A decrease of neuroprotector effect of ganglioside GM1 on PC12 cells under conditions of oxidative stress in the presence of inhibitor of tyrosine kinase of Trk-receptors

Effects of inhibitors of tyrosine kinases (K-252a, genistein) and of phospholipase A2 (bromophenacetyl bromide) on viability of PC12 cells are studied in the presence of hydrogen peroxide and ganglioside GM1. The degree of inhibition of hydrogen peroxide cytotoxic effect by ganglioside GM1 amounted to 52.8 +/- 4.3 %. However, in the presence in the medium of 0.1 and 1 microM inhibitors of tyrosine kinase of Trk-receptors (K-252a) it was as low as 32.7 +/- 6.5 % and 11.7 +/- 9.8 %, respectively. GM1 prevented Na+, K+-ATPase produced by H2O2, but in the presence of 1 microM K-252a this effect was practically not pronounced. In the presence of another inhibitor of tyrosine kinases--genistein, a tendency for a decrease of the GM1 protective effect was observed at its concentrations 0.1 and 1 microM, whereas at a higher concentration 10 microM genistein depressed the GM1 neuroprotective effect statistically significantly. It was found that inhibitor of phospholipase A2 bromophenacetyl bromide did not affect the action of GM1 aimed at increasing the viability of cells under action of hydrogen peroxide on them. It seems that this enzyme is not involved in the cascade of reactions participating in realization of the ganglioside protective effect. Thus, inhibitor of tyrosine kinase of Trk-receptors K-252 decreases or practically prevents the ganglioside GM1 neuroprotective effect of PC12 cells under stress conditions; the same ability is characteristic of genistein--an inhibitor of tyrosine kinases of the wider spectrum of action.     

PC12 cells transfected with human mutant gene causing one of Alzheimer’s disease forms have a high sensitivity to oxidative stress

Used in this work are PC12 cells transfected with human gene expressing amyloid-precursor protein of β-peptide and carrying the so-called “Swedish mutation” leading to the appearance of one of Alzheimer’s disease family forms. It has been shown that the PC12 cells transfected with this mutant gene, at action of various hydrogen peroxide concentrations, die to the significant greater degree than the used for comparison PC12 cells transfected with analogous human gene of the wild type or than vector-transfected cells. It has been found that ganglioside GM1 at micro-or nanomolar concentrations is able to increase viability of the PC12 cells transfected with the mutant gene causing a significant accumulation of endogenous amyloid β-peptide. The obtained data confirm an important role of oxidative stress in injury and death of brain nerve cells in Alzheimer’s disease.     

Exendin-4对甲基乙二醛诱导PC12细胞氧化应激的影响

目的：探讨肠促胰岛素类似物（Ex-4）对甲基乙二醛（MG）诱导PC12细胞氧化应激的影响及其机制。方法：传代培养PC12细胞,不同浓度MG（0、0.25、0.50、0.75、1.0、2.0 mmol/l）处理PC12细胞12~48 h,或用不同浓度Ex-4（25、50、100、200 nmol/L）预处理24 h后加用MG（0.75 mmol/L）干预24 h后,MTT比色法检测细胞存活率;荧光探针法检测活性氧（ROS）含量,黄嘌呤氧化酶法检测超氧化物歧化酶（SOD）活力。Ex-4（100nmol/L）预处理PC12细胞24h加用MG（0.75 mmol/L）干预1 h后,Western blot检测蛋白P-IκB-α、IκB-α表达情况。结果：随着MG浓度的增加和作用时间的延长,PC12细胞存活率逐渐降低;加用不同浓度Ex-4预处理后,PC12细胞存活率较单独MG处理组逐渐升高。100 nmol/L的Ex-4预处理PC12细胞后,ROS表达量较MG单独处理组下降65.30%（P<0.01）; NAC预处理组（阳性对照）ROS表达量下降107.40%（P<0.01）;Ex-4预处理组SOD活力增加5.30 U/mg prot（P<0.01）;NAC预处理组SOD活力增加8.53 U/mg prot（P<0.01）。Ex-4预处理组P-IκB-α/IκB-α表达比例下降25.50%（P<0.01）; NAC预处理组P-IκB-α/IκB-α表达比例下降35.14%（P<0.01）。结论：Ex-4浓度依赖性地增加MG诱导的PC12细胞的存活率。Ex-4能够减轻MG诱导的PC12细胞的氧化应激,其机制可能涉及抑制蛋白IκB-α的活化。     

Unglycosylated Trk protein does not co-localize nor associate with ganglioside GM1 in stable clone of PC12 cells overexpressing Trk (PCtrk cells)

Our previous studies have shown that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. GM1 is also known to be a major constituent of caveola or glycosphingolipid-enriched microdomain (GEM) of the plasma membrane. In order to study the effect of the glycosylation of Trk on the formation of GM1-Trk complex and subcellular distribution of this protein, we generated PC12 cells stably overexpressing Trk (PCtrk). Pretreatment of this stable clones with tunicamycin, a potent inhibitor of N-glycosylation, caused the appearance of unglycosylated Trk core protein. These unglycosylated Trk can hardly respond to its ligand, NGF. Sucrose density gradient analysis revealed that unglycosylated Trk core protein was recovered in high density fractions, whereas most of GM1 is present in low density fractions corresponding to caveola or GEM fractions. Moreover, these unglycosylated Trk proteins lose their ability to form a complex with GM1, although GM1 is present in the same high density fractions. These data strongly suggest that spatial segregation of GM1 from the Trk protein by the inhibition of the glycosylation of Trk might be an important molecular mechanism for the unresponsiveness to NGF. Moreover, the binding site of GM1 in the Trk protein might act as an important determinant for the normal trafficking of the Trk protein within the cells.     

Protective effects of ellagic and chlorogenic acids against oxidative stress in PC12 cells

Following exposure of differentiated neuronal PC12 cells to either t-BHP, hydrogen peroxide (H₂O₂) or FeSO₄ various kinds of reactive oxygen species (ROS) are generated leading to oxidative injury. The protective effects of two plant polyphenols, ellagic (EC) and chlorogenic acid (CGA), as well as of two metabolites, caffeic acid (CA) and ferulic acid (FA), were investigated in preincubation and coincubation experiments with respect to the following parameters: prevention of cell death, GSH depletion, lipid peroxidation and ROS formation.

The polyphenols more efficiently suppressed cytotoxicity and loss of GSH caused by peroxides than by iron, particularly in preincubation. Lipid peroxidation which increased much stronger in response to FeSO₄ was counteracted completely by the polyphenols. In case of iron, however, only coincubation was effective. EA and CGA and the metabolites CA and FA showed excellent elimination of ROS induced by all stressors. These findings suggest that two dietary antioxidants, EA and CGA, may have protective properties against oxidative stress induced in CNS.     

Protective effects of ellagic and chlorogenic acids against oxidative stress in PC12 cells

Following exposure of differentiated neuronal PC12 cells to either t-BHP, hydrogen peroxide (H₂O₂) or FeSO₄ various kinds of reactive oxygen species (ROS) are generated leading to oxidative injury. The protective effects of two plant polyphenols, ellagic (EC) and chlorogenic acid (CGA), as well as of two metabolites, caffeic acid (CA) and ferulic acid (FA), were investigated in preincubation and coincubation experiments with respect to the following parameters: prevention of cell death, GSH depletion, lipid peroxidation and ROS formation.

The polyphenols more efficiently suppressed cytotoxicity and loss of GSH caused by peroxides than by iron, particularly in preincubation. Lipid peroxidation which increased much stronger in response to FeSO₄ was counteracted completely by the polyphenols. In case of iron, however, only coincubation was effective. EA and CGA and the metabolites CA and FA showed excellent elimination of ROS induced by all stressors. These findings suggest that two dietary antioxidants, EA and CGA, may have protective properties against oxidative stress induced in CNS.     

Physiological stress and nerve growth factor treatment regulate stress-activated protein kinase activity in PC12 cells

The stress-activated protein kinases (SAPKs) are differentially activated by a variety of cellular stressors in PC12 cells. SAPK activation has been linked to the induction of apoptotic cell death upon serum withdrawal from undifferentiated cells or following nerve growth factor (NGF) withdrawal of neuronally differentiated PC12 cells. However, withdrawal of trophic support from differentiated cells led to only a very modest elevation of SAPK activity and led us to investigate the basis of the relative insensitivity of these enzymes to stressors. NGF-stimulated differentiation of the cells resulted in the elevation of basal SAPK activity to levels four- to sevenfold greater than in untreated cells, which was correlated with an approximate fivefold increase in SAPK protein levels. Paradoxically, in NGF-differentiated PC12 cells, exposure to cellular stressors provoked a proportionately smaller stimulation of SAPK activity than that observed in naive cells, despite the presence of much higher levels of SAPK protein. The insensitivity of SAPK to activation by stressors was reflective of the activity of the SAPK activator SEK, whose activation was also diminished following NGF differentiation of the cells. The data demonstrate that SAPKs are subject to complex controls through both induction of SAPK expression and the regulation mediated by upstream elements within this pathway. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 537–549, 1998     

Stress-mediated signaling in PC12 cells - the role of the small heat shock protein,Hsp27, and Akt in protecting cells from heat stress and nerve growth factor withdrawal

We have investigated the role of stress-activated signaling pathways and the small heat shock protein, Hsp27, in protecting PC12 cells from heat shock and nerve growth factor (NGF) withdrawal-induced apoptosis. PC12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to heat shock. This resulted in the rapid activation of Akt followed by p38 mitogen-activated protein kinase (MAPK) signaling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 was found to form an immunoprecipitable complex with Akt and p38 MAPK in both non-stimulated and heat shocked cells, although after heat shock there was a gradual dissociation of Akt and p38 from the Hsp27. Cells were differentiated with NGF and then subjected to NGF withdrawal, a treatment which results in substantial cell death over 24-72 h. Hsp27 was shown to be protective against this treatment, since HSPC cells which overexpress Hsp27 showed significantly less cell death than the parental PC12 cells. In addition, we observed that phosphorylation of Akt was maintained in HSPC cells subjected to heat shock and NGF withdrawal compared with the parental cells. Taken together, our results suggest that Hsp27 may protect Akt from dephosphorylation and may also act in stabilizing Akt.     

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

There have been obtained evidences that not only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of cells of the neuronal line PC12 under action of H₂O₂. By the example of GM1 and GD1a, gangliosides have been shown to produce a protective effect on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. For the first time, GM1 at nanomolar concentrations was shown to decrease the H₂O₂-induced formation of reactive oxygen species (ROS). It was found that in the presence of inhibitor of tyrosine kinase Trk of receptors K-252a, GM1 at concentrations of 10 μM and 10 nM lost its ability to produce such metabolic effects as a decrease of ROS accumulation and of the degree of oxidative inactivation of Na⁺,K⁺-ATPase in PC12 cells, as well as ceased to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of gangliosides GM1 in PC12 cells treated with H₂O₂ on modulation of activity of activity of tyrosine kinase Trk receptors (i.e., from the same signal system) agrees with concept about the essential role of oxidant effect of GM1 in its increase of cell viability.     

Protective effect of Mori Cortex radicis extract against high glucose-induced oxidative stress in PC12 cells

ABSTRACT

This study was undertaken to investigate the neuroprotective effect of an ethanolic extract of Mori Cortex radicis (MCR) against high glucose (HG)-induced oxidative damage in PC12 cells. Cell cytotoxicity was examined using MTT and lactate dehydrogenase assays. To examine the antioxidative effects, intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels and the activities of antioxidant enzymes were measured. The expressions of apoptosis-associated proteins were assessed. MCR was found to increase the viabilities of HG-induced PC12 cells and to inhibit ROS and MDA production and to promote antioxidative enzyme activities. Furthermore, MCR reduced apoptosis by upregulating p-Akt and Bcl-2/Bax ratio and reducing cytochrome c level. The main flavonoids in MCR were identified by HPLC to be kuwanon G and morusin. These results suggest the antioxidative effects of MCR protect against HG-induced oxidative stress and that MCR has potential therapeutic use for the prevention and treatment of diabetic neuro-degeneration.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

Role of tyrosine kinase of Trk-Receptors in realization of antioxidant effect of ganglioside GM1 in PC12 cells

Ganglioside GM1 has been shown to increase viability of PC12 cells at their induction of oxidative stress by hydrogen peroxide. However, in the presence of inhibitor of tyrosine kinase Trkreceptors K-252a this GM1 effect decreases or virtually disappears. To understand mechanism of the protective effect, there was studied action of H₂O₂, GM1, and inhibitor K-252a on formation of reactive oxygen species (ROS). It has been shown that ganglioside GM1 decreases significantly the H₂O₂-induced ROS accumulation in PC12 cells; however, in the presence of inhibitor of tyrosine kinase of Trk-receptors, this GM1 effect is not revealed. It has been found that inhibitors of each of protein kinases present at the signal realization stages following the stages of activation of tyrosine kinase Trk-receptors—Erk 1/2, PI3-kinases, and PKC, decreased the GM1 ability to reduce the H₂O₂-induced ROS accumulation, while at the combined use of inhibitors of these three protein kinases, the GM1 effect was completely absent. Thus, the ganglioside GM1 antioxidant effect on PC12 is mediated by activation of tyrosine kinase Trk-receptors and protein kinases perceiving signal from this enzyme.     

神经节苷脂对脂多糖损伤大鼠嗜铬细胞瘤细胞的保护作用

目的：研究内源性神经节苷脂对大鼠嗜铬细胞瘤细胞株（PC12）脂多糖（LPS）损伤后的作用及机制。方法：培养PC12细胞,建立LPS损伤模型,采用MTT法检测不同浓度LPS对PC12细胞存活率的改变、葡萄糖神经酰胺合成酶抑制剂D（D-PDMP）耗竭内源性神经节苷脂时LPS对PC12细胞存活率的改变以及添加外源性神经节苷脂GM1后对PC12细胞存活率的保护作用;倒置显微镜和荧光显微镜观察细胞状态;RT-PCR检测NF-κB的相对表达含量。结果：LPS能导致PC12细胞形态学的改变及存活率下降,且随着LPS浓度的增加细胞存活率逐渐降低;神经节苷脂GM1能明显改善LPS所致的细胞形态学以及存活率的改变;工具药D-PDMP耗竭内源性神经节苷脂后,LPS对PC12细胞的损伤更严重,添加外源性神经节苷脂GM1,细胞形态学及存活率得到明显改善,细胞存活率上升;LPS损伤时细胞内NF-κB含量增加;D-PDMP预先耗竭内源性神经节苷脂时NF-κB含量增加更多;添加外源性神经节苷脂GM1后NF-κB含量降低。结论：内源性神经节苷脂对PC12细胞LPS损伤具有保护作用,可能是通过NF-κB信号通路来发挥作用的。     

Reversible effects of vitamins C and E combination on oxidative stress-induced apoptosis in melamine-treated PC12 cells

Abstract

Due to its high nitrogen content, melamine was deliberately added to raw milk for increasing the apparent protein content. Previous studies showed that melamine-induced apoptosis and oxidative damage on PC12 cells and rats’ hippocampus. Several evidences suggested that vitamin antioxidant reduced oxidative stress and improved organic function. Whether treatments with antioxidant vitamins C or E, otherwise combination of them can attenuate oxidative stress after melamine administration remains to be elucidated. In this study, the reversible effects of vitamin antioxidants was investigated on melamine-induced neurotoxicity in cultured PC12 cells, an in vitro model of neuronal cells. When comparing vitamin C and E, the combination of both statistically increased PC12 cells viability. The results further showed that vitamin complex has effectively reduced the formation of reaction oxygen species, decreased the level of malondialdehyde, and elevated the activities of antioxidative enzymes. Hoechst 33342 staining and flow cytometric analysis of apoptosis showed that vitamin combination treatment effectively prevented PC12 cells from this melamine-induced apoptosis. It revealed the apoptotic nuclear features of the melamine-induced cell death. Additionally, a combination treatment of vitamins effectively inhibited apoptosis via blocking the increased activation of caspase-3. In summary, the vitamin E and C combination treatment could rescue PC12 cells from the injury induced by melamine through the downregulation of oxidative stress and prevention of melamine-induced apoptosis.     

Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway

Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H₂O₂) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H₂O₂ and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H₂O₂ by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H₂O₂, which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H₂O₂ via activating the PI3K/Akt signal pathway.     

Protective Effects of Paeonia lactiflora Pall on Hydrogen Peroxide-Induced Apoptosis in PC12 Cells

This study was conducted to examine the antioxidative and neuroprotective effects of Paeonia lactiflora pall (PLE). Total phenolic content of PLE was 89.65 mg of gallic acid equivalent per gram of PLE. IC₅₀ values for reducing power, hydrogen peroxide scavenging activity, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity were 297.57, 3.33, and 32.74 μg, respectively. The protective effect of PLE against H₂O₂-induced oxidative damage to PC12 cells was investigated by an 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assay. After 2 h of cell exposure to 0.5 mM H₂O₂, a marked reduction in cell survival was observed. However, this reduction was significantly prevented by 10–100 μg/ml of PLE. H₂O₂ also induced severe apoptosis of the PC12 cells, which was indicated by a flow cytometric analysis. Interestingly, the H₂O₂-stressed PC12 cells that had been incubated with PLE had greatly suppressed apoptosis. The results suggest that PLE could be a candidate for a new antioxidant against neuronal diseases.