Paraoxonase-1介导硫化氢的抗甲醛神经毒性作用

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

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

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

Neuroprotective and Anti-Apoptotic Propensity of Bacopa monniera Extract Against Sodium Nitroprusside Induced Activation of iNOS,Heat Shock Proteins and Apoptotic Markers in PC12 Cells

Sodium nitroprusside (SNP) is a widely used nitric oxide (NO) donor, known to exert nitrative stress by up-regulation of inducible nitric oxide synthase (iNOS). Nω-nitro-l-arginine-methyl esther (L-NAME) is a NO inhibitor, which inhibits iNOS expression, is used as positive control. The present study was designed to assess neuroprotective propensity of Bacopa monniera extract (BME) in SNP-induced neuronal damage and oxido-nitrative stress in PC12 cells via modulation of iNOS, heat shock proteins and apoptotic markers. Our results elucidate that pre-treatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP (200 μM) as evidenced by MTT and LDH assays. BME pre-treatment inhibited NO generation by down regulating iNOS expression. BME replenished the depleted antioxidant status induced by SNP treatment. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic protein biomarkers such as Bax, Bcl-2, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. Q-PCR results further elucidated up-regulation of neuronal cell stress markers like HO-1 and iNOS and down-regulation of BDNF upon SNP exposure was attenuated by BME pre-treatment. By considering all these findings, we report that BME protects PC12 cells against SNP-induced toxicity via its free radical scavenging and neuroprotective mechanism.     

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.     

Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells

In the present study, we investigated the effects of tetramethylpyrazine (TMP) on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease in Bcl-2/Bax protein ratio, release of cytochrome c to cytosol and the activation of caspase-3. TMP not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation and eventually protected against H2O2-induced apoptosis. These results indicated that TMP blocked H2O2-induced apoptosis by the regulation of Bcl-2 family members, suppression of cytochrome c release, and caspase cascade activation in PC12 cells.     

Neuroprotective Effects of Salidroside in the PC12 Cell Model Exposed to Hypoglycemia and Serum Limitation

The hypoglycemia and serum limitation-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., a traditional Chinese medicinal plant, and has displayed a broad spectrum of pharmacological properties. In this study, MTT assay, Hoechst 33342 staining, and flow cytometry with annexin V/PI staining collectively showed that pretreatment with salidroside attenuated, in a dose-dependent manner, cell viability loss, and apoptotic cell death in cultured PC12 cells induced by hypoglycemia and serum limitation. RT-PCR, Western blot analysis, and enzymatic colorimetric assay indicated the changes in expression levels of Bcl-2, Bax, and caspase3 in PC12 cells on exposure to hypoglycemia and serum limitation with and without salidroside pretreatment, respectively. Rhodamine 123 staining and flow cytometry with 2′,7′-Dichlorofluorescin diacetate staining revealed the changes in the mitochondrial membrane potential and radical oxygen species (ROS) production in PC12 cells on exposure to hypoglycemia and serum limitation with and without salidroside pretreatment, respectively. The experimental results suggest that salidroside protects the PC12 cells against hypoglycemia and serum limitation-induced cytotoxicity possibly by the way of the modulation of apoptosis-related gene expression, the restoration of the mitochondrial membrane potential, and the inhibition of the intracellular ROS production. Our findings might raise a possibility of potential therapeutic applications of salidroside for preventing and treating cerebral ischemic and neurodegenerative diseases.     

Neuroprotective Effect of Crocin on Acrylamide-induced Cytotoxicity in PC12 cells

Acrylamide (ACR) is a potent neurotoxic in human and animal models. In this study, the effect of crocin, main constituent of Crocus sativus L. (Saffron) on ACR-induced cytotoxicity was evaluated using PC12 cells as a suitable in vitro model. The exposure of PC12 cells to ACR reduced cell viability, increased DNA fragmented cells and phosphatidylserine exposure, and elevated Bax/Bcl-2 ratio. Results showed that ACR increased intracellular reactive oxygen species (ROS) in cells and ROS played an important role in ACR cytotoxicity. The pretreatment of cells with 10–50 μM crocin before ACR treatment significantly attenuated ACR cytotoxicity in a dose-dependent manner. Crocin inhibited the downregulation of Bcl-2 and the upregulation of Bax and decreased apoptosis in treated cells. Also, crocin inhibited ROS generation in cells exposed to ACR. In conclusion, our results indicated that pretreatment with crocin protected cells from ACR-induced apoptosis partly by inhibition of intracellular ROS production.     

Rotenone-induced oxidative stress and apoptosis in human liver HepG2 cells

Rotenone, a commonly used pesticide, is well documented to induce selective degeneration in dopaminergic neurons and motor dysfunction. Such rotenone-induced neurodegenration has been primarily suggested through mitochondria-mediated apoptosis and reactive oxygen species (ROS) generation. But the status of rotenone induced changes in liver, the major metabolic site is poorly investigated. Thus, the present investigation was aimed to study the oxidative stress-induced cytotoxicity and apoptotic cell death in human liver cells-HepG2 receiving experimental exposure of rotenone (12.5–250 μM) for 24 h. Rotenone depicted a dose-dependent cytotoxic response in HepG2 cells. These cytotoxic responses were in concurrence with the markers associated with oxidative stress such as an increase in ROS generation and lipid peroxidation as well as a decrease in the glutathione, catalase, and superoxide dismutase levels. The decrease in mitochondrial membrane potential also confirms the impaired mitochondrial activity. The events of cytotoxicity and oxidative stress were found to be associated with up-regulation in the expressions (mRNA and protein) of pro-apoptotic markers viz., p53, Bax, and caspase-3, and down-regulation of anti-apoptotic marker Bcl-2. The data obtain in this study indicate that rotenone-induced cytotoxicity in HepG2 cells via ROS-induced oxidative stress and mitochondria-mediated apoptosis involving p53, Bax/Bcl-2, and caspase-3.     

Corilagin prevents tert-butyl hydroperoxide-induced oxidative stress injury in cultured N9 murine microglia cells

Oxidative stress plays an important role in neurodegenerative diseases. Reactive oxygen species (ROS)-mediated stress in microglia in vivo could result in cellular injuries and preferentially induces neuronal injury. Corilagin, a novel member of the phenolic tannin family, has been shown to possess antioxidant properties. In this study, we investigated the effects of corilagin on tert-butyl hydroperoxide (TBHP)-induced injury in cultured N9 murine microglial cells and the underlying mechanisms by a methyltetrazolium assay and oxidative damage assay. We found that exposure of N9 cells to TBHP induced cytotoxicity as demonstrated by cell shrinkage, loss of cell viability, increased lactate dehydrogenase (LDH) leakage, and increased intracellular levels of ROS. By contrast, TBHP reduced both superoxide dismutase activity and total cell anti-oxidation capacity, but glutathione was not reduced. Moreover, TBHP treatment was associated with the loss of mitochondrial membrane potential, and it induced cell apoptosis through the mitochondrial-mediated pathway involving the down-regulation of Bcl-2 expression and up-regulation of the Bax/Bcl-2 ratio. Interestingly, pre-treatment with corilagin reversed these reactions. These data collectively indicated that corilagin could attenuate TBHP-induced oxidative stress injury in microglial cells, and its protective effects may be ascribed to its antioxidant and antiapoptotic properties. Our findings suggest that corilagin should be a potential candidate for the treatment of oxidative stress-induced neurodegenerative diseases.     

Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.     

Hydroxysafflor Yellow A Protects PC12 Cells Against the Apoptosis Induced by Oxygen and Glucose Deprivation

Hydroxysafflor yellow A (HSYA) was reported neuroprotective under several ischemic models in vivo. In this study, the direct effect of HSYA against oxygen–glucose deprivation (OGD) inducing acute neuronal injury and the underling mechanisms in vitro were investigated. Four-hour oxygen and glucose deprivation (OGD) followed by 20 h reperfusion (adding back oxygen and glucose, OGD-R) was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. HSYA (1, 10, and 100 μmol/l) was added to the cultures 30 min prior to the ischemic insult and was present during OGD and reoxygenation phases. The survival rate of PC12 cells was detected by MTT assay. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) activity were elevated by biochemical method. Hoechst 33258 staining and flow cytometric analysis were used to detect apoptosis; western blotting was used to detect the expression of Bcl-2, Bax, and Cytochrome C protein. The activity of caspase-3 was assessed by colorimetry. HSYA concentration-dependently attenuated neuronal damage with characteristics of increasing injured neuronal absorbance of MTT, decreasing cell apoptosis, and antagonizing decreases in SOD activity and increase in MDA level induced by OGD-R. Moreover, the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol and the consequent activation of caspase-3 were reversed by HSYA in a dose-dependent manner. These results suggest that apoptosis is an important characteristic of OGD-R-induced PC 12 death and that treatment of PC12 cells with HSYA can block OGD-R-induced apoptosis through suppression of intracellular oxidative stress and mitochondria dependent caspase cascade.     

The Role of Klotho Protein Against Sevoflurane-Induced Neuronal Injury

The effects of general anesthetics on the developing brain have aroused much attention in recent years. Sevoflurane, a commonly used inhalation anesthetic especially in pediatric anesthesia, can induce developmental neurotoxicity. In this study, the differentially expressed mRNAs in the hippocampus of newborn rats exposed to 3% sevoflurane for 6 h were detected by RNA-Sequencing. Those data indicated that the mRNA of Klotho was increased after exposure to sevoflurane. Moreover, the protein expression of Klotho was assayed by Western Blot. Besides over-expression and under-expression of Klotho protein, we also detected changes of cell proliferation, ROS, JC-1, and Bcl-2/Bax ratio in PC12 cells exposed to sevoflurane. After exposure to 3% sevoflurane, the expression of Klotho protein increased in the hippocampus of neonatal rats. In PC12 cells, exposure to sevoflurane could increase cellular ROS level, reduce mitochondrial membrane potential and Bcl-2/Bax ratio. While overexpression of Klotho alleviated the above changes, knockdown of Klotho aggravated the injury of sevoflurane. Klotho protein could reduce oxidative stress and mitochondrial injury induced by sevoflurane in the neuron.

     

Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate

Hydrogen sulfide (H(2)S) has been shown to exert cardioprotective effects. However, the roles of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in H(2)S-induced cardioprotection have not been completely elucidated. In this study, cobalt chloride (CoCl(2)), a chemical hypoxia mimetic agent, was applied to treat H9c2 cells to establish a chemical hypoxia-induced cardiomyocyte injury model. The results showed that pretreatment with NaHS (a donor of H(2)S) before exposure to CoCl(2) attenuated the decreased cell viability, the increased apoptosis rate, the loss of mitochondrial membrane potential (ΔΨm), and the intracellular accumulation of reactive oxygen species (ROS) in H9c2 cells. Exposure of H9c2 cells to CoCl(2) or hydrogen peroxide (H(2)O(2)) upregulated expression of phosphorylated (p) ERK1/2, which was reduced by pretreatment with NaHS or N-acetyl-L-cysteine, a ROS scavenger. More importantly, U0126, a selective inhibitor of ERK1/2, mimicked the above cytoprotection of H(2)S against CoCl(2)-induced injury in H9c2 cells. In conclusion, these results indicate that H(2)S protects H9c2 cells against chemical hypoxia-induced injury partially by inhibiting ROS-mediated activation of ERK1/2.     

Protective effects of Peroxiredoxin 6 overexpression on amyloid β-induced apoptosis in PC12 cells

Abstract

Oxidative stress triggered by amyloid beta (Aβ) accumulation contributes substantially to the pathogenesis of Alzheimer's disease (AD). In the present study, we examined the involvement of the antioxidant activity of peroxiredoxin 6 (Prdx 6) in protecting against Aβ_25–35-induced neurotoxicity in rat PC12 cells. Treatment of PC12 cells with Aβ_25-35 resulted in a dose- and time-dependent cytotoxicity that was associated with increased accumulation of intracellular reactive oxygen species (ROS) and mitochondria-mediated apoptotic cell death, including activation of Caspase 3 and 9, inactivation of poly ADP-ribosyl polymerse (PARP), and dysregulation of Bcl-2 and Bax. This apoptotic signaling machinery was markedly attenuated in PC12 cells that overexpress wild-type Prdx 6, but not in cells that overexpress the C47S catalytic mutant of Prdx 6. This indicates that the peroxidase activity of Prdx 6 protects PC12 cells from Aβ_25-35-induced neurotoxicity. The neuroprotective role of the antioxidant Prdx 6 suggests its therapeutic and/or prophylactic potential to slow the progression of AD and limit the extent of neuronal cell death caused by AD.     

Hydrogen sulfide protects H9c2 cells against doxorubicin-induced cardiotoxicity through inhibition of endoplasmic reticulum stress

The roles of hydrogen sulfide (H(2)S) and endoplasmic reticulum (ER) stress in doxorubicin (DOX)-induced cardiotoxicity are still unclear. This study aimed to dissect the hypothesis that H(2)S could protect H9c2 cells against DOX-induced cardiotoxicity by inhibiting ER stress. Our results showed that exposure of H9c2 cells to DOX significantly inhibited the expression and activity of cystathionine-γ-lyase (CSE), a synthetase of H(2)S, accompanied by the decreased cell viability and the increased reactive oxygen species (ROS) accumulation. In addition, exposure of cells to H(2)O(2) (an exogenous ROS) mimicked the inhibitory effect of DOX on the expression and activity of CSE. Pretreatment with N-acetyl-L: -cysteine (NAC) (a ROS scavenger) attenuated intracellular ROS accumulation, cytotoxicity, and the inhibition of expression and activity of CSE induced by DOX. Notably, the ER stress-related proteins, including glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were obviously upregulated in DOX-treated H9c2 cells. Pretreatment with sodium hydrosulfide (NaHS, a H(2)S donor) before DOX exposure markedly suppressed DOX-induced overexpressions of GRP78 and CHOP, cytotoxicity and oxidative stress. In conclusion, we have demonstrated that ROS-mediated inhibition of CSE is involved in DOX-induced cytotoxicity in H9c2 cells, and that exogenous H(2)S can confer protection against DOX-induced cardiotoxicity partly through inhibition of ER stress.     

S. choloroleuca, S. mirzayanii and S. santolinifolia protect PC12 cells from H(2)O (2)-induced apoptosis by blocking the intrinsic pathway

Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer's disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran-S. choloroleuca, S. mirzayanii and S. santolinifolia-against H(2)O(2)-induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases.     

Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Homer proteins, a new member of the postsynaptic scaffolding proteins, regulate glutamatergic signaling and intracellular calcium mobilization in the central nervous system. Here we investigated the effects of down-regulating Homer1b/c, a constitutively expressed long form of Homer proteins, on glutamate excitotoxicity-induced neuronal injury. In our in vitro excitotoxic models, we demonstrated that glutamate insults led to a dose-dependent neuronal injury, which was mediated by the intracellular calcium-dependent reactive oxygen species (ROS) production. We found that down-regulation of Homer1b/c with specific small interfering RNA (siRNA) improved neuronal survival, inhibited intracellular ROS production, and reduced apoptotic cell death after neurotoxicity. Homer1b/c knockdown decreased the intracellular calcium overload through inhibition of the group I metabotropic glutamate receptor (mGluR)/inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) in injured neurons. In addition, Homer1b/c siRNA transfection attenuated the activation of eukaryotic initiation factor 2α (eIF2α), RNA-dependent protein kinase-like ER kinase (PERK) and caspase-12, and inhibited the up-regulation of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) after glutamate treatment. Homer1b/c knockdown also preserved the mitochondrial membrane potential (MMP), reduced cytochrome c (Cyt. c) release, and partly blocked the increase of capase-9 activity and Bax/Bcl-2 ratio. Taken together, these results suggest that down-regulation of Homer1b/c protects cortical neurons against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the inhibition of calcium-dependent ROS production and the preservation of the ER and mitochondrial function.     

Neuroprotective Effect of Fucoidan on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis in PC12 Cells Via Activation of PI3K/Akt Pathway

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. In this study, we investigated the neuroprotective effect of fucoidan on H(2)O(2)-induced apoptosis in PC12 cells and the possible signaling pathways involved. The results showed that fucoidan inhibited the decrease of cell viability, scavenged ROS formation and reduced lactate dehydrogenase release in H(2)O(2)-induced PC12 cells. These changes were associated with an increase in superoxide dismutase and glutathione peroxidase activity, and reduction in malondialdehyde. In addition, fucoidan treatment inhibited apoptosis in H(2)O(2)-induced PC12 cells by increasing the Bcl-2/Bax ratio and decreasing active caspase-3 expression, as well as enhancing Akt phosphorylation (p-Akt). However, the protection of fucoidan on cell survival, p-Akt, the Bcl-2/Bax ratio and caspase-3 activity were abolished by pretreating with phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In consequence, fucoidan might protect the neurocytes against H(2)O(2)-induced apoptosis via reducing ROS levels and activating PI3K/Akt signaling pathway.     

Fasudil Mesylate Protects PC12 Cells from Oxidative Stress Injury via the Bax-Mediated Pathway

We previously reported that fasudil mesylate (FM) improves neurological deficit and neuronal damage in rats with ischemia following middle cerebral artery occlusion and reperfusion in vivo. In this study, the properties of FM on hydrogen peroxide (H₂O₂)-induced oxidative stress insult in cultured PC12 cells as well as the underlying mechanisms were investigated in vitro. Pretreatment with FM (5, 10 μM) prior to H₂O₂ exposure significantly elevated cell viability, reduced cell apoptosis by MTT assay, LDH assay, Hoechst 33258 dye staining, and FM also decreased the accumulation of reactive oxygen species (ROS) by DCFH-DA staining and NBT test. Furthermore, FM also reversed the upregulation of Bax/Bcl-2 ratio, the downstream cascade following ROS. FM protected PC12 cells from oxidative stress insult via down-regulating the Bax/Bcl-2 ratio. These findings indicate that a direct effect of fasudil mesylate on PC12 cells may be partly responsible for its protective effect against oxidative stress injury.