry milk in PBS], washed 3 times with PBS containing 0.05% Tween-20 (PBST), and then probed overnight with 1:10 000 dilutions of anti-PPARγ and anti-Nrf2 antibodies (Cell Signaling Technology, Beverly, MA, U.S.A.) and

Figure 3. Effect of GD on SH-SY5Y cell apoptosis induced by OGD/R. SH-SY5Y cells were subjected to 6 h of OGD, followed by 24 h of reperfusion with or without GD, and then double-stained with AV−FITC and PI. Dot plots show the intensity of AV−FITC fluorescence on the x axis and PI fluorescence on the y axis. GD, glyceryl 1,3-dipalmitate; OGD, oxygen−glucose deprivation. 7928

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Journal of Agricultural and Food Chemistry anti-NF-κB, anti-HO-1, anti-Bcl-2, and anti-Bax antibodies (Abcam, Cambridge, MA, U.S.A.) at 4 °C. Blots probed at a dilution of 1:1000 with a mouse monoclonal antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cell Signaling Technology) or Lamin B purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Burlingame, CA, U.S.A.) were used as a control for the loading of the protein level. The membrane was washed 3 times in PBST for 5 min, incubated with diluted secondary antibody solution (conjugated to horseradish peroxidase) for 1 h at room temperature with shaking, and washed 3 times with PBST (each for 5 min). Protein bands were detected using an enhanced chemiluminescence reagent from Millipore (Billerica, MA, U.S.A.). Relative band intensities were analyzed using a Gel-Pro Analyzer 4 (Media Cybernetics, Inc., Rockville, MD, U.S.A.). 2.10. Statistical Analysis. The triplicate results were averaged and displayed as means ± standard deviation (SD), and analysis was carried out using the Statistical Package for the Social Sciences (SPSS) software (version 19.0, Chicago, IL, U.S.A.). Statistical analysis was analyzed using one-way analysis of variance (ANOVA), followed by post-hoc Duncan’s multiple range test. Duncan’s multiple range test was used to determine significant differences (p < 0.05) between means of the treatment groups.

this allowed us to further investigate the neuroprotective effects of GD. 3.2. Defining the Optimal GD Dose for Protection of SH-SY5Y Cells upon OGD. SH-SY5Y cells were preconditioned with GD at doses between 0.25 and 2 μM for 24 h. Pretreatment with 2 μM GD significantly increased cell viability (75% compared to 50% in cells exposed only to OGD/R; p < 0.01; Figure 1C). The protective effect was also seen at lower concentrations of GD but was not concentration-dependent. We therefore chose 0.5, 1, and 2 μM GD for further experiments. In addition, pretreatment with GD alone was not cytotoxic to SH-SY5Y cells (data not shown). 3.3. GD Ameliorates OGD/R-Induced Oxidative Stress. Reperfusion following OGD induced significantly increased ROS accumulation. Therefore, we next tested the effects of GD on the OGD/R-induced generation of ROS in SH-SY5Y cells. ROS accumulated rapidly following reperfusion and increased steadily before peaking at 4 h post-reperfusion (nearly 1.6-fold compared to the control). Subsequently, ROS progressively declined to pretreatment levels (Figure 2A). Pretreatment with 2 μM GD significantly suppressed ROS when compared to the OGD/R group (Figure 2B). 3.4. GD Inhibits OGD/R-Induced SH-SY5Y Cell Death. To determine that cell death induced by OGD/R was due to either necrosis or apoptosis, SH-SY5Y cells were simultaneously stained with AV−FITC and PI, followed by flow cytometry analysis. Under normal conditions, a low level of neuronal apoptosis and necrosis was noted. After the OGD/R insult, the percentage of apoptotic cells (AV−FITC+/PI−) increased

3. RESULTS 3.1. OGD Kills SH-SY5Y Cells in a Time-Dependent Manner. SH-SY5Y cells were exposed to the OGD for 0−6 h periods that were then followed by 24 h of reperfusion. There was a time-dependent reduction in viability, which reached 55% following 6 h of OGD when compared to the control group (p < 0.001; Figure 1B). We selected the 6 h OGD treatment for subsequent experiments, because we found that the amount of cell death can still be modulated pharmacologically at this time;

Figure 4. GD upregulates Nrf2, PPARγ, and HO-1 expression. (A) Nuclear Nrf2 and PPARγ and HO-1 were evaluated by Western blot and densitometric analysis of (B) PPARγ, (C) Nrf2, and (D) HO-1 levels, normalized to the level of Lamin B or GAPDH. Data are the mean ± SD of experiments performed in triplicate. The letters above bars indicate significant difference between the groups (p < 0.05), obtained using Duncan’s multiple range test. GD, glyceryl 1,3-dipalmitate; OGD/R, oxygen−glucose deprivation and reperfusion. 7929

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Journal of Agricultural and Food Chemistry from 0.2 to 14.7%. However, the number of necrotic cells (AV−FITC+/PI+) was unchanged, regardless of the treatment. In contrast, pretreatment with 2 or 1 μM GD suppressed the apoptosis rates to 4.6 and 10.1%, respectively. Thus, the results of flow cytometry analysis indicated that GD protected SH-SY5Y cells from OGD/R-induced apoptosis (Figure 3). 3.5. GD Modulates the Expression of PPARγ, Nrf2, and HO-1 after OGD/R Insult. We first evaluated nuclear PPARγ protein expression. While OGD/R rapidly reduced expression of PPARγ, pretreatment with 2 μM GD significantly reversed this effect (p < 0.05; panels A and B of Figure 4). Therefore, whether GD exerts antioxidant effects by increasing the expression of Nrf2 and its downstream targets, such as the antioxidant enzyme HO-1, was analyzed. After OGD/R insult, the level of nuclear Nrf2 decreased; further, expression of HO-1, a downstream target factor of Nrf2, was also elevated following GD pretreatment (panels A, C, and D of Figure 4). These findings suggest that GD might protect SH-SY5Y cells against oxidative injury via Nrf2. 3.6. GD Modulates the Expression of NF-κB, Bax, Bcl-2, and Cleaved Caspase-3 after OGD/R Insult. Next, the influence of OGD/R insult on the induction of the NF-κB p65 subunit was analyzed. p65 levels increased rapidly after OGD/R insult in the SH-SY5Y cells compared to the control group. Pretreatment with GD reversed this effect, wherein p65 returned to basal (no OGD/R treatment) levels (Figure 5). OGD/R treatment produced high Bax and low Bcl-2 levels. Strikingly, 2 μM GD significantly attenuated Bax and the Bax/ Bcl-2 ratio and cleaved caspase-3 induction (Figure 6). Therefore, it could be inferred that GD may exert some of its anti-apoptotic effects by preventing the induction of proapoptotic Bax and altering the level of pro-survival Bcl-2 family proteins. 3.7. GD Treatment Increases Plasma Membrane Ca2+ ATPase (PMCA) Expression after OGD/R Insult. The PMCA level in SH-SY5Y cells was measured after OGD/R insult. As shown in Figure 7, a statistically significant increase in PMCA protein expression was noted in cells pretreated with 2 μM GD compared to that in cells exposed only to OGD/R (p < 0.05). Therefore, it could be suggested that this GD-dependent upregulation of PMCA may counteract the increased Ca2+ load that occurs after OGD/R, thereby protecting against cell death.

Figure 5. GD regulates NF-κB expression. (A) NF-κB was evaluated by Western blot and (B) densitometric analysis of NF-κB levels, normalized to the level of GAPDH. Data are the mean ± SD of experiments performed in triplicate. The letters above bars indicate significant difference between the groups (p < 0.05), obtained using Duncan’s multiple range test. GD, glyceryl 1,3-dipalmitate; OGD/R, oxygen−glucose deprivation and reperfusion.

changes. However, the mechanism underlying the neuroprotective action of dodecylglycerol remains unknown.21 Docosahexaenoate is the major ω-3 fatty acid family member. Pretreatment with docosahexaenoate could prevent concentration-dependent reduction of cell viability,22 and docosahexaenoate conferred neuroprotection via downregulation of pro-apoptotic (Bax) and pro-inflammatory factors (tumor necrosis factor-α, interleukin 1β, and cyclooxygenase-2) and upregulation of the anti-apoptotic Bcl-2 proteins.23 In this study, we found that GD ameliorates OGD/R-induced apoptosis in SH-SY5Y cells by upregulating nuclear PPARγ and Nrf2, thereby attenuating ROS generation and NF-κB expression. PPARγ plays an important role in mediating oxidative stress, inflammation, and apoptosis and confers significant protection against OGD injury in neurons.24 Moreover, PPARγ activation is associated with the attenuation of symptoms in several diseases associated with stroke, such as inflammation, oxidative damage, edema, blood−brain barrier preservation, and excitotoxicity.7 Transcription factor Nrf2 attenuates intracellular stress, in part by inducing the expression of HO-1, an antioxidant enzyme.25 In addition, Nrf2 and PPARγ pathways converge to mount an antioxidant response to limit oxidant-induced damage. For example, the 15-deoxy-Δ12,14-prostaglandin J2-PPARγ agonists induce HO-1 expression through mechanisms independent of PPARγ regulation of Nrf2.8 Moreover, Cho et al. reported that PPARγ acts on upstream signaling pathways that lead to Nrf2 activation.26 Activating the Nrf2 pathway to ameliorate oxidative stress may have therapeutic potential in treating stroke.27 Furthermore, the Nrf2/HO-1 pathway is now viewed as a key

4. DISCUSSION Ischemic stroke can be precipitated by multiple pathologies and manifests as energy failure in the brain neural cells as a result of disruption of mitochondrial adenosine triphosphate (ATP) synthesis. This results in membrane depolarization, activation of glutamate receptors, and induction of a massive calcium influx, further increasing production of free radicals.17 Oxidative stress, inflammatory response, and apoptosis are the predominant pathways that induce cell death in response to OGD/R. Curcuma oil (isolated from powdered rhizomes of Curcuma longa Linn.) possesses antioxidant and neuroprotective activities because of its ability to suppress lipid peroxidation and DNA damage.18 This is encouraging from the human health perspective because curcuma oil is a safe natural product that is used in culinary practice and traditional medicine.19 Glycerol has been reported to attenuate ischemia injury by decreasing edema formation in the brain and reducing lipid peroxidation.20 Pretreatment with dodecylglycerol for 24 h before glutamate treatment caused significant neuroprotection, as observed by decreasing LDH activity and morphological 7930

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Figure 6. GD regulation of Bax and Bcl-2 expression. (A) Bax and Bcl-2 were evaluated by Western blot, densitometric analysis of (B) Bax and (C) Bcl-2 levels, normalized to the level of GAPDH, (D) ratio of Bax and Bcl-2, and (E) cleaved caspase-3 levels, normalized to the level of GAPDH Results are expressed as the mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05). GD, glyceryl 1,3-dipalmitate; OGD/R, oxygen−glucose deprivation and reperfusion.

determinant of the cellular response to neuronal injury.25,28,29 In this study, for OGD/R injury, the ROS production was dramatically increased. Consequently, there was no significant accumulation of nuclear Nrf2, and expression of HO-1 was downregulated after the reperfusion injury. Apart from GD, several other potential neuroprotective agents, such as curcumin,29 flavanol,9 and sulforaphane,28 have shown important beneficial effects in the treatment of OGD/R-related neuronal disease; this is thought to be through activation of the Nrf2/HO-1 pathway. Therefore, we suggest that pretreatment with GD may strongly potentiate the adaptive antioxidant ability of the cell. Zhang et al. observed that the induction of Nrf2 may not only increase anti-apoptotic protein Bcl-2 expression but also inhibit the translocation of Bax to mitochondria.30 Cerebral ischemia activates intrinsic apoptosis, and the ratio of Bax/Bcl-2 is an important determinant of this pathway.31 In this study, we have shown that pro-apoptotic Bax expression increases and antiapoptotic Bcl-2 expression decreases after OGD/R insult and that GD pretreatment attenuates the increased Bax/Bcl-2 ratio. Bax not only exerts broad protective effects against ischemic neuronal injury but also regulates intracellular calcium signaling

in neurons.32 The intracellular calcium concentration is a key factor during neurodegeneration observed upon ischemic/reperfusion injury. Furthermore, enhanced calcium entry into neurons following ischemia has also been linked to ion channels and transporters, such as PMCA.33 As a result of its high affinity for calcium, PMCA plays a central role by maintaining basal intracellular calcium levels.34 The PMCA expression level can determine the survival of PC12 cells following exposure to increased intracellular calcium.35 Our study reveals that GD pretreatment significantly reduced the level of Bax upon OGD/R insult while concomitantly upregulating PMCA. Therefore, we suggest that GD regulates intracellular calcium by downregulating Bax and PMCA levels. Pro-inflammatory cytokines released in the ischemia-injured brain are regulated by NF-κB. NF-κB consists of a family of transcription factors that are important in the inflammatory response as well as in the ROS response.36 Activated PPARγ may directly bind to the p50 and p65 subunits of NF-κB, resulting in NF-κB inactivation.37 Moreover, PPARγ may be inhibited indirectly by NF-κB through Nrf2 activation, which reduces the levels of pro-oxidative molecules that are required 7931

DOI: 10.1021/acs.jafc.7b02728 J. Agric. Food Chem. 2017, 65, 7926−7933

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between PPARγ, Nrf-2, and NF-κB pathways, which highlights the therapeutic potential of this extract. The effect of GD preconditioning in SH-SY5Y cells is summarized in Figure 8. GD dictates the balance between oxidative stress, inflammatory response, and the intracellular calcium concentration during OGD/R challenge. Taken together, these results provide an important advance in understanding the use of GD (and potential novel derivatives) to formulate improved neuroprotective agents for the treatment of stroke. Moreover, the NTU 101-fermented products may be used to develop health foods to prevent cerebral ischemia.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +886-2-33664519, ext. 10. Fax: +886-2-33663838. E-mail: [email protected]. ORCID

Tzu-Ming Pan: 0000-0002-9865-1893 Funding

This work was financially supported by a grant from the Ministry of Science and Technology of Taiwan, Republic of China (MOST 104-2320-B-002-055). Notes

The authors declare no competing financial interest.

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Figure 7. GD upregulates PMCA expression. (A) PMCA was evaluated by Western blot and (B) densitometric analysis of PMCA levels, normalized to the level of GAPDH. Data are the mean ± SD of experiments performed in triplicate. The letters above bars indicate significant difference between the groups (p < 0.05), obtained using Duncan’s multiple range test. GD, glyceryl 1,3-dipalmitate; OGD/R, oxygen−glucose deprivation and reperfusion.

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Figure 8. Proposed mechanism by which GD protects cells from OGD/R-dependent death.

for NF-κB activation.10 The PPARγ agonist, L-796,449, exerts effects that are independent of PPARγ and could directly inhibit NF-κB activation,38 whereas carbaprostacyclin and ciglitazone suppress NF-κB-dependent induction of pro-inflammatory mediators.39 Our present study suggested that pretreatment with GD might inhibit NF-κB via activation of PPARγ, which would effectively reduce the levels of pro-inflammatory mediators. We further suggest that the GD/PPARγ axis might exert neuroprotective action through suppression of inflammatory response induced by OGD/R-induced ischemia-like injury. We showed that GD is neuroprotective in an in vitro ischemic stroke model. This is likely due to the GD-dependent cross-talk 7932

DOI: 10.1021/acs.jafc.7b02728 J. Agric. Food Chem. 2017, 65, 7926−7933

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DOI: 10.1021/acs.jafc.7b02728 J. Agric. Food Chem. 2017, 65, 7926−7933