سوپرفود NBS اثرات ضد آپوپتوز و تکثیر بر سلول های بنیادی عصبی

Abstract

Background: The importance of neural stem cell (NSC) survival in the appropriate brain function, synapsis formation, and neural cell communication is to the degree that any perturbation in this process leads to many neurodegenerative diseases. Numerous therapeutic approaches have recently been developed to prolong the survival of NSCs, so that these multipotent stem cells could differentiate into other neural cells and improve the function of the brain.

Methods: In this study, we aimed to investigate whether the treatment of isolated NSCs from the Wistar rats with the nutrition bio-shield (NBS) supplement could boost the survival of NSCs.

Results: Of note, we found that the extract had negative effects neither on the biochemical and hematological parameters nor on the morphology of hepatocytes of the rats, suggestive of the lack of toxicity of the extract. Our results also suggested that the extract robustly increased the survival and the proliferative capacity of NSCs by elevating the metabolic activity of the cells. The NBS supplement also prevented the induction of apoptotic cell death in NSCs, as revealed by the decrease in the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells as compared to the control group.

Conclusion: Overall, this study highlighted the potent efficacy of the NBS supplement in prolonging the survival of NSCs and proposed the possibility of the clinical use of the extract in the treatment of different neural diseases. However, further investigations in this field are required to provide the extract’s mechanism of action.

Introduction

Annually, a considerable number of people lose their lives due to neurodegenerative diseases, and the future for other patients is dismal and disappointing,¹ as no proper treatment strategies have so far been explained for these diseases. Despite the advances in neuroscience, the treatment of many neurodegenerative diseases is still not possible due to the lack of agents to properly penetrate the blood–brain barrier. Moreover, the toxicity of the agents for other tissues and some reported off-target effects have put an obstacle to the entrance of such agents in the treatment protocol of diseases.^2,3 For example, it has been reported that stimulating the phosphoinositide inositol-3 kinase (PI3K)/Akt signaling axis in patients with Alzheimer’s disease (AD) could prevent the formation of amyloid β and tau hyperphosphorylation.⁴ Nevertheless, it should not be forgotten that the overactivation of the PI3K/Akt signaling axis could also increase the risk of cancer development, so the enthusiasm for using PI3K modulator in AD patients has been muted.⁵ In recent years, exogenous neural stem cells (NSCs)-based transplantation has been suggested to be a practical approach for replacing the damaged cells and evolving an immunomodulatory shield. However, this treatment approach’s promising effect has also been jeopardized by the little change of engraftment and the lack of survival of NSCs.⁶ As the survival and proliferative capacity of NSCs play a crucial role in the success of this approach, finding strategies that could boost the survival of NSCs and prevent the induction of apoptotic cell death could bring a ray of hope for the successful treatment of neurodegenerative diseases.

Among the endless list of synthesized compounds that could interact with numerous signaling pathways to halt apoptosis in NSCs, the nutrition bio-shield (NBS) supplement seems to be one of the most potent. First, the presence of a tight correlation between nutrition supplements and the incidence of neural damage has been explained in several reports.^7,8 In a study conducted by Bianchi et al., it has been reported that malnutrition and lack of vitamins could exacerbate the incidence of the neurodegeneration process and eventually lead to the formation of diseases such as Parkinson’s disease (PD).⁹ Moreover, the NBS supplement showed beneficial therapeutic effects in the treatment of fatty liver disease through reducing the lipid profile.¹⁰ Moreover, in another study, it has been claimed that the NBS supplement could boost the immune system by shifting the ratio of neutrophils to lymphocytes.¹¹ Given the safety profile of the NBS supplement as well as its beneficial therapeutic effects, it was of great interest to dissect the effect of this extract on the survival of isolated NSCs from rats to investigate its potential in bypassing apoptotic signals and boosting the survival of neural cells.

Material and Methods

Animal and ethical statements

To evaluate the therapeutic value of the NBS supplement including antiapoptotic effects, Wistar rats were kept at 22–25°C at the laboratory with a 12 h light–dark cycle. Rats were housed in the special cages, and they had free access to water and food supplies. For analyzing the extract’s safety, a total of 20 rats were divided into four equal groups: the control group and rats treated with 500, 1,000, and 1,500 mg/kg of extract, respectively. After 28 days, rats were anesthetized with xylazine ketamine as the most common method (4 mg/mL xylazine mixed with 30 mg/mL ketamine, 4 mL/kg, intraperitoneally), and 3–5 mL of blood was drawn from their hearts to perform biochemical and hematologic tests, including fasting blood sugar (FBS), blood urea nitrogen (BUN), creatinine (Cr), albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), hemoglobin (Hb), hematocrit (HCT), white blood cells (WBC), red blood cells (RBC), and platelet (PLT).¹²

The effects of live and healthy dietary supplement extract on liver pathology

Twenty-eight days after treatment with the NBS supplement and blood sample collection, liver tissue was removed and fixed in 10% formalin for 24 h. The fixed samples were then prepared using the high-pressure silica gel and three phylogenetic bases. The specimens were cut into 4–6 mm, and each slice was stained with hematoxylin–eosin. The morphology of hepatocytes was studied by light microscopy at a magnification of 10.

Isolation of neural stem cells from rats

We have chosen five Wistar rats from the Green Drug Research Knowledge Company to isolate NSCs. Rats, kept at optimum laboratory conditions, were then anesthetized, and their hippocampal segments were surgically removed. Then, the tissues were crushed twice and were incubated with accutase and collagenase for 30 min at 37°C. To isolate NSCs, the prepared suspension was filtered by 70 µM nylon mesh and was centrifuged at 3,000 rpm for 10 min.

Cell culture and live and healthy dietary supplement extract treatment

The isolated NSCs were grown in a suspension in Dulbecco's modified eagle medium (DMEM)/F12 medium supplemented with 2 ng basic fibroblast growth factor, 2% B27, epithelial growth factor, 5% fetal bovine serum, and 100 U/mL penicillin-streptomycin in a humidified 5% CO₂ atmosphere at 37°C. The culture media was changed daily for 5 days until the NSCs were attached to the bottom of the flask. After centrifugation, the floating neurospheres were then collected via sampler and transferred to another flask containing DMEM/F12 medium. After reaching confluency (70–80%), cells were treated with the NBS supplement at 200, 400, 600, and 800 µg/mL concentrations for 48 h.

MTT assay

The effects of the NBS supplement on the survival and the proliferative capacity of NSCs were studied using an MTT assay. Briefly, cells were collected from the flasks using trypsin and were seeded at the density of 3,000 cells/well for 24 h. Cells were then treated with the designated concentrations of the extract for 48 h. After removing the media, MTT solution (5 mg/mL in PBS) was added to each well, and cells were incubated for 3 h at 37°C. Cells were then centrifuged, and the media was discarded again. The resulting formazan was solubilized with dimethyl sulfoxide. The absorption was measured at 570 nm in an enzyme-linked immunosorbent assay reader.

TUNEL test

To study the effect of the extract on the induction of apoptotic cell death in NSCs, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) test was used, which is a test for monitoring the morphological changes and DNA fragmentation in apoptotic cells. After incubation of NSCs with the NBS supplement at the concentration of 800 µg/mL for 48 h, cells were treated with 10% ethanol to induce apoptotic cell death. Of note, the control group was only treated with 10% ethanol. Then, cells were washed three times with PBS and exposed to 4% paraformaldehyde for 30 min. Fixed cells were incubated for 10 min with blocking solution (3% H₂O₂ in methanol), washed with PBS, and exposed to permeabilization solution. Afterward, cells were incubated with a reaction mixture for 1 h and 50 1 Converter-POD solution for 30 min. After 50 min incubation with DAB-substrate, 20 µL of diaminobenzidine-chromogen was added to the mixture. The alteration in the morphology of the cells and DNA fragmentation were then analyzed using light microscopy.

Statistical analysis

To statistically analyze our data, we first used the Kolmogorov–Smirnov test to ensure that data distribution was normal. Experiments were done in triplicate, and the results are presented as the mean ± standard deviation. Analysis of variance test was used to evaluate the significance of the data with an acceptable probability level of P < 0.05. SPSS and GraphPad Prism software were used for data analysis.

Results

Evaluating the safety of dietary supplement extract on rats

NBS superfood is a supplementary food processed from wheat extract, which contains micronutrients including vital vitamins and minerals ( Table 1). To shed light on the safety profile of the NBS supplement, four groups of Wistar rats were treated with increasing concentrations of the extract (500, 1,000, and 1,500 mg/kg) for 28 days. Blood samples were randomly collected from two rats of each group to check different biochemical and hematological parameters. As presented in Tables 2 and 3, dietary supplement extract had no effects on both biochemical and hematological parameters. These parameters remained almost similar in dietary supplement extract-treated rats and the untreated control group rats. This finding proposed that probably, the extract could be well-tolerated in the tested rats. This finding was

further confirmed by the results of pathologic studies on the liver tissues of rats, which revealed a normal hepatocyte and liver tissue structure without any signs of necrosis or tissue damage ( Fig. 1).

FIG. 1. The effect of the healthy and alive dietary supplement extract on the hepatocyte structure. Treatment of Wistar rats with increasing concentrations of the extract (500, 1,000, and 1,500 mg/kg) had no effects on the structure of hepatocytes. No signs of necrosis or tissue damage could be detected in the liver tissue.

NBS supplement exerted antiapoptotic effects on neural stem cells

Having established the lack of toxicity of the NBS supplement, it was of particular interest to evaluate whether this extract could increase the survival and the lifespan of the neonatal SCs isolated from rat hippocampus. To do so, we treated NSCs with increasing extract concentrations ranging from 200 to 800 µg/mL for 48 h. Evaluating the metabolic activity of the cells using MTT assay, we found that treatment with the extract led to a considerable increase in survival and the proliferative capacity of the cells compared to the control group. As presented in Fig. 2, 48 h of treatment with the NBS supplement resulted in a clear-cut increase in the proliferative potential of NSCs by nearly 1.14- and 1.42-fold change at the concentrations of 600 and 800 µg/mL, respectively. Given the maximum effects of the extract at the concentration of 800 µg/mL on the survival of the NSCs, we, therefore, chose this concentration for further analysis.

FIG. 2. The healthy and alive dietary supplement extract increased the survival of the proliferative capacity of NSCs. The results of the MTT assay revealed that in the presence of the increasing concentrations of the extract, the metabolic activity of the cell increased significantly as compared to the control group. Values are given as mean ± standard deviation of three independent experiments. *P ≤ 0.05 represented significant changes from the control.

NBS supplement increased the survival of NSCs through suppression of apoptotic cell death

It is well-established that the induction of apoptosis holds a respectable share in reducing the survival of NSCs. This event eventually results in many neurodegenerative diseases.¹³ To investigate whether the effect of dietary supplements on the survival of NSCs is mediated through suppression of apoptosis, isolated NSCs were treated with the NBS supplement at the concentrations of 800 µg/mL, and the induction of apoptosis was evaluated using TUNEL assay. Analyzing the morphological alternations and DNA fragmentations showed that the number of TUNEL positive cells was greater in the control group than extract-treated NSCs cells ( Fig. 3). This finding suggested that while the induction of apoptotic cell death significantly diminished the number of cells in the control group, the NBS supplement robustly increased the number of viable cells by halting the propagation of apoptosis.

FIG. 3. The effect of the healthy and alive dietary supplement extract on the induction of apoptosis in isolated NSCs from Wistar rats. Analyzing the morphological alternations and DNA fragmentations using the TUNEL test showed that the extract could robustly prevent the induction of apoptotic cell death in NSCs as compared to the control group.

Discussion

The survival of the neural cells holds a respectable share in the maintenance of the function and the health of the nervous system to the degree that the loss of neural cells survival is considered to be the main reason for the formation of many neurodegenerative diseases such as AD, PD, Huntington’s disease, and ischemia.¹⁴ A considerable number of attempts have been made to recognize the mechanisms that are responsible for diminishing the lifespan of neural cells, and the results of all these efforts suggested that the loss of neural cells could be mediated due to the induction of apoptosis, necrosis, or excitotoxicity.¹⁴ Identification of the footprint of apoptosis in neural cells introduces another layer of complexity in the term of neurodegenerative diseases as this event not only has a tight cross-talk with other forms of cell death but also could be easily manipulated by therapeutic approaches to enhance the survival of the neural cells.^15,16 Thus far, numerous compounds have been recognized, which could prevent apoptosis in neural cells.^17,18 In an effort to find an efficient as well as safe approach for prolonging the survival of neural cells, this study aimed to evaluate the effect of the NBS supplement, which had previously been shown to have therapeutic value in the treatment of fatty liver disease,¹⁰ on the survival of NSCs collected from Wistar rats. Of note, our results shed light on the safety of this extract, as the treatment of Wistar rats with increasing concentrations of the extract had effects neither on the biochemical and hematological parameters nor on the morphology of hepatocytes.

In keeping with the extract’s safety, our results also showed the capacity of the NBS supplement to increase the survival and proliferation of NSCs isolated from the Wistar rats. The MTT assay results suggested that compared to the control group, those NSCs treated with different concentrations of extract had more metabolic activity. It is well-established that proper metabolic activity is a powerful tool for promoting neurogenesis in the central nervous system (CNS).¹⁹ Recently, it has been indicated that metabolic regulation could be a fundamental approach for promoting the survival of NSCs.²⁰ The increase in the survival of NSCs is of particular importance, as these multipotent cells have the ability to differentiate into a wide range of neural cells, including neurons, astrocytes, or oligodendrocytes in the CNS. In accordance with our study, Chu et al. also used melatonin, a normal hormone of the pineal gland, to increase the proliferation, differentiation, and survival of NSCs. The results of their study suggested that melatonin probably interacts with MAPK/ERK signaling pathway to modulate the expression of apoptotic-related genes in NSCs and thereby prevent the induction of cell death.²¹ In another study, Siebzehnrubl et al. indicated that histone deacetylase inhibitor could increase the differentiation ability of NSCs of the forebrain to oligodendrocytes by elevating the expression of NeuroD, Cyclin D2, and B-lymphocyte translocation gene 3.²² Taken together and as the most straightforward interpretation of our results, we suggest that treatment with the NBS supplement probably could be a valuable approach to boost the metabolic activity of the NSCs. This event could thereby guarantee the energy supply that is required for the maintenance of the survival and the proliferative capacity of the cells.

So far, numerous mediators have been identified to threaten the survival of the NSCs via disturbing the function of mitochondria and thereby induction of apoptotic cell death. Oxidative stress, for example, is one of the most important ones that could reduce the survival of the NSCs via elevating the intracellular amount of reactive oxygen species (ROS), which, in turn, could attenuate the function of mitochondria to produce the necessary energy for cell proliferation.¹⁷ Moreover, ROS could integrate with various signaling pathways such as PI3K, MAPK, and ERK to increase the expression of apoptotic-related genes, and thereby diminish the survival of NSCs.²³ Of particular interest, our results showed that the NBS supplement had a remarkable ability to prevent the induction of apoptosis in neural cells. Our data showed that when NSCs were treated with the extract, the number of TUNEL-positive cells reduced more significantly than the cells that were only treated with ethanol as an agent to induce apoptosis. Indeed, our findings are in agreement with a large number of dependent observations.^24–26 The same results were obtained when NSCs were treated with semaglutide, which prevented the incidence of stroke in rats by hampering the induction of apoptotic cell death in NSCs through altering the balance between Bcl-2 and BAX, two important proteins involved in the induction of apoptosis.²⁷ Ginsenoside Rg1 is another compound reported to increase the survival of NSCs in ischemia through exerting anti-inflammatory and antioxidative effects. It has also been reported that Ginsenoside Rg1 can elevate the expression of Bcl2 in NSCs and subsequently hamper the apoptotic signals.²⁸ Indeed, these findings represent valuable clues of NBS’s antiapoptotic effects. We, therefore, suggest that further studies should be conducted to investigate the process of antiapoptotic in NBS supplement. Overall, the obtained results from this study highlighted the potent efficacy of the NBS supplement in prolonging the survival of NSCs and opened a new gate into the treatment perspective of many neurodegenerative diseases.

Conclusion

Based on the pharmacologic safety of the extract and its broad clinical value, our study proposed that the NBS supplement could be used as a tool in the treatment of different neural diseases; however, further investigations in this field are required to provide the mechanism of action of the extract in several diseases.

Authorship Confirmation Statement: The authors confirm that their research is supported by Islamic Azad University of Mashhad and Tehran University of Medical Sciences that are primarily involved in education or research.

Authors’ Disclosure Statement: The authors declare that they have no conflict of interest.

Funding Statement: This study was supported by the Faculty of Basic Sciences of the Islamic Azad University, Mashhad Branch. The protocol was reviewed and approved by the institutional review board of Islamic Azad University of Mashhad (IR.IAU.MSHD.REC.1398.233).

Acknowledgments

The authors would like to praise their gratitude to the faculty of basic sciences of the Islamic Azad University of Mashhad for supporting this study.