|Year : 2019 | Volume
| Issue : 1 | Page : 41-47
Phoenix dactylifera L. Fruits Date Fruit Ameliorate Oxidative Stress in 3-NP Intoxicated PC12 Cells
Musthafa M Essa1, Vandita Singh1, Nejib Guizani1, Tamilarasan Manivasagam2, Arokiasamy J Thenmozhi2, Abid Bhat3, Bipul Ray3, Saravana B Chidambaram3
1 Department of Food Science and Human Nutrition, Sultan Qaboos University, Muscat; Ageing and Dementia Research Group, Sultan Qaboos University, Oman
2 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, Tamil Nadu, India
3 Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education and Research (JSS AHER), Mysuru, Karnataka, India
|Date of Web Publication||30-Apr-2019|
Musthafa M Essa
Food Science and Nutrition Sultan Qaboos University
Saravana B Chidambaram
Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education and Research (JSS AHER), Mysuru, Karnataka, 570015
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Date palm fruits (DFs) are reported to possess antimutagenic, antiviral, antifungal, and anti-inflammatory properties. Effect of date fruits in the management of Huntington’s is yet to be studied. Methods: The protective effects of DF were measured in terms of superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and reduced glutathione (rGSH), malondialdehyde (MDA) and nitrate/nitrite (NO2/NO3) content in cells. Cellular adenosine triphosphate (ATP) content was also measured. Cytotoxicity assay revealed that DF has the ability to protected cellular viability against 3-NP intoxication. Results: DFs increased the SOD and GPx activities and rGSH content. On the other hand, DF decreased MDA and NO2/NO3 levels in 3-NP intoxicated cells. Interestingly, DF increased ATP content in pheochromocytoma (PC) cells. Conclusion: DF has the ability to encounter 3-NP intoxication induced biochemical changes and improves cellular ATP contents, hence may be an interestingly candidate for further investigations.
Keywords: 3-Nitropropionic acid, date fruits, Huntington’s, disease, PC12 cells
|How to cite this article:|
Essa MM, Singh V, Guizani N, Manivasagam T, Thenmozhi AJ, Bhat A, Ray B, Chidambaram SB. Phoenix dactylifera L. Fruits Date Fruit Ameliorate Oxidative Stress in 3-NP Intoxicated PC12 Cells. Int J Nutr Pharmacol Neurol Dis 2019;9:41-7
|How to cite this URL:|
Essa MM, Singh V, Guizani N, Manivasagam T, Thenmozhi AJ, Bhat A, Ray B, Chidambaram SB. Phoenix dactylifera L. Fruits Date Fruit Ameliorate Oxidative Stress in 3-NP Intoxicated PC12 Cells. Int J Nutr Pharmacol Neurol Dis [serial online] 2019 [cited 2020 Jan 25];9:41-7. Available from: http://www.ijnpnd.com/text.asp?2019/9/1/41/257486
| Introduction|| |
Huntington’s disease (HD) is an inherited autosomal dominant, progressive, fatal, neuro-degenerative disorder caused due to cytosine-adenine-guanine (CAG) repeat in the huntingtin (Htt) gene. HD is clinically characterized with advanced motor dysfunction, decreased cognition, and psychiatric instabilities. Aggregation of Htt proteins causes excitotoxicity, oxidative stress, and neuronal death, in striatum. Excitotoxicity initiates inflammatory reactions, that is, it overactivates microglial and astroglial cells and triggers apoptotic cell death in various neurodegenerative diseases including HD.,,, Currently, only tetrabenazine is approved by US-FDA for the symptomatic management of HD. Other drugs such as neuroleptics, energy metabolites, glutamergic modifying drugs, etc., have moderate-to-weak evidences in the treatment/management of HD. However, these classes of drug have limitations for chronic use because of their serious adverse effects such as depression, hepatotoxicity, gastrointestinal disturbances, insomnia, etc. Hence, it is need of the hour for the development of safer and effective regimens in the management of HD.
Plants rich in phytochemicals such as flavonoids, polyphenols act as potent antioxidants and are reported to slow-down the progression of neurodegeneration. Many of these phyto-principles have the ability to restore cognitive functions. In many instances, particularly in chronic neurodegenerative diseases, use of modern medicines poses severe untoward effects and even worsen the quality of life. There is a large interest in the studying the role of naturally occurring substance that are rich in antioxidants, such as fruits, vegetables, and nuts are of paramount importance in chronic ailment.
Phoenix dactylifera L. fruits, commonly known as date fruits, are major staple food in North-African, Middle (DF) Eastern, and South-west Asian countries. Date fruits (DF) are highly nutritious and are reported to have significant antioxidant property.,, Presence of bioactive principles, such as polyphenols, anthocyanins, flavonoid-glycosides, and procyanidins present, are responsible for the antioxidant activities., Earlier studies showed that date palm fruits (DFs) possess antimutagenic, antiviral activity, antifungal, anti-inflammatory, antihyperlipidemic and hepatoprotective, nephroprotective, anticancer, and neuroprotective activities.,, The effects of date fruits on HD are still to be studied scientifically. In the present study, we investigated the role of ethanolic extract of date fruits on 3-nitropropionic acid (3-NP) chemical model of HD using PC12 cell lines.
| Materials and Methods|| |
PC12 cell line (pheochromocytoma cell line) was procured from ATCC, Rockville, USA. Cells were grown in roswell park memorial institute medium (RPMI) 1640 medium supplemented with 10% (v/v) donor horse serum, 5% fetal bovine serum in a 75 cm2 culture flask. Cells used for experimentations were seeded at a density of 4.5 × 105 cells/well on a six-well culture plate. Cell differentiation was allowed in a differentiation medium (RPMI 1640 medium, 1% donor horse serum, 100 ng/mL nerve growth factor (NGF), 50 ng/mL cyclic adenosine monophosphate (cAMP)) for 4 days.
Hydroethanolic extraction of date fruits
Extraction of date fruits was conducted as described earlier with slight modification (Singh et al.). Briefly, 100 g of edible part of date fruit at tamar stage was pitted, chopped in to small pieces and dry blended for 3 min using a blender Braun, Turbo, 500 W, Spain: Chopped fruits were cold macerated using 300-mL ethanol—100 mL of water (4:1, v/v) as a solvent at room temperature (25 ± 2°C) for 24 h. The flask was stirred continuously with the help of a magnetic stirrer. After 24 h, flask was centrifuged at 6000 relative centrifugal force (RCF) for 30 min at 4°C and the supernatant filtered and concentrated under reduced pressure at 40°C for 6 to 8 h with the help of a rotary evaporator to obtain the hydroalcoholic extract. The extract was stored in an air-tight container and stored at 2 to 4°C till further use.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The effect of hydroalcoholic date fruits extract on cell viability was tested with various concentrations (1–1000 μg/mL). Then the role of date fruits on cell viability as assessed in cells exposed to 3-NP. PC12 cells were pretreated with DF extract (50 µg/mL), and then exposed to 5 mM of 3-NP for 24 h. The cells were incubated with MTT (0.25 mg/mL) for 4 h at 37°C once the medium was aspired-off. The amount of MTT formazan formed was determined in a microplate reader at wavelength of 570 and 630 nm.
Lactate dehydrogenase (LDH) assay
Lactate dehydrogenase (LDH) levels in media was measured using diagnostic kit in semiautomatic biochemical analyzer. The results were expressed as U/L.
Determination of reactive oxygen species formation using DCF-DA staining
In intact cells, Dichloro-dihydro-fluorescein diacetate (DCFH-DA) penetrates the cell membrane and hydrolyzed by cytosolic esterases to nonfluorescent DCFH. Reactive oxygen species (ROS) initiates oxidation of DCFH into a highly fluorescent DCF. Thus, the fluorescent intensity is directly proportional to ROS level in cells. PC12 cells were pretreated with DFs extract (50 μg/mL), and then exposed to 5 mM of 3-NP for 24 h. The cells were incubated with 10 mM DCFH-DA for 30 min and were washed to eliminate the extracellular DCFH-DA. The fluorescence intensity was measured using (CytoFluor 4000, Applied Biosystems, Framingham, Massachusetts USA) fluorescence plate reader (excitation: 485 and emission 535 nm).
Estimation of malondialdehyde content
The extent of lipid peroxidation was measured as malondialdehyde (MDA) content using commercial assay kit (Cayman Chemical Co, Ann Arbor, Michigan, USA). To 50 μL of cell lysate, 50 μL sodium dodecyl sulfate (SDS) solution, and 1 mL thiobarbituric acid (TBA) were added and boiled at 100°C for 1 h. Reaction was terminated by placing the samples on ice platform for 10 min and centrifuged at 1600 rpm for 10 min to remove cell debris. The absorbance was read at 540 nm using microplate reader (BioRad, Hercules, California, USA). The results were expressed in MDA (μM)/mg protein.
Determination of nitric oxide (NO) content
NO content was measured in term of amount of nitrite/nitrate (NO2/NO3) accumulated in the media using Griess reagent. To 100 μL of supernatants, 100 μL of Griess reagent was added and kept in dark for 10 min at room temperature. Absorbance was read at 550 nm using microplate reader (BioRad). Freshly prepared NaNO2 was used as a standard (200–1000 ug/ml). The results were expressed in ηmole/mg protein.
Measurement of superoxide dismutase (SOD) activity
Superoxide dismutase (SOD) activity was measured using colorimetric assay kit (Cayman Chemical Co). One unit of SOD is defined as the quantity of enzyme required to show 50% dismutation of the superoxide radical. The absorbance was read at 450 nm using microplate reader (BioRad). The results were expressed in SOD activity (U/mg protein).
Measurement of glutathione peroxidase (GPx) activity
Glutathione peroxidase (GPx) activity was measured using colorimetric assay kit (Cayman Chemical Co). In this assay, oxidized glutathione (GSSG) is reduced to glutathione by GPx with concomitant oxidation of Nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+. The oxidation of NADPH was measured using microplate reader (BioRad) at 340 nm. The results were expressed in nmol/min/mg protein.
Estimation of glutathione (GSH) content
GSH content was measured using commercial assay kit (Cayman Chemical Co). In this assay, O-phthalaldehyde reacts with GSH present in the sample, and the fluorescence intensity (ex. 340 nm, em. 420 nm) was calculated after every 30 s for a total of 60 min using Fluostar Optima Fluorometer, FLUOstar® Omega, BMG LABTECH (NY, USA). The results were expressed in μM/mg protein.
Measurement of intracellular ATP content
Cell lysates were obtained by centrifugation, and intracellular adenosine triphosphate (ATP) was measured in lumino-meter (BD Biosciences, San Jose, California, USA) using ATP Bioluminescence assay kit HS II (Roche Molecular Biochemicals, USA). To 50 µL of reaction mixture, 50 µL of cell lysate was added and the luminosity was measured. The results were expressed in ATP (μM)/mg protein.
Estimation of total proteins content
Total protein content in cell lysates were determined by Bradford method.
In each group (n=3) data are expressed as mean± standard error of the mean (SEM). Statistical differences between the groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison. Statistics was performed using GraphPad Prism software 5.0 version, San Diego, USA. The statistical significance at P≤0.05 was considered as significant
| Results|| |
Dates produced a concentration-dependent cytotoxic effect on PC12 cells with an half maximal inhibitory concentration (IC50) value of 276.40 μg/mL. These cells when pretreated with dates significantly retained the cell numbers and morphological characteristics [Figure 1].
|Figure 1 Cytotoxicity potential of dates extract measured in PC12 cells using MTT assay with various concentrations (1–1000 μg/mL)|
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Dates decreased LDH
Pretreatment with dates showed significant (P ≤ 0.01) decrease in the levels of LDH when compared with the vehicle-treated 3-NP intoxicated cells. Dates control cells didn’t show any significant effect [Figure 2].
|Figure 2 The effect of dates on LDH levels in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); ## denotes 0.01 (comparison between normal and positive cells); * denotes P < 0.05 (comparison between positive control and other treatment cells)|
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Dates decreased intracellular reactive oxygen species (ROS) formation
The vehicle-treated 3-NP intoxicated cells showed significant (P < 0.01) increase in the intracellular ROS content when compared to control cells. Exposure with dates significantly (P < 0.01) decreased the levels of ROS when compared with vehicle-treated 3-NP intoxicated cells. Control cells pretreated with dates didn’t show any significant effect when compared with control cells [Figure 3].
|Figure 3 The effect of dates on ROS levels in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); ## denotes 0.01 (comparison between normal and positive control); ** denotes P < 0.01 (comparison between positive control and other treatment cells)|
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Dates produce decline in MDA content
Dates extract showed to produce significant (P ≤0.01) decline in MDA content in 3-NP intoxicated cells when compared with vehicle treated 3-NP intoxicated cells. Dates control did not show any significant effect [Figure 4].
|Figure 4 The effect of dates on MDA levels in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); ## denotes 0.01 (comparison between normal and positive control); * denotes P < 0.05 (comparison between positive control and other treatment cells)|
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Dates reduce the nitric oxide content
The vehicle-treated 3-NP intoxicated cells significantly increased (P < 0.05) nitrite/nitrate levels when compared with control cells. Pretreatment of cells with dates extract significantly (P < 0.01) decreased nitrite/nitrate level as compared to vehicle-treated 3-NP cells. Dates control did not show any significant effect when compared with control cells [Figure 5].
|Figure 5 The effect of dates on NO content in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); ## denotes 0.01 (comparison between normal and positive control); * denotes P < 0.05 (comparison between positive control and other treatment cells)|
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Dates improves the levels of superoxide dimutase
Pretreatment of 3-NP intoxicated cells with dates extract significantly (P ≤ 0.01) improved the levels of SOD compared to vehicle-treated 3-NP cells. Vehicle treated 3-NP intoxicated cells exhibited significant (P ≤ 0.05) decrease in the SOD content [Figure 6].
|Figure 6 The effect of dates on SOD levels in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); ## denotes 0.01 (comparison between normal and positive control); * denotes P < 0.05 (comparison between positive control and other treatment cells)|
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Dates increased the levels of glutathione peroxidase
GPx levels significantly (P ≤ 0.01) decreased in vehicle-treated 3-NP intoxicated cells. Pretreatment with dates showed a significant (P ≤ 0.01) elevation in GPx levels in 3-NP intoxicated cells. Dates control cells didn’t show any significant effect [Figure 7].
|Figure 7 The effect of dates on GPx levels in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); # denotes 0.05 (comparison between normal and positive control); * denotes P < 0.05 (comparison between positive control and other treatment cells)|
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Effect of dates extract on reduced glutathione levels
Dates significantly (P ≤ 0.01) improved the levels of GSH in 3-NP intoxicated cells. Vehicle treated intoxicated cells showed a significant (P ≤ 0.01) decline in GSH content. Dates control cells did not show any significant change [Figure 8].
|Figure 8 The effect of dates on GSH levels in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); # denotes 0.05 (comparison between normal and positive control); * denotes P < 0.05 (comparison between positive control and other treatment cells)|
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Dates improved the intracellular ATP production
Dates pretreatment showed significant (P ≤0.05) increase in ATP levels in 3-NP intoxicated cells. Vehicle treated 3-NP intoxicated cells exhibited a significant (P ≤0.05) decline in ATP levels. Dates control cells did not show any substantial effect as compared to normal cells [Figure 9].
|Figure 9 The effect of dates on intracellular ATP content in PC12 cells exposed to 3-NP. Results were expressed in mean ± SEM (N = 3); ## denotes 0.01 (comparison between normal and positive control); ** denotes P < 0.01 (comparison between positive control and other treatment cells)|
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| Discussion|| |
Dates are one of the ideal nutrients a lot of potential health benefits. Dates fruits are rich in carbohydrates (70%), of which glucose and fructose form the major portions, dietary fiber, and also contains essential minerals such as iron, potassium, calcium, selenium, copper, and magnesium. Proteins and fats are present in microquantities. Dates contain a large amount of flavonoids including quercetin, apigenin, p-coumaric acid, ferulic acid, and sinapic acids which possess potent antioxidant activity.,,
The compound 3-NP is a widely used animal model that mimics HDs phenotype. 3-NP causes striatal lesions and cell death through excessive generation of free radicals. It alters the mitochondrial respiratory chain complex II by irreversibly inhibiting succinate dehydrogenase and also cause excitotoxicity via activation of N-Methyl-D-aspartic acid (NMDA) receptor., These processes culminate to the impaired production of cellular ATP. In contrast, 3-NP stimulates Ca2+ release from mitochondrial membrane, which increases the membrane potential (ΔΨm) and release of cytochrome c. These multifacet pathological cascade needs multitarget therapeutic approach; obviously it is impractical with conventional modern medicines. Food supplements of natural origin contain bioactive principles which impart many health benefits. Particularly, polyphenols such as quercetin, apigenin ameliorate mitochondrial functions by reducing the mitochondrial membrane potential (ΔΨm). In the present study, bioactive principles such as polyphenols and flavonoids present in date might have ameliorated mitochondrial membrane potential and improved ATP content in 3-NP intoxicated cells.
Antioxidative enzymes such as SOD (manganese superoxide dismutase (MnSOD)) and GPx detoxify ROS. But excess ROS generation can overwhelm the capacity of these defense mechanism, leading to mitochondrial damage. Flavonoids such as apigenin and quercetin have multiple actions such as SOD-mimetic, facilitates the replacement of lost SOD activity, or by enhancing a de novo synthesis of essential repair enzymes. Date fruits increased SOD activity in 3-NP intoxicated cell which could be possibly through the mechanism corroborated to flavonoids actions.
GPx catalyses the GSH-dependent reduction of H2O2 and also involved detoxifying metabolic processes., Exposure of cells to 3-NP decreased cellular GSH and GPx indicating that lipid membrane are at high vulnerability and undergo damage. Increase in the levels of lipid peroxides directly affects membrane fluidity, which results in altered membrane protein activity. Accumulation of lipid peroxidation (LPO products promotes in the progression of neurodegenerative diseases. Flavonoids and isoflavonoids possess B-ring conjugated chemical structures with many hydroxyl groups. These compounds inactivate superoxide anions, oxygen lipid peroxide radicals by donating electrons. The presence of bioactive flavonoids in date fruits might have neutralized in free radicals generated by 3-NP and which could be corroborated to decreased lipid peroxide (MDA) and increased GSH and GPx levels.
3-NP triggers ROS generation and prompts mitochondrion release of Ca2+. This activates nitric oxide synthase or nitric oxide synthase type I and causes subsequent release of nitric oxide (NO). In turn, NO transforms to peroxy-nitrite (ONOO−) after reacting with superoxide anion (O2−). Amelioration of mitochondrial function suppress the superoxide anions and hence NO. Further, the phyto-principles particularly polyphenols are reported to directly neutralize NO to nitrite and nitrate by donating electron to the peroxy-nitrite. Increased LDH levels have been found in the brain of HD induced mice. DF decreased LDH level in 3-NP intoxicated cells, but the mechanism behind the same is unclear at this moment.
Oxidative stress plays a crucial role in the progression of HD by depleting the levels of antioxidant enzymes which have a prominent action in maintaining the homeostasis. Increase in the production of ROS leads to the mitochondrial dysfunction by promoting the fission and reducing in the production of ATP. This compromised antioxidant status and decreased mitochondrial bioenergetics leads neuronal death. Flavanoids such as quercetin and apigenin present in the date fruit can protect the neuronal cell death by combating the oxidative stress and improving the overall antioxidant strength. Quercetin and apigenin also ameliorate mitochondrial alterations and improve the mitochondrial biogenesis. Quercetin enhances the expression of peroxisome proliferator-activated receptor gamma coactivator (PGC1α) a master regulator in the biogenesis of mitochondria.
Microessential minerals such as iron, potassium, calcium, selenium, copper, and magnesium present in the dates act as a cofactor of enzymes to regulate cellular activities. Deficiency of these metals has been associated with various neurological diseases. DFs are rich in antioxidants, and their regular consumption might be helpful to ameliorate or slow down the neurodegenerative process in various neurological disorders including HD.
| Conclusion|| |
DF rich in polyphenols and flavonoids could have the potential to restore ATP production and ameliorate oxidative stress in 3-NP intoxicated cells. Hence, it can be considered for further extensive investigations in HD research.
The authors acknowledge the funding support provided by SQU in the form of an internal grant to MME (IG/AGR/FOOD/14/01 and 17/02).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]