• Journal of Periodontal and Implant Science
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• 제34권4호
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• pp.807-818
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• 2004

산화질소는 생리적 농도에서 세포내 신호전달자로 작용하지만 높은 농도에서는 세포독성을 일으킨다. 최근 치은 섬유아세포와 치주인대 섬유아세포는 산화질소 합성효소를 가지고 있고 세균의 lipopolysaccharide나 cytokine에 의해 대량의 높은 농도의 산화질소가 합성된다는 보고가 있음에도 지금까지 치은 조직에서 산화질소의 세포독성에 대한 연구는 아직 이루어 지지않고 있다. 본 연구는 사람의 치은 섬유아세포에서, 산화질소유도세포 고사기전을 밝히는데 목적이 있다. 세포 생장력은 MTT 방법으로 측정하였고, 세포의 형태적 변화는 Diff-Quick 염색법으로 조사하였다. Bcl-2 famly와 Fas 발현 정도는 RT-PCR 방법에 의해 확인하였으며, caspase-3, -8 와 -9의 활성은 spectrophotometer로 reactive oxygen species (ROS)는 형광분광계에 의해 측정되었다. 미토콘드리아에서 세포질로 분비된 cytochrome c는 western blot으로 조사하였다. 산화질소 유리제인 sodium nitroprusside (SNP) 처리는 사람 섬유아세포의 생존률을 시간과 농도 의존적으로 감소시켰고, 세포용적축소, 염색사 용축, DNA 절편화를 일으켰다. 또한, SNP 처리로 미토콘드리아에서 세포질로 유리되는 cytochrome c 양이 증가되었고, caspase-9 과 caspase-3 의 활성이 증가되었다. 한편, SNP 처리에 의해 death receptor 구성요소인 Fas 발현이 증가되었고, caspase-8의 활성이 증가되었다. Bcl-2 family 에 대한 RT-PCR 분석결과, 세포고사를 억제하는 Bcl-2 발현은 감소되었으나 세포고사를 자극하는 Bax와 Bid의 발현은 증가되었다. Soluble guanylate cyclase 억제제인 ODQ는 SNP에 의한 세포 생존율 감소를 차단하지 못했다. 따라서, 본 실험의 결과들은 사람 섬유아세포에서 산화질소유도 세포고사에 Bcl-2 family나 ROS가 매개하는 미토콘드리아 의존 및 death receptor 의존 세포고사기전이 관여함을 시사하였다.

• 한국환경과학회지
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• 제20권12호
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• pp.1509-1519
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• 2011

The protective effect of nitric oxide (NO) on the antioxidant system under paraquat(PQ) stress was investigated in leaves of 8-week-old lettuce (Lactuca sativa L.) plants. PQ stress caused a decrease of leaf growth including leaf length, width and weight. Application of NO donor, sodium nitroprusside (SNP), significantly alleviated PQ stress induced growth suppression. SNP permitted the survival of more green leaf tissue preventing chlorophyll content reduction and of higher quantum yield for photosystem II than in non-treated controls under PQ exposure, suggesting that NO has protective effect on chloroplast membrane in lettuce leaves. Flavonoids and anthocyanin were significantly accumulated in the leaves upon PQ exposure. However, the rapid increase of these compounds was alleviated in the SNP treated leaves. PQ treatment resulted in lipid peroxidation and induced accumulation of hydrogen peroxide ($H_2O_2$) in the leaves, while SNP prevented PQ induced increase in malondialdehyde (MDA) and $H_2O_2$. These results demonstrate that SNP serves as an antioxidant agent able to scavenge $H_2O_2$ to protect plant cells from oxidative damage. The activities of two antioxidant enzymes that scavenge reactive oxygen species, superoxide dismutase (SOD) and catalase (CAT) in lettuce leaves in the presence of NO donor under PQ stress were higher than those under PQ stress alone. Application of 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, to the lettuce leaves arrested SNP mediated protective effect on leaf growth, photosynthetic pigment and antioxidant systems. However, PTIO had little effect on lettuce leaves under PQ stress compared with that of PQ stress alone. The obtained data suggest that the damage caused by PQ stress is in part due to increased generation of active oxygen by maintaining increased antioxidant enzyme activities and SNP protects plants from oxidative stress. From these results it is suggested that NO might act as a signal in activating active oxygen scavenging system that protects plants from oxidative damage induced by PQ stress and thus confer PQ tolerance.

• 대한한방내과학회지
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• 제21권1호
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• pp.46-54
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• 2000

Objectives : Talmyung-san(TMS) has been used for treatment of brain diseases in Chinese traditional medicine. However, little is known about the mechanism by which TMS rescues brain cells from ischemic damages. To elucidate the protective mechanisms of TMS, we execute experiments. Methods : The effects of TMS on ischemia/reperfusion-induced cytotoxicity and generation of nitric oxide(NO) are investigated in primary neonatal myocardial cells and A7rS, aortic smooth muscle cell line. Results : Ischemia/reperfusion itself induces severe myocardial cell death in vitro. However, treatment of the cells with TMS significantly reduces both ischemia/reperfusion-induced myocardial cell death and LDH release. In addition, pretreatment of TMS before reperfusion recovers the lose of beating rates alter ischemia/reperfusion. For a while, the water extract of TMS stimulates myocardial cells to produce NO in a dose dependent manner and it protects the damage of ischemia/reperfusion-induced myocardial cells. Furthermore, the protective effects of the water extract of TMS is mimicked by treatment of sodium nitroprusside, an exogenous NO donor. NG-monomethyl-L-arginine (NGMMA), a specific inhibitor of nitric oxide synthase(NOS), significantly blocks the protective effects of TMS on the cells after ischemia/reperfusion. In addition, on ischemia the water extract of TMS induce NO in A7r5 cell. Conclusions : Taken together, we suggest that the protective effects of TMS against ischemia/reperfusion-induced myocardial damages may be mediated by NO production of myocardial and vascular smooth muscle cell during ischemic condition.

• 한국식품영양과학회지
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• 제43권5호
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• pp.668-674
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• 2014

채소즙이란 생채소를 마쇄하여 인체가 영양소를 흡수하기 쉬운 상태로 제조된 즙으로 생채소의 영양 섭취 효율을 높일 수 있는 식품이라 할 수 있다. 본 연구에서는 재배방법에 따라 생산된 유기농 및 일반농 케일을 착즙하여 다양한 실험을 통하여 항산화 활성을 비교하였다. DPPH radical, NO, $O_2{^-}$, ${\cdot}OH$ 라디칼 소거능에서 유기농 케일 착즙액은 일반농케일 착즙액보다 더 우수한 효과를 나타내었다. LLC-PK1 세포를 이용하여 산화적 스트레스 개선효과에서 유기농 케일 착즙액은 일반농 케일 착즙액에서보다 NO, $O_2{^-}$와 $ONOO^-$에 의해 유발된 산화적 스트레스에 대한 세포 생존율을 증가시키고, 지질과산화물을 억제시켜 유리라디칼에 대한 보호효과를 유의적으로 나타내었다. 위의 결과로 보아 유기농케일 착즙액은 일반농 케일 착즙액보다 산화적 스트레스에 대한 개선효과가 뛰어난 것으로 사료되며 재배방법의 차이에 따라 그 효과도 차이가 있었다.

• IMMUNE NETWORK
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• 제6권3호
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• pp.117-122
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• 2006

Background: Caveolin, a family of integral membrane proteins are a principal component of caveolae membranes. In this study, we investigated the effect of p38 kinase on differentiation and on inflammatory responses in sodium nitroprusside (SNP)-treated chondrocytes. Methods: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. SNP was used as a nitric oxide (NO) donor. In this experiments measuring SNP dose response, primary chondrocytes were treated with various concentrations of SNP for 24h. The time course of the SNP response was determined by incubating cells with 1mM SNP for the indicated time period $(0{\sim}24h)$. The cyclooxygenase-2 (COX-2) and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin $E_2\;(PGE_2)$ assay was used to measure the COX-2 activity. The tyrosine phosphorylation of caveolin-1 was determined by immunoblot analysis and immunostaining. Results: SNP treatment stimulated tyrosine phosphorylation of caveolin-1 and activation of p38 kinase. SNP additionally caused dedifferentiation and inflammatory response. We showed previously that SNP treatment stimulated activation of p38 kinase and ERK-1/-2. Inhibition of p38 kinase with SB203580 reduced caveolin-1 tyrosine phosphorylation and COX-2 expression but enhanced dedifferentiation, whereas inhibition of ERK with PD98059 did not affect caveolin-1 tyrosine phosphorylation levels, suggesting that ERK at least is not related to dedifferentiation and COX-2 expression through caveolin-1 tyrosine phosphorylation. Conclusion: Our results indicate that SNP in articular chondrocytes stimulates dedifferentiation and inflammatory response via p38 kinase signaling in association with caveolin-1 phosphorylation.

• International Journal of Oral Biology
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• 제39권4호
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• pp.229-236
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• 2014

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions ($O{_2}^{{\cdot}_-}$) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of $O{_2}^{{\cdot}_-}$ and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP ($10{\mu}M$) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-${\beta}$-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.

• Journal of Korean Neurosurgical Society
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• 제30권9호
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• pp.1059-1064
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• 2001

Objective : 6R-Tetrahydrobiopterin(BH4) is a cofactor for the aromatic amino acid hydroxylases which is essential for the biosynthesis of catecholamines and serotonin. It also acts as a cofactor for nitric oxide synthase, and stimulates the release of some neurotransmitters such as dopamine, serotonin, acetylcholine and glutamate. Recently, it has been reported that BH4 could induce cellular proliferation and enhance neuronal survival. This study was performed to investigate the antioxidative effect of BH4 on the various oxidative insults in mouse cerebral cortical cell cultures. Methods : Iron ion(FeCl2), zinc ion(ZnCl2), sodium nitroprusside(SNP) and buthionine sulfoximine(BSO, a glutathione depletor) were used as oxidants. Cell death was assessed by measurement of lactate dehydrogenase efflux to bathing media at the end of exposure. Result : All 4 oxidants induced neuronal cell death associated with cell body swelling, which was markedly inhibited by trolox($100{\mu}M$), a vitamin E analog. BH4($10-100{\mu}M$) markedly inhibited the neuronal cell death induced by all 4 oxidants($20{\mu}M\;Cu^{2+}$, $20{\mu}M\;Zn^{2+}$, $1{\mu}M$ SNP or 1mM BSO). However, BH4 failed to inhibit the neuronal cell death induced by 24hr exposure to $20{\mu}M$ NMDA. Conculsion : These results suggest that BH4 has antioxidative action independently of any actions of enzyme cofactor.

• 대한의생명과학회지
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• 제7권4호
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• pp.181-190
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• 2001

This study was designed to investigate the effects of reactive oxygen species (ROS) on DNA stability in human spermatozoa. To verify human spermatozoa were incubated with xanthine-xanthine oxidase (X 100$\mu$M-XO 50 mlU ~ 400 mIU), $H_2O_2$ (125 $\mu$M ~ 1 mM), sodium nitroprusside (SNP 0.1 $\mu$M ~ 100 $\mu$M) or lymphocyte. Otherwise, spermatozoa were incubated under low $O_2$ (5%) condition. Damage of sperm DNA was analyzed by single cell electrophoresis (Comet assay) and flow cytometry after acridine orange staining. In the presence of ROS, there was increase in DNA damage. The rate of DNA single strand breakage (9.0$\pm$1.0% ~ 46.0$\pm$4.6%) and DNA fragmentation (7.51$\pm$1.0% ~ 29.5$\pm$4.6%) were similar regardless of the kinds of ROS and exposure time. DNA damage in the lower $O_2$ condition (5%) was lower than ambient $O_2$ condition (20%). Taken together, it suggested that sperm DNA might be damaged by ROS. In the presence of ROS, increase in DNA damage and chromatin instability was obvious in spite of short exposure. Although present study reconfirmed that sperm incubation in the low concentration of ROS have the benefit m the induction of capacitation and Ah, the increase in DNA damage by ROS and possible genetic problem should be considered before the human trials.

• Plant Biotechnology Reports
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• 제2권2호
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• pp.113-122
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• 2008

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and $N{\omega}-nitro-{\text\tiny{L}}-arginine$ methyl ester hydrochloride (${\text\tiny{L}}-NAME$), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of $O_2{^{{\cdot}-}}$, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and $O_2{^{{\cdot}-}}$ anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of $H_2O_2$ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of $O_2{^{{\cdot}-}}$ by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in $O_2{^{{\cdot}-}}$ generation through NADPH oxidase and subsequent root growth is discussed.

• Natural Product Sciences
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• 제20권3호
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• pp.216-225
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• 2014

The present study was designed to investigate whether ethylacetate (EtOAc) fraction extracted from Rubus coreanum affect the contractility of the isolated thoracic aortic strips and blood pressure of normotensive rats. The EtOAc fraction ($400{\mu}g/mL$) significantly depressed both phenylephrine (PE, $10{\mu}M$)- and high $K^+$ (56 mM)-induced contractile responses of the isolated thoracic aortic strips in a concentration-dependent fashion. In the simultaneous presence of L-NAME (an inhibitor of NO synthase, $300{\mu}M$) and EtOAc ($400{\mu}g/mL$), both PE- and high $K^+$-induced contractile responses were recovered to the corresponding control level in comparison with inhibition of EtOAc-treatment alone. Moreover, in the simultaneous presence of EtOAc after pretreatment with 0.4% CHAPS, both PE- and high $K^+$-induced contractile responses were recovered to the corresponding control level compared to the inhibitory response of EtOAc-treatment alone. Also, in anesthetized rats, EtOAc fraction (0.3~3.0 mg/kg) injected into a femoral vein dose-dependently produced depressor responses. This hypotensive action of EtOAc fraction was greatly inhibited after treatment with phentolamine (1 mg/kg), chlorisondamine (1 mg/kg), L-NAME (3 mg/kg/30 min) or sodium nitroprusside ($30{\mu}g/kg/30 min$). Intravenous infusion of EtOAc fraction (1.0~10.0 mg/kg/30 min) markedly inhibited norepinehrine-induced pressor responses. Taken together, these results demostrate that EtOAc causes vascular relaxation in the isolated rat thoracic aortic strips as well as hypotensive action in anesthetized rats. These vasorelaxation and hypotension of EtOAc seem to be mediated at least by the increased NO production through the activation of NO synthase of vascular endothelium, and the inhibitory adrenergic modulation.