• 한국수정란이식학회지
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• 제30권4호
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• pp.289-298
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• 2015

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals ($H_2O_2$). Reactive oxygen species (ROS) such as $H_2O_2$ can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda ($10{\mu}M$), $H_2O_2$ ($200{\mu}M$), and Eda+$H_2O_2$ treated group, respectively. Rate of blastocyst development was significantly increased (P<0.05) in the Eda treated group compared with only $H_2O_2$ treated group. And, we measured intracellular levels of ROS by DCF-DA staining methods and investigated numbers of apoptotic nuclei by TUNEL assay analysis is in porcine blastocyst, respectively. Both intracellular ROS levels and the numbers of apoptotic nucleic were significantly decreased (P<0.05) in porcine blastocysts cultured with Eda ($10{\mu}M$). More over, the total cell number of blastocysts were significantly increased (P<0.05) in the Eda-treated group compared with untreated group and the only $H_2O_2$ treated group. Based on the results, Eda was related to regulate as antioxidant-like function according to the reducing ROS levels during preimplantation periods. Also, Eda is beneficial for developmental competence and preimplantation quality of porcine embryos. Therefore, we concluded that Eda has protective effect to ROS derived apoptotic stress in preimplantation porcine embryos.

• 생명과학회지
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• 제23권8호
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• pp.1057-1063
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• 2013

진피(Citri Pericarpium)의 항암작용 기전 해석을 위하여 U937 백혈병 세포의 apoptosis 유발에 미치는 메탄올 추출물(EMCP)의 영향을 조사하였으며, apoptosis 조절에 중요한 몇 가지 유전자들의 발현 및 활성 변화, ROS의 생성 변화를 조사하였다. EMCP 처리에 의한 U937 세포의 증식 억제는 apoptosis 유도와 연관성이 있음을 DAPI 염색을 통한 apoptotic body 출현의 증가 및 Flow cytometry 분석에 의한 Sub-G1기 세포 빈도의 증가로 확인하였다. EMCP 처리에 의한 apoptosis 유도에서 Bax 발현 증가, caspases의 활성 및 PARP의 단편화 등이 동반되었으며, ROS 생성의 증가와 연관성이 있었다. 산화적 손상에 대해 세포나 조직을 보호하는 역할을 하는 것으로 알려진 세포 내 항산화 효소인 HO-1의 발현이 EMCP의 처리에 의해 증가되었으나 ROS 생성 억제제인 NAC의 전처리에 의해 감소된 HO-1의 발현은 전사인자인 Nrf2의 핵으로의 이동 억제와 관련되어 있었다. 이상의 결과에서 EMCP 처리에 의한 U937 세포의 apoptosis 유발에는 ROS 생성의 증가와 pNrf2에 의해 조절되는 HO-1의 발현 증가가 중요한 기전으로 작용한다는 것을 알 수 있었다. 이러한 자료는 진피의 항암기전 해석을 이해하는데 중요한 기초자료로서 활용될 수 있을 것으로 생각된다.

• 대한화장품학회지
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• 제31권3호
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• pp.265-271
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• 2005

Citrus essential oil (bergamot, grapefruit, lemon, mandarin, petigrain)이 B16 melanoma 세포에서 melanin 생성에 미치는 영향과 RBL 2H3 비만세포에서 ROS 생성에 미치는 영향을 관찰하였다 5 종류의 citrus essential oil은 DPPH 라디칼 소거와 세포 증식 및 독성에서 대조군에 비해 유의한 변화를 일으키질 않았다. 정제한 tyrosinase 활성에 있어서 mandarin과 petigrain essential oil은 유의하게 tyrosinase 활성을 억제하였으나 bergamot은 전혀 억제하지 않았다. 이러한 결과는 mandarin과 petigrain essential oil이 tyrosinase 효소 억제 기전은 설명하지 못하지만 tyrosinase 효소에 직접적으로 작용하여 억제하는 것으로 보인다. B16 melanoma 세포에서 MSH에 의한 melanin 생성이 5 종류의 citrus essential oil에 의해 모두 농도 의존적으로 억제되었다. Bergamot essential oil은 tyrosinase 효소 활성을 직접적으로 억제하지는 못했지만 melanoma 세포에서 MSH에 의한 melanin 생성은 억제하는 것으로 나타났다. 다른 4종류의 citrus essential oil도 모두 tyrosinase 효소 활성을 억제하였으며, MSH에 의한 melanin 생성도 억제하였다. 한편 DCF-DA를 이용한 ROS 생성에 있어서는 madarin essential oil은 ROS 생성에 이렇다 할 영향을 주지 않았지만 다른 4개의 essential oil (Bergamot, Grapefruit, Lemon, Petigrain)은 농도 의존적으로 ROS 생성을 증가시켰다. 이상의 결과를 종합하여 볼 때 citrus essential oil은 MSH에 의한 melanin 생성을 억제하는 것으로 보아 미백제로서의 개발 가능성이 있는 것으로 사료된다.

• International Journal of Oral Biology
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• 제31권4호
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• pp.127-133
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• 2006

[ $H_2O_2$ ], a member of reactive oxygen species (ROS), is known to be involved in the mediation of physiological functions in a variety of cell types. However, little has been known about the physiological role of $H_2O_2$ in exocrine cells. Therefore, in the present study, the effect of $H_2O_2$ on cholecystokinin (CCK)-evoked $Ca^{2+}$ mobilization and amylase release was investigated in rat pancreatic acinar cells. Stimulation of the acinar cells with sulfated octapeptide form of CCK (CCK-8S) induced biphasic increase in amylase release. Addition of $30\;{\mu}M\;H_2O_2$ enhanced amylase release caused by 10 pM CCK-8S, but inhibited the amylase release induced by CCK-8S at concentrations higher than 100 pM. An ROS scavenger, $10\;{\mu}M$ Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, increased amylase release caused by CCK-8S at concentrations higher than 100 pM, although lower concentrations of CCK-8S-induced amylase release was not affected. To examine whether the effect of $H_2O_2$ on CCK-8S-induced amylase release was exerted via modulation of intracellular $Ca^{2+}$ signaling, we measured the changes in intracellular $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ in fura-2 loaded acinar cells. Although $30\;{\mu}M\;H_2O_2$ did not induce any increase in $[Ca^{2+}]_i$ by itself, it increased the frequency and amplitude of $[Ca^{2+}]_i$ oscillations caused by 10 pM CCK-8S. However, $30\;{\mu}M\;H_2O_2$ had little effect on 1 nM CCK-8S-induced increase in $[Ca^{2+}]_i$. ROS scavenger, 1 mM N-acetylcysteine, did not affect $[Ca^{2+}]_i$ changes induced by 10 pM or 1 nM CCK-8S. Therefore, it was concluded that $30\;{\mu}M\;H_2O_2$ enhanced low concentration of CCK-8S-induced amylase release probably by increasing $[Ca^{2+}]_i$ oscillations while it inhibited high concentration of CCK-8S-induced amylase release.

• 한국동물생명공학회지
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• 제34권1호
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• pp.10-19
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• 2019

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: $79.9{\pm}3.8%$ vs G2: $57.5{\pm}4.6%$) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO ($0.1{\mu}M$, MT) treatment (G2: $68.4{\pm}3.2%$ vs G2 + MT: $73.9{\pm}1.4%$). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

• 대한의생명과학회지
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• 제11권4호
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• pp.441-446
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• 2005

The molecular mechanism of ischemia/reperfusion injury remains unclear. Reactive oxygen species (ROS) are implicated in cell death caused by ischemia/reperfusion in vivo or hypoxia in vitro. Poly (ADP-ribose) polymerase (PARP) activation has been reported to be involved in hydrogen peroxide-induced cell death in renal epithelial cells. This study was therefore undertaken to evaluate the role of P ARP activation in chemical hypoxia in opossum kidney (OK) cells. Chemical hypoxia was induced by incubating cells with antimycin A, an inhibitor of mitochondrial electron transport. Exposure of OK cells to chemical hypoxia resulted in a time-dependent cell death. In OK cells subjected to chemical hypoxia, the generation of ROS was increased, and this increase was prevented by the $H_2O_2$ scavenger catalase. Chemical hypoxia increased P ARP activity and chemical hypoxia-induced cell death was prevented by the inhibitor of PARP activation 3-aminobenzamide. Catalase prevented OK cell death induced by chemical hypoxia. $H_2O_2$ caused PARP activation and $H_2O_2-induced$ cell death was prevented by 3-aminobenzamide. Taken together, these results indicate that chemical hypoxia-induced cell injury is mediated by PARP activation through H202 generation in renal epithelial cells.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1364-1367
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• 2008

Oxidative stress damages all cellular constituents, and therefore, cell has to possess various defense mechanisms to cope. Saccharomyces cerevisiae, widely used as a model organism for studying cellular responses to oxidative stress, contains three glutathione peroxidase (Gpx) proteins. Among them, Gpx3 plays a major defense role against oxidative stress in S. cerevisiae. In this study, in order to identity the new interaction proteins of Gpx3, we carried out two-dimensional gel electrophoresis after immunoprecipitation (IP-2DE), and MALDI-TOF mass spectrometry. The results showed that several proteins including protein disulfide isomerase, glutaredoxin 2, and SSY protein 3 specifically interact with Gpx3. These findings led us to suggest the possibility that Gpx3, known as a redox sensor and ROS scavenger, has another functional role by interacting with several proteins with various cellular functions.

• 한방재활의학과학회지
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• 제27권4호
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• pp.1-9
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• 2017

Objectives Oxidative stress, in which antioxidant proteins and scavenger protection are overwhelmed by reactive oxygen species (ROS) production, is recognized as one of central causes of disuse muscle atrophy. In this study, the hypothesis that oral treatment with Dangguibohyul-tang (Dangguibuxuetang) could attenuate immobilization-induced skeletal muscle atrophy was tested. Methods The hindlimb immobilization was performed with casting tape to keep the left ankle joint in a fully extended position. The Rats in Dangguibohyul-tang treated group (DGBHT) (n=10) were orally administrated Dangguibohyul-tang water extract, and rats of Control group (n=10) were given with saline only. After 2 weeks of immobilization, the morphology of right and left gastrocnemius muscles in both DGBHT and Control groups were assessed by hematoxylin and eosin staining. Results Dangguibohyul-tang water extract represented the significant protective effects against the reductions of the left gastrocnemius muscles weight and average cross section area to compared with Control group. Moreover, the treatment with Dangguibohyul-tang extract significantly enhanced the Cu/Zn-SOD activities in gastrocnemius muscle compared with Control group. Conclusions Thses results suggest that Dangguibohyul-tang has protective effects against immobilization-induced muscle atrophy by increasing the Cu/Zn-SOD activities in gastrocnemius muscle.

• 대한한의학회지
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• 제25권4호
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• pp.61-78
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• 2004

Objective : This study was undertaken to determine whether Gamibaegi-eum (BGU) in vitro and in vivo exerts a beneficial effect against cell injury induced by reactive oxygen species (ROS) in the human intestine. Methods : Effects of BGU in vitro on cell injury were examined using Caco-2 cells, cultured human intestinal cell line. Exposure of cells to H₂O₂ induced increases in the loss of cell viability in a time and dose-dependent fashion. Results : BGU prevented H₂O₂-induced cell death and its effect was dose-dependent over a concentration range of 0.05­1%. H₂O₂-induced cell death was prevented by catalase, the hydrogen peroxide scavenger enzyme, and deferoxamine, the iron chelator. However, the potent antioxidant DPPD did not affect H₂O₂-induced cell death. H₂O₂ increased lipid peroxidation, which was inhibited by BGU and DPPD. H₂O₂ caused DNA damage in a dose-dependent manner, which was prevented by BGU, catalase, and deferoxamine, but not DPPD. BGU restored ATP depletion induced by H₂O₂. BGU inhibited generation of superoxide and H₂O₂ and scavenged directly H₂O₂. Oral administration of mepirizole in vivo at a dose of 200mg/kg resulted in ulcer lesions in the stomach and the proximal duodenum. Pretreatment of BGU(0.1%/kg, orally) and catalase (800Units/kg, i.v.) significantly decreased the size of ulcers. Mepirizole increased lipid peroxidation in the mucosa of the duodenum, suggesting an involvement of ROS. Pretreatment of BGU and catalase significantly inhibited lipid peroxidation induced by mepirizole. Morphological studies showed that mepirizole treatment causes duodenal injury and its effect is prevented by BGU. Conclusion : These results indicate that BGU exerts a protective effect against cell injury in vitro and in vivo through antioxidant action. The present study suggests that BGU may playa therapeutic role in the treatment of human gastrointestinal diseases mediated by ROS.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2569-2574
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• 2015

Necroptosis, also known as "programmed necrosis", has emerged as a critical factor in a variety of pathological and physiological processes and is considered a cell type-specific tightly regulated process with mechanisms that may vary rather greatly due to the change of cell line. Here we used HT-29, a human colon cancer cell line, to establish a necroptosis model and elucidate associated mechanisms. We discovered that cobalt chloride, a reagent that could induce hypoxia-inducible $factor-1{\alpha}(HIF1{\alpha})$ expression and therefore mimic the hypoxic microenvironment of tumor tissue in some aspects induces necroptosis in HT-29 cells when caspase activity is compromised. On the other hand, apoptosis appears to be the predominant death form when caspases are functioning normally. HT-29 cells demonstrated significantly increased RIPK1, RIPK3 and MLKL expression in response to cobalt chloride plus z-VAD treatment, which was accompanied by drastically increased $IL1{\alpha}$ and IL6 expression, substantiating the notion that necrosis can induce profound immune reactions. The RIPK1 kinase inhibitor necrostatin-1 and the ROS scavenger NAC each could prevent necrosis in HT-29 cells and the efficiency was enhanced by combined treatment. Thus by building up a necroptosis model in human colon cancer cells, we uncovered that mechanically RIP kinases collaborate with ROS during necrosis promoted by cobalt chloride plus z-VAD, which leads to inflammation. Necroptosis may present a new target for therapeutic intervention in cancer cells that are resistant to apoptotic cell death.