Caffeic acid phenethyl ester blocks free radical generation and 6-hydroxydopamine-induced neurotoxicity

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS). Antioxidants protect neurons against 6-OHDA-induced neurotoxicity by inhibiting free radical generation. In this study, we investigated whether or not caffeic acid phenethyl ester (CAPE) could protect neurons against 6-OHDA-induced neurotoxicity in cultured rat rostral mesencephalic neurons (RMN) and cerebellar granule neurons (CGN). We now report that exposure of RMN and CGN to 6-OHDA (40 microM for RMN and 70 microM for CGN) resulted in significant increases in free radical production and death of both neuron types. Pretreatment with CAPE (10 microM) for 2 h prevented both 6-OHDA-induced free radical generation and neurotoxicity. Furthermore, CAPE also attenuated H(2)O(2)-induced neurotoxicity. Our results strongly suggest that CAPE blocks 6-OHDA-induced neuronal death possibly by inhibiting 6-OHDA-induced free radical generation and blocking free radical-induced neurotoxicity in neurons. Both the antioxidative and neuroprotective effects of CAPE may be beneficial in the therapy for Parkinson's disease and other neurodegenerative diseases.     

Molecular mechanisms of 6-hydroxydopamine-induced cytotoxicity in PC12 cells: involvement of hydrogen peroxide-dependent and -independent action

The neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to generate an experimental model of Parkinson's disease. It has been reported that reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide (H2O2), generated from 6-OHDA are involved in its cytotoxicity; however, the contribution and role of ROS in 6-OHDA-induced cell death have not been fully elucidated. In the present study using PC12 cells, we observed the generation of 50 microM H2O2 from a lethal concentration of 100 microM 6-OHDA within a few minutes, and compared the sole effect of H2O2 with 6-OHDA. Catalase, an H2O2-removing enzyme, completely abolished the cytotoxic effect of H2O2, while a significant but partial protective effect was observed against 6-OHDA. 6-OHDA induced peroxiredoxin oxidation, cytochrome c release, and caspase-3 activation. Catalase exhibited a strong inhibitory effect against the peroxiredoxin oxidation, and cytochrome c release induced by 6-OHDA; however, caspase-3 activation was not effectively inhibited by catalase. On the other hand, 6-OHDA-induced caspase-3 activation was inhibited in the presence of caspase-8, caspase-9, and calpain inhibitors. These results suggest that the H2O2 generated from 6-OHDA plays a pivotal role in 6-OHDA-induced peroxiredoxin oxidation, and cytochrome c release, while H2O2- and cytochrome c-independent caspase activation pathways are involved in 6-OHDA-induced neurotoxicity. These findings may contribute to explain the importance of generated H2O2 and secondary products as a second messenger of 6-OHDA-induced cell death signal linked to Parkinson's disease.     

Isoliquiritigenin isolated from licorice Glycyrrhiza uralensis prevents 6-hydroxydopamine-induced apoptosis in dopaminergic neurons

Licorice (Glycyrrhiza uralensis) is a medicinal herb containing various bioactive components implicated in antioxidative, anti-inflammatory, antiviral, and neuroprotective effects, but the effects of licorice against Parkinson's disease (PD)-related dopaminergic cell death have not been studied. In this study, we investigated the protective effects of isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a dopaminergic cell line, SN4741. ISL (1 μM) significantly attenuated 6-OHDA (50 μM)-induced reactive oxygen species (ROS) and nitric oxide (NO) generation and apoptotic cell death. ISL pretreatment effectively suppressed 6-OHDA-mediated upregulation of Bax, p-c-Jun N-terminal kinase (JNK), p-p38 mitogen-activated protein (MAP) kinase, cytochrome c release, and caspase 3 activation. In addition, ISL significantly attenuated 6-OHDA-induced Bcl-2, brain-derived neurotrophic factor (BDNF), and mitochondrial membrane potential (MMP) reduction. Pharmacological inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway reversed ISL-mediated neuroprotection against 6-OHDA toxicity in SN4741 cells. These results provide the first evidence that ISL can protect dopaminergic cells under oxidative stress conditions by regulating the apoptotic process.     

Immortalized dopamine neurons: A model to study neurotoxicity and neuroprotection

6-Hydroxydopamine (6-OHDA) causes selective degeneration of dopaminergic neurons in the rat brain and has been used to produce an animal model of Parkinsonism. Recently, a clonal line of immortalized dopamine (DA) neurons (1RB3AN27), which expresses varying levels of tyrosine hydroxylase, dopamine transporter, neuron specific enolase, and nestin, was established. These DA neurons reduce behavioral deficits in 6-OHDA-lesioned rats. The relative sensitivity of fetal and adult neurons to potential neurotoxins is not well defined. The availability of immortalized DA neurons provides a unique opportunity to compare the relative neurotoxicity of 6-OHDA in differentiated and undifferentiated DA neurons in vitro and identify neuroprotective agents. Our results showed that 6-OHDA treatment for 24 hr decreased the viability of undifferentiated and differentiated immortalized DA neurons in vitro, as determined by the MTT assay, and increased the rate of apoptosis in differentiated DA neurons. The differentiated DA neurons (IC50 = 33 microM) were about 2-fold more sensitive to 6-OHDA than undifferentiated DA neurons (IC50 = 75 microM) in cell culture. Similarly, the differentiated DA neurons were more sensitive to another neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), which is commonly used to induce Parkinsonism in animal models, than were the undifferentiated DA neurons in culture. Among growth factors tested, only glial cell line-derived neurotrophic factor (GDNF) partially protected differentiated DA neurons against 6-OHDA-induced toxicity. These results suggest that undifferentiated and differentiated immortalized DA neurons can be a useful experimental model to study relative sensitivity to neurotoxins and neuroprotective agents that could have relevance to fetal and adult neurons.     

Apomorphine attenuates 6-hydroxydopamine-induced apoptotic cell death in SH-SY5Y cells

Enhanced oxidative stress is implicated in the pathogenesis of Parkinson's disease. The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS), leading to neuronal cell death. On the other hand, apomorphine, a dopamine D1/D2 receptor agonist and known as a potent antioxidant, has been reported to have a neuroprotective effect. In the present study, we investigated the effect of apomorphine on 6-OHDA-induced apoptotic cell death using the human dopaminergic neuroblastoma cell line, SH-SY5Y. The co-treatment of cells with apomorphine significantly attenuated 6-OHDA-induced ROS generation, the phosphorylation of c-Jun N-terminal kinase (JNK), DNA fragmentation and subsequent apoptotic cell death. In addition, pretreatment with apomorphine for 24 h and the following concomitant treatment enhanced the protective effects against 6-OHDA-induced toxicity except for the attenuation of JNK phosphorylation. We also demonstrated that pretreatment alone with apomorphine for 24 h prior to the exposure confers resistance against 6-OHDA-induced cell toxicity. These findings suggested that apomorphine acts principally as a radical scavenger to suppress the level of ROS and ROS-stimulated apoptotic signaling pathway, whereas the other mechanisms might be involved in the protective effects.     

Neuroprotective effects of Astragaloside IV in 6-hydroxydopamine-treated primary nigral cell culture

Parkinson's disease (PD) is caused by a progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress and neural degeneration are suggested to be involved in the pathogenesis of Parkinson's disease. In the present study, Astragaloside IV (AS-IV) extracted from the dried root of Astragalus membranaceus, a well-known Chinese medicine used for the treatment of neurodegenerative diseases, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. By examining the effect of AS-IV on 6-hydroxydopamine (6-OHDA)-induced loss of dopaminergic neurons in primary nigral culture, we found that AS-IV pretreatment significantly and dose-dependently attenuated 6-OHDA-induced loss of dopaminergic neurons. Neuronal fiber length studies showed that massive neuronal cell death with degenerated neurons was observed in those cultures incubated with 6-OHDA, whereas in AS-IV co-treatments most dopaminergic neurons were seen to be intact and sprouting. In flow cytometric analysis, AS-IV resulted in a marked and dose-dependent rescue in tyrosine hydrolase (TH)-immunopositive cells from 6-OHDA-induced degeneration of dopaminergic neurons. Double immunofluorescence revealed that AS-IV treatment alone at concentrations of 100 and 200 μM increased the level of TH and NOS (nitrite oxide synthase) immunoreactivities; however, the protective effect of AS-IV on TH and NOS immunopositive cells in 6-OHDA treated nigral cell cultures was only seen at a concentration of 100 μM. These findings show that AS-IV can protect dopaminergic neurons against 6-OHDA-induced degeneration. Besides the neuroprotective effect, AS-IV alone promoted neurite outgrowth and increased TH and NOS immunoreactive of dopaminergic neurons. The neuroprotective and neurosprouting effects of AS-IV are specific for dopaminergic neurons and it has therapeutic potential in the treatment of PD.     

Neuroprotective Effects of Chalcones from <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> on 6-Hydroxydopamine-Induced Cytotoxicity in Rat Mesencephalic Cells

In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal plant, Myracrodruon urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells. In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml) reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as Parkinson’s disease.     

Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease

The aim of this study was to investigate changes in protein profiles during the early phase of dopaminergic neuronal death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were identified whose expression was significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). In particular, we detected oxidative modification of thioredoxin-dependent peroxidases (peroxiredoxins; PRX) in treated MN9D cells. Oxidative modification of PRX induced by 6-OHDA was blocked in the presence of N-acetylcysteine, suggesting that reactive oxygen species (ROS) generated by 6-OHDA induce oxidation of PRX. These findings were confirmed in primary cultures of mesencephalic neurons and in rat brain injected stereotaxically. Overexpression of PRX1 in MN9D cells (MN9D/PRX1) exerted neuroprotective effects against death induced by 6-OHDA through scavenging of ROS. Consequently, generation of both superoxide anion and hydrogen peroxide following 6-OHDA treatment was decreased in MN9D/PRX1. Furthermore, overexpression of PRX1 protected cells against 6-OHDA-induced activation of p38 MAPK and subsequent activation of caspase-3. In contrast, 6-OHDA-induced apoptotic death signals were enhanced by RNA interference-targeted reduction of PRX1 in MN9D cells. Taken together, our data suggest that the redox state of PRX may be intimately involved in 6-OHDA-induced dopaminergic neuronal cell death and also provide a molecular mechanism by which PRX1 exerts a protective role in experimental models of Parkinson disease.     

Pyrroloquinoline Quinone is a Potent Neuroprotective Nutrient Against 6-Hydroxydopamine-Induced Neurotoxicity

Pyrroloquinoline quinone (PQQ), which is an essential nutrient, has been shown to act as an antioxidant. Reactive oxygen species (ROS) are thought to be responsible for neurotoxicity caused by the neurotoxin 6-hydroxydopamine (6-OHDA). In this study, we investigated the ability of PQQ to protect against 6-OHDA-induced neurotoxicity using human neuroblastoma SH-SY5Y. When SH-SY5Y cells were exposed to 6-OHDA in the presence of PQQ, PQQ prevented 6-OHDA-induced cell death and DNA fragmentation. Flow cytometry analysis using the ROS-sensitive fluorescence probe, dihydroethidium, revealed that PQQ reduced elevation of 6-OHDA-induced intracellular ROS. In contrast to PQQ, antioxidant vitamins, ascorbic acid and α-tocopherol, had no protective effect. Moreover, we showed that PQQ effectively scavenged superoxide, compared to the antioxidant vitamins. Therefore, our results suggest the protective effect of PQQ on 6-OHDA-induced neurotoxicity is involved, at least in part, in its function as a scavenger of ROS, especially superoxide.     

Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants

6-Hydroxydopamine (6-OHDA) neurotoxicity has often been related to the generation of free radicals. Here we examined the effect of the presence of iron (Fe(2+) and Fe(3+)) and manganese and the mediation of ascorbate, L-cysteine (CySH), glutathione (GSH), and N-acetyl-CySH on hydroxyl radical (*OH) production during 6-OHDA autoxidation. In vitro, the presence of 800 nM iron increased (> 100%) the production of *OH by 5 microM 6-OHDA while Mn(2+) caused a significant reduction (72%). The presence of ascorbate (100 microM) induced a continuous generation of *OH while the presence of sulfhydryl reductants (100 microM) limited this production to the first minutes of the reaction. In general, the combined action of metal + antioxidant increased the *OH production, this effect being particularly significant (> 400%) with iron + ascorbate. In vivo, tyrosine hydroxylase immunohistochemistry revealed that intrastriatal injections of rats with 6-OHDA (30 nmol) + ascorbate (600 nmol), 6-OHDA + ascorbate + Fe(2+) (5 nmol), and 6-OHDA + ascorbate + Mn(2+) (5 nmol) caused large striatal lesions, which were markedly reduced (60%) by the substitution of ascorbate by CySH. Injections of Fe(2+) or Mn(2+) alone showed no significant difference to those of saline. These results clearly demonstrate the role of ascorbate as an essential element for the neurotoxicity of 6-OHDA, as well as the diminishing action of sulfhydryl reductants, and the negligible effect of iron and manganese on 6-OHDA neurotoxicity.     

Isoliquiritigenin Isolated from Licorice Glycyrrhiza uralensis Prevents 6-Hydroxydopamine-Induced Apoptosis in Dopaminergic Neurons

Licorice (Glycyrrhiza uralensis) is a medicinal herb containing various bioactive components implicated in antioxidative, anti-inflammatory, antiviral, and neuroprotective effects, but the effects of licorice against Parkinson’s disease (PD)-related dopaminergic cell death have not been studied. In this study, we investigated the protective effects of isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a dopaminergic cell line, SN4741. ISL (1 μM) significantly attenuated 6-OHDA (50 μM)-induced reactive oxygen species (ROS) and nitric oxide (NO) generation and apoptotic cell death. ISL pretreatment effectively suppressed 6-OHDA-mediated upregulation of Bax, p-c-Jun N-terminal kinase (JNK), p-p38 mitogen-activated protein (MAP) kinase, cytochrome c release, and caspase 3 activation. In addition, ISL significantly attenuated 6-OHDA-induced Bcl-2, brain-derived neurotrophic factor (BDNF), and mitochondrial membrane potential (MMP) reduction. Pharmacological inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway reversed ISL-mediated neuroprotection against 6-OHDA toxicity in SN4741 cells. These results provide the first evidence that ISL can protect dopaminergic cells under oxidative stress conditions by regulating the apoptotic process.     

Apomorphine attenuates 6-hydroxydopamine-induced apoptotic cell death in SH-SY5Y cells

Abstract

Enhanced oxidative stress is implicated in the pathogenesis of Parkinson's disease. The catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) induces the production of reactive oxygen species (ROS), leading to neuronal cell death. On the other hand, apomorphine, a dopamine D1/D2 receptor agonist and known as a potent antioxidant, has been reported to have a neuroprotective effect. In the present study, we investigated the effect of apomorphine on 6-OHDA-induced apoptotic cell death using the human dopaminergic neuroblastoma cell line, SH-SY5Y. The co-treatment of cells with apomorphine significantly attenuated 6-OHDA-induced ROS generation, the phosphorylation of c-Jun N-terminal kinase (JNK), DNA fragmentation and subsequent apoptotic cell death. In addition, pretreatment with apomorphine for 24 h and the following concomitant treatment enhanced the protective effects against 6-OHDA-induced toxicity except for the attenuation of JNK phosphorylation. We also demonstrated that pretreatment alone with apomorphine for 24 h prior to the exposure confers resistance against 6-OHDA-induced cell toxicity. These findings suggested that apomorphine acts principally as a radical scavenger to suppress the level of ROS and ROS-stimulated apoptotic signaling pathway, whereas the other mechanisms might be involved in the protective effects.     

Protective effect of (+/-) isoborneol against 6-OHDA-induced apoptosis in SH-SY5Y cells.

Oxidative stress caused by dopamine (DA) may play an important role in the pathogenesis of Parkinson's disease (PD). (+/-) Isoborneol is a monoterpenoid alcohol present in the essential oils of numerous medicinal plants and is a known antioxidant. In this study, we investigated the neuroprotective effect of isoborneol against 6-hydroxydopamine (6-OHDA)-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with isoborneol significantly reduced 6-OHDA-induced generation of reactive oxygen species (ROS) and 6-OHDA-induced increases in intracellular calcium. Furthermore, apoptosis induced by 6-OHDA was reversed by isoborneol treatment. Isoborneol protected against 6-OHDA-induced increases in caspase-3 activity and cytochrome C translocation into the cytosol from mitochondria. Isoborneol prevented 6-OHDA from decreasing the Bax/Bcl-2 ratio. We also observed that isoborneol decreased the activation of c-Jun N-terminal kinase and induced activation of protein kinase C (PKC) which had been suppressed by 6-OHDA. Our results indicate that the protective function of isoborneol is dependent upon its antioxidant potential and strongly suggest that isoborneol may be an effective treatment for neurodegenerative diseases associated with oxidative stress.     

Dietary oxyresveratrol prevents parkinsonian mimetic 6-hydroxydopamine neurotoxicity

Oxyresveratrol (OXY) is a polyhydroxylated stilbene existing in mulberry. Increasing lines of evidence have shown its neuroprotective effects against Alzheimer disease and stroke. However, little is known about its neuroprotective effect in Parkinson disease (PD). Owing to its antioxidant activity, blood-brain barrier permeativity, and water solubility, we hypothesized that OXY may exert neuroprotective effects against parkinsonian mimetic 6-hydroxydopamine (6-OHDA) neurotoxicity. Neuroblastoma SH-SY5Y cells have long been used as dopaminergic neurons in PD research. We found that both pretreatment and posttreatment with OXY on SH-SY5Y cells significantly reduced the release of lactate dehydrogenase, the activity of caspase-3, and the generation of intracellular reactive oxygen species triggered by 6-OHDA. Compared to resveratrol, OXY exhibited a wider effective dosage range. We proved that OXY could penetrate the cell membrane by HPLC analysis of cell extracts. These results suggest that OXY may act as an intracellular antioxidant to reduce oxidative stress induced by 6-OHDA. Western blot analysis demonstrated that OXY markedly attenuated 6-OHDA-induced phosphorylation of JNK and c-Jun. Furthermore, we proved that OXY increased the basal levels of SIRT1, which may disclose new pathways accounting for the neuroprotective effects of OXY. Taken together, our results suggest OXY, a dietary phenolic compound, as a potential nutritional candidate for protection against neurodegeneration in PD.     

Pharmacological and physiological properties of serofendic acid, a novel neuroprotective substance isolated from fetal calf serum

Excess activation of glutamate receptors and production of large amount of free radicals including nitric oxide (NO) may be responsible for neuronal death associated with neurodegenerative disorders, but endogenous defense systems that protect neurons from these insults are poorly understood. In the course of studies to explore neuroprotective substance in mammalian origin, we isolated a neuroprotective factor from an ether extract of fetal calf serum based on the ability to protect rat primary cortical neurons against NO-induced cytotoxicity. A novel lipophilic low-molecular-weight substance that exerted potent neuroprotective actions at submicromolar concentrations was named "serofendic acid". Mass spectrometry and nuclear magnetic resonance spectroscopy revealed the chemical structure of serofendic acid (15-hydroxy-17-methylsulfinylatisan-19-oic acid) as a sulfur-containing atisane type diterpenoid, which is unique among known endogenous substances. Synthetic serofendic acid exhibited potent protective actions on cortical neurons against cytotoxicity of a NO donor as well as of glutamate, although it did not affect glutamate receptor-mediated responses in these neurons. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on NO but was capable of inhibiting the generation of hydroxyl radical, a presumed 'executor' radical in the nitric oxide-mediated neurotoxic cascade. These findings suggest that serofendic acid is a low-molecular-weight neuroprotective factor that attenuates free radical-mediated damage triggered by excessive stimulation of neuronal glutamate receptors.     

Enriched environment protects the nigrostriatal dopaminergic system and induces astroglial reaction in the 6-OHDA rat model of Parkinson's disease

Enriched environment (EE) is neuroprotective in several animal models of neurodegeneration. It stimulates the expression of trophic factors and modifies the astrocyte cell population which has been said to exert neuroprotective effects. We have investigated the effects of EE on 6-hydroxydopamine (6-OHDA)-induced neuronal death after unilateral administration to the medial forebrain bundle, which reaches 85–95% of dopaminergic neurons in the substantia nigra after 3 weeks. Continuous exposure to EE 3 weeks before and after 6-OHDA injection prevents neuronal death (assessed by tyrosine hydroxylase staining), protects the nigrostriatal pathway (assessed by Fluorogold retrograde labeling) and reduces motor impairment. Four days after 6-OHDA injection, EE was associated with a marked increase in glial fibrillary acidic protein staining and prevented neuronal death (assessed by Fluoro Jade-B) but not partial loss of tyrosine hydroxylase staining in the anterior substantia nigra. These results robustly demonstrate that EE preserves the entire nigrostriatal system against 6-OHDA-induced toxicity, and suggests that an early post-lesion astrocytic reaction may participate in the neuroprotective mechanism.     

Tetracycline derivatives and ceftriaxone, a cephalosporin antibiotic, protect neurons against apoptosis induced by ionizing radiation

DNA damage induced by low doses of ionizing radiation causes apoptosis, which is partially mediated via the generation of free radicals. Both free radicals and apoptosis are involved in the majority of brain diseases, including stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Because previous studies have shown that tetracycline derivatives doxycycline and minocycline have anti-inflammatory effects and are protective against brain ischemia, we studied whether minocycline and doxycycline or ceftriaxone, a cephalosporin antibiotic with the potential to inhibit excitotoxicity, protect neurons against ionizing radiation in primary cortical cultures. A single dose of 1 Gy significantly increased lactate dehydrogenase release, induced DNA fragmentation in neurons and triggered microglial proliferation. Treatment with minocycline (20 nM), doxycycline (20 nM) and ceftriaxone (1 microM) significantly reduced irradiation-induced lactate dehydrogenase release and DNA fragmentation. The most efficient protection was achieved by minocycline treatment, which also inhibited the irradiation-induced increase in microglial cell number. Our results suggest that some tetracycline derivatives, such as doxycycline and minocycline, and ceftriaxone, a cephalosporin derivative, protect neurons against apoptotic death.     

Catechin attenuates 6-hydroxydopamine (6-OHDA)-induced cell death in primary cultures of mesencephalic cells

Past studies have shown the protective effects of tea catechins on oxidative cell damage induced by 6-OHDA in PC12 cells. In this study we verified whether or not catechin prevents 6-OHDA-induced oxidative cell damage in primary cultures of rat mesencephalic cells. On exposure to 6-OHDA (200 microM), the cultures showed a marked decrease in cell viability, disturbances in lipid peroxidation, and an increased generation of NO, as assayed by MTT, TBARS and nitrite assays, respectively. Introduction of catechin significantly attenuated the cell death caused by 6-OHDA at concentrations of 3.4, 34 and 340 microM in a dose-related manner. Catechin produced no marked changes on 6-OHDA-induced increases in NO, but caused a significant inhibition of lipid peroxidation. These results suggest that catechins offer similar cytoprotection against 6-OHDA-induced oxidative cell damage in mesencephalic cell cultures, as previously described in PC12 cells. The cytoprotective function of catechin results from its antioxidant property and is not due to the inhibition of nitric oxide synthase. These findings further support and substantiate traditional consumption of catechin rich green/black tea as protection against neurodegenerative diseases like Parkinsonism.     

Tacrine(2)-ferulic acid, a novel multifunctional dimer, attenuates 6-hydroxydopamine-induced apoptosis in PC12 cells by activating Akt pathway

Oxidative stress is closely related to the pathogenesis of neurodegenerative disorders such as Parkinson’s disease (PD). In this study, we investigated the neuroprotective effect of tacrine–ferulic acid dimers linked by an alkylenediamine side chain (TnFA, n = 2−7), a series of novel acetylcholinesterase inhibitors, against 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells. Among these dimers, pre-treatment of tacrine(2)–ferulic acid (T2FA, 3−30 μM) attenuated 6-OHDA-induced apoptosis in a concentration-dependent manner. The activations of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) were observed after the treatment of 6-OHDA. Both SB415286 (an inhibitor of GSK3β) and PD98059 (an inhibitor of ERK kinase) reduced the neurotoxicity induced by 6-OHDA, indicating that GSK3β and ERK are involved in 6-OHDA-induced apoptosis. T2FA was able to inhibit the activation of GSK3β, but not ERK, in an Akt-dependent manner. Furthermore, LY294002, a phosphoinositide 3-kinase inhibitor, abolished the neuroprotective effect of T2FA. Collectively, these results suggest that T2FA prevents 6-OHDA-induced apoptosis possibly by activating the Akt pathway in PC12 cells.     

Ginsenoside Rg1 protects against 6-OHDA-induced neurotoxicity in neuroblastoma SK-N-SH cells via IGF-I receptor and estrogen receptor pathways

Our previous studies have demonstrated that ginsenoside Rg1 is a novel class of potent phytoestrogen and can mimic the action of estradiol in stimulation of MCF-7 cell growth by the crosstalk between insulin-like growth factor-I receptor (IGF-IR)-dependent pathway and estrogen receptor (ER)-dependent pathway. The present study was designed to investigate the neuroprotective effects of ginsenoside Rg1 against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human neuroblastoma SK-N-SH cells and the possible mechanisms. Pre-treatment with ginsenoside Rg1 resulted in an enhancement of survival, and significant rescue occurred at the concentration of 0.01 μM on cell viability against 6-OHDA-induced neurotoxicity. These effects could be completely blocked by IGF-IR antagonist JB-1 or ER antagonist ICI 182780. 6-OHDA arrested the cells at G₀G₁ phase and prevented S phase entry. Rg1 pre-treatment could reverse the cytostatic effect of 6-OHDA. Ginsenoside Rg1 also could attenuate 6-OHDA-induced decrease in mitochondrial membrane potential. These effects could also be completely blocked by JB-1 or ICI 182780. Furthermore, 6-OHDA-induced up-regulation of Bax and down-regulation of Bcl-2 mRNA and protein expression could be restored by Rg1 pre-treatment. Rg1 pre-treatment could reverse the down-regulation of ERα protein expression induced by 6-OHDA treatment. Cells transfected with the estrogen responsive element (ERE)-luciferase reporter construct exhibited significantly increased ERE-luciferase activity in the Rg1 presence, suggesting that the estrogenic effects of Rg1 were mediated through the endogenous ERs. These results suggest that ginsenoside Rg1 may attenuate 6-OHDA-induced apoptosis and its action might involve the activation of IGF-IR signaling pathway and ER signaling pathway.