Protective effect of orexin-A on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

doi:10.1016/j.neuint.2013.09.022

Neurochemistry International

Volume 63, Issue 8, December 2013, Pages 719-725

https://doi.org/10.1016/j.neuint.2013.09.022 Get rights and content

Highlights

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Orexin-A has protective effect during neuronal damage.
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Parkinson’s disease is a progressive neurodegenerative disease.
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The loss or dysfunction of orexin neurons has been reported in some neurodegenerative disease.
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Pretreatment of SH-SY5Y cells with orexin-A elicited protective effect against 6-OHDA toxicity.
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Protective effect of orexin is accompanied by its anti-apoptotic properties.

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by progressive and selective death of midbrain dopaminergic neurons. Pharmacologic treatment of PD can be divided into symptomatic and neuroprotective therapies.

Orexin-A (hypocretin-1) is a hypothalamic peptide that exerts its biological effects by stimulation of two specific, membrane-bound orexin receptors. Recent studies have shown that orexin-A has a protective role during neuronal damage.

Here, we investigated the effects of orexin-A on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson’s disease. Cell damage was induced by 150 μM 6-OHDA and the cells viability was examined by MTT assay. Intracellular reactive oxygen species (ROS) was determined by fluorescence spectrophotometry method. Immunoblotting and DNA analysis were also employed to determine the levels of biochemical markers of apoptosis in the cells.

The data showed that 6-OHDA could decrease the viability of the cells. In addition, intracellular ROS, activated caspase 3, Bax/Bcl-2 ratio, cytochrome c as well as DNA fragmentation were significantly increased in 6-OHDA-treated cells. Pretreatment of cells with orexin-A (80 pM) elicited protective effect and reduced biochemical markers of cell death.

The results suggest that orexin-A has protective effects against 6-OHDA-induced neurotoxicity and its protective effects are accompanied by its antioxidant and anti-apoptotic properties and contribute to our knowledge of the pharmacology of orexin-A.

Introduction

Parkinson’s disease (PD) is a chronic neurodegenerative disorder which is due to the progressive death of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of this disorder is not fully known, many pathological mechanisms such as oxidative stress, mitochondrial and lysosomal dysfunctions, neuroinflammatory processes, and the formation of pathologic inclusions have been reported as possible causes (Tolleson and Fang, 2013).

Pharmacologic treatment of PD can be divided into symptomatic and neuroprotective therapies. The aim of symptomatic strategy is to counteract deficiency of dopamine in the basal ganglia or to block muscarinic receptors. Neuroprotective therapy aims to slow, block, or reverse disease progression. However, such therapies are defined as those that slow underlying loss of dopaminergic neurons. At this time, there is no completely proven neuroprotective or disease-modifying therapy.

6-Hydroxydopamine (6-OHDA) is a widely used neurotoxin that can be utilized to selectively damage dopaminergic neurons in vivo and in vitro (Schober, 2004, Bove et al., 2005). In addition, human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for the study of PD and to determine the effect of protective and therapeutic agents. These cells have become a popular research cell model for PD, because this cell line posses many characteristics of dopaminergic neurons. The SH-SY5Y cells selectively express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter (Xie et al., 2010).

Neuropeptides orexins (A and B) are predominantly expressed in a few thousand hypothalamic neurons that project throughout the central nervous system. Outside the hypothalamus, orexin containing fibers have been observed in the thalamus, cortex, brain stem, amygdala, hippocampus, medulla, spinal cord, and olfactory bulbs (Tsunematsu and Yamanaka, 2012).

It has been documented that moderate orexin-A contents (100–250 fmol/mg protein) are found in the substantia nigra (Mondal et al., 1999). However, the presence of functional orexin receptors in this area is controversial and has not been fully clarified. Some investigators reported that orexin-immunoreactive fibers exist in the substantia nigra (Peyron et al., 1998, Korotkova et al., 2002). Furthermore, Korotkova et al. (2002) showed that dopaminergic neurons in substantia nigra pars compacta are unaffected by orexins (Korotkova et al., 2002). Single-cell PCR experiments demonstrated that orexin receptors are also expressed in mesocorticolimbic dopaminergic neurons in the ventral tegmental area (Korotkova et al., 2003).

Generally, orexin receptors are expressed in numerous brain regions as well as in peripheral tissues and may be involved in a large number of diverse biological processes. The well known actions of orexins include the regulation of sleep, wakefulness, feeding, energy homeostasis and breathing (Baumann and Bassetti, 2005, Tsuneki et al., 2012).

Orexins bind to specific G-protein coupled receptors, OX1R and OX2R, and activate multiple signaling cascades. orexin-mediated signaling pathways include adenylyl cyclase (AC)/cyclic AMP, phospholipase C/intracellular Ca²⁺, phospholipase D/phosphatidic acid (PA), phospholipase A2/arachidonic acid release and a cascade of mitogen-activated protein kinases (MAPKs).

However, the most characteristic response to orexins is an increase in intracellular Ca²⁺ concentration which is mediated via different signaling mechanisms including adenylyl cyclase (AC), phospholipase C (PLC) and PKC signaling pathways as well as the co-involvement of PLC and PKC pathways (Ozcan et al., 2010).

Recently, the loss or dysfunction of orexin neurons has been reported in some neurodegenerative disease such as multiple sclerosis (Oka et al., 2004) Huntington disease (Gabery et al., 2010), Alzheimer’s (Fronczek et al., 2012) and Parkinson’s disease (Fronczek et al., 2007, Fronczek et al., 2008). Drouot et al. (2003) reported that in PD patients orexins levels decreased with the severity of the disease. In addition, a decreased in orexin-A containing neurons has been demonstrated in a rat model of 6-hydroxydopamine-induced Parkinson’s disease (Cui et al., 2010).

The neuropeptide orexin-A consists of 33 amino acids with N-terminal pyroglutamyl residue, amidated C-terminus and two intrachain disulfide bonds. Recently, Yuan and colleagues reported that orexin-A has a neuroprotective effect against cerebral ischemia–reperfusion injury in rats (Yuan et al., 2011). Furthermore, central administration of orexin-A significantly reduces the brain infarct area of rats subjected to middle cerebral artery occlusion-reperfusion brain injury model and produces a profound neuroprotective effect (Kitamura et al., 2010).

The antiapoptotic and neuroprotective effects of orexin-A have been demonstrated in a novel immortalized primary embryonic rat hypothalamic cell line (Butterick et al., 2012). Additionally, it has been reported that orexin-A stimulates the proliferation and viability of 3T3-L1 preadipocytes and protects them from apoptosis (Skrzypski et al., 2012).

Since numerous evidences suggest that the occurrence of aberrant cell death in PD and activation of orexin receptors have protective and anti-apoptotic effects and also PD is associated with a decreased in orexinergic neuronal component and function, the present study was designed to investigate the effects of orexin-A in 6-OHDA-induced SH-SY5Y cells toxicity as an in vitro model of Parkinson’s disease.

Section snippets

Materials

Cell culture reagents, penicillin–streptomycin solution, trypsin EDTA, fetal bovine serum (FBS) and heat-inactivated horse serum (HS) were obtained from Biosera Co. (East Sussex, UK). Culture flasks and dishes were acquired from SPL Lifesciences Inc. (Gyeonggi-Do, South Korea). 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT), 6-hydroxydopamine (6-OHDA) and 2,7-dichlorofluorescein diacetate and orexin-A were purchased from Sigma–Aldrich (St Louis, MI, USA). Primary

Analysis of cell viability

At first the effect of different concentrations of 6-OHDA were analyzed on SH-SY5Y cells viability using the MTT assay. Fig. 1A shows that 6-OHDA could decrease the viability of SH-SY5Y cells and this toxicity was dose-dependent. A significant toxic effect was observed in groups that received 100, 150, 200 and 250 μM 6-OHDA. However, 150 μM of 6-OHDA which resulted in 55.5 ± 1.38% of relative cell viability was selected for inducing cell damage and evaluating the protective effects of the orexin-A.

Discussion

The neuropeptides orexins are involved in many physiological processes, such as the regulation of sleep, wakefulness, feeding, energy homeostasis and breathing. In the present study, the possible protective effect of orexin-A in a cellular model of Parkinson’s disease (PD) was investigated. The results showed that orexin incubation significantly preserves the viability of SH-SY5Y cells and down-regulates apoptotic signals in the 6-OHDA-induced cell apoptosis pathway, suggesting that orexin-A

Conclusion

Our results demonstrate that orexin-A can protect SH-SY5Y cells against apoptosis induced by 6-OHDA. It seems that orexin-A protective ability may be mediated via its antioxidant properties and apoptosis pathway modulation. However, further studies are needed to explore the details of its protective pathway and the role of orexin receptor subtypes signaling in this phenomenon.

Conflict of interest

No conflict to disclose.

Acknowledgments

The authors would like to thank the three anonymous reviewers for their helpful and critical comments on an earlier version of the manuscript. This work was supported by Kerman Neuroscience Research Center (KNRC/91). The funding source had no role in study design, in the collection, analysis and interpretation of data, in the writing of the report; and in the decision to submit the article for publication

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Citation Excerpt :
The application of orexin-B in midbrain culture alleviates the death of tyrosine-hydroxylase-positive dopaminergic neurons, and the IP3 receptor and RyR channel may be implicated in this cellular process (Guerreiro et al., 2015). Therefore, the antioxidant and anti-apoptotic properties of orexin-A, the induction of HIF-1α and the upregulation of BDNF may attenuate the degeneration of dopaminergic neurons in the cellular model of PD (Esmaeili-Mahani et al., 2013; Feng et al., 2014; Liu et al., 2018). However, the exact signalling pathways associated with the neuroprotective effects of orexins are still not well known.
Parkinson's disease (PD) is the second most common neurodegenerative disease caused by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. PD is characterized by motor dysfunctions as well as non-motor disorders. Orexin (also known as hypocretin) is a kind of neuropeptide involved in the regulation of motor control, the sleep/wake cycle, learning and memory, gastric motility and respiratory function. Several lines of evidence suggest that the orexinergic system is involved in the manifestations of PD, especially the non-motor disorders. Recent studies have revealed the protective actions and potential therapeutic applications of orexin in both cellular and animal models of PD. Here we present a brief overview of the involvement of the orexinergic system in PD, including the pathological changes in the lateral hypothalamus, the loss of orexinergic neurons and the fluctuation of orexin levels in CSF. Furthermore, we also review the neuroprotective effects of orexin in cellular and animal models of PD.
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Recently, the effect of Orexin-A on cells has been a matter of debate. Some studies demonstrated that Orexin-A has protective effects [22,23], but others proved that Orexin-A can induce cell death in some cell types, for example in hippocampal neurons and neuroblastoma cells [24–26]. In our study, we found that Orexin-A had harmful effects on Aβ-treated SH-SY5Y cells, possibly because Orexin-A participates in the pathogenesis of AD.
Alzheimer’s disease (AD) is a progressive neurodegenerative disease which is characterized by the accumulation of amyloid-β peptide (Aβ). Orexin-A is a neuropeptide which has been reported to participate in the pathogenesis of AD. Thus, we aimed to investigate the possible mechanism by which Orexin-A acts in AD. APP/PS1 transgenic mice, an animal model of AD, were intracerebroventricularly injected with Orexin-A. Aβ-treated SH-SY5Y cells were used as a cell model of AD and treated with Orexin-A. The Morris water maze test, fluorescence microscopy, enzyme-linked immunosorbent assay (ELISA), electron microscopy, real-time PCR, and other biochemical assays were conducted. The Morris water maze test showed that Orexin-A aggravated cognitive deficit in APP/PS1 mice. Using thioflavine-S staining and ELISA, we found that Orexin-A promoted Aβ accumulation in APP/PS1 mice. By evaluating mitochondrial morphology, cytochrome c oxidase activity, ATP level, mitochondrial DNA copy number, and reactive oxygen species, we found that Orexin-A aggravated mitochondrial impairment in APP/PS1 mice and Aβ-treated SH-SY5Y cells. Our results indicate that Orexin-A exacerbates AD by inducing mitochondrial impairment. This is a new mechanism that explains how Orexin-A participates in the pathogenesis of AD.
The ability of three African herbal remedies to offer protection against an in vitro model of Parkinson's disease
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The 6-OHDA induced mitochondrial membrane depolarisation by 54.2% (Fig. 3A, B) and ROS generation by 217.7%, (Fig. 3C, D), however, unexpectedly increased GSH by 102.1% (Fig. 3E, F). The literature supports the depolarisation observed, as 6-OHDA interferes with the ETC by blocking mitochondrial complex I with subsequent free radical production (Mazzio et al., 2004; Lopes et al., 2010; Esmaeili-Mahani et al., 2013). Twenty-four-hour exposure to 50 μM 6-OHDA has been reported to increase GSH levels, possibly due to adaptive de novo synthesis (Tirmenstein et al., 2005) to attenuate the ROS generation observed.
Parkinson’s disease, characterised by loss of dopaminergic neurons in the substantia nigra of the brain, is attributed to oxidative stress and mitochondrial dysfunction. As no cure is available, and dopamine-replacement therapy only offers symptomatic relief, other avenues of treatment are sought. Acokanthera oppositifolia, Boophone disticha and Xysmalobium undulatum are used ethnomedicinally for the treatment of neurological disorders, however, these plants have not been assessed in vitro for cytoprotective activity. The aim of the study was to assess the cytoprotective activity of these three plants in an in vitro SH-SY5Y cellular model of Parkinson’s disease induced by 6-hydroxydopamine (6-OHDA).
Plant material was extracted using acetone and methanol ultrasonic maceration. Cytotoxicity was induced by exposing cells to 33.3 μM 6-OHDA for 2 h, followed by 24 h incubation with the crude extracts. Ultra-performance liquid chromatography high definition mass spectrometry was used for tentative identification of phytochemicals. Cytoprotection was initially assessed using the sulforhodamine B staining assay to determine concentration ranges. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, reduced glutathione (GSH) content, intracellular Ca²⁺ flux and ATP levels were assessed using the JC-1 ratiometric, dihydrodichlorofluorescein cleavage, monochlorobamine adduct formation, Fura-2AM and bioluminescence assays, respectively. Cell morphology was visualized using polarisation-optical transmitted light differential interference contrast microscopy.
Several phytochemicals were tentatively identified that are known markers in the plant species, however, little difference was noted between the acetone and methanol extracts qualitatively. Extracts reduced cell density by 91%, increased ROS (217.7%) and GSH (102.1%) levels. Mitochondrial depolarisation (54.2%) was evident. Crude extracts attenuated cytotoxicity by reducing ROS and sustaining ATP production, however, no alteration to MMP was observed. Furthermore, Ca²⁺ effects were maintained by B. disticha and X. undulatum, but reduced by A. oppositifolia. Morphological changes, characteristic of cytotoxicity, was observed when exposed to 6-OHDA-in the micrographs. Intermediate-polarity extracts reduced the detrimental effects associated with 6-OHDA-induced cytotoxicity. Xysmalobium undulatum (5 μg/mL) displayed the greatest level of cytoprotection, however, the inherent cytotoxicity may limit the usefulness of extracts during treatment of disease.
Although none of the plant extracts showed potential to reverse in vitro characteristics of Parkinson’s disease completely, concomitant use with dopamine replacement therapy should be investigated as a possible treatment modality. Adjunct use of the crude extracts with traditional dopamine replacement therapy may offer an alternative approach, however, future studies are required to elucidate the feasibility of such a combination.
Agaropentaose protects SH-SY5Y cells against 6-hydroxydopamine-induced neurotoxicity through modulating NF-κB and p38MAPK signaling pathways
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Agaro-oligosaccharides, hydrolysates of agarose isolated from red algae, were reported to possess various biological functions and activities, including anti-bacteria, anti-inflammatory and anti-oxidative properties. In this study, the potential neuroprotective effects and underlying molecular mechanisms of agaro-oligosaccharides against neurotoxicity induced by 6-hydroxydopamine in SH-SY5Y cells were explored. The results indicated that pretreatment with agaro-oligosaccharide monomers especially agaropentaose (AOP) considerably enhanced the cell viability and the activities of antioxidant enzymes in SH-SY5Y cells treated by 6-hydroxydopamine. In addition, AOP markedly reduced intracellular reactive oxygen species level and inhibited the loss of mitochondrial membrane potential. Moreover, NF-κB and p38MAPK signaling pathways associated with neuroinflammation were significantly down-regulated by pretreatment with AOP. These findings reflected the protective effects and mechanisms of AOP against neurologic damage in SH-SY5Y cells induced by 6-hydroxydopamine. Hence the marine-derived agaropentaose merits further study as a novel functional food additive or therapeutic agent against some neuronal diseases.
Neuroprotective and antihyperalgesic effects of orexin-A in rats with painful diabetic neuropathy
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Citation Excerpt :
It has been revealed that central administration of orexin-A significantly reduces the brain infarct area of rats subjected to middle cerebral artery occlusion-reperfusion brain injury model and produces a profound neuroprotective effect (Kitamura et al., 2010). In addition, it has been reported that orexin-A has a protective effect against 6-OHDA-induced SH-SY5Y cell apoptosis by decreasing the caspase-3 activation and Bax/Bcl2 ratio (Esmaeili-Mahani et al., 2013). In several investigations it has been shown that free radical generation and oxidative stress play critical roles in the development of neuronal apoptosis and neuropathy in diabetes (Brownlee, 2001).
Diabetes mellitus is related to the development of neuronal tissue injury in different peripheral and central nervous system regions. A common complication of diabetes is painful diabetic peripheral neuropathy (PDN). We have studied the neuroprotective and anti-nociceptive properties of neuropeptide orexin-A in an animal experimental model of diabetic neuropathy.
All experiments were carried out on male Wistar rats (220-250 g). Diabetes was induced by a single intraperitoneal injection of 55 mg/kg (i.p.) streptozotocin (STZ). Orexin-A was chronically administrated into the implanted intrathecal catheter (0.6, 2.5 and 5 nM/L, daily, 4 weeks). The tail-flick and rotarod treadmill tests were used to evaluate the nociceptive threshold and motor coordination of these diabetic rats, respectively. Cleaved caspase-3, Bax, Bcl2 and the Bax/Bcl-2 ratio, as the biochemical indicators of apoptosis, were investigated in the dorsal half of the lumbar spinal cord tissue by western blotting method.
Treatment of the diabetic rats with orexin-A (5 nM/L) significantly attenuated the hyperalgesia and motor deficit in diabetic animals. Furthermore, orexin-A (5 nM/L) administration suppressed pro-apoptotic cleaved caspase-3 and Bax proteins. Also, orexin-A (5 nM/L) reduced the expression of Bax/Bcl-2 ratio in spinal cord dorsal half of rats with PDN.
Altogether our data suggest that the orexin-A has anti-hyperalgesic and neuroprotective effects in rats with PDN. Cellular mechanisms underlying the observed effects may, at least partially, be related to reducing the neuronal apoptosis.
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Mitochondrial dysfunction is associated with a large number of human diseases, including neurological and muscular degeneration, cardiovascular disorders, obesity, diabetes, aging and rare mitochondrial diseases. Replacement of dysfunctional mitochondria with functional exogenous mitochondria is proposed as a general principle to treat these diseases. Here we found that mitochondria isolated from human hepatoma cell could naturally enter human neuroblastoma SH-SY5Y cell line, and when the mitochondria were intravenously injected into mice, all of the mice were survived and no obvious abnormality appeared. The results of in vivo distribution suggested that the exogenous mitochondria distributed in various tissues including brain, liver, kidney, muscle and heart, which would benefit for multi-systemically mitochondrial diseases. In normal mice, mitochondrial supplement improved their endurance by increase of energy production in forced swimming test; and in experimental Parkinson's disease (PD) model mice induced by respiratory chain inhibitor MPTP, mitochondrial replacement prevented experimental PD progress through increasing the activity of electron transport chain, decreasing reactive oxygen species level, and preventing cell apoptosis and necrosis. Since effective drugs remain elusive to date for mitochondrial diseases, the strategy of mitochondrial replacement would provide an essential and innovative approach as mitochondrial therapy.

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Protective effect of orexin-A on 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y human dopaminergic neuroblastoma cells

Highlights

Abstract

Introduction

Section snippets

Materials

Analysis of cell viability

Discussion

Conclusion

Conflict of interest

Acknowledgments

J. Biol. Chem.

Sleep Med. Rev.

NeuroRx

Neurosci. Lett.

J. Immunol. Methods

Neurobiol. Aging

Biochem. Biophys. Res. Commun.

Neurosci. Res.

Regul. Pept.

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

Neurosci. Lett.

J. Biol. Chem.

J. Biol. Chem.

Free Radical Biol. Med.

FEBS Lett.

Neurosci. Lett.

Vitam. Horm.

Neurobiol. Dis.

Orexin A suppresses the growth of rat C6 glioma cells via a caspase-dependent mechanism

J. Mol. Neurosci.