The presence of ascorbate induces expression of brain derived neurotrophic factor in SH-SY5Y neuroblastoma cells after peroxide insult, which is associated with increased survival

Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the mechanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.     

Effect of oxytocin on neuroblastoma cell viability and growth

Oxytocin, released in response to different physiological stimuli, could play a key role in reducing stress reaction. It was suggested that it has protective effect against inflammation and consequences of oxidative stress. Mechanisms how oxytocin effects mediated in the brain tissue are unclear. In this study, oxytocin effect on cell growth and neuronal viability was examined. Human neuroblastoma (SH-SY5Y and SK-N-SH) and glioblastoma (U87MG) cells were exposed to different concentrations of oxytocin for 12-96 h. Potential protective effect of oxytocin treatment was investigated after exposing cells to oxidative stress using hydrogen peroxide (50 mM, 2 h) or 6-hydroxydopamine (25 μM, 24 h). Cell proliferation was measured by cell counting and cell viability was examined by MTT assay. Protein expression of selected neurotrophic factors was measured as an additional parameter. Oxytocin (1 μM) significantly increased cell number in all three cell types. Viability of SH-SY5Y cells was increased in the presence of oxytocin without significant effect of dose (0.01-1 μM). Cell death induced by hydrogen peroxide was not prevented by incubation with oxytocin. Oxytocin pretreatment blunted neurotoxin 6-OHDA reduction of cell viability in SH-SY5Y cells. Oxytocin (1 μM, 12 h) elevated amount of total proteins without increasing levels of brain-derived neurotrophic factor and neurotrophic growth factor. In conclusion, oxytocin increases growth and viability of neuroblastoma and glioblastoma cells without activation of neurotrophic factors. Oxytocin does not have protective effect in oxidative stress; however, it might be important for neuroprotection to dopaminergic neurons. Its proliferative effect might be important in native cell life, euplastic processes, and tumor progression.     

Side-chain modified vitamin D analogs require activation of both PI 3-K and erk1,2 signal transduction pathways to induce differentiation of human promyelocytic leukemia cells

Synthetic analogs of vitamin D for potential use in differentiation therapy should selectively regulate genes necessary for differentiation without inducing any perturbations in calcium homeostasis. PRI-1906, an analog of vitamin D2, and PRI-2191, an analog of vitamin D3 bind nuclear vitamin D receptor (nVDR) with substantially lower affinity than 1,25-dihydroxyvitamin D3 (1,25-D3), but have higher differentiation-inducing activity as estimated in HL-60 leukemia cellmodel. To examine how their increased differentiation-inducing activity is regulated we tested the hypothesis that membrane-mediated events, unrelated to nVDR, take part in the differentiation in response to PRI-1906 and PRI-2191. The induction of leukemia cell differentiation in response to the analogs of vitamin D was inhibited by LY294002 (phosphatidylinositol 3-kinase inhibitor), PD98059 (inhibitor of MEK1,2, an upstream regulator of extracellular-signal regulated kinase) and rapamycin (p70S6K inhibitor) pointing out that activation of signal transduction pathways unrelated to nVDR is necessary for differentiation. On the other hand, inhibition of cytosolic phospholipase A2 accelerated the differentiation of HL-60 cells induced by either 1,25-D3 or by the vitamin D analogs suggesting possible existence of a feedback loop between extracellular-signal regulated kinases and phospholipase A2.     

Adiponectin is Protective against Oxidative Stress Induced Cytotoxicity in Amyloid-Beta Neurotoxicity

Beta-amyloid (Aβ ) neurotoxicity is important in Alzheimer’s disease (AD) pathogenesis. Aβ neurotoxicity causes oxidative stress, inflammation and mitochondrial damage resulting in neuronal degeneration and death. Oxidative stress, inflammation and mitochondrial failure are also pathophysiological mechanisms of type 2 diabetes (T₂DM) which is characterized by insulin resistance. Interestingly, T₂DM increases risk to develop AD which is associated with reduced neuronal insulin sensitivity (central insulin resistance). We studied the potential protective effect of adiponectin (an adipokine with insulin-sensitizing, anti-inflammatory and anti-oxidant properties) against Aβ neurotoxicity in human neuroblastoma cells (SH-SY5Y) transfected with the Swedish amyloid precursor protein (Sw-APP) mutant, which overproduced Aβ with abnormal intracellular Aβ accumulation. Cytotoxicity was measured by assay for lactate dehydrogenase (LDH) released upon cell death and lysis. Our results revealed that Sw-APP transfected SH-SY5Y cells expressed both adiponectin receptor 1 and 2, and had increased AMP-activated protein kinase (AMPK) activation and enhanced nuclear factor-kappa B (NF-κB) activation compared to control empty-vector transfected SH-SY5Y cells. Importantly, adiponectin at physiological concentration of 10 µg/ml protected Sw-APP transfected SH-SY5Y cells against cytotoxicity under oxidative stress induced by hydrogen peroxide. This neuroprotective action of adiponectin against Aβ neurotoxicity-induced cytotoxicity under oxidative stress involved 1) AMPK activation mediated via the endosomal adaptor protein APPL1 (adaptor protein with phosphotyrosine binding, pleckstrin homology domains and leucine zipper motif) and possibly 2) suppression of NF-κB activation. This raises the possibility of novel therapies for AD such as adiponectin receptor agonists.     

Indirubin-3-Oxime Prevents H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3β and the ERK Pathway

Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H₂O₂)-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H₂O₂ exposure led to the increased activities of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3β cascade and the ERK pathway induced by H₂O₂. In addition, both GSK3β and mitogen-activated protein kinase inhibitors significantly prevented H₂O₂-induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H₂O₂-induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H₂O₂-induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.     

Induction of endogenous glutathione by the chemoprotective agent, 3H-1,2-dithiole-3-thione, in human neuroblastoma SH-SY5Y cells affords protection against peroxynitrite-induced cytotoxicity

Substantial evidence suggests that peroxynitrite generated from the bi-radical reaction of nitric oxide and superoxide is critically involved in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Reaction with sulfhydryl (SH)-containing molecules has been proposed to be a major detoxification pathway of peroxynitrite in biological systems. This study was undertaken to determine if chemically elevated intracellular reduced glutathione (GSH), a major SH-containing biomolecule, affords protection against peroxynitrite-mediated toxicity in cultured neuronal cells. Incubation of human neuroblastoma SH-SY5Y cells with the unique chemoprotectant, 3H-1,2-dithiole-3-thione (D3T), led to a significant elevation of cellular GSH in a concentration-dependent fashion. To examine the protective effects of D3T-induced GSH on peroxynitrite-mediated toxicity, SH-SY5Y cells were pretreated with D3T and then exposed to either the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), or the authentic peroxynitrite. We observed that D3T-pretreated cells showed a markedly increased resistance to SIN-1- or authentic peroxynitrite-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of SIN-1- or authentic peroxynitrite-mediated cytotoxicity. To further demonstrate the causal role for GSH induction in D3T-mediated cytoprotection, SH-SY5Y cells were co-treated with BSO to abolish D3T-induced GSH elevation. Co-treatment of the cells with BSO was found to significantly reverse the protective effects of D3T on SIN-1- or authentic peroxynitrite-elicited cytotoxicity. Taken together, this study demonstrates for the first time that D3T can induce GSH in cultured SH-SY5Y cells, and that the D3T-augmented cellular GSH defense affords a marked protection against peroxynitrite-induced toxicity in cultured human neuronal cells.     

The ATM kinase inhibitor KU-55933 provides neuroprotection against hydrogen peroxide-induced cell damage via a γH2AX/p-p53/caspase-3-independent mechanism: Inhibition of calpain and cathepsin D

The role of the kinase ataxia-telangiectasia mutated (ATM), a well-known protein engaged in DNA damage repair, in the regulation of neuronal responses to oxidative stress remains unexplored. Thus, the neuroprotective efficacy of KU-55933, a potent inhibitor of ATM, against cell damage evoked by oxidative stress (hydrogen peroxide, H₂O₂) has been studied in human neuroblastoma SH-SY5Y cells and compared with the efficacy of this agent in models of doxorubicin (Dox)- and staurosporine (St)-evoked cell death. KU-55933 inhibited the cell death induced by H₂O₂ or Dox but not by St in undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells, with a more pronounced effect in the latter cell phenotype. Furthermore, this ATM inhibitor attenuated the Dox- but not H₂O₂-induced caspase-3 activity in both UN- and RA-SH-SY5Y cells. Although KU-55933 inhibited the H₂O₂- and Dox-induced activation of ATM, it attenuated the toxin-induced phosphorylation of the proteins H2AX and p53 only in the latter model of cell damage. Moreover, the ATM inhibitor prevented the H₂O₂-evoked increases in calpain and cathepsin D activity and attenuated cell damage to a similar degree as inhibitors of calpain (MDL28170) and cathepsin D (pepstatin A). Finally, we confirmed the neuroprotective potential of KU-55933 against the H₂O₂- and Dox-evoked cell damage in primary mouse cerebellar granule cells and in the mouse hippocampal HT-22 cell line. Altogether, our results extend the neuroprotective portfolio of KU-55933 to a model of oxidative stress, with this effect not involving inhibition of the γH2AX/p-p53/caspase-3 pathway and instead associated with the attenuation of calpain and cathepsin D activity.     

Amelioration of oxygen and glucose deprivation-induced neuronal death by chloroform fraction of bay leaves (Laurus nobilis)

The purpose of this study was to determine whether the Laurus nobilis chloroform fraction (LNCF) protects against cerebral ischemia neuronal damage. Human neuroblastoma SH-SY5Y cells and brain slices from rats were subjected to oxygen and glucose deprivation (OGD), followed by reoxgenation with and without LNCF. The viabilities of SH-SY5Y cells and brain slices from the rats were 58.5±4.9% and 79.7±5.9% in the group subjected to OGD, and 80.4±0.4% and 97.2±1.9% at 4 μg/ml of LNCF, respectively. LNCF also significantly inhibited death-associated protein kinase (DAPK) dephosphorylation. Pretreatment with LNCF at 4 mg/kg significantly decreased infarct size by 79% of vehicle control in the middle cerebral artery occlusion (MCAO) in vivo model. LNCF is a neuroprotective drug candidate against cerebral ischemia neuronal damage.     

Loganin protects against hydrogen peroxide-induced apoptosis by inhibiting phosphorylation of JNK, p38, and ERK 1/2 MAPKs in SH-SY5Y cells

We investigated the mechanisms underlying the protective effects of loganin against hydrogen peroxide (H(2)O(2))-induced neuronal toxicity in SH-SY5Y cells. The neuroprotective effect of loganin was investigated by treating SH-SY5Y cells with H(2)O(2) and then measuring the reduction in H(2)O(2)-induced apoptosis using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays. Following H(2)O(2) exposure, Hoechst 33258 staining indicated nuclear condensation in a large proportion of SH-SY5Y cells, along with an increase in reactive oxygen species (ROS) production and an intracellular decrease in mitochondria membrane potential (MMP). Loganin was effective in attenuating all the above-stated phenotypes induced by H(2)O(2). Pretreatment with loganin significantly increased cell viability, reduced H(2)O(2)-induced LDH release and ROS production, and effectively increased intracellular MMP. Pretreatment with loganin also significantly decreased the nuclear condensation induced by H(2)O(2). Western blot data revealed that loganin inhibited the H(2)O(2)-induced up-regulation of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3, increased the H(2)O(2)-induced decrease in the Bcl-2/Bax ratio, and attenuated the H(2)O(2)-induced release of cytochrome c from mitochondria to the cytosol. Furthermore, pretreatment with loganin significantly attenuated the H(2)O(2)-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results suggest that the protective effects of loganin against H(2)O(2)-induced apoptosis may be due to a decrease in the Bcl-2/Bax ratio expression due to the inhibition of the phosphorylation of JNK, p38, and ERK 1/2 MAPKs. Loganin's neuroprotective properties indicate that this compound may be a potential therapeutic agent for the treatment of neurodegenerative diseases.     

Reduction of Muscarinic Receptor Density and of Guanine Nucleotide-Stimulated Phosphoinositide Hydrolysis in Human SH-SY5Y Neuroblastoma Cells Following Long-Term Treatment with 12-O-Tetradecanoylphorbol 13-Acetate or Mezerein

The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca2+ signals were examined in the human neuroblastoma SH-SY5Y cell line. Pretreatment of SH-SY5Y cells with 50 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N-[3H]methylscopolamine and [3H]-scopolamine binding, and a significantly larger reduction (48%) in agonist-stimulated 3H-inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4 alpha-phorbol 12,13-didecanoate was without effect on both antagonist binding and agonist-stimulated phosphoinositide (PPI) turnover. A decline (approximately 50%) in the agonist-mediated rise in cytoplasmic Ca2+ and a substantial loss of protein kinase C activity also were observed following pretreatment with TPA or mezerein. The ability of fluoride, an agent capable of direct activation of guanine nucleotide binding proteins, to stimulate 3H-inositol phosphate release was significantly reduced in SH-SY5Y cells treated with these agents. Furthermore, pretreatment of SH-SY5Y neuroblastoma cells with TPA or mezerein impaired 3H-inositol phosphate formation induced by the addition of either guanosine 5'-O-(3-thiotriphosphate) or carbamylcholine to digitonin-permeabilized cells, but not that elicited by the addition of 2 mM CaCl2. Although cells cultured in the presence of serum-free media also exhibited neuronal differentiation, no significant alteration in either muscarinic receptor number or agonist-stimulated PPI hydrolysis was observed. The results suggest that TPA and mezerein decrease agonist-stimulated PPI hydrolysis and Ca2+ signaling in SH-SY5Y cells not only by a reduction in muscarinic receptor number but also through an inhibition of guanine nucleotide-stimulated PPI turnover.     

Effects of Tissue Transglutaminase on β -Amyloid1-42-Induced Apoptosis

Tissue transglutaminase (TGase) has been implicated in both cell survival and apoptosis. Here we investigate the role of TGase in β-amyloid-induced neurotoxicity using retinoic acid (RA)-differentiated, neuronal SH-SY5Y cells. We show that β-amyloid-induced cell death was reduced in RA-differentiated SH-SY5Y cells treated with the TGase inhibitor monodansyl cadaverine. Expression of wild-type TGase enhanced β-amyloid_1-42-induced apoptosis, whereas transamidation-defective TGase did not. These effects were specific for β-amyloid-treated cells, as TGase reversed the neurotoxic effects caused by hydrogen peroxide treatment. Enhancement of β-amyloid_1-42-induced cell death by TGase was accompanied by marked increases in TGase activity in the membrane fractions and translocation of TGase to the cell surface. Overall, these findings suggest that the ability of TGase to exhibit pro-survival versus pro-apoptotic activity is linked to its cellular localization, with β-amyloid-induced recruitment of TGase to the cell surface accentuating neuronal toxicity and apoptosis.     

PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.     

不同ω-3/ω-6构成比的配伍红花籽油对SH-SY5Y细胞氧化损伤的预防效应

探讨不同ω-3/ω-6构成比的配伍红花籽油(Compatibility Safflower Seed Oil,CSSO)预防神经细胞氧化损伤的作用。通过过氧化氢(hydrogen peroxide,H2O2)氧自由基供体诱导,建立人神经母细胞瘤SH-SY5Y细胞氧化损伤模型;以不同浓度和ω-3/ω-6构成比的CSSO进行细胞药物干预,利用四甲基偶氮唑蓝(methyl thiazolyltetrazolium,MTT)和流式细胞仪检测细胞活力变化和细胞凋亡率。我们建立了H2O2诱导的SH-SY5Y细胞氧化损伤模型,其IC50值为1089.54μmol/L H2O2;随着ω-3相对含量递减,CSSO预防细胞氧化损伤的效应增加,且当ω-3/ω-6比例为1∶6.68和有效浓度范围为375～750μg CSSO/mL时,其药物干预组细胞活力(84.1%)显著高于模型组(61.1%),而药物干预组细胞凋亡率(12.6%)明显低于模型组(25.9%)。从以上结果可以推测,CSSO能够保护细胞并预防氧自由基诱导的细胞损伤,其效果可能与CSSO中ω-3/ω-6构成比密切相关。     

Salvianolic acid A protects human SH-SY5Y neuroblastoma cells against H₂O₂-induced injury by increasing stress tolerance ability

Salvianolic acid A (Sal A) is a polyphenol extracted from the root of the Salvia miltiorrhiza bunge. Hydrogen peroxide (H(2)O(2)) is a major reactive oxygen species (ROS), which has been implicated in stroke and other neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. In this study, we investigated the neuroprotective effects of Sal A in human SH-SY5Y neuroblastoma cells against H(2)O(2)-induced injury. Our results showed that cells pretreated with Sal A exhibited enhanced neuronal survival and that this protection was associated with an increase in adenosine triphosphate (ATP) and the stabilization of mitochondrial membrane potential. In addition, Sal A markedly decreased the excessive activation AMP-activated protein kinase (AMPK) and the serine-threonine protein kinase, Akt, in SH-SY5Ycells induced by H(2)O(2). In conclusion, our results demonstrated that Sal A protects SH-SY5Y cells against H(2)O(2)-induced oxidative stress and these protective effects are related to stress tolerance and not energy depletion via inhibition of the AMPK and Akt signaling pathway.     

Cytotoxic and cytoprotective effects of tryptamine-4,5-dione on neuronal cells: a double-edged sword

Serotonin (5-hydroxytryptamine) is a putative substrate for myeloperoxidase, which may convert it into the reactive quinone tryptamine-4,5-dione (TD). In this study, we found that the viability of human SH-SY5Y neuroblastoma cells treated with 25?μM TD was increased to approximately 117%. On the other hand, the cell viability was significantly decreased by exposure to TD (150–200?μM), with an increase in intracellular reactive oxygen species (ROS). Interestingly, pre-treatment of SH-SY5Y cells with 100?μM TD prevented cell death and suppressed intracellular ROS generation evoked by the addition of hydrogen peroxide (H₂O₂). Expression of the phase-II antioxidant enzyme NAD(P)H: quinone oxidoreductase 1 and haem oxygenase 1 were upregulated by TD at a concentration of 50–100?μM. Nuclear factor erythroid 2-related factor 2 (Nrf2), the regulator of these enzyme, was translocated from the cytosol to the nucleus by 100?μM TD. In summary, moderate concentrations of TD may increase the self-defence capacity of neuronal cells against oxidative stress.     

Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway

Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

     

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells.

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.     

Water-soluble fractions from defatted sesame seeds protect human neuroblast cells against peroxyl radicals and hydrogen peroxide-induced oxidative stress

Oxidative stress is involved in the development of aging-related diseases, such as neurodegenerative diseases. Dietary antioxidants that can protect neuronal cells from oxidative damage play an important role in preventing such diseases. Previously, we reported that water-soluble fractions purified from defatted sesame seed flour exhibit good antioxidant activity in vitro. In the present study, we investigated the protective effects of white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively) against 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H₂O₂) induced oxidative stress in human neuroblast SH-SY5Y cells. Pretreatment with WS-wsf and GS-wsf did not protect cells against AAPH-induced cytotoxicity, while simultaneous co-treatment with AAPH significantly improved cell viability and inhibited membrane lipid peroxidation. These results suggest that WS-wsf and GS-wsf protect cells from AAPH-induced extracellular oxidative damage via direct scavenging of peroxyl radicals. When oxidative stress was induced by H₂O₂, pretreatment WS-wsf and GS-wsf significantly enhanced cell viability. These results suggest that in addition to radical scavenging, WS-wsf and GS-wsf enhance cellular resistance to intracellular oxidative stress by activation of the Nrf-2/ARE pathway as confirmed by the increased Nrf2 protein level in the nucleus and increased heme oxygenase 1 (HO-1) mRNA expression. The roles of ferulic and vanillic acids as bioactive antioxidants in these fractions were also confirmed. In conclusion, our results indicated that WS-wsf and GS-wsf, which showed antioxidant activity in vitro, are also efficient antioxidants in a cell system protecting SH-SY5Y cells against both extracellular and intracellular oxidative stress.