• Archives of Pharmacal Research
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• 제28권2호
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• pp.195-202
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• 2005

Free radicals and reactive oxygen species (ROS) caused by UV exposure or other environmental factors are critical players in cellular damage and aging. In order to develop a new antiphotoaging agent, this work focused on the antioxidant effects of the extract of tinged autumnal leaves of Acer palmatum. One compound was isolated from an ethyl acetate soluble fraction of the A. palmatum extract using silica gel column chromatography. The chemical structure was identified as apigenin-8-C-beta-D-glucopyranoside, more commonly known as vitexin, by spectral analysis including LC-MS, FT-IR, UV, $^{1}H-$, and $^{13}C-NMR$. The biological activities of vitexin were investigated for the potential application of its anti-aging effects in the cosmetic field. Vitexin inhibited superoxide radicals by about $70\%$ at a concentration of $100\;{\mu}g/mL$ and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by about $60\%$ at a concentration of $100\;{\mu}g/mL$. Intracellular ROS scavenging activity was indicated by increases in dichlorofluorescein (DCF) fluorescence upon exposure to UVB $20\;mJ/cm^2$ in cultured human dermal fibroblasts (HDFs) after the treatment of vitexin. The results show that oxidation of 5-(6-)chloromethyl-2',7'-dichlo-rodihydrofluorescein diacetate ($CM-H_{2}DCFDA$) is inhibited by vitexin effectively and that vitexin has a potent free radical scavenging activity in UVB-irradiated HDFs. In ROS imaging using a confocal microscope we visualized DCF fluorescence in HDFs directly. In conclusion, our findings suggest that vitexin can be effectively used for the prevention of UV-induced adverse skin reactions such as free radical production and skin cell damage.

• International Journal of Oral Biology
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• 제40권2호
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• pp.103-109
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• 2015

Growing evidence suggests that mitochondrial reactive oxygen species (ROS) are involved in various pain states. This study was performed to investigate whether ROS-induced changes in neuronal excitability in trigeminal subnucleus caudalis are related to ROS generation in mitochondria. Confocal scanning laser microscopy was used to measure ROS-induced fluorescence intensity in live rat trigeminal caudalis slices. The ROS level increased during the perfusion of malate, a mitochondrial substrate, after loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF-DA$), an indicator of the intracellular ROS; the ROS level recovered to the control condition after washout. When pre-treated with phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), malate-induced increase of ROS level was suppressed. To identify the direct relation between elevated ROS levels and mitochondria, we applied the malate after double-loading of $H_2DCF-DA$ and chloromethyl-X-rosamine (CMXRos; MitoTracker Red), which is a mitochondria-specific fluorescent probe. As a result, increase of both intracellular ROS and mitochondrial ROS were observed simultaneously. This study demonstrated that elevated ROS in trigeminal subnucleus caudalis neuron can be induced through mitochondrial-ROS pathway, primarily by the leakage of ROS from the mitochondrial electron transport chain.

• 대한의생명과학회지
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• 제15권2호
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• pp.153-160
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• 2009

Photodynamic therapy(PDT) is a treatment utilizing the generation of singlet oxygen and other reactive oxygen species(ROS), which selectively accumulate in target cells. The aim of present work is to investigate the photodynamic therapy mechanism of 9-HpbD-a-mediated PDT in HeLa cell lines. We studied the general reactive oxygen species(G-ROS) activation after 9-HpbD-a PDT using fluorescence stain with $H_2DCF-DA$. G-ROS activation observed after 9-HpbD-a PDT and higher activation condition was 1 hour after PDT at 0.5 ${\mu}g/ml$ 9-HpbD-a concentration. Sodium azide and reduced glutathione(the singlet oxygen quencher) could protect HeLa cells from cell death induced by 9-HpbD-a PDT. But D-mannitol(the hydroxyl radical scavenger) could not protect cell death. Singlet oxygen played a decisive role in 9-HpbD-a PDT induced HeLa cell death. Type II reaction was the main type of ROS formation at 9-HpbD-a PDT.

• 한국치위생학회지
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• 제14권5호
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• pp.775-781
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• 2014

Objectives : The purpose of the study is to investigate the protective effect of coenzyme $Q_{10}$ on cytotoxicity effect of dental monomers in odontoblast(MDPC-23). Methods : MDPC-23 was incubated with the(co)monomers triethylene glycol dimethacrylate (TEGDMA) with and without addition of coenzyme $Q_{10}$. The cell proliferation and survival was determined using WST-1 assay. The level of reactive oxygen species(ROS) was measured by immunofluorescent staining for DCF-DA. Results : TEGDMA treatment decreased the cell proliferation by dose dependently(0.1, 1, 2.5, 5, 10 mM) on the growth of MDPC-23 cells. Coenzyme $Q_{10}$ showed cell proliferation from 5 to $500{\mu}M$ by WST-1 assay. Pre-treatment coenzyme $Q_{10}$ showed the antioxidant effect on proliferation and viability of MDPC-23 after 48h(p<0.05). The positive cells were observed in non-coenyme $Q_{10}$ treatment group(group 2) in comparison with coenyme $Q_{10}$ pre-treatment group(group 1) by DCF-DA. The fluorescence positive cells showed 14.715(group 1) and 19.788(group 2) using image J system. Conclusions : TEGDMA induced cytotoxicity. The MDPC-23 cell death was associated with the increasing ROS. Coenyme $Q_{10}$ showed the antioxidant effects by decreasing ROS. This effects may contribute to the treatment of periodontal disease induced by TEGDMA after operation.

• The Korean Journal of Physiology and Pharmacology
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• 제21권3호
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• pp.327-334
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• 2017

Epidemiologic interest in particulate matter (PM) is growing particularly because of its impact of respiratory health. It has been elucidated that PM evoked inflammatory signal in pulmonary epithelia. However, it has not been established $Ca^{2+}$ signaling mechanisms involved in acute PM-derived signaling in pulmonary fibroblasts. In the present study, we explored dust particles PM modulated intracellular $Ca^{2+}$ signaling and sought to provide a therapeutic strategy by antagonizing PM-induced intracellular $Ca^{2+}$ signaling in human lung fibroblasts MRC5 cells. We demonstrated that PM10, less than $10{\mu}m$, induced intracellular $Ca^{2+}$ signaling, which was mediated by extracellular $Ca^{2+}$. The PM10-mediated intracellular $Ca^{2+}$ signaling was attenuated by antioxidants, phospholipase blockers, polyADPR polymerase 1 inhibitor, and transient receptor potential melastatin 2 (TRPM2) inhibitors. In addition, PM-mediated increases in reactive oxygen species were attenuated by TRPM2 blockers, clotrimazole (CLZ) and N-(p-amylcinnamoyl) anthranilic acid (ACA). Our results showed that PM10 enhanced reactive oxygen species signal by measuring DCF fluorescence and the DCF signal attenuated by both TRPM2 blockers CLZ and ACA. Here, we suggest functional inhibition of TRPM2 channels as a potential therapeutic strategy for modulation of dust particle-mediated signaling and oxidative stress accompanying lung diseases.

• 생명과학회지
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• 제28권5호
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• pp.532-539
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• 2018

백모 발생은 연령이 증가함에 따라 모낭에 있는 melanocyte 수가 감소됨과 동시에 세포활성이 저하되고 동시에 과산화수소($H_2O_2$)가 축적되어 melanin 합성이 감소되어 진행된다. 본 연구의 목적은 흑미 주정추출물인 Black oryzasativa ethanolic extract (BLEE)가 백모에서 흑모로 전환되는데 관련되어 있는 melanin 합성에 어떠한 영향을 미치는지 알아보고자 하였다. 본 연구에서 사용된 BLEE는 DPPH radical scavenging assay 및 환원력 실험에서 $64{\mu}g/ml$에서 항산화 효과가 있는 것으로 나타났으며, tyrosinase 활성 및 melanin 생성 촉진효과는 $16{\mu}g/ml$ 이상에서 효과가 나타났다. DCF fluorescence assay에서 세포내 $H_2O_2$를 소거하는 효과가 있는 것으로 나타났고, $H_2O_2$를 처리한 세포에서 melanin 생성 촉진효과가 있는 것으로 나타났다. 그러나 Western blot 분석에서는 catalase의 발현을 감소시키는 것으로 나타났으며 melanin 생성에 관여하는 tyrosinase, TRP-1, TRP-2, 효소 및 MITF 전사인자의 발현촉진에는 영향이 없는 것으로 나타났다. 그러므로 BLEE의 melanin 생성 촉진효과는 tyrosinase 활성 증가와 세포내 $H_2O_2$의 생성을 감소시켜 melanin 생성을 촉진시키는 것으로 나타났다. 결론적으로 BLEE는 melanin 생성을 촉진시키는데 있어서 중요한 역할을 하고 있는 것으로 확인되었으며, 본 연구에서 개발된 BLEE는 흑모성장과 연관이 있는 melanin생성 촉진과 관련된 모발화장품 개발분야에서 하나의 잠재적인 기능성 소재로 응용 될 수 있다는 것을 시사하고 있다.

• International Journal of Oral Biology
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• 제38권1호
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• pp.5-12
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• 2013

Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ($[Ca^{2+}]_i$) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF$-DA). NaOCl-induced depolarization was not blocked by pretreatment with external $Ca^{2+}$ free solution or by the addition of nifedifine. However, when slices were pretreated with the $Ca^{2+}$ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the $Ca^{2+}$-sensitive fluorescence dye fura-2, the $[Ca^{2+}]_i$ was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.

• 한국수정란이식학회지
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• 제32권3호
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• pp.249-255
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• 2017

In this experiment, we determined the effect of curcumin supplementation in freezing buffer for miniature pig sperm cryopreservation. Each ejaculate was diluted with modified Modena B extender and mixed with lactose-egg yolk (LEY extender, 80% v/v lactose solution [310 mM], 20% v/v egg yolk, and $100{\mu}g/mL$ kanamycin sulfate) and LEY-glycerol Orvus ES Paste (LEYGO, 89.5% v/v LEY, 5% v/v glycerol, 1.5% v/v Orvus ES Paste), 100 mM trehalose supplemented with 0, 10, 50, 100, and $500{\mu}M$ of curcumin from turmeric, respectively. Following equilibration, the 0.5 mL French straws were frozen and plunged into $LN_2$ tank for 7 days at least. Sperm parameter and oxidative byproducts were determined by the computer assisted sperm motility analysis (CASA) and fluorescence-activated cell sorting (FACS) as compared with each groups. Supplementation of curcumin had no effect on sperm motility, progressive motility and curvilinear velocity. However, average-path velocity and straight-line velocity were significantly higher in $10{\mu}M$ curcumin group ($100.9{\pm}8.8{\mu}m/s$, $61.7{\pm}2.9{\mu}m/s$, respectively) than control group ($77.8{\pm}3.9{\mu}m/s$, $46.4{\pm}3.0{\mu}m/s$, respectively) (p < 0.05). In addition, the level of the O2 radical and H2O2 were comparatively decreased in curcumin groups by evaluation of ethidium and DCF fluorescence. According to the results, curcumin can improve sperm kinetic variables and alleviate ROS induced cryoinjury to pig sperm.

• Nutrition Research and Practice
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• 제9권6호
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• pp.586-591
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• 2015

BACKGROUND/OBJECTIVES: Reactive oxygen species (ROS) formation is closely related to miconazole-induced heart dysfunction. Although rhamnetin has antioxidant effects, it remained unknown whether it can protect against miconazole-induced cardiomyocyte apoptosis. Thus, we investigated the effects of rhamnetin on miconazole-stimulated H9c2 cell apoptosis. MATERIALS/METHODS: Cell morphology was observed by inverted microscope and cell viability was determined using a WelCount$^{TM}$ cell proliferation assay kit. Miconazole-induced ROS production was evaluated by fluorescence-activated cell sorting with 6-carboxy-2',7'-dichlorofluoroscein diacetate ($H_2DCF$-DA) stain. Immunoblot analysis was used to determine apurinic/apyrimidinic endonuclease 1 (APE/Ref-1) and cleaved cysteine-aspartic protease (caspase) 3 expression. NADPH oxidase levels were measured using real-time polymerase chain reaction. RESULTS: Miconazole (3 and $10{\mu}M$) induced abnormal morphological changes and cell death in H9c2 cells. Rhamnetin enhanced the viability of miconazole ($3{\mu}M$)-treated cells in a dose-dependent manner. Rhamnetin (1 and $3{\mu}M$) treatment downregulated cleaved caspase 3 and upregulated APE/Ref-1 expression in miconazole-stimulated cells. Additionally, rhamnetin significantly reduced ROS generation. CONCLUSIONS: Our data suggest that rhamnetin may have cytoprotective effects in miconazole-stimulated H9c2 cardiomyocytes via ROS inhibition. This effect most likely occurs through the upregulation of APE/Ref-1 and attenuation of hydrogen peroxide levels.

• 생명과학회지
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• 제18권3호
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• pp.311-317
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• 2008

Pathophysiological oxidative stress results in neuronal cell death mainly due to the generation reactive oxygen species (ROS). In low oxygen situation such as hypoxia and ischemia, excessive ROS is generated. Coptidis Rhizoma (CR) is a traditional medicine used for the incipient stroke. In this report we show that CR water extracts $(1\;{\mu}g/ml)$ exhibited protective effects of neuronal cell death in a hypoxic model (2% $O_2/5%\;CO_2,\;37^{\circ}C,$ 3 hr) of cultured rat cortical cells. We further show that CR water extracts significantly reduced the intensity of green fluorescence after staining with $H_2DCF-DA$ on one hour and three days after hypoxic shock and in normoxia as well. Our results indicate that CR water extracts prevent neuronal death by suppressing ROS generation.