Synthesis and protective effects of aralkyl alcoholic 2-acetamido-2-deoxy-β-D-pyranosides on hypoglycemia and serum limitation induced apoptosis in PC12 cell

Neuroprotective agents have been in the focus of attention in the treatment of ischemic stroke. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., possessed a wide range of biological activities, especially neuroprotection. In an attempt to improve neuroprotective effects of new salidroside analogs for ischemic stroke, a series of novel aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides were synthesized and their protective activities against the hypoglycemia and serum limitation induced cell death in rat pheochromocytoma cells (PC12 cells) were studied. Most compounds showed strong neuroprotective effects, especially for 4g and 4h, which exhibited a great potency superior to salidroside. MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed that pretreatment with 4g and 4h attenuated cell viability loss and apoptotic cell death in cultured PC12 cells. Caspase-3 colorimetric assay and Rhodamine 123 staining revealed the changes in expression levels of caspase-3 and mitochondrial membrane potential in PC12 cells on exposure to hypoglycemia and serum limitation with and without 4g and 4h pretreatment, respectively. All the results suggested that 4g and 4h protects the PC12 cells against hypoglycemia and serum limitation induced apoptosis possibly by modulation of apoptosis-related gene expression and restoration of the mitochondrial membrane potential. Therefore, these novel findings may provided a new framework for the design of new aralkyl alcoholic 2-acetamido-2-deoxy-β-d-pyranosides as neuroprotective agents for treating cerebral ischemic stroke and neurodegenerative diseases.     

Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line

Glucose metabolism plays a pivotal role in many physiological and pathological conditions. To investigate the effect of hypoglycemia (obtained by glucose deprivation) on PC12 cell line, we analyzed the cell viability, mitochondrial function (assessed by MTT reduction, cellular ATP level, mitochondrial transmembrane potential), and the level of reactive oxygen species (ROS) after glucose deprivation (GD). Upon exposure to GD, ROS level increased and MTT reduction decreased immediately, intracellular ATP level increased in the first 3 hours, followed by progressive decrease till the end of GD treatment, and the mitochondrial transmembrane potential (ΔΨ_m) dropped after 6 hours. Both necrosis and apoptosis occurred apparently after 24 hours which was determined by nuclei staining with propidium iodide(PI) and Hoechst 33342. These data suggested that cytotoxity of GD is mainly due to ROS accumulation and ATP depletion in PC12 cells.     

Protective Effect of <Emphasis Type="Italic">Nigella sativa</Emphasis> Extract and Thymoquinone on Serum/Glucose Deprivation-Induced PC12 Cells Death

The serum/glucose deprivation (SGD)-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) has been known as a source of antioxidants. In the present study, the protective effects of N. sativa and TQ on cell viability and reactive oxygen species (ROS) production in cultured PC12 cells were investigated under SGD conditions. PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose for 6 and 18 h. Cells were pretreated with different concentrations of N. sativa extract (15.62–250 μg/ml) and TQ (1.17–150 μM) for 2 h. Cell viability was quantitated by MTT assay. Intracellular ROS production was measured by flow cytometry using 2′,7′-dichlorofluorescin diacetate (DCF-DA) as a probe. SGD induced significant cells toxicity after 6, 18, or 24 h (P < 0.001). Pretreatment with N. sativa (15.62–250 μg/ml) and TQ (1.17–37.5 μM) reduced SGD-induced cytotoxicity in PC12 cells after 6 and 18 h. A significant increase in intracellular ROS production was seen following SGD (P < 0.001). N. sativa (250 μg/ml, P < 0.01) and TQ (2.34, 4.68, 9.37 μM, P < 0.01) pretreatment reversed the increased ROS production following ischemic insult. The experimental results suggest that N. sativa extract and TQ protects the PC12 cells against SGD-induced cytotoxicity via antioxidant mechanisms. Our findings might raise the possibility of potential therapeutic application of N. sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders.     

Claulansine F suppresses apoptosis induced by sodium nitroprusside in PC12 cells

Abstract

Reactive oxygen species (ROS) are known to be involved in many neurodegenerative diseases. This study assessed the effect of Claulansine F, a new carbazole isolated from Clausena lansium, on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. First, it was found that Claulansine F showed more potential on inhibiting the programmed death of PC12 cells than edaravone by cell viability, morphologic observation, and flow cytometric analysis. Further results also showed that Claulansine F attenuated the production of total intracellular ROS formation and lipid peroxidation in PC12 cells, inhibited the mitochondrial membrane potential (MMP) loss, and prevented the programmed cell death event via the P53/Bcl-2 family pathway. Its protective effect was likely medicated by the hydroxyl radical (·OH) scavenging ability, as it appeared to be not involved in the natural antioxidant system. These results suggested a promising potential for Claulansine F as a ROS scavenger in pathologies, where an oxidative stress is involved.     

D-β-Hydroxybutyrate Prevents MPP+-Induced Neurotoxicity in PC12 Cells

Numerous studies show that D-β-Hydroxybutyrate (DβHB) is neuroprotective. The present study was to explore the neuroprotective effects of DβHB against the cell death and apoptosis induced by 1-methyl-4-phenylpyridinium ion (MPP+) in PC12 cells. PC12 cells were pretreated with DβHB and followed by MPP+ exposure. The cell viability was determined by MTT assay. The morphological characteristics of apoptosis was observed by Acridine Orange (AO) staining and apoptotic rates were measured by flow cytometer. The product of lipid peroxidation, malondialdehyde (MDA), was measured using thiobarbituric acid method. The mitochondrial membrane potential (MMP), intracellular ROS and total glutathione were detected by microplate reader. In PC12 cells, pretreatment with DβHB significantly reduced MPP+-induced the decrease of cell viability. AO staining and flow cytometric analysis found DβHB inhibited MPP+-induced apoptosis. The measurement of MDA formation showed that DβHB alleviated lipid peroxidation induced by MPP+. The loss of MMP induced by MPP+ was preventive by DβHB. The changes of intracellular ROS and total glutathione induced by MPP+ were reversed by DβHB. DβHB protected PC12 cells against MPP+-induced death and apoptosis.     

Effect of Purple Sweet Potato Anthocyanins on β-Amyloid-Mediated PC-12 Cells Death by Inhibition of Oxidative Stress

Amyloid-beta peptide (Aβ) is known to induce the redox imbalance, mitochondrial dysfunction and caspase activation, resulting in neuronal cell death. Treatment with antioxidants provided a new therapeutic strategy for Alzheimer’s disease (AD) patients. Here we investigate the effects of purple sweet potato anthocyanins (PSPA), the known strong free radical scavengers, on Aβ toxicity in PC12 cells. The results showed that pretreatment of PC12 cells with PSPA reduced Aβ-induced toxicity, intracellular reactive oxygen species (ROS) generation and lipid peroxidation dose-dependently. In parallel, cell apoptosis triggered by Aβ characterized with the DNA fragmentation and caspase-3 activity were also inhibited by PSPA. The concentration of intracellular Ca²⁺ and membrane potential loss associated with cell apoptosis were attenuated by PSPA. These results suggested that PSPA could protect the PC-12 cell from Aβ-induced injury through the inhibition of oxidative damage, intracellular calcium influx, mitochondria dysfunction and ultimately inhibition of cell apoptosis. The present study indicates that PSPA may be a promising approach for the treatment of AD and other oxidative-stress-related neurodegenerative diseases.     

丙戊酸钠对抗鱼藤酮诱导的SH-SY5Y细胞损伤的线粒体机制

研究丙戊酸钠（sodiumvalproate,VPA）对抗鱼藤酮（Rotenone）诱导的SH-SY5Y细胞损伤的作用及线粒体机制。以l,10μmol／LVPA预处理SH－SY5Y细胞3h,再加入400nmol／LRotenone作用24h。MTT法检测与相差显微镜观察相结合,分析VPA对抗Rotenone损伤的作用;JC－1染色法与Mito－Tracker染色法分析线粒体膜电位及线粒体数量的变化;Clark氧电极法检测细胞呼吸功能;DCFH-DA探针法检测细胞中Ros的含量;并在离体线粒体上观察VPA对Ca^2＋诱导的线粒体肿胀的影响。结果发现,1,10p．mol／LVPA预处理SH．SY5Y细胞3h可对抗400nmol／LRotenoneI起的细胞损伤,并且可以提高损伤细胞中线粒体的膜电位,增加线粒体的数量,此外,还可以增强损伤细胞的呼吸功能,降低细胞中ROS的含量,但VPA并不能直接作用于离体的线粒体发挥神经保护作用。由此,VPA具有良好的神经保护作用,其机制与增强线粒体功能和数量、从而改善细胞功能有关,这为其应用于帕金森病的预防与治疗提供了实验依据。     

Betaine Protects Against Rotenone-Induced Neurotoxicity in PC12 Cells

Rotenone is an inhibitor of mitochondrial complex I-induced neurotoxicity in PC12 cells and has been widely studied to elucidate the pathogenesis of Parkinson’s disease. We investigated the neuroprotective effects of betaine on rotenone-induced neurotoxicity in PC12 cells. Betaine inhibited rotenone-induced apoptosis in a dose-dependent manner, with cell viability increasing from 50 % with rotenone treatment alone to 71 % with rotenone plus 100-μM betaine treatment. Flow cytometric analysis demonstrated cell death in the rotenone-treated cells to be over 50 %; the number of live cells increased with betaine pretreatment. Betaine pretreatment of PC12 cells attenuated rotenone-mediated mitochondrial dysfunction, including nuclear fragmentation, ATP depletion, mitochondrial membrane depolarization, caspase-3/7 activation, and reactive oxygen species production. Western blots demonstrated activation of caspase-3 and caspase-9, and their increased expression levels in rotenone-treated cells; betaine decreased caspase-3 and caspase-9 expression levels and suppressed their activation. Together, these results suggest that betaine may serve as a neuroprotective agent in the treatment of neurodegenerative diseases.     

d-β-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress

Oxidative stress has an important role in neurodegenerative diseases and cerebral ischemic injury. It is reported that d-β-hydroxybutyrate (DβHB), the major component of ketone bodies, is neuroprotective in recent studies. Therefore, in the present work the neuroprotective effects of DβHB on H₂O₂-induced apoptosis mediated by oxidative stress was investigated. PC12 cells were exposed to H₂O₂ with different concentrations of H₂O₂ for different times after DβHB pretreatment. MTT assay, apoptotic rates, intracellular reactive oxygen species (ROS) level, GSH content, mitochondrial membrane potential (MMP) and caspase-3 activity were determined. The results showed that DβHB inhibited the decrease of cell viability induced by H₂O₂ in PC12 cells. DβHB decreased the apoptotic rates induced by H₂O₂. The changes of intracellular ROS, GSH, MMP and caspase-3 activity due to H₂O₂ exposure were partially reversed in PC12 cells. So DβHB inhibited the apoptosis of PC12 cells induced by H₂O₂ via inhibiting oxidative stress.     

Acrolein-induced cell death in PC12 cells: role of mitochondria-mediated oxidative stress

Oxidative stress has been implicated in acrolein cytotoxicity in various cell types, including mammalian spinal cord tissue. In this study we report that acrolein also decreases PC12 cell viability in a reactive oxygen species (ROS)-dependent manner. Specifically, acrolein-induced cell death, mainly necrosis, is accompanied by the accumulation of cellular ROS. Elevating ROS scavengers can alleviate acrolein-induced cell death. Furthermore, we show that exposure to acrolein leads to mitochondrial dysfunction, denoted by the loss of mitochondrial transmembrane potential, reduction of cellular oxygen consumption, and decrease of ATP level. This raises the possibility that the cellular accumulation of ROS could result from the increased production of ROS in the mitochondria of PC12 cells as a result of exposure to acrolein. The acrolein-induced significant decrease of ATP production in mitochondria may also explain why necrosis, not apoptosis, is the dominant type of cell death. In conclusion, our data suggest that one possible mechanism of acrolein-induced cell death could be through mitochondria as its initial target. The subsequent increase of ROS then inflicts cell death and further worsens mitochondria function. Such mechanism may play an important role in CNS trauma and neurodegenerative diseases.     

Puerarin protects PC12 cells against beta-amyloid-induced cell injury

beta-Amyloid protein (Abeta), a major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. In this study, the protective effect of puerarin, an isoflavone purified from the radix of the Chinese herb Pueraria lobata, on Abeta-induced rat pheochromocytoma (PC12) cultures was investigated. Although exposure of PC12 cells to 50 microM Abeta25-35 caused significant viability loss and apoptotic rate increase, pretreatment of the cells with puerarin for 24h reduced the viability loss and apoptotic rate. Puerarin (1 microM) significantly inhibited Abeta25-35-induced apoptosis of PC12 cells. Preincubation of the cell with puerarin also restored the ROS and mitochondrial membrane potential levels that had been altered as a result of Abeta25-35 treatment. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce caspase-3 activation. These results suggest that puerarin could attenuate Abeta25-35-induced PC12 cell injure and apoptosis and could also promote the survival of PC12 cells. Therefore, puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta25-35-induced cell injury.     

Salidroside inhibits H2O2-induced apoptosis in PC12 cells by preventing cytochrome c release and inactivating of caspase cascade

We used a rat pheochromocytoma (PC12) cell line to study the effects of salidroside on hydrogen peroxide (H(2)O(2))-induced apoptosis. In PC12 cells, H(2)O(2)-induced apoptosis was accompanied by the down-regulation of Bcl-2, the up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol, and the activation of caspase-3, -8 and -9. However, salidroside suppressed the down-regulation of Bcl-2, the up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol. Moreover, salidroside attenuated caspase-3, -8 and -9 activation, and eventually protected cells against H(2)O(2)-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with salidroside can block H(2)O(2)-induced apoptosis by regulating Bcl-2 family members and by suppressing cytochrome c release and caspase cascade activation.     

Effect of GRP75/mthsp70/PBP74/mortalin overexpression on intracellular ATP level, mitochondrial membrane potential and ROS accumulation following glucose deprivation in PC12 cells

Glucose regulated protein 75 (GRP75) is an important molecular chaperon belonged to the heat shock protein (HSP) family. To evaluate the effect of GRP75 overexpression on PC12 cells under glucose deprivation, cell viability and mitochondrial function of GRP75-overexpressing PC12 cells and the vector transfected control PC12 cells were monitored during glucose deprivation. Upon exposure to glucose deprivation, GRP75-overexpressing PC12 cells exhibited more moderate cell damage than control PC12 cells. Both of the two groups of cells showed a decreased ATP level following an early increase in the condition of glucose deprivation, and the mitochondrial potential were also reduced in the similar manner in the two groups of cells. Control PC12 cells showed an immediate and rapid increase in ROS accumulation after the onset of GD treatment, and this accumulation was slowed and reduced in GRP75-overexpressing PC12 cells. These findings suggested that GRP75 could inhibit the ROS accumulation, and it may be associated with the cytoprotective effect of GRP75 overexpression upon glucose deprivation. (Mol Cell Biochem 268: 45–51, 2005)     

Genistein对卵巢癌铂类耐药细胞株CP70增殖凋亡的影响及与细胞内ROS的相关性

目的:探讨Genistein对卵巢癌铂类耐药细胞CP70增殖、凋亡的影响及与细胞内活性氧水平的关系。方法:采用MTT法检测Genistein对CP70细胞增殖的影响;流式细胞仪分析不同药物处理后对细胞凋亡的影响,线粒体膜电位及细胞内ROS水平的变化情况。结果:Genistein对CP70细胞增殖表现出剂量和时间依赖性的抑制作用,并能诱导其凋亡;Genistein作用于CP70细胞后,可使其线粒体膜电位降低,并引发了细胞内ROS水平的显著升高;ROS抑制剂NAC预处理CP70细胞后,有效抑制了ROS的产生,并降低了细胞凋亡率,与未加NAC组相比差异有显著性(P0.05)。结论:Genistein能抑制铂类耐药卵巢癌细胞CP70的增殖,并促进其凋亡,这与细胞内ROS水平的升高有关,可能是Genistein抗肿瘤诱导细胞凋亡的机制之一。     

Protective effects of resveratrol on hydrogen peroxide-induced apoptosis in rat pheochromocytoma (PC12) cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities. One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), one of the major antioxidative constituents found in the skin of grapes, has been considered to be responsible in part for the protective effects of red wine consumption against coronary heart disease ('French Pardox'). In this study, we have investigated the effects of resveratrol on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by characteristic morphological features, internucleosomal DNA fragmentation and positive in situ end-labeling by terminal transferase (TUNEL staining). Resveratrol pretreatment attenuated hydrogen peroxide-induced cytotoxicity, DNA fragmentation, and intracellular accumulation of ROS. Hydrogen peroxide transiently induced activation of NF-kappaB in PC12 cells, which was mitigated by resveratrol pretreatment. These results suggest that resveratrol has the potential to prevent oxidative stress-induced cell death.     

Betulinic Acid Induces Apoptosis in Differentiated PC12 Cells Via ROS-Mediated Mitochondrial Pathway

Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.     

1,8-Cineole (Eucalyptol) Mitigates Inflammation in Amyloid Beta Toxicated PC12 Cells: Relevance to Alzheimer’s Disease

Inflammatory process has a fundamental role in the pathogenesis of Alzheimer’s disease and insoluble amyloid beta deposits and neurofibrillary tangles provide the obvious stimuli for inflammation. The present study demonstrate the effect of pretreatment of 1,8-cineole (Cin) on inflammation induced by Aβ_(25–35) in differentiated PC12 cells. The cells were treated with Cin at different doses for 24 h and then replaced by media containing Aβ_(25–35) for another 24 h. The cell viability was decreased in Aβ_(25–35) treated cells which was significantly restored by Cin pretreatment. Cin successfully reduced the mitochondrial membrane potential, ROS and NO levels in Aβ_(25–35) treated cells. Cin also lowered the levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 in Aβ_(25–35) treated cells. Moreover, Cin also succeeded in lowering the expression of NOS-2, COX-2 and NF-κB. This study suggests the protective effects of Cin on inflammation and provides additional evidence for its potential beneficial use in therapy as an anti-inflammatory agent in neurodegenerative disease.     

人参皂甙Rd预处理可减轻谷氨酸所致的PC12细胞损伤

目的:研究人参皂甙Rd(Ginsenoside Rd)预处理对谷氨酸所致PC12细胞损伤的影响。方法:将体外培养的PC12细胞分为3组,分别为对照组(Control)、谷氨酸损伤组(Glu)和人参皂甙Rd预处理组(Rd)。Control组细胞正常培养;Glu组细胞暴露于含10mM谷氨酸的DMEM培养基中损伤24 h;Rd组细胞经50μM的人参皂甙Rd预处理30 min后,在谷氨酸浓度为10 mM的DMEM培养基中损伤24 h。采用MTT检测细胞活力和乳酸脱氢酶(LDH)检测试剂盒检测LDH释放量;流式细胞仪检测胞内活性氧(ROS)水平;Western blot检测还原型谷胱甘肽蛋白(GSH)表达;专用试剂盒检测细胞内过氧化氢酶(CAT)和超氧化物歧化酶(SOD)含量,相差显微镜观测细胞形态。结果:50μM的人参皂甙Rd预处理30 min,可明显提高谷氨酸诱导的PC12细胞的活力,降低其LDH释放量、胞内ROS含量,并提高胞内GSH蛋白表达,增加CAT、SOD含量并改善细胞形态。结论:人参皂甙Rd预处理可减轻谷氨酸引起的PC12细胞损伤。     

Metformin protects PC12 cells and hippocampal neurons from H2O 2-induced oxidative damage through activation of AMPK pathway

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H₂O₂-induced cell death. Metformin significantly ameliorated cell death due to H₂O₂ insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H₂O₂. Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H₂O₂. Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.