Involvement of nitric oxide/reactive oxygen species signaling via 8-nitro-cGMP formation in 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells and rat cerebellar granule neurons

To investigate the role of nitric oxide (NO)/reactive oxygen species (ROS) redox signaling in Parkinson's disease-like neurotoxicity, we used 1-methyl-4-phenylpyridinium (MPP⁺) treatment (a model of Parkinson's disease). We show that MPP⁺-induced neurotoxicity was dependent on ROS from neuronal NO synthase (nNOS) in nNOS-expressing PC12?cells (NPC12?cells) and rat cerebellar granule neurons (CGNs). Following MPP⁺ treatment, we found production of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), a second messenger in the NO/ROS redox signaling pathway, in NPC12?cells and rat CGNs, that subsequently induced S-guanylation and activation of H-Ras. Additionally, following MPP⁺ treatment, extracellular signal-related kinase (ERK) phosphorylation was enhanced. Treatment with a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor attenuated MPP⁺-induced ERK phosphorylation and neurotoxicity. In conclusion, we demonstrate for the first time that NO/ROS redox signaling via 8-nitro-cGMP is involved in MPP⁺-induced neurotoxicity and that 8-nitro-cGMP activates H-Ras/ERK signaling. Our results indicate a novel mechanism underlying MPP⁺-induced neurotoxicity, and therefore contribute novel insights to the mechanisms underlying Parkinson's disease.     

Curcumin protects PC12 cells against 1-methyl-4-phenylpyridinium ion-induced apoptosis by bcl-2-mitochondria-ROS-iNOS pathway

The aim of present study is to explore the cytoprotection of curcumin against 1-methyl-4-phenylpridinium ions (MPP⁺)-induced apoptosis and the molecular mechanisms underlying in PC12 cells. Our findings indicated that MPP⁺ significantly reduced the cell viability and induced apoptosis of PC12 cells. Curcumin protected PC12 cells against MPP⁺-induced cytotoxicity and apoptosis not only by inducing overexpression of Bcl-2, but also reducing the loss of mitochondrial membrane potential (MMP), an increase in intracellular reactive oxygen species (ROS) and overexpression of inducible nitric oxide synthase (iNOS). The selective iNOS inhibitor AG partly blocked MPP⁺-induced apoptosis of PC12 cells. The results of present study suggested that the cytoprotective effects of curcumin might be mediated, at least in part, by the Bcl-2-mitochondria-ROS-iNOS pathway. Because of its non-toxic property, curcumin could be further developed to treat the neurodegenerative diseases which are associated with oxidative stress, such as Parkinson’s disease (PD). J. Chen and X. Q. Tang are contributed equally to this work.     

α-Lipoic acid alleviates ferroptosis in the MPP+-induced PC12 cells via activating the PI3K/Akt/Nrf2 pathway

Parkinson's disease (PD) is a typical neurodegenerative disease. α-Lipoic acid (α-LA) can reduce the incidence of neuropathy. The present study explored the role and mechanism of α-LA in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of PD. The PD model was induced via treating PC12 cells with MPP⁺ at different concentrations. MPP⁺ and α-LA effects on PC12 cells were assessed from cell viability and ferroptosis. Cell viability was detected using the cell counting kit-8 assay. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, reactive xygen species (ROS), and glutathione (GSH) concentrations, and ferroptosis-related protein SLC7A11 and GPx4 expressions were used for ferroptosis evaluation. p-PI3K, p-Akt, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels were detected. The PI3K/Akt/Nrf2 pathway inhibitors were applied to verify the role of the PI3K/Akt/Nrf2 pathway in α-LA protection against MPP⁺-induced decreased cell viability and ferroptosis. MPP⁺-reduced cell viability and induced ferroptosis as presented by increased MDA, 4-HNE, iron, and ROS concentrations, and reduced levels of GSH and ferroptosis marker proteins (SLC7A11 and GPx4). α-LA attenuated MPP⁺-induced cell viability decline and ferroptosis. The PI3K/Akt/Nrf2 pathway was activated after α-LA treatment. Inhibiting the PI3K/Akt/Nrf2 pathway weakened the protection of α-LA against MPP⁺ treatment. We highlighted that α-LA alleviated MPP⁺-induced cell viability decrease and ferroptosis in PC12 cells via activating the PI3K/Akt/Nrf2 pathway.     

Paraoxonase-1介导硫化氢的抗甲醛神经毒性作用

我们以往的研究工作证实了硫化氢(hydrogen sulfide,H2S)对甲醛神经毒性和氧化应激具有拮抗作用.Paraoxonase-1(PON-1)是机体重要的内源性抗氧化剂.本研究的目的是探讨PON-1是否可介导H2S的抗甲醛神经毒性作用.采用甲醛损伤PC12细胞为甲醛神经毒性的细胞模型.硫氢化钠(NaHS,一种H2S的供体)不仅可以上调PC12细胞PON-1的活力,还可恢复甲醛对PC12细胞PON-1表达与活力的抑制作用.2-hydroxyquinoline(2-HQ)是一种选择性PON-1抑制剂,它可显著降低H2S对甲醛细胞毒性、凋亡和活性氧(reactive oxygen species,ROS)累积的抑制作用.而且,2-HQ可阻止H2S逆转甲醛激活PC12细胞caspase-3和下调PC12细胞bcl-2表达.结果提示H2S依赖PON-1去保护PC12细胞对抗甲醛的神经毒性.我们的这一发现表明PON-1有希望成为防治甲醛神经损伤的新靶点.     

Role of Aldehyde Dehydrogenase 2 in 1-Methy-4-Phenylpyridinium Ion-Induced Aldehyde Stress and Cytotoxicity in PC12 Cells

Aldehyde stress contributes to molecular mechanisms of cell death and the pathogenesis of Parkinson’s disease (PD). The neurotoxin 1-Methy-4-Phenylpyridinium Ion (MPP⁺) is commonly used to model PD. Aldehyde dehydrogenase 2 (ALDH₂) is an important enzyme detoxifying aldehydes. The aim of this study is to evaluate whether MPP⁺-induced neurotoxicity is involved in aldehyde stress by modulation of ALDH₂. Our results demonstrated that treatment of PC12 cells with MPP⁺ leads to aldehyde stress by increasing in loads of malondialdehyde and 4-hydroxynonenal, which indicated that MPP⁺-induced aldehyde stress contributes to its cytotoxicity in PC12 cells. We also showed that MPP⁺ up-regulates the expression and activity of ALDH₂ in PC12 cells and that inhibition of ALDH₂ by its specific inhibitor daidzin prevents MPP⁺-induced decrease in cell viability and increases in apoptosis, oxidative stress and aldehyde stress in PC12 cells. These findings suggest that aldehyde stress contributes to MPP⁺-induced toxicity in PC12 cells by upregulation of ALDH₂. This study provides a novel insight into the role of ALDH₂ in the neurotoxicity of MPP⁺.     

Neuroprotective Effect of Acute and Chronic Administration of Copper (II) Sulfate against MPP+ Neurotoxicity in Mice

Neurodegenerative effects of MPP⁺, the main metabolite of MPTP include dopamine (DA) depletion and enhanced lipid peroxidation (LPO) in mice striata, both associated to free radicals overproduction. Since copper is related to several antioxidant enzymes, we tested its neuroprotective effect against MPP⁺-induced neurotoxicity (20 g/3 l). CuSO₄ pretreatment was administrated by either acute (2.5 mg/kg, i.p) or chronic (350 or 700 mg/l doses through drinking water, for 30 days) schemes. Acute administration blocked MPP⁺-induced striatal LPO only when administered 16 or 24 hours before MPP⁺, and prevented the DA-depleting effect only at 24 hours. Chronic CuSO₄ prevented the LPO increase, and blocked the DA depletion only at the higher dose used (700 mg/l). Neuroprotective effect of CuSO₄ was dependent on the dose and the time of pretreatment, which suggest that this lag could be related with mechanisms of activation or synthesis of copper-dependent proteins responsible of cellular defense against MPP⁺.     

Protective Effects of Paeoniflorin Against MPP<Superscript>+</Superscript>-induced Neurotoxicity in PC12 Cells

In the present study, we investigated the protective mechanism of paeoniflorin (PF), a monoterpene glycoside extracted from Radix Paeoniae alba roots, on MPP⁺-induced neurotoxicity in cultured rat pheochromocytoma cells (PC12). Our work included examination of cell viability assessment, amounts of released lactic dehydrogenase (LDH), intracellular Ca²⁺ concentration, cell apoptosis, mitochondrial membrane potential, caspase-3 activity, and expression profiling of two apoptosis-related genes (Bcl-2 and Bax). It was shown that, PF functioned as an MPP⁺ antagonist, being able to suppress apoptosis, decrease LDH release and Ca²⁺ concentration, attenuate membrane potential collapse and, inhibit caspase-3 activation, decrease in Bax/Bcl-2 ratio. These observations suggest that PF could protect PC12 cells against MPP⁺-induced injury and the mechanism PF’s neuroprotective effect was closely associated with Bcl-2 up-regulation and Bax down-regulation. PF has neuroprotective effects on MPP⁺-induced apoptosis in PC12 cells via regulating mitochondrial membrane potential and Bcl-2/Bax/caspase-3 signaling pathways, and this new insight will help develop a PF-based therapeutic strategy for treatmenting neurodegenerative diseases and injury.     

Neuroprotective effect of methanol extract of Phellodendri Cortex against 1-methyl-4-phenylpyridinium (MPP)-induced apoptosis in PC-12 cells

Phellodendri Cortex (PC) is a traditional herbal medicine, widely used in Korea and China. The effects of the methanol extract of Phellodendri Cortex (PC extract) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced neuronal apoptosis in PC-12 cells have been investigated. MPP⁺-induced apoptosis in PC-12 cells was accompanied by an increased bax/bcl-2 ratio, release of cytochrome c to the cytosol and activation of caspase-3. PC extract inhibited the downregulation of bcl-2 and the upregulation of bax, as well as the release of mitochondrial cytochrome c into the cytosol. In addition, PC extract attenuated caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP). These results suggest that the PC extract has protective effects against MPP⁺-induced neuronal apoptosis in PC-12 cells.     

Mechanism of 1-Methyl-4-Phenylpyridinium-Induced Dopamine Release from PC12 Cells

The molecular mechanism of 1-methyl-4-phenylpyridinium (MPP⁺), a Parkinsonism-inducing neurotoxin, has been studied in PC12 cells. The cells treated with MPP⁺ (100 μM) induced a rapid increase in phosphorylation of tyrosine residues of several proteins, including synaptophysin, a major 38 kDa synaptic vesicle protein implicated in exocytosis. An accelerated release of dopamine by MPP⁺ correlated with phosphorylation of synaptophysin. Exposing the cells to MPP⁺ triggered reactive oxygen species (ROS) generation within 60 min of treatment and the said effect was blocked by mazindol, a dopamine uptake blocker. In addition, pretreatment with 50–100 μM of selegiline, a selective MAO-B inhibitor, significantly suppressed MPP⁺-mediated ROS generation. These effects of MPP⁺ result in the generation of ROS, which may be involved in neuronal degeneration seen in Parkinson’s disease.     

A microarray study of MPP<Superscript>+</Superscript>-treated PC12 Cells: Mechanisms of toxicity (MOT) analysis using bioinformatics tools

Background

This paper describes a microarray study including data quality control, data analysis and the analysis of the mechanism of toxicity (MOT) induced by 1-methyl-4-phenylpyridinium (MPP⁺) in a rat adrenal pheochromocytoma cell line (PC12 cells) using bioinformatics tools. MPP⁺ depletes dopamine content and elicits cell death in PC12 cells. However, the mechanism of MPP⁺-induced neurotoxicity is still unclear.

Results

In this study, Agilent rat oligo 22K microarrays were used to examine alterations in gene expression of PC12 cells after 500 μM MPP⁺ treatment. Relative gene expression of control and treated cells represented by spot intensities on the array chips was analyzed using bioinformatics tools. Raw data from each array were input into the NCTR ArrayTrack database, and normalized using a Lowess normalization method. Data quality was monitored in ArrayTrack. The means of the averaged log ratio of the paired samples were used to identify the fold changes of gene expression in PC12 cells after MPP⁺ treatment. Our data showed that 106 genes and ESTs (Expressed Sequence Tags) were changed 2-fold and above with MPP⁺ treatment; among these, 75 genes had gene symbols and 59 genes had known functions according to the Agilent gene Refguide and ArrayTrack-linked gene library. The mechanism of MPP⁺-induced toxicity in PC12 cells was analyzed based on their genes functions, biological process, pathways and previous published literatures.

Conclusion

Multiple pathways were suggested to be involved in the mechanism of MPP⁺-induced toxicity, including oxidative stress, DNA and protein damage, cell cycling arrest, and apoptosis.

     

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.     

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (^•OH) scavenging action of nitric oxide (NO^•), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO^• donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro ^•OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP⁺-induced ^•OH production in the mitochondria. We also tested whether co-administration of NO^• donor and MPP⁺ could protect against MPP⁺-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP⁺-induced ^•OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in ^•OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP⁺ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO^•, when administered similarly failed to attenuate MPP⁺-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of ^•OH in the nigrostriatal degeneration caused by MPP⁺, indicate the ^•OH scavenging ability of NO^•, and demonstrate protection by NO^• donors against MPP⁺-induced dopaminergic neurotoxicity in rats.     

S-Allylcysteine, a garlic compound, protects against oxidative stress in 1-methyl-4-phenylpyridinium-induced parkinsonism in mice

S-Allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse has been the most widely used model for assessing neuroprotective agents for Parkinson's disease. 1-Methyl-4-phenylpyridinium (MPP⁺) is the stable metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and it causes nigrostriatal dopaminergic neurotoxicity. Previous studies suggest that oxidative stress, via free radical production, is involved in MPP⁺-induced neurotoxicity. Here, we report on the neuroprotective effect of SAC against oxidative stress induced by MPP⁺ in the striatum of C57BL/6J mice. Mice were pretreated with SAC (125 mg/kg ip) daily for 17 days, followed by administration of MPP⁺ (0.72 mg/kg icv), and were sacrificed 24 h later to evaluate lipid peroxidation, different antioxidant enzyme activities, spontaneous locomotor activity and dopamine (DA) content. MPP⁺ administration resulted in a significant decrease in DA levels in the striatum. Mice receiving SAC (125 mg/kg ip) had significantly attenuated MPP⁺-induced loss of striatal DA levels (32%). The neuroprotective effect of SAC against MPP⁺ neurotoxicity was associated with blocked (100% of protection) of lipid peroxidation and reduction of superoxide radical production — indicated by an up-regulation of Cu-Zn-superoxide dismutase activity — both of which are indices of oxidative stress. Behavioral analyses showed that SAC improved MPP⁺-induced impairment of locomotion (35%). These findings suggest that in mice, SAC attenuates MPP⁺-induced neurotoxicity in the striatum and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

BDNF-TrkB Pathway Mediates Neuroprotection of Hydrogen Sulfide against Formaldehyde-Induced Toxicity to PC12 Cells

Formaldehyde (FA) is a common environmental contaminant that has toxic effects on the central nervous system (CNS). Our previous data demonstrated that hydrogen sulfide (H₂S), the third endogenous gaseous mediator, has protective effects against FA-induced neurotoxicity. As is known to all, Brain-derived neurotropic factor (BDNF), a member of the neurotrophin gene family, mediates its neuroprotective properties via various intracellular signaling pathways triggered by activating the tyrosine kinase receptor B (TrkB). Intriguingly, our previous data have illustrated the upregulatory role of H₂S on BDNF protein expression in the hippocampus of rats. Therefore, in this study, we hypothesized that H₂S provides neuroprotection against FA toxicity by regulating BDNF-TrkB pathway. In the present study, we found that NaHS, a donor of H₂S, upregulated the level of BDNF protein in PC12 cells, and significantly rescued FA-induced downregulation of BDNF levels. Furthermore, we found that pretreatment of PC12 cells with K252a, an inhibitor of the BDNF receptor TrkB, markedly reversed the inhibition of NaHS on FA-induced cytotoxicity and ablated the protective effects of NaHS on FA-induced oxidative stress, including the accumulation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-trans-nonenal (4-HNE), and malondialdehyde (MDA). We also showed that K252a abolished the inhibition of NaHS on FA-induced apoptosis, as well as the activation of caspase-3 in PC12 cells. In addition, K252a reversed the protection of H₂S against FA-induced downregulation of Bcl-2 protein expression and upregulation of Bax protein expression in PC12 cells. These data indicate that the BDNF-TrkB pathway mediates the neuroprotection of H₂S against FA-induced cytotoxicity, oxidative stress and apoptosis in PC12 cells. These findings provide a novel mechanism underlying the protection of H₂S against FA-induced neurotoxicity.     

Involvement of tubulin in MPP+ neurotoxicity on NGF-differentiated PC12 cells.

In vivo, the neurotoxin MPTP is oxidated to MPP⁺, which is toxic to dopaminergic neurons. In this paper, we have used MPP⁺ as a tool to evoke neurotoxicity in the PC12 cell line and investigate the intracellular events that are involved. A cytotoxicity test, performed on undifferentiated and NGF-differentiated PC12 cells, showed that MPP⁺ is much more toxic on differentiated cells and indicated the suitable range of concentrations for studying the starting events evoked by the neurotoxin. By indirect immunofluorescence we have shown that the localisation of α - and β -tubulin in NGF-differentiated cells was modified by a 24 h treatment with 15 μmol/l MPP⁺. A biochemical analysis was performed on cell extracts and the results showed that MPP⁺ treatment induced an increase in α -tubulin levels and a decrease in β -tubulin levels. These results suggest the involvement of the two microtubule proteins in MPP⁺ neurotoxicity on NGF-differentiated PC12 cells.     

Inhibition of Store-Operated Calcium Entry Attenuates MPP+-Induced Oxidative Stress via Preservation of Mitochondrial Function in PC12 Cells: Involvement of Homer1a

The process of store-operated calcium entry (SOCE), whereby the release of intracellular Ca²⁺ from endoplasmic reticulum (ER) activates Ca²⁺ influx channels in the plasma membrane, has been demonstrated to impact a diverse range of cell functions. In the present study, we investigated the potential protective effect of SOCE inhibition against 1-methyl-4-phenylpyridinium (MPP⁺) injury by using pharmacological antagonists or specific small interfering RNA (siRNA) in PC12 cells. The results showed that both antagonists (15 μM MRS-1845 and 50 μM ML-9) and stromal interacting molecule-1 (STIM1) targeted siRNA (Si-STIM1) significantly increased cell viability, decreased apoptotic cell death and reduced intracellular reactive oxygen species (ROS) production and lipid peroxidation in MPP⁺ injured PC12 cells. SOCE inhibition also prevented MPP⁺ induced mitochondrial dysfunction and activation of mitochondrial related apoptotic factors, while had no effect on mitochondrial biogenesis. Moreover, inhibition of SOCE by antagonists and siRNA increased the expression levels of Homer1a mRNA and protein, and knockdown of Homer1a expression by specific siRNA partly reversed the protective effects induced by SOCE inhibition in PC12 cells. All these results indicated that SOCE inhibition protected PC12 cells against MPP⁺ insult through upregulation of Homer1a expression, and SOCE might be an ideal target for investigating therapeutic strategy against neuronal injury in PD patients.     

A novel neuroprotectant PAN-811 protects neurons from oxidative stress

Hydrogen peroxide (H₂O₂), a major non-radical reactive oxygen species (ROS) could elicit intracellular oxidative damage and/or cause extracellular free calcium influx by activating the NMDA receptor or through calcium channels. In the present study, NMDA receptor antagonist MK-801 fully blocked H₂O₂-induced neuronal cell death, whereas green tea (GT) extract containing-antioxidants only partially suppressed the neurotoxicity of H₂O₂. These suggest that majority of ROS overproduction is downstream of H₂O₂-induced calcium influx. A novel neuroprotectant PAN-811 was previously demonstrated to efficiently attenuate ischemic neurotoxicity. PAN-811 hereby fully blocks H₂O₂-elicited neuronal cell death with a more advanced neuroprotective profile than that of GT extract. PAN-811 was also shown to protect against CaCl₂-elicited neurotoxicity. Efficient protection against oxidative stress-induced neurotoxicity by PAN-811 indicates its potential application in treatment of ROS-mediated neurodegenerative diseases. W.P. and C.M.D. had equal contributions to this project     

Antioxidant and Neuroprotective Effects of Scrophularia striata Extract Against Oxidative Stress-Induced Neurotoxicity

In this study, the neuroprotective effect of Scrophularia striata Boiss (Scrophulariaceae) extract, a plant growing in northeastern of Iran, against oxidative stress-induced neurocytotoxicity in PC12 was evaluated. The PC12 cell line pretreated with different concentrations (10, 50, 100, and 200 μg/ml) of the extract and then treated with H₂O₂ to induce oxidative stress and neurotoxicity. Survival of the cells, reactive oxygen species (ROS) generation, and apoptosis were measured using MTT assay, fluorescent probe 2′,7′-dichlorofluorescein diacetate, and annexin V/propidium iodide, respectively. Moreover, the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) was used to evaluate the antioxidant capacity of the plant extract. Phytochemical assay by thin layer chromatography showed that the main components, including phenolic compounds, phenyl propanoids and flavonoids, were presented in the S. striata extract. The extract in concentrations of 50–200 μg/ml protected PC12 cells from H₂O₂-induced toxicity. The survival of the cells at concentration of 200 μg/ml was 64 % compared to that of H₂O₂ alone-treated cells (48 %) (p < 0.001). The extract also dose-dependently reduced intracellular ROS production (p < 0.001). Moreover, the extract showed antioxidative effects and decreased apoptotic cells. Collectively, these findings indicated the ability of S. striata to decrease ROS generation and cell apoptosis and also suggest the presence of the neuroprotective agents in this plant.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

Inhibition of ROS-Activated p38MAPK Pathway is Involved in the Protective Effect of H2S Against Chemical Hypoxia-Induced Inflammation in PC12 Cells

We have demonstrated the neuroprotection of hydrogen sulfide (H₂S) against chemical hypoxia-induced injury by inhibiting p38MAPK pathway. The present study attempts to evaluate the effect of H₂S on chemical hypoxia-induced inflammation responses and its mechanisms in PC12 cells. We found that treatment of PC12 cells with cobalt chloride (CoCl₂, a hypoxia mimetic agent) enhanced IL-6 secretion, nitric oxide (NO) generation and expression levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). L-canavanine, a selective iNOS inhibitor, partly blocked CoCl₂-induced cytotoxicity, apoptosis and mitochondrial insult. In addition, 7-Nitroindazole (7-NI), an inhibitor of nNOS, also partly attenuated the CoCl₂-induced cytotoxicity. The inhibition of p38MAPK by SB203580 (a selective p38MAPK inhibitor) or genetic silencing of p38MAPK by RNAi (Si-p38) depressed not only CoCl₂-induced iNOS expression, NO production, but also IL-6 secretion. In addition, N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, conferred a similar protective effect of SB203580 or Si-p38 against CoCl₂-induced inflammatory responses. Importantly, pretreatment of PC12 cells with exogenous application of sodium hydrosulfide (a H₂S donor, 400 μmol/l) for 30 min before exposure to CoCl₂ markedly attenuated chemical hypoxia-stimulated iNOS and nNOS expression, NO generation and IL-6 secretion as well as p38MAPK phosphorylation in PC12 cells. Taken together, we demonstrated that p38MAPK-iNOS pathway contributes to chemical hypoxia-induced inflammation and that H₂S produces an anti-inflammatory effect in chemical hypoxia-stimulated PC12 cells, which may be partly due to inhibition of ROS-activated p38MAPK-iNOS pathway.