• Journal of Nutrition and Health
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• v.51 no.6
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• pp.507-514
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• 2018

Purpose: Sargassum horneri (S. horneri) is a species of brown macroalgae that is common along the coast of Japan and Korea. The present study investigated the immuno-modulatory effects of different types of S. horneri extracts in RAW264.7 macrophages. Methods: S. horneri was extracted by three different methods, hot water extraction, 50% ethanol extraction, and supercritical fluid extraction. Cell viability was then measured by MTT assay, while the production levels of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-6 (IL-6), and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay and Griess assay, respectively. The expression and activation levels of inducible NO synthase (iNOS), mitogen-activated protein kinase (MAPK) and nuclear factor ${\kappa}B$ ($NF-{\kappa}B$) were examined by western blot analysis. Results: The three different S. horneri extracts were nontoxic against RAW 264.7 cells up to $50{\mu}g/mL$, among which treatment with hot water extract (HWE) of S. horneri significantly enhanced the production of TNF-${\alpha}$, IL-6, and NO in a dose-dependent manner. Hot water extract of S. horneri also increased the expression level of iNOS, suggesting that up-regulation of iNOS expression by HWE of S. horneri was responsible for the induction of NO production. In addition, treatment of RAW 264.7 macrophages with HWE of S. horneri increased the phosphorylation levels of ERK, p38 and JNK. Furthermore, the activation and subsequent nuclear translocation of $NF-{\kappa}B$ was enhanced upon treatment with HWE of S. horneri, indicating that HWE of S. horneri activates macrophages to secrete TNF-${\alpha}$, IL-6 and NO and induces iNOS expression via activation of the $NF-{\kappa}B$ and MAPKs signaling pathways. Conclusion: Taken together, these findings suggest that HWE of S. horneri possesses potential as a functional food with immunomodulatory activity.

• Journal of Life Science
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• v.24 no.11
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• pp.1157-1167
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• 2014

In order to compare the anti-inflammatory effects of five selected cereal grains-proso millet, hwanggeumchal sorghum, foxtail millet, barnyard millet, and adlay-the inhibitory activities of 80% ethanol (EtOH) extracts obtained from the individual grains on lipopolysaccharide (LPS)-induced nitric oxide (NO) generation were investigated in RAW264.7 cells. The EtOH extract of barnyard millet (Echinochloa crus-galli var. frumentacea) grains exhibited more potent anti-inflammatory activity than that of the other grains. When the EtOH extract of barnyard millet grains was sequentially fractionated with n-hexane, methylene chloride (MC), ethyl acetate (EtOAc), and n-butanol, the majority of the anti-inflammatory activity was detected in the MC fraction, followed by the EtOAc fraction. Pretreatment with the MC fraction caused downregulation of the expression levels of iNOS- and COX-2-specific transcripts and proteins, as well as proinflammatory cytokine gene transcripts (IL-$1{\beta}$, IL-6, and TNF-${\alpha}$) in LPS-stimulated RAW264.7 cells. Additionally, the MC fraction could suppress not only the LPS-induced nuclear translocation of cytosolic NF-kB, but also the LPS-induced activation of MAPKs, such as ERK, JNK, and p38MAPK. Further analysis of the MC fraction by HPLC identified kaempferol, biochanin A, and formononetin as the major phenolic components. Both kaempferol and biochanin A, but not formononetin, could exert anti-inflammatory effect at the same concentrations as those of the MC fraction. Consequently, these results indicate that kaempferol and biochanin A are among the most effective anti-inflammatory phenolic components in barnyard millet grains. This finding suggests that barnyard millet grains and the MC extract enriched in kaempferol and biochanin A could be beneficial functional food sources that have an anti-inflammatory effect.

• The korean journal of orthodontics
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• v.33 no.3 s.98
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• pp.169-183
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• 2003

Tooth movement by orthodontic force effects great tissue changes within the periodontium, especially by shifting the blood flow in the pressure side and resulting in a hypoxic state of low oxygen tension. The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3El osteoblasts, the main cells in bone remodeling during orthodontic tooth movement. MC3T3El osteoblasts under hypoxic conditions ($2\%$ orygen) resulted in apoptosis in a time-dependent manner as estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pancaspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, Western blot analysis was performed using anti-caspase-3 or -6 antibodies. The 10-kDa protein, corresponding to the active products of caspase-3, and the 10-kDa protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which the processing of the full length form of caspase-3 and -6 was evident. While a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, which was typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we observed that pre-treatment with SB203580, a selective p38 mitogen activated protein kinase inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203S80 suppressed caspase-3 and -6-like protease activity by hypoxia up to $50\%$. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAPK -JNK/SAPK or ERK- was either only modestly activated or not at all. These results suggest that p38 MAPK is involved in hypoxia-induced apoptosis in MC3T3El osteoblasts.

• Journal of Gastric Cancer
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• v.5 no.3 s.19
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• pp.191-199
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• 2005

Background: Though infections of Helicobacter pylori (H. pylori) are closely associated with activation of host angiogenesis, the underlying mechanisms, as well as the strategy for its prevention, have not been identified. Here, we investigated a causal role of H. pylori infection in angiogenesis of gastric mucosa and a potent inhibitory effect of a gastric proton pump inhibitor (PPI) on the gastropathy. Materials and Methods: A comparative analysis of CD 34 expression in tissues obtained from 20 H. pylori-associated gastritis and 18 H. pylori-negative gastritis patients was performed. Expression of $HIF-1{\alpha}$ and VEGF were tested by using RT-PCR. To evaluate the direct effect of H. pylori infection on differentiation of endothelial HUVEC cells, we carried out an in vitro angiogenesis assay. Results: H. pyfori-associated gastritis tissues showed significantly higher density of $CD34^+$ blood vessels than did H. pylori-negative gastritis tissues, and the levels were well correlated with expressions of $HIF-1{\alpha}$. Conditioned media from H. pylori-infected gastric mucosal cells stimulated a tubular formation of HUVEC cells. We also found a significant inhibitory effect of PPI, an agent frequently used for H. pylori eradication, on H. pylori-induced angiogenesis. This drug effectively inhibited the phosphorylation of MAP kinase ERK1/2, which is a principal signal for H. pylori-induced angiogenesis. Conclusion: The fact that PPls can down-regulate H. pylori-induced angiogenesis suggest that anti-angiogenic treatment using PPI may be a preventive approach for H. pylori-associated carcinogenesis.

• Journal of Life Science
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• v.21 no.9
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• pp.1234-1243
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• 2011

Liriope platyphylla has been used in oriental medicine as an effective medical plant to improve symptoms of cough, sputum production, neurodegenerative disorders, obesity and diabetes for long time. In order to investigate the effects of novel extracts on nerve growth factors (NGF)-stimulated neuritic outgrowth, the alteration of NGF expression and NGF receptor signaling pathway were detected in neuroblastoma cells and C57BL/6 mice. Of a total of 13 novel extracts, 4 extracts (LP-E, LP-M, LP-M50, LP2E17PJ) showed high viability on MTT assay. Also, all of these extracts induced NGF secretion and NGF mRNA expression in neuroblastoma cells. However, the NGF-induced neuritic outgrowth from PC12 cells was only stimulated by LP-E, LP-M and LP-M50. Furthermore, we selected LP-M as a best candidate, based on method and amounts of extraction, in order to verify its effect in mice. C57BL/6 mice were treated with 50 mg/kg of LP-M for 2 weeks and the effects on NGF regulation were analyzed with various methods. The expression of NGF mRNA was significantly increased in LP-M treated mice compared to vehicle treated mice. Also, the signaling pathway of p75NTR was inhibited in the cortex by LP-M treatment, with no change in the hippocampus of brain. However, the signaling pathway of TrkA was dramatically activated in only hippocampus via LP-M treatment. Therefore, these results suggest that the novel four extracts of L. platyphylla may contribute to the regulation of NGF expression and secretion in neuronal cells. LP-M was especially considered to be an excellent candidate for a neurodegenerative disease-therapeutic drug.

• Journal of Life Science
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• v.19 no.7
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• pp.949-955
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• 2009

Mucosal epithelia sense external stress signals and transmit them to the intracellular cascade responses. Ribotoxic stress-producing chemicals such as deoxynivalenol (DON) or other trichothecene mycotoxins have been linked with gastrointestinal inflammatory diseases by Fusarium-contamination. The purpose of this study was to test the hypothesis that DON evokes the epithelial sentinel signals of RNA-dependent protein kinase (PKR) and early growth response gene 1 (EGR-1), which together contribute to the pro-inflammatory cytokine interleukin 8 (IL-8) in human intestinal epithelial cells. PKR suppression by the dominant negative PKR expression attenuated DON-stimulated interleukin-8 production. Moreover, 1L-8 transcriptional activation by DON was also reduced by PKR inhibition in the human intestinal epithelial cells. Treatment with the PKR inhibitor also suppressed EGR-1 promoter activity, mRNA and protein induction, although mitogen-activated protein (MAP) kinases such as extracellular signal-regulated protein kinases (ERK) 1/2, p38, c-Jun N-terminal Kinase (INK) were little affected or even enhanced in presence of a PKR inhibitor. These patterns were also compared in the EGR-1-suppressed cells, which showed much more suppressed production of 1L-8. All things taken into consideration, DON-activated sentinel signals of EGR-1 via PKR mediated interleukin-8 production in human intestinal epithelial cells, which provide insight into the possible general mechanism associated with mucosal inflammation as an intestinal toxic insult by ribotoxic trichothecene mycotoxins.

• BMB Reports
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• v.49 no.2
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• pp.111-115
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• 2016

Caffeine has been proposed to have several beneficial effects on obesity and its related metabolic diseases; however, how caffeine affects adipocyte differentiation has not been elucidated. In this study, we demonstrated that caffeine suppressed 3T3-L1 adipocyte differentiation and inhibited the expression of CCAAT/enhancer binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ, two main adipogenic transcription factors. Anti-adipogenic markers, such as preadipocyte secreted factor (Pref)-1 and Krüppel-like factor 2, remained to be expressed in the presence of caffeine. Furthermore, 3T3-L1 cells failed to undergo typical mitotic clonal expansion in the presence of caffeine. Investigation of hormonal signaling revealed that caffeine inhibited the activation of AKT and glycogen synthase kinase (GSK) 3 in a dose-dependent manner, but not extracellular signal-regulated kinase (ERK). Our data show that caffeine is an anti-adipogenic bioactive compound involved in the modulation of mitotic clonal expansion during adipocyte differentiation through the AKT/GSK3 pathway.

• BMB Reports
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• v.34 no.2
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• pp.123-129
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• 2001

Cellular response to ionizing radiation is affected by cell types, radiation doses, and post-irradiation time. Based on the trypan blue dye exclusion assay in normal oral mucosal cells (OM cells), a 48 h post-irradiation was sufffcient and an adequate time point for the evaluation of radiation sensitivity Its $LD_{50}$ was approximately 1.83 Gy To investigate possible biomarkers useful for the biological radiodosimetry of normal epithelial cells (p53, c-fos, cyclin D1, cdc-2, pRb) EGF receptor phosphorylation and Erk activation were evaluated at different radiation doses and different post-irradiation times. From 0.5 Gy, p53 was accumulated in the nucleus of basal cells of the OM raft culture at 4 h post-irradiation and sustained up to 24 h post-irradiation, which suggests that radiation-induced apoptosis or damage repair was not yet completed. The number of p53 positive cells and biosynthesis of p53 were correlated with radiation doses. Both cyclin D1 and c-fos were only transiently induced within 1 h post-irradiation. Cyclin D1 was induced at all radiation doses. However, cfos induction was highest at 0.1 Gy, approximately 7.3 fold more induction than the control, whose induction was reduced in a reverse correlation with radiation dose. The phosphorylation pattern of cdc-2 and pRb were unaffected by radiation. In contrast to A431 tails overexpressing the EGF receptor approximately 8.5 fold higher than normal epithelial, the OM cells reduced the basal level of the EGF receptor phosphorylation in a radiation dose dependent fashion. In conclusion, among radiation-induced biomolecules, the p53 nuclear accumulation may be considered for the future development of a useful marker far biological radiodosimetry in normal epithelial tissue since it was sustained for a longer period and showed a dose response relationship. Specific c-fos induction at a low dose may also be an important finding in this study It needs to be studied further for the elucidation of its possible connection with the low dose radio-adaptive response.

• Environmental Mutagens and Carcinogens
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• v.26 no.3
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• pp.77-85
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• 2006

2,3,7,8-Tetrachlorodibenzo-$\rho$-dioxin(TCDD) displays high toxicity in animals and has been implicated in human carcinogenesis. Although TCDD is recognized as potent carcinogens, relatively little is known about their role in the tumor promotion and carcinogenesis. It is known that TCDD can increase of cancer risk from various types of tissue by a mechanism possibly involving the aryl hydrocarbon receptor (AhR) activation. In this study, effects of TCDD on cellular proliferation of normal human skin and lung fibroblasts, Detroit551 and WI38 cells were investigated. In addition, to enhance our understanding of TCDD-mediated carcinogenesis, we have investigated process in which expression of Erk1/2, cyclinD1, oncogene such as Ha-ras and c-myc, and their cognate signaling pathway. TCDD that are potent activators of AhR-mediated activity was found to induce significant increase of cytochrome P4501A1 mRNA expression, suggesting a presence of functional AhR. These results support that CYP1A1 enzyme may be involved in the generation of TCDD-induced toxicity. Moreover mitogen-activated protein kinases (MARKs) phosphorylation and cyclin D1 overexpression are induced by TCDD, which corresponded with the progression of cellular proliferation. However, TCDD did not affected Ha-ras and c-myc mRNA expression. Taken together, it seems that TCDD are could be a part of cellular proliferation in non-tumorigenic normal human cells such as Detroit551 and WI38 cells through the upregulation of MAPKs signaling pathway regulating growth of cell population. Therefore, AhR-activating TCDD could potentially contribute to tumor promotion and Detroit551 and WI38 cells have been used as a detection system of tumorigenic effects of TCDD.

• BMB Reports
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• v.45 no.6
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• pp.354-359
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• 2012

We examined that the protective effects of ANX1 on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in animal models using a Tat-ANX1 protein. Topical application of the Tat-ANX1 protein markedly inhibited TPA-induced ear edema and expression levels of cyclooxygenase-2 (COX-2) as well as pro-inflammatory cytokines such as interleukin-1 beta (IL-$1{\beta}$), IL-6, and tumor necrosis factor-alpha (TNF-${\alpha}$). Also, application of Tat-ANX1 protein significantly inhibited nuclear translocation of nuclear factor-kappa B (NF-${\kappa}B$) and phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TPA-treated mice ears. The results indicate that Tat-ANX1 protein inhibits the inflammatory response by blocking NF-${\kappa}B$ and MAPK activation in TPA-induced mice ears. Therefore, the Tat-ANX1 protein may be useful as a therapeutic agent against inflammatory skin diseases.