• Journal of Acupuncture Research
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• v.21 no.3
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• pp.107-119
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• 2004

Objective : The purpose of this study was to investigate the effect of Bee Venom and melittin on the lipopolysaccharide(LPS) and sodium nitroprusside(SNP)-induced expressions of Cell viability, nitric oxide(NO), inducible nitric oxide synthase(iNOS), extra-signal response kinase(ERK), jun N-terminal Kinase(JNK) and p38 kinase(p38)- mitogen activated protein kinase(MAPK) Family- in RAW 264.7 cells, a murine macrophage cell line. Methods : The expressions of cell viability by MTT assay, NO by Nitrite assay and iNOS, ERK, JNK and p38 were determined by Western blotting. Results : 1. Compared with the control group, 0.5, 1, $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin increased cell viability of RAW 264.7 induced by LPS and SNP significantly respectively. 2. Compared with the control group, 0.5, 1, $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin inhibited expression of NO induced by LPS and SNP significantly respectively. 3. Compared with the control group, 1, $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin inhibited expression of iNOS induced by LPS significantly and 0.5, 1, $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin inhibited expression of iNOS induced by SNP significantly. 4. Compared with the control group, the expression of ERK induced by LPS and SNP decreased significantly in the treatment groups of $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin, which of p-ERK by LPS also did in 1, $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin, but which of p-ERK by SNP did not decrease. 5. Compared with the control group, the. expression of JNK induced by LPS and SNP decreased significantly in the treatment groups of 5, $10{\mu}g/m{\ell}$ melittin, which of p-JNK by LPS in 5, $10{\mu}g/m{\ell}$ melittin and by SNP in $1{\mu}g/m{\ell}$ bee venom and $10{\mu}g/m{\ell}$ melittin decreased significantly. 6. Compared with the control group, the expression of p38 induced by LPS did not have significant difference, which induced by SNP decreased significantly in the treatment groups of 1, $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin. p-p38 induced by LPS decreased significantly in the treatment group of $10{\mu}g/m{\ell}$ of melittin, which induced by SNP also decreased significantly in 0.5, 1, $5{\mu}g/m{\ell}$ bee venom and 5, $10{\mu}g/m{\ell}$ melittin.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.756-766
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• 2005

The signaling mechanism underlying aburatubolactam C-induced FasL upregulation was investigated in human Jurkat T cells. After treatment with aburatubolactam C, the src-family PTKs $p56^{lck}\;and\;p59^{fyn}$, and MAP kinases ERK2 and JNK1, were activated prior to FasL upregulation; Both $p56^{lck}\;and\;p59^{fyn}$ were directly activated 2.4- and 2.2-fold, respectively, in vitro by aburatubolactam C. The aburatubolactam C-induced cellular changes, including the activation of ERK2 and INK1, and FasL upregulation, were completely prevented by the PTK inhibitor genistein. The activation of protein kinase C (PKC$\delta,\;\epsilon\;and\;\mu$ was also induced following aburatubolactam C treatment. Although the activation of $p56^{lck}$ and tyrosine phosphorylation of the cellular proteins were not blocked by the PKC inhibitor GFl09203X, the activation of ERK2 was completely abrogated, along with a detectably enhanced JNK1 activation; FasL upregulation, and apoptosis. However, the FasL upregulation and apoptosis were significantly inhibited by the PKC activator PMA, with a remarkable increase in the ERK2 activation. The cytotoxic effect of aburatubolactam C was reduced in the presence of the anti-Fas neutralizing antibody ZB-4. Although ectopic expression of Bcl-2 failed to completely block the cytotoxicity of aburatubolactam C, it was clearly suppressed. The c-Fos mRNA expression was upregulated in a biphasic manner, where the second phasic expression overlapped with the FasL upregulation. Accordingly, these results demonstrate that aburatubolactam C-induced apoptosis is exerted, at least in part, by FasL upregulation dictated by activation of the PTK ($p56^{lck}\;and\;p59^{fyn}$) /JNKI pathway, which is negatively affected by the concurrent activation of the PKC/ERK2 pathway proximal to PTK activation.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.2
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• pp.220-227
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• 2010

Puerariae radix (PR) is a popular natural herb and a traditional food in Asia, which has antithrombotic and anti-allergic properties and stimulates estrogenic activity. One of the major side effects of cisplatin is nephrotoxicity, leading to acute renal failure. Recent study has suggested a role of ROS and p53 in renal cell injury by cisplatin. We studied that protective effects of PR on cisplatin-induced apoptosis in rat mesangial cell. Rat mesangial cell was preincubated with PR (50, 100, 150 and 200 ${\mu}g/m{\ell}$) for 12 hr and then treated with 30 ${\mu}M$ cisplatin for 24 hr. Protective effect of PR on cisplatin-induced apoptosis in ECV304 cells was determined using MTT assay, FDA-PI staining, flow cytometric analysis, caspase-3 activity assay, ROS assay and western blot. Our results showed that PR inhibited in cisplatin-induced apoptosis and ROS production in ECV304 cells. Moreover, PR reduced ERK, p38 and JNK activation that increased in cisplatin-treated rat mesangial cell. Furthermore, activation of p53 by cisplatin in rat mesangial cell was inhibited by PR treatment. These results suggest that protective effect of PR on cisplatin-induced apoptosis in rat mesangial cell may be associated with reduction of ERK, p38, JNK, p53 activation.

• Journal of Acupuncture Research
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• v.37 no.1
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• pp.42-48
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• 2020

Background: Atopic dermatitis (AD) is a chronic inflammatory condition which can be studied using phthalic anhydride (PA) to induce AD. Anti-inflammatory properties of bee venom (BV) wereinvestigated to determine whether it may be a useful treatment for AD. Methods: AD was induced by applying to pical PA to 8-week-old HR-1 mice (N = 50), then treating with (0.1, 0.25, and 0.5 ㎍) or without topical BV. Body weight, ear thickness histology, enzyme-linked immune sorbent assay (serum IgE concentrations), Western blot analysis [inducible nitric oxide synthase, cyclooxygenase-2, IκB-α, phospho-IκB-α, c-Jun N-terminal kinase (JNK), phospho-JNK, p38, phospho-p38, extra cellular signal-regulated kinase (ERK), and phospho-ERK], and the pull down assay for immunoblotting (p50), were used to measure inflammatory mediators. Results: PA + BV (0.1, 0.25, and 0.5 ㎍) significantly decreased ear thickness without altering body weight. IgE concentrations decreased in the PA + BV (0.5 ㎍)-treated groups compared with PAtreatment. Tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, cyclooxygenase-2, phospho-IκB-α, phospho-JNK, p38, phospho-p38, and phospho-ERK, all decreased following treatment with PA + BV compared with the PA-treatment alone. p50 was upregulated in the PA + BV-treated groups compared with the PA-treated group. Furthermore, the number of mast cells decreased in the PA + BV-treated groups compared with the PA-treated group. Epidermal thickness was significantly lower in the PA + BV-treated group compared with PA treatment alone. Conclusion: BV maybe a useful anti-inflammatory treatment for AD.

• Bulletin of the Korean Chemical Society
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• v.35 no.11
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• pp.3169-3174
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• 2014

Numerous studies have examined the role of flavonoids in modulating inflammatory responses in vitro. In this study, we found a novel flavonoid, 3,6,3'-trihydroxyflavone (1), with anti-inflammatory effects. Anti-inflammatory activity and mechanism of action were examined in mouse macrophages stimulated with lipopolysaccharide (LPS). Our results showed that the anti-inflammatory effects of 1 are mediated via p38 mitogen-activated protein kinase (p38 MAPK), Jun-N terminal kinase (JNK), and the extracellular-signal-regulated kinase (ERK) pathway in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Binding studies revealed that 1 had a high binding affinity to JNK1 ($1.568{\times}10^8M^{-1}$) and that the 3- and 6-hydroxyl groups of the C-ring and A-ring of 1 participated in hydrogen bonding interactions with the side chains of Asn114 and Lys55, respectively. The oxygen at the 3' position of the B-ring formed a hydrogen bond with side chain of Met111. Therefore, 1 could be a potential inhibitor of JNKs, with potent anti-inflammatory activity.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2022.09a
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• pp.93-93
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• 2022

Hovenia dulcis, one of the traditional medicinal plants, is currently being used as a functional ingredient for the development of health functional foods that protects the liver from alcohol damage in Korea. A variety of pharmacological effects of Hovenia dulcis have been reported so far, but studies on immune-enhancing activity are insufficient. Thus, in this study, we report that Hovenia dulcis branches (HDB) induce the activation of macrophages. HDB increased the production of immunostimulatory factors and phagocytosis in RAW264.7 cells. TLR4 inhibition blocked HDB-mediated production of immunostimulatory factors. In addition, the JNK inhibition reduced the HDB-mediated production of immunostimulatory factors, and the HDB-mediated JNK activation was blocked by the TLR4 inhibition. HDB increased the level of LC3-II and p62/SQSTM1. TLR4 inhibition blocked HDB-mediated increase in the level of LC3-II and p62/SQSTM1. These findings indicate that HDB may induce TLR4/JNK-dependent macrophage activation and TLR4-dependent macrophage autophagy.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.3
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• pp.187-197
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• 2009

Objective: Phosphorylation and dephosphorylation of proteins are important in regulating cellular signaling pathways. Bead-based multiplex phosphorylation assay was conducted to detect the phosphorylation of seven proteins to maximize the information obtained from a single lysate of stage-specific mouse oocytes at a time. Methods: Cumulus-oocyte complexes (COCs) were cultured for 2 h, 8 h, and 16 h, respectively to address phosphorylation status of seven target proteins during oocyte maturation process. We analyzed the changes in phosphorylation at germinal vesicle (GV, 0 h), germinal vesicle breakdown (GVBD, 2 h), metaphase I (MI, 8 h), and metaphase II (MII, 16 h in vitro or in vivo) mouse oocytes by using Bio-Plex phosphoprotein assay system. We chose seven target proteins, namely, three mitogen-activated protein kinases (MAPKs), ERK1/2, JNK, and p38 MAPK, and other 4 well known signaling molecules, Akt, GSK-$3{\alpha}/{\beta}$, $I{\kappa}B{\alpha}$, and STAT3 to measure their phosphorylation status. Western blot analysis and kinase inhibitor treatment for ERK1/2, JNK, and Akt during in vitro maturation of oocytes were conducted for the confirmation. Results: Phosphorylation of ERK1/2, JNK, p38 MAPK and STAT3 was increased over 3 folds up to 20 folds, while phosphorylation of the other three signal molecules, Akt, GSK-$3{\alpha}/{\beta}$, and $I{\kapa}B{\alpha}$ was less than 3 folds. All of these results except for Akt were statistically significant (p<0.05). Conclusion: This is the first report on the new and valuable method measuring many phosphoproteins simultaneously in one minute sample such as oocyte lysates. All of the three MAPKs, ERK1/2, JNK, and p38 MAPK are involved in the process of mouse oocyte maturation. In addition, STAT3 might be important regulator of oocyte maturation, while Akt phosphorylation at Serine 473 may not be involved in the regulation of oocyte maturation.

• Journal of the Korean Applied Science and Technology
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• v.34 no.4
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• pp.769-777
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• 2017

To research the effects of marigold extract, which is used mixed with calendula extract, on collagen growth and MMP-1 expression in human fibroblast, we measured cytotoxicity, collagen growth and MMP-1 expression by using HDF cells. The result of measurement showed over 80% cell survival rate in $5{\sim}100{\mu}g/mL$ concentration of marigold extract and calendula extract for HDF cells, which indicates there is no cytotoxicity. The result of measuring collagen synthetic abilities showed both types of extract had collagen synthetic ability increase dose dependently, by 25% in $100{\mu}g/mL$ concentration of marigold extract, and by 7% in $100{\mu}g/mL$ concentration of calendula extract. The result of experimenting the effect on MMP-1 expression showed that both types of extract suppress MMP-1 expression. The result of observing phosphorylation of p-JNK and p-ERK, which are known to be involved with MMP-1 expression, revealed that marigold extract effectively suppresses MMP-1 expression through signaling pathway of p-JNK and p-ERK. The above results confirm the wrinkle improvement effect of marigold extract, and furthermore, it can be used as a cosmetic ingredient for anti-aging.

• Nutrition Research and Practice
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• v.11 no.2
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• pp.90-96
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• 2017

BACKGROUND/OBJECTIVES: Although the antioxidative effects of lycopene are generally known, the molecular mechanisms underlying the anti-inflammatory properties of lycopene are not fully elucidated. This study aimed to examine the role and mechanism of lycopene as an inhibitor of inflammation. METHODS/MATERIALS: Lipopolysaccharide (LPS)-stimulated SW 480 human colorectal cancer cells were treated with 0, 10, 20, and $30{\mu}M$ lycopene. The MTT assay was performed to determine the effects of lycopene on cell proliferation. Western blotting was performed to observe the expression of inflammation-related proteins, including nuclear factor-kappa B ($NF-{\kappa}B$), inhibitor kappa B ($I{\kappa}B$), mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 (p38 MAP kinase). Real-time polymerase chain reaction was performed to investigate the mRNA expression of tumor necrosis factor ${\alpha}$ ($TNF-{\alpha}$), interleukin-1 beta ($IL-1{\beta}$), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Concentrations of nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) were determined via enzyme-linked immunosorbent assays. RESULTS: In cells treated with lycopene and LPS, the mRNA expression of $TNF-{\alpha}$, $IL-1{\beta}$, IL-6, iNOS, and COX-2 were decreased significantly in a dose-dependent manner (P < 0.05). The concentrations of $PGE_2$ and NO decreased according to the lycopene concentration (P < 0.05). The protein expressions of $NF-{\kappa}B$ and JNK were decreased significantly according to lycopene concertation (P < 0.05). CONCLUSIONS: Lycopene restrains $NF-{\kappa}B$ and JNK activation, which causes inflammation, and suppresses the expression of $TNF-{\alpha}$, $IL-1{\beta}$, IL-6, COX-2, and iNOS in SW480 human colorectal cancer cells.

• Archives of Pharmacal Research
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• v.26 no.12
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• pp.1055-1060
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• 2003

Lysophosphatidic acid (LPA) is a well-known mitogen in various cell types. However, we found that LPA inhibits melanocyte proliferation. Thus, we further investigated the possible signaling pathways involved in melanocyte growth inhibition. We first examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by LPA. The activations of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) were observed in concert with the inhibition of melanocyte proliferation by LPA, whereas p38 MAP kinase and Akt were not influenced by LPA. However, the specific inhibition of the ERK or JNK pathways by PD98059 or D-JNKI1, respectively, did not restore the antiproliferative effect. We next examined changes in the expression of cell cycle related proteins. LPA decreased cyclin $D_1 and cyclin D_2$ levels but increased $p21^{WAF1/CIP1}$ (p21) and $p27^{KIP1}$ (p27) levels, which are known inhibitors of cyclin-dependent kinase. Flow cytometric analysis showed the inhibition of DNA synthesis by a reduction in the S phase and an increase in the $G_0/G_1$ phase of the cell cycle. Our results suggest that LPA induces cell cycle arrest by regulating the expressions of cell cycle related proteins.