• The Korean Journal of Malacology
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• v.29 no.1
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• pp.1-6
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• 2013

This study aimed to evaluate the effects of shaking stress in the hemolymph of the Manila clam Ruditapes philippinarum by quantification of nitric oxide (NO) levels. The clams were divided into 3 groups as follows: clams placed in a plain container (control), clams injected with nitro-L-arginine methyl ester (NAME, an NO inhibitor), and clams in a container filled with nylon fiber at a density of $1kg/m^3$. Subsequently, each group was placed in sea water and shaken at 100 rpm for 6 h. The concentration of NO was quantified by using DAF assay and Griess assay. Both the assays showed that while shaking significantly increased the NO concentration, the NO inhibitor reduced the NO concentration in the hemolymph of the clams tested. In addition, the nylon fiber, which was used as a filler, effectively prevented the increase in NO concentration. This result suggests that measurement of NO concentration is a useful tool for evaluation of physiological stress in marine bivalves. In addition, it should be considered that a filler is necessary when dredge fishing or the suspended clam culture method is developed.

• The Korean Journal of Food And Nutrition
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• v.32 no.2
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• pp.106-113
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• 2019

The objective of this study was to evaluate the effect of fermented Kalopanax pictus (KP-F) on macrophage activation and its effect as a competitive inhibitor of LPS and inhibitory effect on endotoxemia. The results showed that KP-F could activate macrophage in a dose-dependent manner, and KP-F was confirmed to act as a ligand for TLR4. Also, it was found that KP-F did not exhibit the same biotoxicity as LPS in intraperitoneal injection, and that it could suppress the neutrophil migration induced by LPS administration. In normal mice, the body weight, tissue weight, and amount of nitrite and pro-inflammatory cytokines in serum showed no significant changes with KP-F diet for 2 weeks, confirming that administration of KP-F in normal mice did not lead to over activation of immune response and biotoxicity. In the mouse model of endotoxemia induced by LPS and D-galactosamine(D-GalN) in sub-lethal dose, the diet of KP-F effectively inhibited the amount of nitrite and cytokines in the blood, and thus was found to be able to relieve the hepatic and kidney injury. In addition, in the endotoxemia mouse model induced by LPS and D-GalN of lethal dose, the survival rate was increased by KP-F diet in a dose-dependent manner.

• Journal of Integrative Natural Science
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• v.15 no.3
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• pp.131-136
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• 2022

Cell migration is essential for diverse cellular processes including wound healing, immune response, development, and cancer metastasis. Pi3-kinase (PI3K) is a key regulator for actin cytoskeleton and phosphorylates phosphatidylinositol (4,5)-diphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). High levels of PIP3 by PI3Ks are associated with increased levels of F-actin and pseudopod extension at the leading edge of migrating cells such as neutrophils and Dictyostelium. LY294002 is a well-known PI3K specific inhibitor. Here, we investigated the effect of LY294002 on cell migration. First, we evaluated the appropriate concentration of dimethyl sulfoxide (DMSO) for using as a solvent for LY294002. DMSO is a highly polar organic reagent and one of the most common solvent for organic and inorganic chemicals. Cell morphology and cell migration were unaffected at the concentrations less than 0.1 % DMSO. Therefore, stock solution of LY294002 was prepared so that the final concentration of DMSO was 0.1 % or less when treated. When cells were treated with LY294002, cell migration was increased in a concentration-dependent manner. The maximum speed was detected in the presence of 30 µM LY294002. These results suggest that PI3Ks play a inhibitory role in regulating cell migration in our experimental conditions.

• IMMUNE NETWORK
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• v.20 no.3
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• pp.26.1-26.9
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• 2020

Cereblon (CRBN), a negative modulator of AMP-activated protein kinase (AMPK), is highly expressed in the retina. We confirmed the expression of CRBN in ARPE-19 human retinal cells by Western blotting. We also demonstrated that CRBN knock-down (KD) could effectively downregulate IL-6 and MCP-1 protein and gene expression in LPS-stimulated ARPE-19 cells. Additionally, CRBN KD increased the phosphorylation of AMPK/acetylcoenzyme A carboxylase (ACC) and the expression of heme oxygenase-1 (HO-1) in ARPE-19 cells. Furthermore, CRBN KD significantly reduced LPS-induced nuclear translocation of NF-κB p65 and activation of NF-κB promoter activity. However, these processes could be inactivated by compound C (inhibitor of AMPK) and zinc protoporphyrin-1 (ZnPP-1; inhibitor of HO-1). In conclusion, compound C and ZnPP-1 can rescue LPS-induced levels of proinflammatory cytokines (IL-6 and MCP-1) in CRBN KD ARPE-19 cells. Our data demonstrate that CRBN deficiency negatively regulates proinflammatory cytokines via the activation of AMPK/HO-1 in the retina.

• Journal of dental hygiene science
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• v.17 no.1
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• pp.81-86
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• 2017

Bacterial infection and smoking are an important risk factors involved in the development and progression of periodontitis. However, the signaling mechanism underlying the host immune response is not fully understood in periodontal lesions. In this study, we determined the expression of janus kinase (JAK)/signal transducer and activator of transcription (STAT) on Porphyromonas gingivalis lipopolysaccharide (LPS)- and nicotine-induced cytotoxicity and the production of inflammatory mediators, using osteoblasts. The cells were cultured with 5 mM nicotine in the presence of $1{\mu}g/ml$ LPS. Cell viability was determined using MTT assay. The role of JAK on inflammatory mediator expression and production, and the regulatory mechanisms involved were assessed via enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and Western blot analysis. LPS- and nicotine synergistically induced the production of cyclooxgenase-2 (COX-2) and prostaglandin $E_2$ ($PGE_2$) and increased the protein expression of JAK/STAT. Treatment with an JAK inhibitor blocked the production of COX-2 and $PGE_2$ as well as the expression of pro-inflammatory cytokines, such as tumor necrosis factor-${\alpha}$, interleukin-$1{\beta}$ ($IL-1{\beta}$), and IL-6 in LPS- and nicotine-stimulated osteoblasts. These results suggest that JAK/STAT is closely related to the LPS- and nicotine-induced inflammatory effects and is likely to regulate the immune response in periodontal disease associated with dental plaque and smoking.

• Fisheries and Aquatic Sciences
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• v.18 no.3
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• pp.273-281
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• 2015

The follistatin (FST) gene encodes a monomeric glycoprotein that plays a role in binding and inhibiting the functions of members of the transforming growth factor (TGF)-${\beta}$ superfamily. Thus, FST facilitates a wide variety of functions, ranging from muscle growth, to inflammation and immunity. In this study, we sought to characterize an FST counterpart, RbFST, which was identified from rock bream Oplegnathus fasciatus. The RbFST cDNA sequence (2,419 bp) contains a 933-bp open reading frame (ORF) that encodes a putative amino acid sequence for RbFST (35 kDa). The putative amino acid sequence contains a Kazal-type serine protease inhibitor domain (51-98 residues) and an EF-hand, calcium-binding domain (191-226 residues). Additionally, this sequence shares a high identity (98.7%) with the Siniperca chuatsi FST sequence, with which it also has the closest evolutionary relationship according to a phylogenetic study. Omnipresent distribution of RbFST transcripts were detected in the gill, liver, spleen, head kidney, kidney, skin, muscle, heart, brain, and intestine of healthy animals, with significantly higher expression levels in the heart, followed by the liver tissue. Under pathogenic stress caused by two bacterial pathogens, Streptococcus iniae and Edwardsiella tarda, RbFST transcription was found to be significantly up-regulated. Altogether, our findings suggest the putative role of RbFST in immune related responses against pathogenic infections, further prefiguring its significance in rock bream physiology.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.4
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• pp.199-205
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• 2003

Osteoblasts are affected by TNF-${\alpha}$ overproduction by immune cells during inflammation. It has been suggested that functional $NF-{\kappa}B$ sites are involved in TNF-${\alpha}$-induced bone resorption. Thus, we explored the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor $(NF-{\kappa}B)$, on the induction of TNF-${\alpha}$-induced activation of JNK/SAPK, AP-1, cytochrome c, caspase and apoptosis in MC3T3E1 osteoblasts. Pretreatment of the cells with PDTC blocked TNF-${\alpha}$-induced $NF-{\kappa}B$ activation. TNF-${\alpha}$-induced activation of AP-1, another nuclear transcription factor, was suppressed by PDTC. The activation of c-Jun N-terminal kinase, implicated in the regulation of AP-1, was also down regulated by PDTC. TNF-${\alpha}$-induced apoptosis, release of cytochrome c and subsequent activation of caspase-3 were abolished by PDTC. TNF-${\alpha}$-induced apoptosis was partially blocked by Ac-DEVD-CHO, a caspase-3 inhibitor, suggesting that caspase-3 is involved in TNF-${\alpha}$-mediated signaling through $NF-{\kappa}B$ in MC3T3E1 osteoblasts. Thus, these results demonstrate that PDTC, has an inhibitory effect on TNF-${\alpha}$-mediated activation of JNK/SAPK, AP-1, cytochrome c release and subsequent caspase-3, leading to the inhibition of apoptosis. Our study may contribute to the treatment of TNF-${\alpha}$-associated immune and inflammatory diseases such as rheumatoid arthritis and periodontal diseases.

• Parasites, Hosts and Diseases
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• v.54 no.6
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• pp.711-717
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• 2016

Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines, which are important for innate immunity. NLRs, i.e., nucleotide-binding oligomerization domain (NOD)-like receptors, play a crucial role as innate immune sensors and form multiprotein complexes called inflammasomes, which mediate caspase-1-dependent processing of $pro-IL-1{\beta}$. To elucidate the role of inflammasome components in T. gondiiinfected THP-1 macrophages, we examined inflammasome-related gene expression and mechanisms of inflammasome-regulated cytokine $IL-1{\beta}$ secretion. The results revealed a significant upregulation of $IL-1{\beta}$ after T. gondii infection. T. gondii infection also upregulated the expression of inflammasome sensors, including NLRP1, NLRP3, NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and NAIP, in a time-dependent manner. The infection also upregulated inflammasome adaptor protein ASC and caspase-1 mRNA levels. From this study, we newly found that T. gondii infection regulates NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and neuronal apoptosis inhibitor protein (NAIP) gene expressions in THP-1 macrophages and that the role of the inflammasome-related genes may be critical for mediating the innate immune responses to T. gondii infection.

• Molecules and Cells
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• v.25 no.2
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• pp.253-257
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• 2008

Acrolein is a highly electrophilic ${\alpha},{\beta}$-unsaturated aldehyde present in a number of environmental sources, especially cigarette smoke. It reacts strongly with the thiol groups of cysteine residues by Michael addition and has been reported to inhibit nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) activation by lipopolysaccharide (LPS). The mechanism by which it inhibits $NF-{\kappa}B$ is not clear. Toll-like receptors (TLRs) play a key role in sensing microbial components and inducing innate immune responses, and LPS-induced dimerization of TLR4 is required for activation of downstream signaling pathways. Thus, dimerization of TLR4 may be one of the first events involved in activating TLR4-mediated signaling pathways. Stimulation of TLR4 by LPS activates both myeloid differential factor 88 (MyD88)- and TIR domain-containing adapter inducing $IFN{\beta}$ (TRIF)-dependent signaling pathways leading to activation of $NF-{\kappa}B$ and IFN-regulatory factor 3 (IRF3). Acrolein inhibited $NF-{\kappa}B$ and IRF3 activation by LPS, but it did not inhibit $NF-{\kappa}B$ or IRF3 activation by MyD88, inhibitor ${\kappa}B$ kinase $(IKK){\beta}$, TRIF, or TNF-receptor-associated factor family member-associated $NF-{\kappa}B$ activator (TANK)-binding kinase 1 (TBK1). Acrolein inhibited LPS-induced dimerization of TLR4, which resulted in the down-regulation of $NF-{\kappa}B$ and IRF3 activation. These results suggest that activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain chemicals with a structural motif that enables Michael addition.

• IMMUNE NETWORK
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• v.10 no.6
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• pp.188-197
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• 2010

Background: Lichen-derived glucans have been known to stimulate the functions of immune cells. However, immunostimulatory activity of glucan obtained from edible lichen, Umbilicaria esculenta, has not been reported. Thus we evaluated the phenotype and functional maturation of dendritic cells (DCs) following treatment of extracted glucan (PUE). Methods: The phenotypic and functional maturation of PUE-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. PUE-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity. Finally we detected the activation of MAPK and NF-${\kappa}B$ by immunoblot. Results: Phenotypic maturation of DCs was shown by the elevated expressions of CD40, CD80, CD86, and MHC class I/II molecules. Functional activation of DCs was proved by increased cytokine production of IL-12, IL-$1{\beta}$, TNF-${\alpha}$, and IFN-${\alpha}/{\beta}$, decreased endocytosis, and enhanced proliferation of allogenic T cells. Polymyxin B, specific inhibitor of lipopolysaccharide (LPS), did not affect PUE activity, which suggested that PUE was free of LPS contamination. As a mechanism of action, PUE increased phosphorylation of ERK, JNK, and p38 MAPKs, and enhanced nuclear translocation of NF-${\kappa}B$ p50/p65 in DCs. Conclusion: These results indicate that PUE induced DC maturation via MAPK and NF-${\kappa}B$ signaling pathways.