• International Journal of Oral Biology
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• v.34 no.2
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• pp.97-103
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• 2009

L-trans-pyrrolidine-2,4-dicarboxylate (PDC) is a potent inhibitor of glutamate transporters. In our current study, we investigated whether the neuronal death induced by PDC involves mechanisms other than excitotoxicity in mixed mouse cortical cultures. Cortical cultures at 13-14 days in vitro were used and cell death was assessed by measuring the lactate dehydrogenase efflux into bathing media. Glutamate and PDC both induced neuronal death in a concentration-dependent manner but the neurotoxic effects of glutamate were found to be more potent than those of PDC. Treatment with 10, 100 and 200 ${\mu}$M PDC equally potentiated 50 ${\mu}$M glutamate-induced neuronal death. The neuronal death induced by 75 ${\mu}$M glutamate was almost abolished by treatment with the NMDA antagonists, MK-801 and AP-5, but was unaffected by NBQX (an AMPA antagonist), trolox (antioxidant), BDNF or ZVAD-FMK (a pan-caspase inhibitor). However, the neuronal death induced by 200 ${\mu}$M PDC was partially but significantly attenuated by single treatments with MK-801, AP-5, trolox, BDNF or ZVAD-FMK but not NBQX. Combined treatments with MK-801 plus trolox, MK-801 plus ZVAD-FMK or MK-801 plus BDNF almost abolished neuronal death, whereas combined treatments with trolox plus ZVADFMK, trolox plus BDNF or ZVAD-FMK plus BDNF did not enhance the inhibitory action of any single treatment with these drugs. These results demonstrate that the neuronal death induced by PDC involves not only in the excitotoxicity induced by the accumulation of glutamate but also the oxidative stress induced by free radical generation. This suggests that apoptotic neuronal death plays a role in PDCinduced oxidative neuronal injury.

• The Journal of Internal Korean Medicine
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• v.40 no.3
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• pp.425-442
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• 2019

Objectives: This study aimed to reveal the pharmacological properties of the newly prescribed herbal mixture, Chenmadansamgamibokhap-tang(CDBT), against hypoxia-induced neuronal cell injury (especially mouse hippocampal neuronal cell line, HT-22 cells) and their corresponding mechanisms. Methods: A cell-based in vitro experiment, in which a hypoxia condition induced neuronal cell death, was performed. Various concentrations of the CDBT were pre-treated to the HT-22 cells for 4 h before 18 h in the hypoxia chamber. The glial cell BV-2 cells were stimulated with $IFN{\gamma}$ and LSP to produce inflammatory cytokines and reactive oxygen species. When the neuronal HT-22 cells were treated with this culture solution, the drug efficacy against neuronal cell death was examined. Results: CDBT showed cytotoxicity in the normal condition of HT-22 cells at a dose of $125{\mu}g/mL$ and showed a protective effect against hypoxia-induced neuronal cell death at a dose of $31.3{\mu}g/mL$. CDBT prevented hypoxia-induced neuronal cell death in a dose-dependent manner in the HT-22 cells by regulating $HIF1{\alpha}$ and cell death signaling. CDBT prevented neuronal cell death signals and DNA fragmentation due to the hypoxia condition. CDBT significantly reduced cellular oxidation, cell death signals, and caspase-3 activities due to microglial cell activations. Moreover, CDBT significantly ameliorated LPS-induced BV-2 cell activation and evoked cellular oxidation through the recovery of redox homeostasis. Conclusions: CDBT cam be considered as a vital therapeutic agent against neuronal cell deaths. Further studies are required to reveal the other functions of CDBT in vivo or in the clinical field.

• BMB Reports
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• v.35 no.1
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• pp.67-86
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• 2002

Three routes have been identified triggering neuronal death under physiological and pathological conditions. Excess activation of ionotropic glutamate receptors cause influx and accumulation of $Ca^{2+}$ and $Na^+$ that result in rapid swelling and subsequent neuronal death within a few hours. The second route is caused by oxidative stress due to accumulation of reactive oxygen and nitrogen species. Apoptosis or programmed cell death that often occurs during developmental process has been coined as additional route to pathological neuronal death in the mature nervous system. Evidence is being accumulated that excitotoxicity, oxidative stress, and apoptosis propagate through distinctive and mutually exclusive signal transduction pathway and contribute to neuronal loss following hypoxic-ischemic brain injury. Thus, the therapeutic intervention of hypoxic-ischemic neuronal injury should be aimed to prevent excitotoxicity, oxidative stress, and apoptosis in a concerted way.

• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.1
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• pp.117-125
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• 2007

Neuronal ischemia is a pathological process caused by a lack of oxygen (anoxia) and glucose (hypoglycemia), resulting in neuronal death. It is believed that apoptosis is one of the mechanisms involved in ischemic cell death. Neuronal apoptosis is a process characterized by nuclear DNA fragmentation, changes of plasma membrane organization. To elucidate the mechanism of neuronal death following ischemic insult and to develop neuroprotective effects of Daebowonjeon(DBWJ) against ischemic damage, in vitro models are used. In vitro models of cell death have been devloped with pheochromocytoma (PC12) cell, which have become widely used as neuronal models of oxidative stress, trophic factor, serum deprivation and chemical hypoxia. Using a special ischemic device and PC12 cultures, we investigated an in vitro model of ischemia based on combined Oxygen and Glucose Deprivation (OGD) insult, followed by reoxygenation, mimicking the pathological conditions of ischemia. In this study, Daebowonjeon rescued PC12 cells from Oxygen-Glucose Deprivation (OGD)-induced cell death in a dose-dependent manner The nuclear staining of PC12 cells clearly showed that DBWJ attenuated nuclear condensation and fragmentation which represent typical neuronal apoptotic characteristics. DBWJ also prevents the LDH release and induction of Hypoxia Inducing Factor (HIF)-1 by OGD-exposed PC12 cells. Furthermore, DBWJ reduced the activation of polyADP-ribose polymerase (PARP) by OGO-exposed PC12 cells. These results suggest that apoptosis is an important characteristic of OGD-induced neuronal death and that oriental medicine, such as DBWJ, may prevent PC12 cell from OG D-induced neuronal death by inhibiting the apoptotic process.

• Molecules and Cells
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• v.37 no.4
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• pp.345-355
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• 2014

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing ($GFAP^+$) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, $GFAP^+$ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including $S100{\beta}$, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.4
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• pp.265-271
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• 2009

Nitric oxide (NO) has both neuroprotective and neurotoxic effects depending on its concentration and the experimental model. We tested the effects of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, and aminoguanidine, a selective inducible NOS (iNOS) inhibitor, on kainic acid (KA)-induced seizures and hippocampal CA3 neuronal death. L-NAME (50 mg/kg, i.p.) and/or aminoguanidine (200 mg/kg, i.p.) were administered 1 h prior to the intracerebroventricular (i.c.v.) injection of KA. Pretreatment with L-NAME significantly increased KA-induced CA3 neuronal death, iNOS expression, and activation of microglia. However, pretreatment with aminoguanidine significantly suppressed both the KA-induced and L-NAME-aggravated hippocampal CA3 neuronal death with concomitant decreases in iNOS expression and microglial activation. The protective effect of aminoguanidine was maintained for up to 2 weeks. Furthermore, iNOS knockout mice ($iNOS^{-1-}$) were resistant to KA-induced neuronal death. The present study demonstrates that aminoguanidine attenuates KA-induced neuronal death, whereas L-NAME aggravates neuronal death, in the CA3 region of the hippocampus, suggesting that NOS isoforms play different roles in KA-induced excitotoxicity.

• Animal cells and systems
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• v.12 no.4
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• pp.211-217
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• 2008

Bax, a pro-apoptotic member of Bcl-2 family proteins, plays a central role in the mitochondria-dependent apoptosis. Apoptotic signals induce the translocation of Bax from cytosol into the mitochondria, which triggers the release of apoptogenic molecules such as cytochrome C and apoptosis-inducing factor, AIF. Bax-inhibiting peptide(BIP) is a cell permeable peptide comprised of five amino acids designed from the Bax-interaction domain of Ku70. Because BIP inhibits Bax translocation and Bax-mediated release of cytochrome C, BIP suppresses Bax-dependent apoptosis. In this study, we observed that BIP inhibited staurosporine-induced neuronal death in cultured cerebral cortex and cerebellar granule cells, but BIP failed to rescue granule cells from trophic signal deprivation-induced neuronal death, although both staurosporine-induced and trophic signal deprivation-induced neuronal death are dependent on Bax. These findings suggest that the mechanisms of the Bax activation may differ depending on the type of cell death induction, and thus BIP exhibits selective suppression of a subtype of Bax-dependent neuronal death.

• The Korea Journal of Herbology
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• v.21 no.4
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• pp.77-84
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• 2006

Objectives : To evaluate the neuroprotective effects of modified Bo-Yang-Hwan-O-Tang (BHT), we investigated the neuronal death protection effects to oxidative damages in N2a neuroblastoma cells. Methods : To study the cytotoxic effect of BHT on N2a neuronal cells, the cell viability was determined by MTT assay. To investigate the neuronal death protection of BHT, N2a neuronal cells were induced oxidative damages by H2O2, and assayed the cell viability and DNA fragmentation. We also investigated DPPH free radical scavenging effects of BHT by tube test. Results : In MTT assay, $500{\mu}g/ml$ of BHT was not showed cytotoxic effect on N2a neuronal cells. BHT protected N2a neuronal cells from H2O2-induced cell death in a dose-dependent manner. BHT also protected N2a neuronal cells from H2O2-induced DNA fragmentation. BHT scavenged DPPH free radicals in a dose-dependent manner. Conclusion : These data suggest that BHT may have strong anti-oxidant effects through the free radical scavenging and neuroprotective effects in neuronal cells.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.6
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• pp.239-246
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• 2007

Expressions of endoplasmic reticulum stress response (ERSR) genes were examined during the neuronal differentiation of rat fetal cortical precursor cells (rCPC) and rat pheochromocytoma PC12 cells. When rCPC were differentiated into neuronal cells for 7 days, early stem cell marker, nest in, expression was decreased from day 4, and neuronal markers such as neurofilament-L, -M and Tuj1 were increased after day 4. In this condition, expressions of BIP, ATF6, and phosphorylated PERK as well as their down stream signaling molecules such as CHOP, ATF4, XBP1, GADD34, Nrf2 and $p58^{IPK}$ were significantly increased, suggesting the induction of ERSR during neuronal differentiation of rCPC. ERSR was also induced during the differentiation of PC12 cells for 9 days with NGF. Neurofilament-L transcript was time-dependently increased. Both mRNA and protein levels of Tuj1 were increased after the induction, and the significant increase in NeuN was observed at day 9. Similar to the expression patterns of neuronal markers, BIP/GRP78 and CHOP mRNAs were highly increased at day 9, and ATF4 mRNA was also increased from day 7. These results strongly suggest the induction and possible role of ERSR in neuronal differentiation process. Further study to identify targets responsible for neuronal induction will be necessary.

• The Journal of Internal Korean Medicine
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• v.24 no.2
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• pp.348-357
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• 2003

Objectives : The purpose of this investigation is to evaluate the effects of Aucklandiae Radix Moschus(木香 麝香)and to study the mechanism for neuronal death protection in hypoxia with Embryonic day 20 (E20) cortical cells of a rat (Sprague Dawley). Methods : E20 cortical cells used in this investigation were dissociated in Neurobasal media and grown for 14 days in vitro (DIV). On 14 DIV, Aucklandiae Radix Moschus(木香 麝香) was added to the culture media for 72 hrs. On 17 DIV, cells were given a hypoxic shock and further incubated in normoxia for another three days. On 20 DIV, Moschus(麝香)'s effects for neuronal death protection were evaluated by LDH assay and the mechanisms were studied by Bcl-2, Bak, Bax, caspase family. Results : This study indicate that Aucklandiae Radix(木香)'s effects for neuronal death protection in normoxia and Scutellariae Radix(麝香)'s effects for neuronal death protection in hypoxia were confirmed by LDH assay in culture method of Embryonic day 20(E20) cortical neuroblast. Moschus(麝香)'s mechanism for neuronal death protection in hypoxia is to increase the anti-apoptosis protein Bcl-2. Conclusions : It may be reasonable to propose that Moschus(麝香) protects delayed neuronal death in hypoxia by increasing Bcl-2, thereby reducing mitochondrial permeability transition(PT) pores, the cytochrome c channels.