• Korean Journal of Environmental Biology
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• v.27 no.1
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• pp.66-72
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• 2009

Diabetic Retinopathy (DR) is a leading cause of blindness among adults in the western countries. Hyperglycemia is a condition, that induces apoptotic cell death in a variety of cell types in diabetes, but the mechanism remains unclear. The aim of the study is to understand the effects of high Glucose on Human Retinal Endothelial Cells. Retinal endothelial cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 5, 25 and 50 mM Glucose, incubated for 24, 36 and 48 hours in humidified 5 % CO$_2$ incubator at 37$^{\circ}C$. Human Retinal Endothelial Cell Line (HREC) were characterized for morphology with different treatment by phase contrast microscopic analysis. Number of dead and viable cells was counted by trypan blue exclusion and supported by MTT assay. The intracellular Hydrogen peroxide (H$_2$O$_2$), a Reactive Oxygen Species (ROS) generation in high glucose conditions was assessed by FOX II assay and apoptosis by caspase-3 assay. The high glucose treated cells undergoing DNA fragmentation was witnessed by Agarose gel electrophoresis. We found that the cells incubated with 25 and 50 mM glucose containing medium for 48 hours altered the morphology of the cell, induced apoptosis and DNA fragmentation. The dead cell number were high in 25 and 50 mM when compared to the cells incubated with 5 mM glucose for 24, 36, and 48 hours. Also, the H$_2$O$_2$ levels and the activity of caspase-3 were increased in high glucose treated cells. Conclusions/interpretation: Our results demonstrated that elevated glucose induces apoptosis in cultured HREC. The hyperglycemia-induced increase in apoptosis may be dependent on caspase activation. The association between ROS generation and caspase-3 activation on high glucose treated cells is yet to be investigated.

• Journal of the Korean Society of Food Science and Nutrition
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• v.44 no.10
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• pp.1458-1469
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• 2015

This study was carried out to investigate the nitrite scavenging activities (NSA) of nine kinds of wild vegetables in a $NaNO_2$ model system and nitrite of Chinese cabbage as well as the inhibitory effect of kimchi containing a mixture of wild vegetables (MWV) with nitrite scavenging activity on brain neuronal cell death. NSA was higher at pH 1.2 than pH 4.2 in all samples. NSA of extracts from sprouts of Oenothera laciniata and Aster scaber (AS) was above 90% at pH 1.2. AS, Codonopsis lanceolate (CL), Adenophora triphylla (AT), Platycodon grandiflorum (PG), and Taraxacum officinale (TO) extracts showed significantly higher levels of NSA than those from other extracts at pH 4.2. CL, AT, PG, and TO extracts showed high NSA on nitrite of Chinese cabbage. In addition, the effects of MWV on antioxidant and brain neuronal cell death induced by oxidative stress were investigated in human brain neuroblastoma SK-N-SH cells. MWV extract attenuated $H_2O_2$-induced cell death and reactive oxygen species (ROS) generation in SK-N-SH cells. MWV extract showed significantly higher DPPH radical scavenger activity when compared to normal kimchi extract. MWV extract showed an inhibitory effect on brain neuronal cell death against oxidative stress by antioxidant activities.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.2
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• pp.529-541
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• 2003

Yukmijihwang-tang has been widely used as an and-aging herbal medicine for hundred years in Asian countries. Numerous studies show that Yukmijihwangtang has anti-oxidative effect both in vivo and in vitro. It has been reported that Moutan Cortex Radicis extract (MCR) was the most effective herb in Yukmijihwang-tang on undifferentiated PC12 cells upon oxidative-stressed with hydrogen peroxide. The purpose of this study is to; 1) evaluate the recovery of neuronal damage by assessing the anti-oxidant effect of MCR on PC12 cells differentiated with nerve growth factor (NGF), 2) identify candidate genes responsible for anti-oxidative effect on differentiated PC12 cells by oligonucleotide chip microarray. PC12 cells, which were differentiated by treating with NGF, were treated without or with hydrogen peroxide in the presence or absence of various concentration of MCR. Cell survival was determined by using MTS assay. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2DCFDA assay The viability of cells treated with MCR was significantly recovered from stressed PC12 cell. In addition, wide rage of concentrations of MCR shows dose-dependent inhibitory effect on ROS production in oxidative-stressed cells. Total RNAs of cells without treatment(Control group), only treated with H₂O₂ (stressed group) and treated with both H₂O₂ and of MCR (MCR group) were isolated, and cDNAs was synthesized using oligoT7(dT) primer. The fragmented cRNAs, synthesized from cDNAs, were applied to Affymetrix GeneChip Rat Neurobiology U34 Array. mRNA of Calcium/calmodulin-dependent protein kinase II delta subunit(CaMKII), neuron glucose transporter (GLUT3) and myelin/oligodendrocyte glycoprotein(MOG) were downregulated in Stressed group comparing to Control group. P2X2-5 receptor (P2X2R-5), P2X2-4 receptor (P2X2R-4), c-fos, 25 kDa synaptosomal attachment protein(SNAP-25a) and GLUT3 were downregulated, whereas A2 adenosine receptor (A2AR), cathechol-O-methyltransferase(COMT), glucose transporter 1 (GLUT1), EST223333, heme oxygenase (HO), VGF, UI-R-CO-ja-a-07-0-Ul.s1 and macrophage migration inhibitory factor (MIF) were upregulated in MCA group comparing to Control group. Expression of Putative potassium channel subunit protein (ACK4), P2X2A-5, P2X2A-4, Interferon-gamma inducing factor isoform alpha precursor (IL-18α), EST199031, P2XR, P2X2 purinoceptor isoform e (P2X2R-e), Precursor interleukin 18 (IL-18) were downregulated, whereas MOO, EST223333, GLUT-1, MIF, Neuronatin alpha, UI-R-C0-ja-a-07-0-Ul.s1, A2. adenosine receptor, COMT, neuron-specific enolase (NSE), HO, VGF, A rat novel protein which is expressed with nerve injury (E12625) were upregulated in MCR group comparing to Stressed group. The results suggest that decreased viability and AOS production of PC12 cell by H₂O₂ may be, at lease, mediated by impaired glucose transporter expression. It is implicated that the MCR treatment protect PC12 cell from oxidative stress via following mechanisms; improving glucose transport into the cell, enhancing expression of anti-oxidative genes and protecting from dopamine cytotoxicity by increment of COMT and MIF expression. The list of differentially expressed genes may implicate further insight on the action and mechanism behind the anti-oxidative effects of herbal extract Moutan Cortex Radicis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.8
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• pp.979-984
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• 2006

Although iron is essential for many physiological processes, excess iron can lead to tissue damage by promoting the generation of reactive oxygen species (ROS). There is increasing evidence that ROS might play an important role in the pathogenesis of cardiovascular disease. However, the effects of iron excess on platelet function and the thrombotic response to vascular injury are not well understood. We examined the effects of iron excess-induced oxidative stress and the antioxidants on platelet aggregation. Oxidative stress was accessed by either free iron $(Fe^{+2})$ or hydrogen peroxide $(H_2O_2)$, as well as their combination on washed rabbit platelets (WPs) in vitro. When WPs were stimulated with either $Fe^{+2}$ alone or a subthreshold concentration of collagen, which gave an aggregatory curve with a little effect, and a dose dependent increase in platelet aggregation was observed by increasing concentrations of $Fe^{+2}$ with $H_2O_2$. This aggregation was associated with the iron-catalyzed formation of hydroxyl radicals from $H_2O_2$, and were inhibited by NAD/NADP (proton acceptor), catalase $(H_2O_2\;scavenger)$, tiron (iron chelator), mannitol (hydroxyl radical scavenger), and indomethacin (cyclooxygenase inhibitor), but not by NADH/NADPH (proton donor), superoxide mutase, and aspirin. However, NADH/NADPH, an essential cofactor for the antioxidant capacity by the supply of reducing potentials, showed the effect of an enhanced radical formation, suggesting a role for NADH/NADPH-dependent oxidase. These results suggest that iron $(Fe^{+2})$ can directly interact with washed rabbit platelets and this aggregation be mediated by OH formation as in the Fenton reaction, inhibited by radical scavengers.

• Journal of Nutrition and Health
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• v.54 no.6
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• pp.618-630
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• 2021

Purpose: The ginger rhizome (Zingiber officinale) is widely cultivated as a spice for its aromatic and pungent components. One of its constituents, 6-hydroxydopamine (6-OHDA) is usually thought to cross the cell membrane through dopamine uptake transporters, and induce inhibition of mitochondrial respiration and the generation of intracellular reactive oxygen species (ROS). This study examines the neuroprotective effect and acetylcholinesterase (AChE) inhibitory activity of fermented ginger extracts (FGEs) on 6-OHDA induced toxicity in SH-SY5Y human neuroblastoma cells. Methods: Ginger was fermented using 2 species of Bacillus subtilis, with or without enzyme pretreatment. Each sample was extracted with 70% ethanol. Neurotoxicity was assessed by applying the EZ-Cytox cell viability assay and by measuring lactic dehydrogenase (LDH) release. Morphological changes of apoptotic cell nuclei were observed by Hoechst staining. Cell growth and apoptosis of SH-SY5Y cells were determined by Western blotting and enzyme activity analysis of caspase-3, and AChE enzymatic activity was determined by the colorimetric assay. Results: In terms of cell viability and LDH release, exposure to FGE showed neuroprotective activities against 6-OHDA stimulated stress in SH-SY5Y cells. Furthermore, FGE reduced the 6-OHDA-induced apoptosis, as determined by Hoechst staining. The occurrence of apoptosis in 6-OHDA treated cells was confirmed by determining the caspase-3 activity. Exposure to 6-OHDA resulted in increased caspase-3 activity of SH-SY5Y cells, as compared to the unexposed group. However, pre-treatment with FGE inhibited the activity of caspase-3. The neuroprotective effects of FGE were also found to be caspase-dependent, based on reduction of caspase-3 activity. Exposure to FGE also inhibited the activity of AChE induced by 6-OHDA, in a dose-dependent manner. Conclusion: Taken together, our results show that FGE exhibits a neuroprotective effect in 6-OHDA treated SH-SY5Y cells, thereby making it a potential novel agent for the prevention or treatment of neurodegenerative disease.

• Journal of Life Science
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• v.29 no.11
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• pp.1273-1280
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• 2019

Age-related macular degeneration (AMD) is one of the leading causes of blindness in the elderly population, and damage to retinal pigment epithelial (RPE) cells due to oxidative stress contributes to the development of AMD. Glycyrrhiza uralensis Fischer is one of the most widely used herbal medicines for the treatment of various diseases in Asian countries. Although recent studies indicated that treatment with G. uralensis can protect cells from oxidative stress, its mechanisms in RPE cells remain unknown. We evaluated the effect of a G. uralensis ethanol extract (GU) on $H_2O_2$-induced oxidative injury in ARPE-19 RPE cells. The GU pretreatment attenuated reactive oxygen species (ROS) generation induced by $H_2O_2$, which was associated with induced expression of nuclear factor erythroid-derived-2-like 2 (Nrf2) and heme oxygenase-1 (HO-1). GU also suppressed $H_2O_2$-induced DNA damage and mitochondrial dysfunction. The inhibitory effect of GU on $H_2O_2$-induced apoptosis was associated with the protection of caspase-3 activation. Overall, GU appeared to protect RPE cells from oxidative injury by inhibiting DNA damage and reducing apoptosis. Further studies are needed to determine the regulation of Nrf2-mediated HO-1 expression, but our results suggest the possibility of using GU to reduce the risk of AMD.