Protective effect of Saccharomyces cerevisiae in Rattus norvegicus Ischemic Stroke Model
Dedy Budi Kurniawan1, Mokhamad Fahmi Rizki Syaban2, Arinal Mufidah2,
Muhammad Unzila Rafsi Zulfikri2, Wibi Riawan3
1Mastes of Biomedical Science Program, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.
2Undergraduated Student, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.
3Department of Biochemistry, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.
*Corresponding Author E-mail: mokhamadfahmi@gmail.com
ABSTRACT:
Stroke is one of the leading causes of morbidity and mortality in all ages. Ischemic stroke activates excitotoxic glutamate cascade leading to brain tissue injury. Saccharomyces cerevisiae is a unicellular yeast widely found in nature. S. cerevisiae is neuroprotective and able to increase the differentiation of hematopoietic stem cells (HSCs) into neuronal cells. it may increase levels of neuroprotectant BDNF in the brain tissue, therefore increase the protection of neurons. BDNF may prevent glutamate-driven excitotoxicity by reducing glutamate levels. This study uses a randomized post-test only controlled group design. In this in vivo study, rodent models of ischemic stroke were divided into five groups comprising of the negative control group, positive control group, intervention group 1 (18mg/kgBW), intervention group 2 (36mg/kgBW) and intervention group 3 (72 mg/kgBW). Groups treated with Saccharomyces cerevisiae extract showed significantly increased BDNF levels in the brain tissue, and the expression of the glutamate level was significantly reduced (P <0.05) compared to the positive control group. Thus Saccharomyces cerevisiae has a promising potential to become a therapy against ischemic stroke disease. however further research is needed regarding the efficacy and toxicity of Saccharomyces cerevisiae.
KEYWORDS: Saccharomyces cerevisiae, Ischemic Stroke, Brain-Derived Neurotrophic Factor, Glutamate.
INTRODUCTION:
Stroke is the second leading causejof mortality among 6.7 million peoplehworldwide, according to the World Health Organization. In Indonesia, stroke ranks 3rd as a cause of death, according to the Research Agencies and Health Ministries of the Republic of Indonesia in 2013. Stroke may occur in all ages but are more likely in people aged 65 years old and above. Stroke also has a high disability rate and can cause limitations on daily activities1. The high rate of disability is exacerbated by the lack of awareness which causes treatment delay so that rehabilitation therapy is needed2. There are two types ofgstroke, ischemic strokefand hemorrhagic stroke, both types of stroke can affect patient’s quality of life equally3,4.
However, 80% of strokes are ischemic type. Ischemic stroke occurs when blood circulations in the brain are clogged by a thrombotic blood-clot or fat. The blood vessel swelling caused by obstructed blood flow will compress the surrounding brain tissue causing ischemia5. Neurotoxic excitatory glutamate cascades are activated in response to ischemic attack; therefore, more glutamates are released in the synaptic cleft and are bonded to NMDA receptors. Ca2+ channels will open, and neurons will receive an influx of Ca2+ ions. This condition can lead to cell death by excitotoxicity6.
The brain releases endogenous brain-derived neurotrophicgfactor (BDNF), which is a neuroprotective cytokine released during an ischemic attack. Other studies had proven that exogenous BDNF was a remarkable neuroprotectant against ischemic attack7. BDNF is synthesized endogenously by the hypothalamus, cortex, amygdala, and peripheral tissues. BDNF is also produced by the microglia8. Hypoxic-ischemic attack on neuronal tissues will stimulate BDNF and bind to tyrosine kinase receptor type B (TrkB). The activation of the TrkB receptor has both promotion and inhibition effects, which is advantageous for the post-ischemic brain condition. BDNF generally promotes neuronal growth, differentiation, and regeneration in central nervous system9. BDNF also inhibits neurotoxicity, neuronal apoptosis, and inflammation, as normally seen in ischemic stroke10. Thus, by targeting an increase in the production of neurotropic factors such as BDNF, it is permissible to be used as rehabilitation care for patients after acute hypoxia due to ischemia11.
Saccharomyces cerevisiae is a unicellular yeast found easily in nature, the wall is composed of polysaccharide fraction (85%–90%) and protein fraction (10%–15%). Mannans and mannoproteins comprise between 30% and 40% of dry weight of yeast, β-1,3- glucan (30%–50%), highly branched β- 1,6-glucan (approximately 10%), while chitin content does not exceed 1%. β-glucan is a polysaccharide extracted from the cell walls of Saccharomyces cerevisiae, a unicellular yeast found easily in nature. β-glucan is neuroprotective and able to increase the differentiation of hematopoietic stem cells (HSCs) into neuronal cells12. Based on the previous study conducted in silico, β-glucan binds to the active site of threonine 83, a tyrosine kinase receptor. This study examines the use of β- glucan extracted from the cell wallkof Saccharomyces cerevisiae and the production of BDNF as a neuroprotector against glutamate-induced neuronal apoptosis.
MATERIAL AND METHODS:
Study Design:
Theeexperimental using randomized post-test only controlled group design study was conducted infMalang, thirty male rats (Rattus norvegicus) were used. They were divided into five groups comprised of negative control group (without any intervention), positive control group (rodent models of ischemic stroke without intervention), and three intervention groups at dosages 18mg/kgBW (P1), 36mg/kgBW (P2), and 72 mg/kgBW (P3) for 48 days. The animals were put on normal diets and observed at the Laboratory of Pharmacology, Faculty of Medicine Brawijaya University. Rats were given pars diet, put with a room temperature and a diurnal 12-hour light/dark cycle. All experimental protocols described in this study were approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Brawijaya. Number: No. 373/UN`0.F08.10/PN/2018,
Ischemicfstroke condition was induced by the MiddlegCerebral Artery Occlusion (MCAO) method. The rats were doubled- anaesthetized by intravenous xylazine- ketamine. A 10 mm incision was created along the middle cervical artery. The arterial vessels were ligated then blocked for 30-90 minutes in 37°C environment setting
Saccharomyces cerevisiae Extraction:
Saccharomyces cerevisiae was centrifuged and filtrated five times with lysis buffer. The re-suspension process was conducted at 0oC. Cell walls were lysed by vortexing with glass beads using the Omni Mixer for 30 seconds. The suspensionfwas centrifuged at 3000 G for 10 minutes. The cellgwall fraction (pellet) was collected, and the cytoplasmic solution fraction (supernatant) was removed. The pellets were washed with 5% NaCl and centrifuged five times at 3000 G under 0oC for 3 minutes then kept in the freezer. Saccharomyces extract is given orally once day by sonde.
The brain tissues were fixed using 4% PFA for 15 minutes, washedgthree times with PBS for 10 minutes, incubated with a blocking solution forf30 minutes and washed three times with PBS for 10 minutes. The brain tissues were then incubated with annexin V-FITC (1:100) in annexin binding buffer for 60 minutes in a dark space then washedgthree times with PBS for 10 minutes. The tissues were incubated with blocking solutions for 30 minutes and washed three times with PBS for 10 minutes. The tissues were then incubated with a primary antibody and washed with PBS for 10 minutes. Tissue biopsies were observed using a light microscope at 1000x magnification with oil drop.
The data was analyzed using the normality and homogeneity test using SPSS 18 software. One-Way Anova test wasdused for normallyfdistributed data and the non- parametric Kruskal Wallis test used for non-normally distributed data. The data analysis conducted used a probability value of 0.05 (p=0.05) and a 95% confidence interval (α=0.05).
RESULTS AND DISCUSSION:
The Measurement of BDNF Expression Immunohistochemistry Assay
Figure 1. BDNF expression in immunohistochemistry assay. a P < 0,05 compared to positive control group
The assay showed that the data was normal and homogenous (p >0.05). One WayfANOVA test revealed the neurotrophin value was significant, p = 0.000 (p <0.05). Besides, post hoc multiple comparison Tukey found a significant differencegbetween negative control and positive control group (p = 0.000, p < 0.05). The result showedfthat BDNF expression increased significantly in the P2 group (36mg/kgBW) and the P3 group (72mg/kgBW). That shows that BDNF could protect the neuron by inhibiting glutamate-induced apoptosis. Thus, BDNF in high concentrations may reduce the risk of neuronal apoptosis. BDNF has a neuroprotective effect by stabilized calcium ion and inhibits excessive calcium influx by reduced neurotoxicity. β-glucan might also increase BDNF expression in the brain tissue, particularly in high dose administration. Extracts obtained from yeast cell walls have many benefits. In previous literature studies, it has been found that the administration of exogenous saccharides obtained from various sources has neurological functions such as improving cognitive, synaptic plasticity, and neuroprotective effects13. In this study, we assumed that the administration of Saccharomyces cerevisiae extract could increase BDNF expression in the brain tissue of mice after ischemic stroke. BDNF has a role in neuroprotectivefmechanisms against ischemic brain injury, including itsginvolvement in promoting nerve regeneration, angiogenesis in ischemic penumbra and inhibition of inflammatory processes, neurotoxicity, and apoptosis7. In this study, we also assumed that the administration of Saccharomyces cerevisiae extract could decrease the expression of glutamate in post-ischemic stroke brain tissue, therefore reduced the excitotoxicity of neurons process.
In ischemic stroke, there is a decrease infoxygen availability and an increase indpro-inflammatory cytokines. This can lead to the upregulation of BDNF and the production of other neurotrophic factors such as increased NT produced by cerebral endothelial cells14. In addition, microglia can also increase the upregulation of BDNF and NT if there is neuronal damage15. However, the increase in NT in the ischemic stroke group tends to be less significant, so it is not sufficient for the process of neurodegeneration and neuroplasticity. Saccharomyces Cerevisiae can also stimulate microglia and astrocyte cells to produce neurotrophic factors. Microglia have receptors that have the same structure as TLR216. These receptors can be activated by Saccharomyces Cerevisiae, and activation of these receptors can increase the production of neurotrophic factors to inhibit the process of apoptosis and increase neuroregeneration17. The BDNF and TrkB pathways have the effect of regulating cell survival and other biological processes. BDNF is important for neuron and axonal growth and is needed for the survival and development of dopaminergic, GABAergic, serotonergic, and cholinergic neurons. Activation of the TrkB pathway has been shown to improve cognition and correlates to increased synaptic density. BDNF and TrkB are regulated in a place with neuronal plasticity18.
There is an association between plasma BDNF levels and stroke severity in the acute stroke stage (4 hours post-embolization)19. A study was conducted in a small group (n = 10) of stroke patients. This study reported exceptional stability in plasma BDNF levels from hospital admission to 4 days, as well as a lack of correlation between plasma BDNF levels and lesion size or clinical score20. But in other study, BDNF activity enhanced neurogenesis, suppressed apoptosis and modulation in synaptic activity by various signalling cascades. s Ischemic stroke results in a reduction of peripheral BDNF levels, partly compensated by rehabilitation treatment. Rehabilitation also improves the clinical condition. Functional training in motor tasks is accompanied by increasing serum BDNF levels, which may be related to the learned motor skills' consolidation21.
The Measurement of Glutamate Expression Immunohistochemistry Assay:
Based on the in vivo experiment, the administration of Saccharomyces cerevisiae could decrease the glutamate-induced excitotoxicity effect.
The statistical test showed that the data was normal and homogeneous (p > 0.05). Post hoc Tukey multiple comparison tests, which were used to find the difference among the groups, revealed that there was a significantfdifference between positive control group compared to negative and intervention 1 group (P1) (p = 0.000, and p = 0.001, p < 0.05). However, there were no significant differences between the negative control group when compared to P2 and P3 intervention groups (p = 1.000, p > 0.05). The assay showed increased glutamate expression ingthe positive control (K+) and intervention 1 group (P1).
The immunohistochemical results after treatment that is in the preparation of treatment 1 (dose 18 mg/kg bb) compared with positive control seen a decrease in the distribution of glutamate is characterized by reduced brownish colour in the brain tissue. At the treatment dose 2 (dose 36 mg/kg), the brown colour was seen to be less than the positive control and treatment 1. At the treatment dose 3 (72 mg/kg), the glutamate distribution was significantly decreased compared to the positive control and treatment 1 and 2. This data shows that Saccharomyces Cerevisiae extract can significantly reduce glutamate distribution at an effective dose of 72 mg/kg.
Figure 2. Glutamate expression in immunohistochemistry assay a P <0,05 compared to positive control group
Temporary or permanent blockages in blood vessels can cause ischemic stroke, which results in permanent damage at the cellular and tissue level in the brain22. The blockage of blood vessels causes hypoxia and increased activity of ROS (Reactive Oxygen Species) due to oxidative stress process, which causes Ca2+ influx into cells to increase23. Increased Ca2+ influx into cells caused cell oedema and increased GABA activity, which results in excessive glutamate. Accumulation of glutamate in the brain tissue can defect neurons. It also stimulates excessive calcium influx through NMDA receptors leading to cell swelling and apoptosis. Moreover, it causes neurodegenerative effects and neurotoxicity. Therefore, the glutamate level should be suppressed to avoid neuronal cell death24.
Glutamate cascades are activated in response to ischemic attack; therefore, more glutamates are released in the synaptic cleft and are bonded to NMDA receptors. Ca2+ channels will open, and neurons will receive an influx of Ca2+ ions. This condition can lead to cell death by excitotoxicity25. Excitotoxicity is a description of the process of an excess number of glutamate neurotransmitters, causing excessive activation of NMDAR and inducing neuronal toxicity26. That is consideredfas one of the mechanisms underlying brain death. Saccharomyces Cerevisiae has potential as a new drug and was developed as a candidate for neuroprotectants through the mechanism of reducing the occurrence of brain cell death in ischemic stroke Saccharomyces Cerevisiae can significantly reduce glutamate levels in the therapy group 3 compared with positive controls having very significant difference.
CONCLUSION:
According to the data collected in this experiment, it showed that Saccaromyces cerevisae extract might increase BDNF release and decrease glutamate levels in the stroke ischemic rat model. Besides, 72 mg/kg BW dose was the most effective in modulating BDNF and glutamate levels. In conclusion, this experiment exhibits the possibility of Saccaromyces cerevisae to be used as a therapy for stroke ischemic patients but however further research is needed regarding the efficacy and toxicity of Saccaromyces cerevisae.
ACKNOWLEDGEMENT:
This work was supported by Fakultas Kedokteran Universitas Brawijaya and Health Research Forum
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 05.07.2020 Modified on 29.10.2020
Accepted on 22.02.2021 © RJPT All right reserved
Research J. Pharm. and Tech 2021; 14(11):5785-5789.
DOI: 10.52711/0974-360X.2021.01006