• The Journal of Internal Korean Medicine
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• v.21 no.4
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• pp.535-542
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• 2000

The water extracts of Samul-tang(SMT) has been used for treatment of ischemic brain damage in Oriental traditional medicine. However, little is known about the mechanism by which the water extracts of SMT rescues brain cells from ischemic damages. To elucidate the protective mechanism on ischemic induced cytotoxicity, I investigate the regulation of LPS and PMA induced iNOS expression in C6 glial cells. LPS and PMA treatment for 72 h in C6 glial cells markedly induce nitric oxide(NO), but treatment of the cells with the water extracts of SMT decrease. dose dependently nitrite formation. In addition, LPS and PMA treatment for 72 h induce severe cell death and LDH release in C6 glial cells. However treatment of the cells with the water extracts of SMT dose not induce significant changes compare to control cells. Furthermore, the protective effects of the water extracts of SMT is mimicked by treatment of $N^{G}MMA$, a specific inhibitor of NOS. LPS and PMA induced iNOS activation in C6 glial cells cause chromosomal condensation and fragmentation of nuclei by caspase activation. The treatment of the cells with the water extracts of SMT may suppress apoptosis via caspase inhibition by regulation of iNOS expression. Taken together, I suggest that the protective effects of the water extracts of SMT against ischemic brain damages may be mediated by regulation of iNOS during ischemic condition.

• Korean Journal of Medicinal Crop Science
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• v.25 no.5
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• pp.315-321
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• 2017

Background: Glycyrrhiza uralensis Radix (GR) is a crude drugs used in Asian countries that has been reported to prevent the progression of neurodegenerative diseases such as Alzheimer's disease. The present study examined whether GR and its active compounds, glycyrrhizic acid (GA) and isoliquiritigenin (IL), exerted protective effects on $H_2O_2$-induced oxidative damage in C6 glial cells. Methods and Results: We exposed C6 glial cells to hydrogen peroxide ($H_2O_2$) for 24 h and investigated the cellular response to GR and its active compounds by evaluating cell viability, reactivie oxygen species (ROS) production, and apoptosis-related protein expression. GR successfully mitigated the reduced cell viability and ROS production induced by $H_2O_2$ in C6 glial cells, IL and GA significantly increased the cell viability and decreased ROS production. In addition, IL and GA down-regulated apoptotic Baxdependent caspase-3 activation, but each compound exerted different mechanisms, i.e., IL dose-dependently decreased ROS production and, GA up-regulated anti-apoptotic Bcl-2 expression. Conclusions: These results demonstrated that GR and its active components, IL and GA, exhibit potential for use as natural neurodegenerative agents for the modulation of apoptosis in C6 glial cells.

• The Journal of Internal Korean Medicine
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• v.31 no.4
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• pp.693-705
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• 2010

Objectives : The water extract of Daechilgi-tang(DCGT) has traditionally been used for treatment of qi stagnation(氣滯), which is considered to be one of the important causes of neuronal disease in oriental medicine. However, little is known about the mechanism by which DCGT protects neuronal cells from brain cell damages. Methods and Results : The author tested the mechanism of the cytoprotective effect of DCGT on glutamate -stimulated rat C6 glial cells. DCGT significantly protected C6 glial cells from glutamate in MTT assay. Pre-treatment of C6 glial cells with DCGT markedly inhibited the DNA fragmentation of C6 cells induced by glutamate. Glutamate increased the generation of reactive oxygen species(ROS) and intracellular calcium level in C6 glial cells. However, pre-treatment with DCGT markedly suppressed the increase of ROS generation and intracellular calcium accumulation induced by glutamate. Among apoptosis signaling mediators, DCGT markedly increased the expression level of Bcl2 in glutamate-treated cells. It also inhibited the cleavage of caspase-3 and PARP proteins by glutamate in C6 glial cells. Conclusions : These results suggest that DCGT protects brain cells from glutamate cytotoxicity through inhibition of ROS generation and activation of apoptosis signaling pathway as well as induction of the anti-oxidant system.

• Toxicological Research
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• v.16 no.3
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• pp.179-185
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• 2000

Zinc is known to generate reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide ($H_2O_2$), which eventually contribute to cytotoxicity in a variety of cell types. Here in, we demonstrated that zinc decreased the viability of C6 glial cells in a time and dose-dependent manner, which was revealed as apoptosis characterized by ladder-pattern fragmentation of genomic DNA. chromatin condensation and DNA fragmentation in Hoechst dye staining. Zinc-induced apoptosis of C6 glial cells was prevented by the addition of catalase and antioxidants including reduced glutathione (GSH), N-acetyl-L-cysteine (NAC) and pyrrolidinedithiocarbamate (PDTC). Wefurther confirmed that zinc decreased intrac-ellular levels of GSH and generated $H_2O_2$in C6 glial cells. Moreover, antioxidants also decreased the generation of zinc-induced $H_2O_2$ in C6 glial cells. These data indicated that zinc-induced the apoptotic death of C6 glial cells via generation of reactive oxygen species such as $H_2O_2$.

• The Journal of Internal Korean Medicine
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• v.33 no.3
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• pp.272-283
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• 2012

Objectives : The purpose of this study was to investigate the mechanism of protective effect of Jinmu-tang (JMT, Zhenwu-tang) extract on $H_2O_2$-induced cell death in C6 glial cells. Methods : Cultured C6 glial cells of white mice were pretreated with JMT extract and exposed to $H_2O_2$ for inducing cell death. We measure the cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and investigate the cell morphology using a light microscope after crystal violet (CV) staining. Reactive oxygen species (ROS) formation was analyzed using a flow cytometer and a fluorescent microscope after staining with 2'7'-dichlorofluorescein diacetate (DCF-DA). DNA fragmentation was analyzed using a flow cytometer after propidium iodide (PI) staining and nuclei morphology was investigated using a fluorescent microscope after 2-[4-amidinophenyl]-6-indo-lecarbamidine dihydrochloride (DAPI) staining. We analyzed expression of Bax, processing of procaspase-3 and poly (ADP-ribose) polymerase (PARP), and activation of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) by western blot method. Tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) secretion was analyzed using Quantikine kit. Results : We determined the elevated cell viability by JMT extract on $H_2O_2$-induced C6 glial cell death. ROS formation, DNA fragmentation, $I{\kappa}B{\alpha}$ phosphorylation, NF-${\kappa}B$ activation, and secretion of TNF-${\alpha}$ induced by $H_2O_2$ are inhibited by JMT extract pre-treatment. JMT extract inhibits Bax expression, processing of caspase-3 and PARP that are critical biochemical markers of apoptotic cell death. Conclusions : These results suggest that JMT extract has a protective effect on $H_2O_2$-induced C6 glial cell death in various pathways.

• Korean Journal of Agricultural Science
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• v.47 no.3
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• pp.497-508
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• 2020

In this study, we investigated the protective effects of 'Donganme' (Sorghum bicolor L. Moench) against oxidative stress under in vitro conditions and in a cellular system using C6 glial cells. The radical scavenging activities were observed using the substrates 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl (•OH) radicals. The Donganme extract had an •OH radical scavenging activity of 82.66% at a concentration of 100 ㎍·mL^-1. Additionally, when DPPH was used as the substrate, the Donganme extract exhibited a strong radical scavenging activity in a concentration-dependent manner with an IC₅₀ value of 28.56 ㎍·mL^-1. Furthermore, treating C6 glial cells with hydrogen peroxide (H₂O₂) reduced the cell viability and generated reactive of oxygen species (ROS) and lactate dehydrogenase (LDH) compared to the normal levels, indicating that H₂O₂ induced oxidative stress. However, Donganme extracts increased the cell viability and inhibited ROS and LDH production against oxidative stress by H₂O₂ in the C6 glial cells. In particular, it showed effective cell protection with the cell viability, ROS production, and LDH release at 83.50, 88.06, and 14.87%, respectively, which were lower than the control or similar to the normal levels even at a low concentration of 100 ㎍·mL^-1. The present study suggests that the Donganme extract was effective in protecting against oxidative stress in C6 glial cells through its antioxidant activity. Thus, Donganme could be a promising therapeutic agent for neurodegenerative diseases due to oxidative stress.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.5
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• pp.235-239
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• 2002

It has been reported that lovastatin induced cell death and suppressed proliferation in various cell lines. In this study, we examined whether the cytotoxic effects of lovastatin could be prevented by Bcl-2 or $Bcl-_{XL}$ in C6 glial cells. Overexpression of human Bcl-2 or $Bcl-_{XL}$ prevented lovastatin $(25{\mu}M)-induced$ changes such as DNA fragmentation, chromatin condensation, disruption of cell membrane, and cleavage of poly (ADP-ribose) polymerase. Lovastatin-induced inhibition of cell proliferation was unaffected by Bcl-2 or $Bcl-_{XL}$ overexpression. These results suggest that Bcl-2 and $Bcl-_{XL}$ can prevent lovastatin-induced apoptosis in C6 glial cells, though the inhibition of proliferation remains unaffected by these proteins.

• Journal of Applied Biological Chemistry
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• v.63 no.4
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• pp.327-334
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• 2020

Oxidative stress is common cause of neurodegenerative diseases. The purpose of this study is to investigate the in vitro free radical scavenging activity and protective effect of three Glycyrrhiza species including Glycyrrhiza uralensis, G. glabra, and a new variety of Glycyrrihza (Shinwongam, SW) against hydrogen peroxide-induced oxidative stress in C6 glial cells. In vitro assays, radical scavenging activities of G. uralensis, G. glabra, and SW against 2,2-diphenyl-1-picrylhydrazyl, ·OH, and O2- increased as concentration-dependent manner. In addition, the SW was found to contain the highest polyphenol and flavonoid contents. The treatment of H2O2 to C6 glial cell induced oxidative stress, whereas G. uralensis, G. glabra, and SW significantly increased the cell viability as dose-dependent manner. In particular, SW exerted stronger protective effect on H2O2-induced cytotoxicity, than G. uralensis and G. glabra. Furthermore, reactive oxygen species (ROS) formation was significantly elevated by H2O2 in C6 glial cells. However, treatments of G. uralensis, G. glabra, and SW decreased ROS formation. In addition, SW decreased pro-inflammatory related protein expression levels such as inducible nitric oxide synthase and cyclooxygenase-2, compared to H2O2-treated control group. These results indicated that G. uralensis and G. glavra, especially SW, may be useful for preventing from oxidative stress-induced neuronal damage by regulating inflammatory reaction.

• Korean Journal of Pharmacognosy
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• v.46 no.2
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• pp.109-115
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• 2015

This study was investigated the radical scavenging effect and the protective activity of caffeic acid (CA) against oxidative stress. CA showed strong 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and hydroxyl radical ( OH) scavenging activity, showing 42.00% and 87.22% at 5 μM concentration of DPPH and ·OH scavenging activity, respectively. Furthermore, we studied protective activity of CA from amyloid beta (A${\beta}$_25-35) and lipopolysaccharide (LPS) induced neuronal cell damage and neuronal inflammation using C6 glial cells. The treatment of A${\beta}$_25-35 to C6 glial cell showed declines in cell viability and high generation levels of reactive oxygen species (ROS). However, the treatment of CA increased cell viability. The treatment of 5 ${{\mu}M}$ CA led to the elevation of cell viability from 59.28% to 81.22%. In addition, the production of ROS decreased cellular levels of ROS by the treatment of CA. The treatment of LPS to C6 glial cells increased significant elevation of nitric oxide (NO) production, while CA decreased NO production significantly. The production of NO increased by the treatment of LPS to 131.08%, while CA at the concentration of 1 ${{\mu}M}$ declined the NO production to 104.86%. The present study indicated thatCA attenuated A${\beta}$_25-35-induced neuronal oxidative stress and inflammation by LPS, suggesting as a promising agent for the neurodegenerative diseases.

• The Journal of Internal Korean Medicine
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• v.34 no.2
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• pp.178-191
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• 2013

Objectives : In this study, we made an effort to investigate the protective mechanism of Ukgan-san (UGS) extracts on hypoxia-induced C6 glial cell death. Methods : The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MMT) assay and cell morphological changes were analysed with microscope after staining with crystal violet (CV). Reactive oxygen species (ROS) formation was assessed by flow cytometer after staining with 2'7'-dichlorofluorescein diacetate (DCF-DA). We also analyzed expression of hypoxia-inducible factor-1 alpha (HIF-$1{\alpha}$) and p53, processing of procaspase-3 and procyclic acidic repetitive protein (PARP) by western blot method. Results : We estimated the elevated cell viability by UGS extract on $CoCl_2$-induced C6 glial cells. UGS attenuated $CoCl_2$-induced ROS formation in C6 glial cells and also showed a protective activity compared to antioxidants and exhibited abrogation of LDH-released by $CoCl_2$. UGS suppressed the typical apoptotic cell death markers, caspase-3 and PARP activation. UGS inhibited $CoCl_2$-induced HIF-1${\alpha}$ expression which is known as a major regulator for hypoxia-induced cell death, and suppressed p53 expression. Conclusions : These results suggest that UGS extract contains protective constituents for hypoxia-induced C6 glial cell death.