Neuroprotective Effects of Phenolic and Carboxylic Acids on Oxidative Stress-Induced Toxicity in Human Neuroblastoma SH-SY5Y Cells

An increase in oxidative stress is a key factor responsible for neurotoxicity induction and cell death leading to neurodegenerative diseases including Parkinson’s and Alzheimer’s diseases. Plant phenolics exert diverse bioactivities i.e., antioxidant, anti-inflammatory, and neuroprotective effects. Herein, phenolic compounds, namely protocatechuic aldehyde (PCA) constituents of Hydnophytum formicarum Jack. including vanillic acid (VA) and trans-ferulic acid (FA) found in Spilanthes acmella Murr., were explored for anti-neurodegenerative properties using an in vitro model of oxidative stress-induced neuroblastoma SH-SY5Y cells. Exposure of the neuronal cells with H₂O₂ resulted in the decrease of cell viability, but increasing in the level of reactive oxygen species (ROS) together with morphological changes and inducing cellular apoptosis. SH-SY5Y cells pretreated with 5 µM of PCA, VA, and FA were able to attenuate cell death caused by H₂O₂-induced toxicity, as well as decreased ROS level and apoptotic cells after 24 h of treatment. Pretreated SH-SY5Y cells with phenolic compounds also helped to upregulate H₂O₂-induced depletion of the expressions of sirtuin-1 (SIRT1) and forkhead box O (FoxO) 3a as well as induce the levels of antioxidant (superoxide dismutase (SOD) 2 and catalase) and antiapoptotic B-cell lymphoma 2 (Bcl-2) proteins. The findings suggest that these phenolics might be promising compounds against neurodegeneration.     

Isolation, structural elucidation, and neuroprotective effects of iridoids from Valeriana jatamansi

Two new iridoids, jatadoids A (1) and B (2), and two known compounds (3 and 4) were isolated from Valeriana jatamansi. Their structures were elucidated on the basis of extensive spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR). Compound 1 possessed an isovaleroxy group at the C-3 position that has previously been unreported in the class of iridoids. Four compounds were evaluated and compounds 1 and 3 showed moderate neuroprotective effects against MPP⁺-induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells.     

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.     

Gastrodiae rhizoma attenuates brain aging via promoting neuritogenesis and neurodifferentiation

BackgroundGastrodiae Rhizoma (Tianma), the dried tuber of Gastrodia elata Bl. (Orchidaceae), is listed as a top-grade herbal medicine in Shen-nong Ben-ts'ao Jing and has been used for treating headaches, dizziness, vertigo and convulsion. It has a neuroprotective effect and extends the lifespan in mouse models of Huntington's disease and Niemann-Pick type C disease. However, its effect on senescence remains unknown.PurposeThis study aimed to investigate the anti-aging effects and the underlying mechanism of Gastrodiae Rhizoma.MethodsD-galactose (D-gal)- and BeSO₄-induced cellular senescence and senescence-associated β-galactosidase (SA-β-gal) activity were evaluated in SH-SY5Y and PC12 cells. D-gal–induced aging mice were used as an in vivo model. Animal behaviors including nesting and burrowing and Morris water maze were conducted. Neurogenesis in the hippocampus was assessed by immunohistochemistry and confocal microscopy, and the aging-related proteins were assessed by Western blot analysis. The potential neuritogenesis activity of the partially purified fraction of Gastrodiae Rhizoma (TM-2) and its major ingredients were investigated in PC12 cells.ResultsTM-2 could improve D-gal-induced learning and memory impairement by inhibiting oxidative stress, increasing hippocampal neurogenesis and regulating the SH2B1-Akt pathway. Moreover, N⁶-(4-hydroxybenzyl)adenine riboside (T1-11) and parishins A and B, three constituents of TM-2, had anti-aging activity, as did T1-11 and parishin A induced neuritogenesis.ConclusionOur data suggested that TM-2 slowed down D-gal-induced cellular and mouse brain aging. These results indicate that Gastrodiae Rhizoma has a beneficial effect on senescence. It may be used for neuroprotection and promoting neurogenesis.     

Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH₂-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson’s disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27^Kip1 protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27^Kip1 significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27^Kip1 degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.     

Neuroprotective Effects of Ugonin K on Hydrogen Peroxide-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells

Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. Ugonin K, a flavonoid isolated from the rhizomes of Helminthostachys zeylanica, possesses potent antioxidant property. In this study, we investigate the neuroprotective effects of ugonin K on hydrogen peroxide (H₂O₂)-induced apoptosis in SH-SY5Y cells. Incubation of SH-SY5Y cells with H₂O₂ for 24 h induced cell death measured with MTT assay. Hoechst 33258 staining confirmed that the reduced cell viability by H₂O₂ was due to apoptosis. In addition, H₂O₂ increased the expression of 17-kDa cleaved fragment of caspase-3 which could be reversed by pretreatment with ugonin K. Pretreatment with ugonin K attenuated H₂O₂-induced cell death in a dose-dependent manner. Neuroprotective effect of ugonin K was abolished by ERK and PI3K inhibitors. Pretreatment with JNK kinase and p38 MAPK inhibitors had no effect on ugonin K-mediated protection against H₂O₂-induced apoptosis. Western blotting with anti-phospho-ERK1/2 and anti-phospho-Akt (pS473) antibodies showed that ugonin K increased both ERK1/2 and Akt phosphorylation. These results suggest that ugonin K by activation of ERK1/2 and PI3K/Akt signal pathways protects SH-SY5Y cells from H₂O₂-induced apoptosis.     

Neuroprotective effects of triterpenoid saponins from Medicago sativa L. against H2O2-induced oxidative stress in SH-SY5Y cells

Medicago sativa L. is a forage legume plant widely distributed in all continents. Six new triterpenoid saponins, Medicagosides A-F (1–6) and five known ones (7–11) were isolated from M. sativa. Their structures were determined via HRESIMS, 1D and 2D NMR analysis. Biologically, all the isolates displayed neuroprotective activities against H₂O₂-induced damage in SH-SY5Y cells. Among them, compounds 1, 3–5 and 10 exhibited striking neuroprotective activities at 100 μM, restoring cell viability range from 79.66% to 89.03%, relative to 79.46% (100 μM) of Trolox used as the positive control.     

Caffeoylquinic Acid Derivatives Protect SH-SY5Y Neuroblastoma Cells from Hydrogen Peroxide-Induced Injury Through Modulating Oxidative Status

Oxidative stress has been confirmed as a contribution to the pathogenesis and pathophysiology of many neurological disorders such as Alzheimer’s disease and Parkinson’s disease. Caffeoylquinic acids (CQAs) are considered to have anti-oxidative stress ability in a previous study, but the structure–activity relationships (SARs) of CQAs in neuroprotective effects are still unclear. In the present study, we primarily expound the SARs of CQAs in counteracting H₂O₂-induced injury in SH-SY5Y cells. We found that CQAs (1–10) represented the protection of SH-SY5Y cells against H₂O₂-induced injury in varying degrees and malonyl groups could obviously increase the anti-oxidative stress ability of CQAs. Intensive studies of 4,5-O-dicaffeoyl-1-O-(malic acid methyl ester)-quinic acid (MDCQA) indicated that the mechanisms could potentially involve activation of endogenous antioxidant enzymes and the regulation of the phosphorylation of MAPKs and AKT. In conclusion, MDCQA could serve as a neuroprotective agent with a potential to attenuate oxidative stress.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.     

Neuroprotective Effect of <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis> Extract on Human Dopaminergic Cell line,SH-SY5Y

Hydrogen peroxide (H₂O₂) is a major Reactive Oxygen Species (ROS), which has been implicated in many neurodegenerative conditions including Parkinson’s disease (PD). Rosmarinus officinalis (R. officinalis) has been reported to have various pharmacological properties including anti-oxidant activity. In this study, we investigated the neuroprotective effects of R. officinalis extract on H₂O₂-induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that H₂O₂-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with R. officinalis. Moreover, R. officinalis was very effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death induced by H₂O₂. R. officinalis extract effectively suppressed the up-regulation of Bax, Bak, Caspase-3 and -9, and down-regulation of Bcl-2. Pretreatment with R. officinalis significantly attenuated the down-regulation of tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC) gene in SH-SY5Y cells. These findings indicate that R. officinalis is able to protect the neuronal cells against H₂O₂-induced injury and suggest that R. officinalis might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.     

Synthesis and biological evaluation of 3-carbamate smilagenin derivatives as potential neuroprotective agents

Studies indicated that smilagenin, isolated from Anemarrhena asphodeloides Bunge, could improve cognitive impairment and exhibit neuroprotective activity. On the basis of the structure of smilagenin, a series of derivatives were synthesized and evaluated for their neuroprotective effects of H₂O₂-induced, oxygen glucose deprivation-induced neurotoxicity in SH-SY5Y cells and LPS-induced NO production in RAW264.7 cells. Structure activity relationship of derivatives revealed that benzyl-substituted piperazine formate derivatives showed the potent neuroprotective activity such as A12. These findings may provide new insights for the development of neuroprotective agents against Alzheimer’s disease.     

Neuroprotective effects of aucubin on hydrogen peroxide-induced toxicity in human neuroblastoma SH-SY5Y cells via the Nrf2/HO-1 pathway

BackgroundWhen redox balance is lost in the brain, oxidative stress can cause serious damage that leads to neuronal loss, in congruence with neurodegenerative diseases. Aucubin (AU) is an iridoid glycoside and that is one of the active constituents of Eucommia ulmoides, has many pharmacological effects such as anti-inflammation, anti-liver fibrosis, and anti-atherosclerosis.PurposeThe present study aimed to evaluate the inhibitory effects of AU on cell oxidative stress against hydrogen peroxide (H₂O₂)-induced injury in SH-SY5Y cells in vitro.MethodsSH-SY5Y cells were simultaneously treated with AU and H₂O₂ for 24 h. Cell viability was measured by CCK-8. Additionally, mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and cell apoptosis were measured by flow cytometry.ResultsThe results showed that AU can significantly increase the H₂O₂-induced cell viability and the mitochondrial membrane potential, decrease the ROS generation, malondialdehyde (MDA), and increase glutathione (GSH) contents and the superoxide dismutase (SOD) activity. We also found that H₂O₂ stimulated the production of nitric oxide (NO), which could be reduced by treatment with AU through inhibiting the inducible nitric oxide synthase (iNOS) protein expression. In H₂O₂-induced SH-SY5Y cells, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) content and cell apoptosis were significantly reduced by AU treatment through nuclear factor E2-related factor 2/hemo oxygenase-1 (Nrf2/HO-1) activation, inhibiting the expression of p-NF-κB/NF-κB and down-regulating MAPK and Bcl-2/Bax pathways.ConclusionThese results indicate that AU can reduce inflammation and oxidative stress through the NF-κB, Nrf2/HO-1, and MAPK pathways.     

The Neuroprotective Effect of Conditioned Medium from Human Adipose-Derived Mesenchymal Stem Cells is Impaired by N-acetyl Cysteine Supplementation

Oxidative stress is a common feature in neurodegenerative diseases associated with neuroinflammation, and therefore, has been proposed as a key target for novel therapies for these diseases. Recently, adipose-derived stem cell (ASC)-based cell therapy has emerged as a novel strategy for neuroprotection. In this study, we evaluate the therapeutic role of ASC-conditioned medium (ASC-CM) against H₂O₂-induced neurotoxicity in a new in vitro model of ec23/brain-derived neurotrophic factor (BDNF)-differentiated human SH-SY5Y neuron-like cells (SH-SY5Yd). In the presence of ASC-CM, stressed SH-SY5Yd cells recover normal axonal morphology (with an almost complete absence of H₂O₂-induced axonal beading), electrophysiological features, and cell viability. This beneficial effect of ASC-CM was associated with its antioxidant capacity and the presence of growth factors, namely, BDNF, glial cell line-derived neurotrophic factor, and transforming growth factor β1. Moreover, the neuroprotective effect of ASC-CM was very similar to that obtained from treatment with BDNF, an essential factor for SH-SY5Yd cell survival. Importantly, we also found that the addition of the antioxidant agent N-acetyl cysteine to ASC-CM abolished its restorative effect; this was associated with a strong reduction in reactive oxygen species (ROS), in contrast to the moderate decrease in ROS produced by ASC-CM alone. These results suggest that neuronal restorative effect of ASC-CM is associated with not only the release of essential neurotrophic factors, but also the maintenance of an appropriate redox state to preserve neuronal function.     

Arctium lappa L. roots ameliorates cerebral ischemia through inhibiting neuronal apoptosis and suppressing AMPK/mTOR-mediated autophagy

BackgroundArctium lappa L. roots are very popular cultivated vegetables, which possesses various pharmacological activities. Our previous studies have demonstrated that Arctium lappa L. roots exerted protective effects against H₂O₂, glutamate and N-methyl-D-aspartic acid (NMDA)-induced neuronal injury in vitro. However, whether Arctium lappa L. roots could prevent against cerebral ischemia and the underlying mechanism remain unclear.PurposeThe objective of the present study was to investigate the neuroprotective effects of ethyl acetate extract of Arctium lappa L. roots (EAL) and the active ingredient 4,5-O-dicaffeoyl-1-O-[4-malic acid methyl ester]-quinic acid (DCMQA) in EAL against cerebral ischemia and explore the underlying mechanism.Study DesignThe neuroprotective effects of EAL and DCMQA were investigated in rats with permanent middle cerebral artery occlusion (MCAO) and in oxygen glucose deprivation/reoxygenation (OGD/R)-stimulated SH-SY5Y cells, respectively.MethodsThe infarct volume, brain edema and neurological deficits were measured following MCAO. TUNEL and Nissl staining were performed to detect neuronal loss and apoptosis of neurons in rat brains. Cell survival was measured by MTT and LDH assay. In addition, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels were determined by DCFH-DA and JC-1 fluorescent probe, respectively. Hoechst 33342 staining and Annexin V-FITC/PI double staining were performed to evaluate neuronal apoptosis. The expression levels of proteins were evaluated by western blot.ResultsEAL reduced brain infarct volume, ameliorated brain edema and improved neurological deficits in MCAO rats. In addition, EAL inhibited oxidative stress and inflammatory responses following MCAO. Besides, active compound DCMQA alleviated cytotoxicity as well as inhibited over-production of intracellular ROS and loss of MMP induced by OGD/R in SH-SY5Y cells. Moreover, EAL and DCMQA inhibited apoptosis by decreasing the expressions of pro-apoptotic proteins including bax, cytochrome c and cleaved caspase-3 while promoting the bcl-2 expression in MCAO rats and OGD/R-stimulated neurons, respectively. In addition, DCMQA suppressed the production of autophagosomes and down-regulated expression of Beclin 1 and LC3. Furthermore, inhibiting AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway contributed to DCMQA-mediated suppression of autophagy induced by OGD/R.ConclusionOur findings demonstrate that Arctium lappa L. roots protect against cerebral ischemia through inhibiting apoptosis and AMPK/mTOR-mediated autophagy in vitro and in vivo, providing a theoretical basis for the development of CQAs in Arctium lappa L. roots as neuroprotective drugs for the prevention and treatment of ischemic stroke.     

Determination by Gas Chromatography-Mass Spectrometry of [N(5)]Adenine Incorporation into Endogenous Cytokinins and the Effect of Tissue Age on Cytokinin Biosynthesis in Datura innoxia Crown Gall Tissue

In this study gas chromatographic-mass spectrometric techniques have been used to identify and quantify the metabolic incorporation of [¹⁵N₅]adenine into zeatin and its metabolites by 3-week-old Datura innoxia Mill, crown gall tissue. In a parallel study the levels of endogenous cytokinins were also determined by the stable isotope dilution technique using deuterium (²H)-labeled internal standards. Incorporation levels of the [¹⁵N₅]adenine after 8 hours of incubation, expressed as a percentage of the endogenous cytokinins, were as follows: zeatin (1.0%), zeatin riboside (1.5%), and zeatin riboside 5′-phosphate (10.2%). These results are consistent with those observed in complementary experiments using [U-¹⁴C]adenine, and support the proposal that the cytokinin biosynthesis occurs primarily at the nucleotide level. The effect of tissue age on cytokinin biosynthesis, determined by [U-¹⁴C]adenine incorporation into cytokinins by tissues at varying growth stages, indicated a steady increase with time reaching maximal synthesis at five weeks following subculture after which the level of ¹⁴C incorporation into cytokinins declined.     

Protective effects of a wheat germ peptide (RVF) against H2O2-induced oxidative stress in human neuroblastoma cells

RVF (Arg-Val-Phe), a peptide derived from wheat germ, shows antioxidant properties. Here, the neuroprotective efficacies of RVF were investigated in human neuroblastoma cells (SH-SY5Y) that were pretreated with RVF (150–250 μM, 4 h) and exposed to H₂O₂ (200 μM). RVF increased viable cell numbers by 37 % and reduced the release of lactate dehydrogenase. Pretreatment with RVF also inhibited H₂O₂-induced accumulation of reactive oxygen species and maintained the mitochondrial transmembrane potential as well as preventing intracellular Ca²⁺ dysregulation during H₂O₂ exposure. Furthermore, pretreatment with RVF increased the Bcl-2/Bax ratio and blocked cleavage poly(ADP-ribose) polymerase by inhibiting caspase-3 activation, thus decreasing apoptosis.     

Cannabinoid Effects on β Amyloid Fibril and Aggregate Formation,Neuronal and Microglial-Activated Neurotoxicity In Vitro

Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ_1–42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ⁹-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ_1–42. Aβ_1–42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.     

Protective effect of augmenter of liver regeneration on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells

Background: Hydrogen peroxide, as other reactive oxygen species (ROS) produced during redox processes, induces lipid membrane peroxidation and protein degeneration causing cell apoptosis. ROS are recently considered as messengers in cell signalling processes, which, through reversible protein disulphide bridges formation, activate regulatory factors of cell proliferation and apoptosis. Disulphide bridges formation is catalysed by sulphydryl oxidase enzymes.

Aim: The neuroprotective effect of ALR protein (Alrp), a sulphydryl oxidase enzyme, on H₂O₂-induced apoptosis in SH-SY5Y cells has been evaluated.

Methods: Cell viability, flow cytometric evaluation of apoptotic cells, fluorescent changes of nuclear morphology, immunocytochemistry Alrp detection, Western blot evaluation of mitochondrial cyt c release and mitochondrial swelling were determined.

Results: Alrp prevents the H₂O₂-induced cell viability loss, apoptotic cell death and mitochondrial swelling in SH-SY5Y cells in culture.

Conclusions: The data demonstrate that Alrp improves SH-SY5Y cells survival in H₂O₂-induced apoptosis. It is speculated that this effect could be related to the Alrp enzymatic activity.     

Discovery of a benzofuran derivative (MBPTA) as a novel ROCK inhibitor that protects against MPP+-induced oxidative stress and cell death in SH-SY5Y cells

Parkinson disease (PD) is a neurodegenerative disease with multifactorial etiopathogenesis. The discovery of drug candidates that act on new targets of PD is required to address the varied pathological aspects and modify the disease process. In this study, a small compound, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1,3,4-thiadiazol-2-yl) acetamide (MBPTA) was identified as a novel Rho-associated protein kinase inhibitor with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced damage in SH-SY5Y neuroblastoma cells. Further investigation showed that pretreatment of SH-SY5Y cells with MBPTA significantly suppressed MPP⁺-induced cell death by restoring abnormal changes in nuclear morphology, mitochondrial membrane potential, and numerous apoptotic regulators. MBPTA was able to inhibit MPP⁺-induced reactive oxygen species (ROS)/NO generation, overexpression of inducible NO synthase, and activation of NF-κB, indicating the critical role of MBPTA in regulating ROS/NO-mediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling, and its cytoprotective effect was abolished by PI3K and Akt inhibitors. The structural comparison of a series of MBPTA analogs revealed that the benzofuran moiety probably plays a crucial role in the anti-oxidative stress action. Taken together, these results suggest that MBPTA protects against MPP⁺-induced apoptosis in a neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling.     

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ_25-35, Aβ_1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ_1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.