• The Korean Journal of Physiology and Pharmacology
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• v.22 no.2
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• pp.215-223
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• 2018

Intracellular $Ca^{2+}$ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide ($H_2O_2$) on cytosolic $Ca^{2+}$ accumulation in mouse parotid acinar cells. Intracellular $Ca^{2+}$ levels were slowly elevated when $1mM\;H_2O_2$ was perfused in the presence of normal extracellular $Ca^{2+}$. In a $Ca^{2+}-free$ medium, $1mM\;H_2O_2$ still enhanced the intracellular $Ca^{2+}$ level. $Ca^{2+}$ entry tested using manganese quenching technique was not affected by perfusion of $1mM\;H_2O_2$. On the other hand, $10mM\;H_2O_2$ induced more rapid $Ca^{2+}$ accumulation and facilitated $Ca^{2+}$ entry from extracellular fluid. $Ca^{2+}$ refill into intracellular $Ca^{2+}$ store and inositol 1,4,5-trisphosphate ($1{\mu}M$)-induced $Ca^{2+}$ release from $Ca^{2+}$ store was not affected by $1mM\;H_2O_2$ in permeabilized cells. $Ca^{2+}$ efflux through plasma membrane $Ca^{2+}-ATPase$ (PMCA) was markedly blocked by $1mM\;H_2O_2$ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected $H_2O_2-induced$ $Ca^{2+}$ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of $H_2O_2$ under pathological conditions may lead to cytosolic $Ca^{2+}$ accumulation and that the primary mechanism of $H_2O_2-induced$ $Ca^{2+}$ accumulation is likely to inhibit $Ca^{2+}$ efflux through PMCA rather than mobilize $Ca^{2+}$ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.

• Environmental Mutagens and Carcinogens
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• v.25 no.2
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• pp.51-59
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• 2005

Overexpression of HSP25 delayed cell growth, increased the level of $p21^{waf}$, reduced the levels of cyclin D1, cylcin A and cdc2, and induced radioresistance in L929 cells. We demonstrated that extracellular regulated kinase (ERK) and MAP kinase/ERK kinase (MEK) expressions as well as their activation (phospho-forms) were inhibited by hsp25 overexpression. To confirm the relationship between ERK1/2 and hsp25-mediated radioresistance, ERK1 or ERK2 cDNA was transiently transfected into the hsp25 overexpressed cells and their radioresistance was examined. HSP25-mediated radioresistance was abolished by overexpression of ERK2, but not by overexpression of ERK1. Alteration of cell cycle distribution and cell cycle related protein expressions (cyclin D, cyclin A and cdc2) by hsp25 overexpression were also recovered by ERK2 cDNA transfection. Increase in Bc1-2 protein by hsp25 gene transfection was also reduced by subsequent ERK2 cDNA-transfection. In addition, HSP25 overexpression reduced reactive oxygen species (ROS) and increased expression of manganese superoxide dismutase (MnSOD) gene. Increased activation of NF-kB (IkB degradation) was also found in hsp25-overexpressed cells. Moreover, transfection of hsp25 antisense gene abrogated all the HSP25-mediated phenomena. To further elucidate the exact relationship between MnSOD induction and NF-kB activation, dominant negative $I-kB\alpha(I-kB\alpha-DN)$ construction was transfected to HSP25 overexpressed cells. $I-kB\alpha-DN$ inhibited HSP25 mediated MnSOD gene expression. In addition, HSP25 mediated radioresistance was blocked by $I-kB\alpha-DN$ transfection. Blockage of MnSOD with antisense oligonucleotides in HSP25 overexpressed cells, prevented apoptosis and returned the ERK1/2 activation to the control level. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated down regulation of ERK1/2.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.31 no.4 s.54
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• pp.305-310
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• 2005

To obtain effective and safe depigmenting agents, we investigated the effects of Scirpi rhizoma, a medicine among Chinese herbs, on melanogenesis. Dried S. rhizoma was refluxed with 70% aqueous ethanol and the extract was evaporated to dryness. To determine the effects as a whitening agent, various in vitro tests were performed such as free radical scavenging activity, melanin formation assay, tyrosinase activity and expression of tyrosinase, TRP-1 and TRP-2(western blot and RT-PCR) in B16 melanoma cells. S. rhizoma showed scavenging activities of free radicals and reactive oxygen species (ROS) with the $IC_{50}\;of\;638{\mu}g/mL$ against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and $21.7{\mu}g/mL$ against superoxide radicals in the xanthine/xanthine oxidase system, respectively. S. rhizoma significantly inhibited melanin production in B16 melanoma cells. S. rhizoma treatment(48 h) suppressed the biosynthesis of melanin up to 27% at 100{\mu}g/mL$ and reduced tyrosinase activity up to 31% at $100{\mu}g/mL$ in B16 melanoma cells. S. rhizoma was also able to significantly inhibit tyrosinase and TRP-1 expression in protein and mRNA level. These results suggest that S. rhizoma inhibited melanin biosynthesis by regulating tyrosinase activity and expression in B16 melanoma cells. Therefore, S. rhizoma may be useful as a new antioxidant and whitening agent to inhibit melanogenesis.

• Animal Bioscience
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• v.36 no.5
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• pp.710-719
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• 2023

Objective: The present study investigated whether protodioscin (PD), a steroidal saponin mainly found in rhizome of Dioscorea species, alleviates oxidative stress-induced damage of porcine oocytes during in vitro maturation. Methods: Oocytes were treated with different concentrations of PD (0, 1, 10, 100, and 200 µM) in the presence of 200 µM H₂O₂ during in vitro maturation. Following maturation, spindle morphology and mitogen-activated protein kinase activity was assessed along with reactive oxygen species level, GSH activity, and mRNA expression of endogenous antioxidant genes at the MII stage. On the day 7 after parthenogenetic activation, blastocyst formation rate was calculated and the quality of embryo and mRNA expression of development-related genes was evaluated. Results: Developmental competence was significantly poorer in the 0 µM PD-treated (control) group than in the non-treated (normal) and 10 µM PD-treated (10PD) groups. Although the reactive oxygen species level did not significantly differ between these three groups, the glutathione level and mRNA expression of antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, nuclear factor erythroid 2-related factor 2 [Nrf2], and hemo oxygenase-1 [HO-1]) were significantly higher in the normal and 10PD groups than in the control group. In addition, the percentage of oocytes with defective spindle and abnormal chromosomal alignment was significantly lower and the ratio of phosphorylated p44/42 to total p44/42 was significantly higher in the normal and 10PD groups than in the control group. The total cell number per blastocyst was significantly higher in the 10PD group than in the control group. The percentage of apoptotic cells in blastocysts was highest in the control group; however, the difference was not significant. mRNA expression of development-related genes (POU domain, class 5, transcription factor 1 [POU5F1], caudal type homeobox 2 [CDX2], Nanog homeobox [NANOG]) was consistently increased by addition of PD. Conclusion: The PD effectively improves the developmental competence and quality of blastocysts by protecting porcine oocytes against oxidative stress.

• Journal of Microbiology and Biotechnology
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• v.33 no.5
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• pp.591-599
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• 2023

Fisetin is a bioactive flavonol molecule and has been shown to have antioxidant potential, but its efficacy has not been fully validated. The aim of the present study was to investigate the protective efficacy of fisetin on C2C12 murine myoblastjdusts under hydrogen peroxide (H₂O₂)-induced oxidative damage. The results revealed that fisetin significantly weakened H₂O₂-induced cell viability inhibition and DNA damage while blocking reactive oxygen species (ROS) generation. Fisetin also significantly alleviated cell cycle arrest by H₂O₂ treatment through by reversing the upregulation of p21WAF1/CIP1 expression and the downregulation of cyclin A and B levels. In addition, fisetin significantly blocked apoptosis induced by H₂O₂ through increasing the Bcl-2/Bax ratio and attenuating mitochondrial damage, which was accompanied by inactivation of caspase-3 and suppression of poly(ADP-ribose) polymerase cleavage. Furthermore, fisetin-induced nuclear translocation and phosphorylation of Nrf2 were related to the increased expression and activation of heme oxygenase-1 (HO-1) in H₂O₂-stimulated C2C12 myoblasts. However, the protective efficacy of fisetin on H₂O₂-mediated cytotoxicity, including cell cycle arrest, apoptosis and mitochondrial dysfunction, were greatly offset when HO-1 activity was artificially inhibited. Therefore, our results indicate that fisetin as an Nrf2 activator effectively abrogated oxidative stress-mediated damage in C2C12 myoblasts.

• Journal of Life Science
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• v.28 no.9
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• pp.1092-1099
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• 2018

This study was orchestrated with the purpose of uncovering new nutraceutical resources possessing biological activities in the plant kingdom. To fulfill our objective, we analyzed several plant extracts and selected five species possessing powerful anti-oxidative activity. The anti-oxidative effect of these five plants, Apios fortunei Maxim., Colubrina arborescens Sarg., Croton caudatus Geiseler, Osmanthus matsumuranus Hayata and Schima noronhae Reinw. ethanol extracts were then evaluated by using in vitro assay, cell model system, and Western blot analysis of target proteins. As the results, all of them possessed the potent scavenging activity against 1,1-diphenyl-2-picryl hydrazyl (DPPH), similar with that of ascorbic acid, used as a common positive control. Moreover, they strongly inhibited hydrogen peroxide ($H_2O_2$)-induced reactive oxygen species (ROS), in a dose-dependent manner, in RAW 264.7 murine macrophage cells. Furthermore, they induced the protein expression of an anti-oxidative enzyme, heme oxygenase 1 (HO-1), and its upstream transcription factor, nuclear factor-E2-related factor 2 (Nrf2). Taken together, these results indicate that these five plants possess potent anti-oxidative activity and thus appear to be useful sources as potential anti-oxidant agents. Therefore, they might be utilized as promising materials in the field of nutraceuticals.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.32 no.3 s.58
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• pp.161-171
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• 2006

In this study, we investigated the whitening and anti-oxidant activity of 37 Jeju island native plants. The active ingredients of the plants were prepared by methanol extraction. Whitening activity of plant extracts was examined from the inhibitory effect of tyrosinase and the inhibition of melanin synthesis of the B16-F1 cell line. Their anti-oxidant activity was measured by electron donating ability of DPPH (1,1-diphenyl-2-picrylhydrazyl) and ROS (reactive oxygen species) scavenging activity in V79-4 lung fibroblast cells using DCF-DA (dichlorofluorescin diacetate). Cytotoxicity of the extracts on cell s based experiments was investigated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Also, the toxicity test using a rabbit and the human skin patch test were carried out for examining the safety of the extracts which showed the high whitening activity. It is interesting that the extracts of Lespedeza cuneata, Ligustrum lucidum (stem), Morus bombycis (stem) and Prunella vulgaris var. lilacina showed both potent whitening and anti-oxidant activities.

• Journal of Korean Medicine Rehabilitation
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• v.24 no.2
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• pp.65-81
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• 2014

Objectives This study was carried out to find out the anti-osteoarthritic effects of Mahwangbujaseshin- tang (Mahuangfuzixixintang ) on the monosodium iodoacetate (MIA)-induced osteoarthritis rats. Methods Osteoarthritis was induced by injecting MIA ($50{\mu}l$) into the knee joint of rats. Rats were divided into a 3 groups (n=7). The injection did not fit the normal group. A week later, after the injection of MIA, while control group took normal saline 2 ml, the extract of Mahwangbujaseshin-tang (Mahuangfuzixixintang ) (MBST) (200 mg/kg) was injected to treated group. After that, we examined hind paw weight bearing ability, functions of liver and kidney, serum TNF-$\alpha$, IL-$1{\beta}$, IL-6, $PGE_2$, $LTB_4$, TIMP-1, MMP-9 and hematology. Volume of cartilage was measured by micro CT arthrography. Injury of synovial tissue was measured by H & E, Safranin-O immunofluorescence. Results 1) DPPH and ABTS free radical scavenging activity of MBST was increased according to concentration of MBST and total phenolic contents were in high level. 2) In RAW 264.7 cells, ROS production was significantly decreased in MBST (at 10, $100{\mu}g/ml$) and NO was also decreased but meaningless in MBST (at $100{\mu}g/ml$). 3) In RAW 264.7 cells, IL-6 production was significantly decreased in MBST (at $100{\mu}g/ml$) and TNF-$\alpha$ and IL-$1{\beta}$ production were also decreased but meaningless in MBST (at $100{\mu}g/ml$). 4) In hind legs weight-bearing measurement, level of weight-bearing was increased. 5) Functions of liver and kidney were not affected. 6) TNF-$\alpha$, IL-$1{\beta}$, IL-6, $PGE_2$, $LTB_4$, MMP-9 and TIMP-1 production were significantly decreased. 7) In hematology, the levels of neutrophils, monocytes were significantly decreased and the levels of white blood cells, lymphocytes were also decreased but meaningless. 8) In micro CT-arthrography, cartilage volume was significantly increased. 9) Histopathologically, injury on cartilage and synovial membrane of MBST group was decreased. Conclusions Based on all results mentioned above, Mahwangbujaseshin-tang (Mahuangfuzixixintang) is believed to be meaningful for suppressing the progress of osteoarthritis. And it is related to inhibiting the activity of inflammatory cytokine and injury of volume in cartilage.

• Journal of Life Science
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• v.22 no.8
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• pp.1018-1023
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• 2012

Snail is a zinc finger transcription factor that induces epithelial-to-mesenchymal transition (EMT), which promotes tumor invasion and metastasis by repressing E-cadherin expression. In addition, Snail restricts the cellular apoptotic response to apoptotic stimuli or survival factor withdrawal; however, its molecular mechanism remains largely unknown. In this study, we have investigated the mechanism underlying Snail-mediated chemoresistance to 5-fluorouracil (5-FU), one of the most widely used anti-cancer drugs. When Snail was overexpressed by doxycycline (DOX) in MCF-7 #5 cells, it inhibited 5-FU-induced apoptotic cell death and switched the cell death mode to necrosis. Snail expression, either by DOX treatment in MCF-7 #5 cells or by the transfection of Snail expression vectors pCR3.1-Snail-Flg, phosphorylation-resistant pCR3.1-S104, and 107A Snail-Flg in MCF-7 cells specifically induced PTEN down-regulation/inactivation and Akt/PKB activation, without affecting ERK1/2 activity. In addition, Snail prominently suppressed 5-FU-induced increases in p53 levels. These findings demonstrate that Snail switches 5-FU-induced apoptosis to necrosis through the activation of Akt/PKB and the down-regulation of p53 levels.

• Journal of Korean Medicine Rehabilitation
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• v.25 no.4
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• pp.1-20
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• 2015

Objectives This study was intended to clarify the anti-inflammation and anti-oxidation effects of gamikyejakjimo-tang herbal acupuncture (GKHA) for osteoarthritis. Methods Osteoarthritis was induced by injection of MIA into right knee joint cavities of rats. Rats were divided into a total of 4 groups (n=8). The 4 groups were normal group, control group, positive comparison group and expeimental group. Indomethacin and GKHA were medicated for a total of 4 weeks. After that, functions of liver and kidney by AST, ALT, creatinine, BUN, DPPH and ABTS free radical scavenging activity, ROS (reactive oxygen species) production, NO (Total Nitric oxide), IL-$1{\beta}$, IL-6, TNF-${\alpha}$ production, weight changes in the hind legs of MIA-induced osteoarthritis rat, serum PGE2, TIMP-1, MMP-2, MMP-9, LTB4, hs-CRP, and white blood cells, neutrophils, lymphocytes, monocytes were measured. The volume of cartilage was observed by micro CT arthrography. H&E and Safranin-O staining were used to examine the injury of synovial tissue. Results 1. In the hind leg weight bearing measurement, level of weight was increased. 2. AST, ALT, BUN, creatinine were decreased. 3. The production of total white blood cell was decreased, and the production of neutrophils, lymphocytes, monocytes were significantly decreased. 4. The production of NO, PGE2, TIMP-1, MMP-2, MMP-9, LTB4 were significantly decreased, and the production of hs-CRP was also decreased but with no significance. 5. The cartilage volume was significantly increased. 6. In H&E staining and Safranin-O staning, the cartilage cell appeared to be proliferated, and proteoglycans appeared to be increased. Conclusions Based on the results above, Gamikyejakjimo-tang Herbal Acupuncture has anti-oxidation and anti-inflammation effects, which leads to suppressing the underlying causes and the progression of osteoarthritis.