EBN Lower Oxidative Stress: Lactoferrin and Ovotransferrin Contribute toward Antioxidative Effects of Edible Bird’s Nest Against Hydrogen Peroxide-Induced Oxidative Stress in Human SH-SY5Y Cells
Hou Zhiping1,3, Mustapha Umar Imam1, Maznah Ismail1,2*, Nur Hanisah Azmi1, Norsharina Ismail1 , Aini Ideris4 and Rozi Mahmud5
1Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia
2Department of Nutrition and Dietetics, Universiti Putra Malaysia, Selangor, Malaysia
3Department of Pathology, Chengde Medical University, Hebei, China
4Department of Veterinary Clinical Studies, Veterinary Medicine Faculty, Universiti Putra Malaysia, Selangor, Malaysia
5Department of Imaging, Medicine and Health Science Faculty, Universiti Putra Malaysia, Selangor, Malaysia
*Corresponding author: Department of Nutrition and Dietetics, Universiti Putra Malaysia, Selangor, Malaysia, Tel: 0086-314-2271157; Fax: 0060-389472188; Email firstname.lastname@example.org
There are reports of improved redox outcomes due to consumption of Edible Bird’s nest (EBN). Many of the functional effects of EBN can be linked to its high amounts of antioxidants. Interestingly, dietary components with high antioxidants have shown promise in the prevention of aging and its related diseases like Alzheimer’s disease. In this study, the antioxidative potentials of EBN and its constituents, Lactoferrin (LF) and Ovotransferrin (OVF), were determined and protective effects against H2O2-induced toxicity on SH-SY5Y cells using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and acridine orange and propidium iodide (AO/PI) staining with microscopy. Results showed that EBN and its constituent’s attenuated hydrogen peroxide (H2O2) induced cytotoxicity and decreased radical oxygen species (ROS) through increased scavenging activity. Furthermore, LF, OVF and EBN produced transcriptional changes in anti-oxidant related genes that tended towards neuroprotection as compared to H2O2 treated group. Overall, the results suggest that LF and OVF may produce synergistic or all-or-none anti-oxidative effects in EBN.
Keywords: Edible Bird’s Nest, lactoferrin, ovotransferrin, Antioxidant, Neuroprotection
AO/PI: Acridine Orange and Propidium Iodide; EBN: Edible Bird’s Nest; LF: Lactoferrin; ORAC: Oxygen Radical Absorbance Capacity; OVF: Ovotransferrin; ROS: Radical Oxygen Species; SOD: Superoxide Dismutase
Aging is a slow and gradual biological process, associated with multiple physiological and pathological changes including altered redox status. The brain cells are normally sensitive to the effects of Reactive Oxygen Species (ROS) because they are a nidus for peroxidative molecules and because of their peculiar energetic demands . In brain senescence, ROS starts to accumulate in neurons before clinically evident signs and symptoms of the disease can be detected . When ROS accumulate, oxidative damage is normally prevented by induction of protective factors like antioxidants, which may not be effective if the insult is too overwhelming. In such cases, apoptotic mechanisms set in to remove neurons deemed irreparable . Loss of neurons through these apoptotic deaths results in severe morphological and functional deficits, which manifest with progressive memory and cognitive decline.
Recently, due to concerns of side effects, researches have been focused on natural substances with neuro-protective potential that can scavenge free radicals and protect cells from oxidative damage, rather than synthetic chemicals . Edible Bird’s Nest (EBN) is produced by swiftlets from their salivary glue, which is a cementing substance. Although EBN mainly contains carbohydrates, amino acids and mineral salts, its major ingredients are glycoproteins . Due to its nutritious and medical properties, EBN has been deemed a precious food tonic in Chinese community ever since Tang (608-907AD) dynasties . Despite the long history of using EBN for medicinal purposes, there has only been limited number of scientific reports on the health benefits of EBN. Recently, EBN has been found to potentiate mitogenic response of human peripheral blood monocytes that stimulate Deoxyribonucleic Acid (DNA) synthesis in 3T3 fibroblasts [7,8]. Furthermore, enzymatic hydrolysis can release active peptides, which have beneficial effects on a variety of biological systems including the cardiovascular, gastrointestinal, immune and nervous systems . The ability of glycoprotein to interact with radical species or to inhibit oxidative reactions could lead to the development of novel food ingredients relevant in health promotion and disease prevention.
Lactoferrin (LF) and Ovotransferrin (OVF) are glycoproteins and family members of transferrin. Different studies have also shown that LF & OVF serve neuroprotective and antioxidant functions and thus showing good sources of functional food ingredients [10,11]. EBN is reported to have antioxidative properties and in view of the properties of LF and OVF. We hypothesize that, they may be contributing towards the overall antioxidant properties of EBN [12,13].
SY-SY5Y cell line can be differentiated by Retinoic Acid (RA) with similar characteristics to brain neurons. Thus, can be used to study neuronal cell activity invitro . Hydrogen peroxide (H2O2) is a major reactive free radical that has been studied in a variety of neurodegenerative diseases that has been implicated as an important mediator of unbalanced redox reactions and apoptosis in various cells including neurons . Consequently, this study was conducted to determine the concentration of LF and OVF in EBN, to evaluate their effectiveness against H2O2-induced oxidative stress on SH-SY5Y cells.
MATERIALS and METHOD
SH-SY5Y human neuroblastoma cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Minimum essential Eagle’s medium, Ham’s nutrientmixture F-12(DMEM/F-12), fetal bovine serum, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, dimethyl sulfixide (DMSO) and all other chemicals were purchased from Sigma (St. Louis, MO, USA). The GenomeLab™ GeXP Start Kit was from Beckman Coulter Inc. (Miami, FL, USA), and the Total ribonucleic acid (RNA) Isolation kit was obtained from RBC Bioscience Corp. (Taipei, Taiwan). The OxiSelectTM Superoxide Dimutase Activity Assay (SOD) and OxiSelectTM Intracellular ROS Assay kit(ROS) were from Cell Biolabs, Inc.(USA). Lactoferrin (L4894) and ovotransferrin (C7786) were purchased from Sigma-Aldrich (St.Louis, Missouri United States)
Preparation of EBN water-soluble protein
House white Edible Bird’s Nest from Malaysia was dried in an oven at 50 ºC for three days and finely ground with a food grinder (Waring Commercial, Torrington, CT, USA). As reported previously, the grounded EBN samples (1.0 g) were dissolved into 1000 mL of dd H2O, followed by ultra-sonication (Power Sonic 505, Hwashin Technology Co. Seoul, Korea) in an ice bath (pulse on, 2 s, pulse off, 4 s, ampl, 80%, 30 min) and centrifugation (10000 rpm for 10 min), the supernatants were desalted and condensed in a dialysis bag with a 3500 cut off molecular weight, then the water-soluble protein stored at -20 °C until use .
Lactoferrin and Ovotransferrin detection
LF and OVF concentration in EBN water extract were detected using Chicken Lactoferrin Elisa kit and Chicken Ovotransferrin Elisa Kit, Biosource (San Diego, California, USA). All the protocols were based on manufacturer instructions.
The human neuroblastoma SH-SY5Y cells were grown in complete culture medium containing DMEM/F-12, 10% fetal bovine serum, 1% MEM (non-essential amino acids) and 50 µg/mL gentamicin. Cells were maintained at 37 °C with 5% CO2 and 95% air.
ABTS radical Cation scavenging assay
ABTS radical cation was generated through oxidation of 7 mM ABTS with 2.45 mM potassium persulfate and incubated overnight in the dark at room temperature. ABTS radical cation solution was then diluted with ethanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm spectrophotometerically (Pharmaspec UV-1700, Shimadzu, Kyoto, Japan). EBN, LF and OVF (50 µL each) were individually mixed with 950 µL of diluted ABTS solution and incubated for 10 min at room temperature. The absorbance was measured under 734 nm. All the determinations were carried out in triplicate and the readings were averaged. Trolox was used as standard and percentage of ABTS radical cation decolorization inhibition was calculated using the formula reported by Norsharina in 2012 . ABTS radical cation scavenging activity of EBN, LF and OVF was expressed in mg Trolox equivalent per gram extracts as described before (mg TE/g extract).
Oxygen radical absorbance capacity assay
The abilities of extracts to scavenge peroxyl radical capacity were determined by Oxygen Radical Absorbance Capacity (ORAC) according to the method described by Huang et al. . Briefly, 150 µL fluorescein and 25 µL sample (1000 µg/ml EBN, 5 µg/ml LF and 10 µg/ml OVF) or standard (Trolox) were added into each well and incubated 37 ºC for 10-15 min. After incubation, fluorescence measurements (Ex. 485 nm, Em. 520 nm) were taken every one min to determine the background signal. After 3 cycles, 25 µL of freshly prepared 2, 2’-Azobis (2-amidinopropane) dihydrochloride (AAPH) at final concentration 81.6 nM was added manually with a multi-channel-pipette. Flourescence readings for each cycle were saved in Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski VT, US), as they would all be used to calculate areas under the curves and trolox equivalents.
SH-SY5Y cells were seeded into 96-well culture plates at density of 2 × 105 cells/mL and were allowed to attach. After 2 days, cells were differentiated with retinoic acid (10 µM) for 7 days prior to treatment. To examine the possible toxic effects, the cells were treated with EBN (10-100000 µg/mL), LF (0.05-500 µg/mL) and OVF (0.1-1000 µg/mL) individually for 24 h. To determine the neuroprotective ability, cells were pre-treated with 1000 µg/mL EBN and the equivalence 5 µg/mL LF and 10 µg/mL OVF diluted in serum-free medium for 24 h and then incubated with H2O2 for another 2 h. Then, MTT was added to all the wells and standardised in incubator for 4 h. The amount of MTT formazan product was dissolved by DMSO and measured absorbance at 540 nm using a Micro plate reader (All the MTT assays were performed in triplicate)
Acridine orange and propidium iodide (AO/PI) staining
SH-SY5Y cells (2 × 105) were seeded into 6-well culture plates, treated with 1000 µg/mL EBN and the equivalence 5 µg/mL LF and 10 µg/mL OVF, then followed by 250 μM H2O2 for 2 h. Then, cells were harvested and stained with AO/PI (10 μL of 1 mg/mL AO and 10 μL of 1 mg/mL PI). They were examined under an inverted fluorescence microscope (Olympus, Tokyo, Japan). Multiple independent images were taken.
SOD and ROS ELISA Assays
SH-SY5Y cells (2 × 105) were seeded into 12 well culture plates, treated with 1000 µg/mL EBN and the equivalence 5 µg/mL LF and 10 µg/mL OVF , then followed by 250 μM H2O2 for 2 h. Protocols for the ELISA tests were based on manufacturer instructions. For SOD assay, absorbance of samples were finally read at 490 nm on the Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski VT, US), while fluorescence for ROS assay were read at 480/530 nm by Synergy H1 Hybrid Multi-Mode Micro plate Reader (BioTek, Winooski VT, US).
RNA extraction, reverse transcription and Multiplex PCR analysis
SH-SY5Y treated cells were extracted by the Total RNA Isolation kit (RBC Bioscience Corp., Taiwan) according to the manufacturer’s instructions. Primer sequences were designed using GenomeLab eXpress Profiler software based on Homo sapiens sequence from the NCBI website (Table1). The primers were supplied by Biosune (Shanghai, China), while the internal control (KanR) was supplied by Beckman Coulter. Reverse transcription and PCR were performed according to the GenomeLab™ GeXP kit instruction (Beckman Coulter) in an XP Thermal Cycler (Bioer Technology, Germany). The PCR products were finally analyzed by the GeXP genetic analysis system and the results were normalized using GeXpress Profiler software based on the manufacturer’s instructions.
All the data (n=3) was conducted by one-way ANOVA, Tukey’s multiple comparisons and Student’s t-test using Statistical Package for Social Science (SPSS) version 20 (SPSS Inc., Chicago, IL). p<0.05 was considered as statistically significant difference.
RESULTS and DISCUSSION
LF and OVF detection in EBN water extraction
The results of LF and OVF concentrations determined using Enzyme-Linked Immuno-Sorbent Assay (ELISA) kits (chicken source), are shown on Table 2. The concentrations of LF and OVF in EBN were almost 0.5 and 1%, respectively. From comparisons made with the reported contents from other sources, the compositions in EBN are considerably high.
Based on the biological functions of LF and OVF reported, we hypothesized that LF and OVF could be contributing towards the overall functional properties of EBN, which prompted further evaluation of their properties [14-18].
EBN, LF and OVF attenuate H2O2 Induced Cytotoxicity on SH-SY5Y Cells
The abilities of EBN, LF and OVF to protect SH-SY5Y cells from H2O2 were determined by MTT assay, an indicator of cell viability. EBN (10-100000 µg/mL) and equivalent concentrations of LF an OVF in EBN (0.05-500 µg/mL and 0.1-1000 µg/mL, respectively) displayed above 80% viability on SH-SY5Y cells and showed no significant differences when compared with the control group (P>0.05) (Figure 1).
Hydrogen Peroxide (H2O2), is an oxidant that can induce intracellular defence mechanisms to attenuate H2O2-induced oxidative damage. As reported in our previous paper, SH-SY5Y cells exposure to 250 µM H2O2 for 2 h resulted in approximately 50% cell death compared with untreated control cells (p<0.01) . As shown on Figure 1B, EBN produced a hormetic effect on the viability of the cells in the presence of H2O2, with 1000 µg/ml EBN producing the best result. Although antioxidants are known to relieve oxidative stress and improve cell survival, it is also suggested that higher concentrations of antioxidants may in fact disrupt the natural mitohormetic capacity of cells resulting in more damage . It is also likely that at higher concentrations, the oxidants simply became provident [22,23]. Therefore, for our subsequent experiments, 1000 µg/ml EBN and equivalent concentrations of LF and OVF (5 µg/ml and 10 µg/ml, respectively) were used.
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation scavenging activities and Oxygen Radical Absorbance Capacity (ORAC) Assay
ORAC assay is a simple, sensitive, and reliable method to quantitate the radical absorbing capacity of antioxidants in serum or other biological fluids, especially natural products . Additionally, ABTS is also widely used for measuring the relative radical scavenging activity of hydrogen donating and chain breaking antioxidants in many fields . To determine antioxidant potentials of EBN, LF and OVF, ABTS and ORAC were performed on the samples (Figure 2). EBN (1000 μg/mL) exhibited the best scavenging effect on ABTS (5.23 ± 0.18 mg Trolox equivalent/g extract), followed by LF (5 μg/mL) and OVF (10 μg/mL) at concentrations similar to what is contained in 1000 μg/mL of EBN (4.67 ± 0.40 and 2.79 ± 0.18 mg Trolox equivalent/g extract, respectively).
Results of the ORAC assay also showed that EBN had the highest antioxidant capacity (3.04 ± 0.23 mg Trolox equivalent/g extract) when compared with LF and OVF (2.14 ± 0.10 and 1.22 ± 0.08 mg Trolox equivalent/g extract, respectively). The results also indicated significant differences (p<0.05) between LF and OVF and also when each is compared with EBN.
Results of EBN, LF and OVF have demonstrated consistent data in scavenging activities by ORAC and ABTS radical scavenging assays. EBN demonstrated the highest antioxidant activity followed by LF and OVF via ORAC and ABTS radical cation scavenging methods (Figure 2). The results, so far suggest that LF and OVF possess antioxidant capacities and even though the capacities of any one of the compounds may not explain the antioxidant capacity of EBN, it is likely that they contribute towards the overall capacity in EBN. Additionally, the overall antioxidant capacity of EBN may be contributed by other compounds in addition to LF and OVF, since LF and OVF do not entirely reflect the same activity as EBN. Different studies have already shown that due to synergistic effects of compounds in crude extracts, crude plant extracts usually exhibit superior health oriented properties than purified compounds . It is also likely that synergy between the different bioactive compounds in EBN may potentiate their activity when they are bound together in a single matrix .
AO and PI are among the most used stains for analyses of cell apoptosis. Using a combination of these stains, cells appear green to indicate they are apoptosis, while intact cells appear orange/red. As shown in Figure 3, control cells appeared green, suggesting normal structure and viability, while H2O2 treated cells appeared orange, representing early apoptosis. As for the EBN-treated cells, majority of the cells were stained green, showing normal appearance of intact cells. Similarly, LF and OVF groups showed mostly normal cells, with little signs of apoptosis.
ROS and Superoxide Dismutase (SOD) assay
SOD belongs to the endogenous antioxidant system that can scavenge ROS. The disruption in the balance between ROS and the endogenous antioxidant systems contributes to oxidative damage of cellular macromolecules ultimately leading to cell death in . In this study, the relative activity of SOD in the SH-SY5Y cells exposed to 250 μM H2O2 for 2 h decreased to 69.6% compared with the control group. Pre-treatment with EBN, LF and OVF for 24 h recovered the SOD activity to 90.7%, 87.8% and 80.9%, respectively (Figure 4A).
To investigate whether EBN, LF and OVF have the effect to scavenge intracellular ROS generation induced by H2O2, intracellular ROS kit was used to examine intracellular H2O2/hydroxyl radical. DCFH-DA (2',7'-dichlorofluorescin diacetate) fluorescence staining indicated that the value of H2O2-treated cells was increased about 2-fold compared with untreated control cells. However, the increase in intracellular H2O2/hydroxyl radical was reduced by EBN, LF and OVF treatment (Figure 4B).
Obviously, these data indicate that LF and OVF have a strong tendency to scavenge ROS and may in fact be contributing to the ROS scavenging ability of EBN. These results corroborate those of the antioxidant capacities of LF and OVF, which indicated that they had high antioxidant capacities that may be contributing to that of EBN.
Effects of EBN, LF and OVF on mRNA levels of antioxidant and apoptosis genes
The mRNA levels of three antioxidant and apoptosis genes (PARP1, SOD1 and SOD2) were studied using Multiplex GeXP genetic analysis system, with KanR as the internal control. The target genes and housekeeping gene are shown on Table1.
As shown in Figure 5, treatment with 250 μm H2O2 down regulated the mRNA levels of SOD1, SOD2 and PARP1 genes. Treatments with 1000 μg/ml EBN water extract, 5 µg/mL LF or 10µg/mL OVF unregulated the expression of the three genes even in the presence of 250 μM H2O2. In the case of SOD1 and SOD2, the EBN, LF and OVF treated-cells unregulated the gene significantly higher than H2O2-treated controls (P<0.01), although the expression levels were lower than in non-treated control cells (P<0.01). The mRNA level of PARP1 in EBN treated cells was higher than H2O2 treated and normal untreated cells (P<0.05), but those of LF and OVF treated cells were not different from H2O2-treated cells, and were lower than in untreated controls (P>005).
Unregulation of antioxidant genes (SOD) is an endogenous mechanism for boosting antioxidant defences in the presence of oxidative insults and could be the basis for the increased SOD activity observed in this study when SH-SY5Y cell were treated with EBN, LF or OVF (Figure 4). Moreover, the mRNA levels for the EBN, LF and OVF groups are reflective of the SOD activities observed in the respective groups, as no significant differences in SOD mRNA levels or activities were observed between the groups. The results, therefore, suggest that EBN boosts SOD levels and its activity through increased transcriptional regulation of the gene and that LF and OVF in EBN contribute to this effect. However, combination of these compounds and others in EBN may not have produced additive effects since there are no differences between EBN, LF and OVF treatments. Furthermore, activation of PARP1 is reported to improve cell survival after oxidative damage from H2O2 . Its increased expression by EBN but not LF or OVF suggest that other constituents in EBN may be contributing to this effect other than LF or OVF, or that their synergistic effect with or without other compounds may be contributing to expression level seen in EBN-treated cells. The overall data from this study indicates that EBN constituents interact in different ways to produce its activity. The presence of multiple compounds may produce additive or synergistic effects as suggested by the antioxidant capacity tests and expression of PARP1, while in some cases it may produce an all-or-none effect as seen with the activity and expression of SOD.
In summary, the present study demonstrated that EBN protects SH-SY5Y cells against H2O2-induced cytotoxicity and cell oxidative stress. LF and OVF contributed to the antioxidative effects of EBN in different ways. They were also able to inhibit early apoptosis in response to H2O2 treatment. The findings indicate that EBN constituents contribute towards producing synergistic antioxidative effects of EBN.
This work was carried out under the financial support of Ministry of Science, Technology and Innovation, e-sciencefund (vote 5450666), Malaysia. The authors wish to thank Mr. Mohd Khairil Othman from Matrix Optics (M) Sdn. Bhd. for the technical assistance in fluorescence microscopy analysis and all staff of Laboratory of Molecular Biomedicine for the help during the study.
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