Protective Effects of Walnut Extract Against Amyloid Beta Peptide-Induced Cell Death and Oxidative Stress in PC12 Cells

Amyloid beta-protein (Aβ) is the major component of senile plaques and cerebrovascular amyloid deposits in individuals with Alzheimer’s disease. Aβ is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Recently, considerable attention has been focused on dietary antioxidants that are able to scavenge reactive oxygen species (ROS), thereby offering protection against oxidative stress. Walnuts are rich in components that have anti-oxidant and anti-inflammatory properties. The inhibition of in vitro fibrillization of synthetic Aβ, and solubilization of preformed fibrillar Aβ by walnut extract was previously reported. The present study was designed to investigate whether walnut extract can protect against Aβ-induced oxidative damage and cytotoxicity. The effect of walnut extract on Aβ-induced cellular damage, ROS generation and apoptosis in PC12 pheochromocytoma cells was studied. Walnut extract reduced Aβ-mediated cell death assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, and release of lactate dehydrogenase (membrane damage), DNA damage (apoptosis) and generation of ROS in a concentration-dependent manner. These results suggest that walnut extract can counteract Aβ-induced oxidative stress and associated cell death.     

Protective Effects of Piperine Against Corticosterone-Induced Neurotoxicity in PC12 Cells

Hyperactivation of the hypothalamic–pituitary–adrenal axis and the associated hippocampal atrophy were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Our previous studies have showed that treating mice with piperine produced antidepressant-like effect in animal models of behavioral despair. This study aimed to examine the protective effect of piperine treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The results showed that piperine co-treatment revealed a differential effect on the cytotoxicity of corticosterone and had its maximum inhibitory effect at 1 μM. Piperine (1 μM) co-treatment also significantly decreased intracellular reactive oxygen species level, and enhanced superoxide dismutase activity and total glutathione level in corticosterone-treated PC12 cells. In addition, piperine (1 μM) co-treatment was found to reverse the decreased brain-derived neurotrophic factor (BDNF) mRNA level caused by corticosterone in PC12 cells. The results suggest that piperine exerts a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress and the upregulation of BDNF mRNA expression. This neuroprotective effect may be one of the acting mechanisms accounts for the in vivo antidepressant activity of piperine.     

Paraquat-Induced Cell Death in PC12 Cells

Paraquat was taken up by PC12 cells in a carrier-mediated, saturable manner. When PC12 cells were permeabilized with digitonin (50 g/ml) lipid peroxidation was observed after paraquat treatment in the presence of NADPH and chelated iron. The fact that lipid peroxidation preceded the appearance of LDH release provides positive evidence that lipid peroxidation may be one of the important factors leading to cytotoxicity of cells. Furthermore, the fact that addition of superoxide dismutase, catalase and promethazine efficiently blocked the malondialdehyde formation and attenuated the cell death indicated the involvement of reactive oxygen radicals in mediating the cytotoxicity induced by paraquat. Taken together the results present in vitro evidence that neurotoxicity of paraquat may be a consequence of cellular lipid peroxidation, which leads to cell death and may have great implications in assessing the risk of exposure to paraquat in Parkinson's disease.     

Neuroprotective Effects of Theaflavins Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Oxidative stress can induce neuronal apoptosis via the production of superoxide and hydroxyl radicals. This process is as a major pathogenic mechanism in neurodegenerative disorders. In this study, we aimed to clarify whether theaflavins protect PC12 cells from oxidative stress damage induced by H₂O₂. A cell model of PC12 cells undergoing oxidative stress was created by exposing cells to 200 μM H₂O₂ in the presence or absence of varying concentrations of theaflavins (5, 10, and 20 μM). Cell viability was monitored using the MTT assay and Hoechst 33258 staining, showing that 10 μM theaflavins enhanced cell survival following 200 μM H₂O₂ induced toxicity and increased cell viability by approximately 40?%. Additionally, we measured levels of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. This suggested that the neuroprotective effect of theaflavins against oxidative stress in PC12 cells is derived from suppression of oxidant enzyme activity. Furthermore, Western blot analyses indicated that theaflavins downregulated the ratio of pro-apoptosis/anti-apoptosis proteins Bax/Bcl-2. Theaflavins also downregulated the expression of caspase-3 compared with a H₂O₂-treated group that had not been treated with theaflavins. Interestingly, this is the first study to report that the four main components of theaflavins found in black tea can protect neural cells (PC12) from apoptosis induced by H₂O₂. These findings provide the foundations for a new field of using theaflavins or its source, black tea, in the treatment of neurodegenerative diseases caused by oxidative stress.     

Apelin-13 Protects PC12 Cells Against Methamphetamine-Induced Oxidative Stress,Autophagy and Apoptosis

Methamphetamine (METH) is a potent psychomotor stimulant that has a high potential for abuse in humans. In addition, it is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to METH causes psychosis and increases the risk of Parkinson’s disease. Apelin-13 is a novel endogenous ligand which studies have shown that may have a neuroprotective effect. Therefore, we hypothesized that Apelin-13 might adequately prevent METH-induced neurotoxicity via the inhibition of apoptotic, autophagy, and ROS responses. In this study, PC12 cells were exposed to both METH (0.5, 1, 2, 3, 4, 6 mmol/L) and Apelin-13 (0.5, 1.0, 2.0, 4.0, 8.0 μmol/L) in vitro for 24 h to measure determined dose, and then downstream pathways were measured to investigate apoptosis, autophagy, and ROS responses. The results have indicated that Apelin-13 decreased the apoptotic response post-METH exposure in PC12 cells by increasing cell viability, reducing apoptotic rates. In addition, the study has revealed Apelin-13 decreased gene expression of Beclin-1 by Real-Time PCR and LC3-II by western blotting in METH-induced PC12 cells, which demonstrated autophagy is reduced. In addition, this study has shown that Apelin-13 reduces intracellular ROS of METH-induced PC12 cells. These results support Apelin-13 to be investigated as a potential drug for treatment of neurodegenerative diseases. It is suggested that Apelin-13 is beneficial in reducing oxidative stress, which may also play an important role in the regulation of METH-triggered apoptotic response. Hence, these data indicate that Apelin-13 could potentially alleviate METH-induced neurotoxicity via the reduction of oxidative damages, apoptotic, and autophagy cell death.

     

Protective Effects of YC-1 Against Glutamate Induced PC12 Cell Apoptosis

Glutamate, one of the major neurotransmitters in the central nervous system, is released into the synaptic spaces and bound to the glutamate receptors which facilitate normal synaptic transmission, synaptic plasticity, and brain development. Past studies have shown that glutamate with high concentration is a potent neurotoxin capable of destroying neurons through many signal pathways. In this research, our main purpose was to determine whether the specific soluble guanylyl cyclase activator YC-1 (3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole) had effect on glutamate-induced apoptosis in cultured PC12 cells. The differentiated PC12 cells impaired by glutamate were used as the cell model of excitability, and were exposed to YC-1 or/and ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) with gradient concentrations for 24 h. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl) assay was used to detect the cellular viability. Radioimmunoassay (RIA) was used to detect the cGMP (cyclic guanosine monophosphate) concentrations in PC12 cells. Hoechst 33258 staining and flow cytometric analysis were used to detect the cell apoptosis. The cellular viability was decreased and the apoptotic rate was increased when PC12 cells were treated with glutamate. Cells treated with YC-1 or/and ODQ showed no significant differences in the cell viability and intracellular cGMP levels compared with those of control group. The specific soluble guanylyl cyclase (sGC) inhibitor ODQ showed an inhibitory effect on cGMP level and aggravated the apoptosis of PC12 cells induced by glutamate. YC-1 elevated cGMP level thus decreased PC12 cell apoptosis induced by glutamate, but this effect could be reversed by ODQ. These results revealed that YC-1 might attenuate glutamate-induced PC12 cell apoptosis via a sGC–cGMP involved pathway.     

锰对PC12 细胞的增殖抑制与凋亡研究

目的:研究锰作用下PC12细胞的增殖抑制作用与凋亡相关的形态学、生化指标改变。方法:用200,400,600,800μmol/LMnCl2的培养液,分别作用对数生长期PC12细胞1,2,3,4d后,用MTT筛选锰的细胞毒性剂量;透射电镜观察细胞形态学变化;琼脂糖凝胶电泳检测MnCl2对PC12细胞基因组DNA的影响。结果:MTT实验显示200-800μmol/L MnCl2作用4天对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600μmol/L MnCl2作用4d对PC12的抑制率可达50%以上。600μmol/L MnCl2作用4d电镜可见细胞凋亡,同样条件下细胞DNA碎片化。结论:PC12细胞在锰作用下发生增殖抑制,原因是锰诱导PC12细胞凋亡。     

Effect of Agmatine Against Cell Death Induced by NMDA and Glutamate in Neurons and PC12 Cells

1. Aims: Agmatine is an endogenous guanido amine and has been shown to be neuroprotective in vitro and in vivo. The aims of this study are to investigate whether agmatine is protective against cell death induced by different agents in cultured neurons and PC12 cells.2. Methods: Cell death in neurons, cultured from neonatal rat cortex, was induced by incubating with (a) NMDA (100 M) for 10 min, (b) staurosporine (protein kinase inhibitor, 100 nM) for 24 h, and (c) calcimycin (calcium ionophore, 100 nM) for 24 h in the presence and absence of agmatine (1 M to 1 mM). Cell death in PC12 cells was induced by exposure to glutamate (10 mM), staurosporine (100 nM), and calcimycin (100 nM). The activity of lactate dehydrogenase (LDH) in the medium was measured as the marker of cell death and normalized to cellular LDH activity.3. Results: Agmatine significantly reduced the medium LDH in NMDA-treated neurons but failed to reduce the release of LDH induced by staurosporin or calcimycin. In PC12 cells, agmatine significantly reduced LDH release induced by glutamate exposure, but not by staurosporine or calcimycin. Agmatine itself neither increased LDH release nor directly inhibited the enzyme activity.4. Conclusion: We conclude that agmatine protects against NMDA excitotoxicity in neurons and PC12 cells but not the cell death induced by protein kinase blockade or increase in cellular calcium.     

Protective Effects of Ginsenoside Rg1 Against Colistin Sulfate-Induced Neurotoxicity in PC12 Cells

The present study aimed to examine the protective effect of ginsenoside Rg1 against colistin-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Ginsenoside Rg1 was shown to elevate cell viability, decrease levels of malondialdehyde and intracellular reactive oxygen species, enhance activity of superoxide dismutase and glutathione, and decrease the release of cytochrome-c, formation of DNA fragmentation in colistin-treated PC12 cells. Ginsenoside Rg1 also reversed the increased caspase-9 and -3 mRNA levels caused by colistin in PC12 cells. These results suggest that ginsenoside Rg1 exerts a neuroprotective effect on colistin-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress, prevention of apoptosis mediated via mitochondria pathway. Co-administration of ginsenoside Rg1 highlights the potential to increase the therapeutic index of colistin.     

Ethanol-Induced Cell Death by Lipid Peroxidation in PC12 Cells

Free radical generation is hypothesized to be the cause of alcohol-induced tissue injury. Using fluorescent cis-parinaric acid and TBARS, lipid peroxidation was shown to be increased in the presence of trace amounts of free ferrous ion in PC12 cells. This increase in lipid peroxidation was enhanced by ethanol in a dose dependent manner and also correlated with loss of cell viability, as measured by increased release of lactate dehydrogenase (LDH). Resveratrol, a potent antioxidant, had a protective effect against lipid peroxidation and cell death. These findings strongly suggest that ethanol-induced tissue injury and cell death is a free radical mediated process, and may be important in alcohol-related premature aging and other degenerative diseases.     

Neuroprotective Effects of n-3 Polyunsaturated Fatty Acid-Enriched Phosphatidylserine Against Oxidative Damage in PC12 Cells

Neurodegenerative diseases are defined by progressive loss of specific neuronal cell populations and are associated with protein aggregates. Oxidative stress has been implicated in their pathological processes. Previous studies revealed that docosahexaenoic acid (DHA) is beneficial in neurodegenerative diseases. Phospholipids (PLs) derived from marine products are rich in DHA and eicosapentaenoic acid (EPA). In the present study, we investigated the neuroprotective effects of DHA-enriched and unenriched phosphatidylcholine (PC) and phosphatidylserine (PS) on oxidative stress induced by hydrogen peroxide (H₂O₂) and tert-butylhydroperoxide in PC12 cells. Cell viability and leakage of lactate dehydrogenase results showed that the neuroprotective effect of PS was superior to that of PC. DHA- and EPA-enriched PC and PS were superior to that without DHA or EPA; in addition, the improvement with n-3 polyunsaturated fatty acid-enriched PS (n-3 PS) was dose dependent. Acridine orange/ethidium bromide staining showed that DHA- and EPA-enriched PS (DHA/EPA-PS) could significantly inhibit apoptosis. Mechanistic studies revealed that EPA-PS and DHA-PS were effective to increase superoxide dismutase (SOD) levels by 48.4 and 58.2 % and total antioxidant capacity (T-AOC) level by 51 and 94 %, respectively, in the H₂O₂ model. Similar results for SOD and T-AOC levels were shown in the t-BHP model. EPA/DHA-PS could downregulate the messenger RNA level of Caspase-3, Caspase-9, and Bax, upregulate Bcl-2, inhibit Bax, and increase Bcl-2 at protein level. In conclusion, EPA/DHA-PS could protect PC12 cells from oxidative stress and prevent mitochondrial-mediated apoptosis. Our findings indicate that the neuroprotective effects of DHA/EPA-PLs depend on the molecular form. Further studies are necessary to reveal detailed mechanisms and structure–effect relationships.     

Protective Effects of Flavonoids Against Oxidative Stress Induced by Simulated Microgravity in SH-SY5Y Cells

Many lines of evidence suggest that microgravity results in increased oxidative stress in the nervous system. In order to protect neuronal cells from oxidative damage induced by microgravity, we selected some flavonoids that might prevent oxidative stress because of their antioxidant activities. Among the 20 flavonoids we examined, we found that isorhamnetin and luteolin had the best protective effects against H₂O₂ or SIN-1-induced cytotoxicity in SH-SY5Y cells. Using a clinostat to simulate microgravity, we found that isorhamnetin and luteolin treatment protected SH-SY5Y cells by preventing microgravity-induced increases in reactive oxygen species (ROS), nitric oxide (NO) and 3-nitrotyrosine (3-NT) levels, and a decrease in antioxidant power (AP). Moreover, isorhamnetin and luteolin treatment downregulated the expression of inducible nitric oxide synthase (iNOS), and oxidative stress was significantly inhibited by an iNOS inhibitor in SH-SY5Y cells exposed to simulated microgravity (SMG). These results indicate that isorhamnetin and luteolin could protect against microgravity-induced oxidative stress in neuroblastoma SH-SY5Y cells by inhibiting the ROS-NO pathway. These two flavonoids may have potential for preventing oxidative stress induced by space flight or microgravity.     

MicroRNA-137-3p Protects PC12 Cells Against Oxidative Stress by Downregulation of Calpain-2 and nNOS

The imbalance between excess reactive oxygen species (ROS) generation and insufficient antioxidant defenses contribute to a range of neurodegenerative diseases. High ROS levels damage cellular macromolecules such as DNA, proteins and lipids, leading to neuron vulnerability and eventual death. However, the underlying molecular mechanism of the ROS regulation is not fully elucidated. Recently, an increasing number of studies suggest that microRNAs (miRNAs) emerge as the targets in regulating oxidative stress. We recently reported the neuroprotective effect of miR-137-3p for brachial plexus avulsion-induced motoneuron death. The present study is sought to investigate whether miR-137-3p also could protect PC12 cells against hydrogen peroxide (H₂O₂) induced neurotoxicity. By using cell viability assay, ROS assay, gene and protein expression assay, we found that PC-12 cells exposed to H₂O₂ exhibited decreased cell viability, increased expression levels of calpain-2 and neuronal nitric oxide synthase (nNOS), whereas a decreased miR-137-3p expression. Importantly, restoring the miR-137-3p levels in H₂O₂ exposure robustly inhibited the elevated nNOS, calpain-2 and ROS expression levels, which subsequently improved the cell viability. Furthermore, the suppressive effect of miR-137-3p on the elevated ROS level under oxidative stress was considerably blunted when we mutated the binding site of calpain-2 targted by miR-137-3p, suggesting the critical role of calpain-2 involving the neuroprotective effect of miR-137-3p. Collectively, these findings highlight the neuroprotective role of miR-137-3p through down-regulating calpain and NOS activity, suggesting its potential role for combating oxidative stress insults in the neurodegenerative diseases.

     

Effects of Fetal Bovine Serum on Ferrous Ion-Induced Oxidative Stress in Pheochromocytoma (PC12) Cells

Ferrous ion (Fe²⁺) has been considered to be a cause of neuronal oxidative injury. Since body fluids contain protein and serum is an essential component of tissue culture medium, we have examined the role of serum protein on Fe²⁺-mediated oxidative stress using PC12 cells and rat cerebral cortices. Fe²⁺ or the combination of ascorbate and Fe²⁺ increased concentrations of thiobarbituric acid reactive substances (TBARS) in PC12 cells and cerebrocortical homogenates in medium (RPMI 1640), but did not increase TBARS when the medium was supplemented with 10% fetal bovine serum. Treatment with ascorbate/Fe²⁺ in serum-free medium reduced endogenous glutathione (GSH) concentration in PC12 cells. However, the medium supplemented with serum did not reduce GSH concentrations. PC12 cell death induced by ascorbate/Fe²⁺ was alleviated by increasing serum or bovine albumin concentrations in the medium. These observations indicated that oxidative injury caused by the transition metal ion could be lessened by adding fetal bovine serum to culture medium.     

花青素能减轻阿尔茨海默症模型细胞的氧化损伤

氧化胁迫在阿尔茨海默症(AD)的发病过程中起重要作用．花青素是一种广泛存在于植物中的黄酮类物质．实验结果表明,100 μmol/L 的花青素可以有效地缓解AD模型细胞氧化胁迫,具体表现为,降低AD模型细胞的H₂O₂易感性,减少胞内ROS和[NO]_i．c-Jun氨基端激酶(JNK)是氧化胁迫导致细胞损伤的重要信号途径．实验结果显示花青素可以有效地抑制氧化胁迫对JNK的激活．提示花青素的抗氧化胁迫作用与JNK信号途径有关．因此,花青素可以作为一种氧化胁迫因子的清除剂来保护AD模型细胞,有望用于AD的辅助治疗．