• International Journal of Oral Biology
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• 제36권4호
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• pp.173-178
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• 2011

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional cytokine that is elevated in inflammatory diseases such as atherosclerosis, diabetes and rheumatoid arthritis. Recent evidence has suggested that ${\beta}2$ adrenergic receptor (${\beta}2AR$) activation in osteoblasts suppresses osteogenic activity. In the present study, we explored whether $TNF{\alpha}$ modulates ${\beta}AR$ expression in osteoblastic cells and whether this regulation is associated with the inhibition of osteoblast differentiation by $TNF{\alpha}$. In the experiments, we used C2C12 cells, MC3T3-E1 cells and primary cultured mouse bone marrow stromal cells. Among the three subtypes of ${\beta}AR$, ${\beta}2$ and ${\beta}3AR$ were found in our analysis to be upregulated by $TNF{\alpha}$. Moreover, isoproterenol-induced cAMP production was observed to be significantly enhanced in $TNF{\alpha}$-primed C2C12 cells, indicating that $TNF{\alpha}$ enhances ${\beta}2AR$ signaling in osteoblasts. $TNF{\alpha}$ was further found in C2C12 cells to suppress bone morphogenetic protein 2-induced alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes including Runx2, ALP and osteocalcin. Propranolol, a ${\beta}2AR$ antagonist, attenuated this $TNF{\alpha}$ suppression of osteogenic differentiation. $TNF{\alpha}$ increased the expression of receptor activator of NF-${\kappa}B$ ligand (RANKL), an essential osteoclastogenic factor, in C2C12 cells which was again blocked by propranolol. In summary, our data show that $TNF{\alpha}$ increases ${\beta}2AR$ expression in osteoblasts and that a blockade of ${\beta}2AR$ attenuates the suppression of osteogenic differentiation and stimulation of RANKL expression by $TNF{\alpha}$. These findings imply that a crosstalk between $TNF{\alpha}$ and ${\beta}2AR$ signaling pathways might occur in osteoblasts to modulate their function.

• 생명과학회지
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• 제29권1호
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• pp.9-17
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• 2019

암조직의 내부에서 hypoxia와 glucose depletion 등의 microenvironmental stress를 받게 되면 necrosis가 유도되고, 실제로 암 조직 내부에서 necrotic core 형성이 관찰된다. Necrotic cells은 high mobility group box 1(HMGB1)를 extracellular space로 방출하는 것으로 알려져 있다. 방출된 HMGB1은 tumor-promoting cytokine으로 작용함으로써 tumor development 시 inflammation, metabolism 및 metastasis에 기여한다. 본 연구에서 non-invasive breast cancer cells MCF-7이 solid tumor의 in vitro model인 multicellular tumor spheroid (MTS) 배양을 통해 완전한 구형의 MTS를 형성하며 MTS가 성장함에 따라 inner region에 necrosis가 유도됨을 밝혔다. 또한 MCF-7 세포의 MTS 배양은 Snail 의존적으로 epithelial-mesenchymal transition (EMT)를 유도함을 관찰하였다. HMGB1의 cell surface receptors인 RAGE, TLR2, TLR4 발현이 MTS 배양에 의해 증가됨을 발견하였다. RAGE, TLR2, TLR4 를 knockdown한 결과 MTS 성장을 억제할 뿐만 아니라 MTS에 의해 증가되는 Snail 발현을 억제함을 밝혔다. 이는 MTS-induced Snail 발현이 RAGE/TLR2/TLR4의존적으로 조절되며 RAGE/TLR2/TLR4-Snail이 MTS 성장에 관여하는 것으로 보인다. 또한 Snail, RAGE, TLR2, TLR4 shRNA는 MTS 배양에 의해 유도되는 EMT를 억제함을 밝혔다. 실제 인간 암조직에서 정상조직에 비해 RAGE, TLR2, TLR4 유전자의 발현이 높음을 관찰하였다. 따라서 HMGB1이 RAGE/TLR2/4-Snail axis를 통해 MTS 배양에 따른 성장 및 EMT에 중요하게 작용할 것으로 예상된다.

• IMMUNE NETWORK
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• 제2권3호
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• pp.133-136
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• 2002

Background: The costimulatory molecule 4-1BB, a member of nerve growth factor receptor/tumor necrosis factor (NGFR/TNFR) super family, is involved in cell survival and death. Methods: In this study, female C57BL/6 ($H-2^b$) mice were used as a recipient, and DBA/2 ($H-2^d$) as a donor to assess a mixed lymphocyte reaction (MLR) and CTL response in vitro, and skin graft survival. IL-2, IFN level was measured by ELISA. Results: Mixed lymphocyte reaction (MLR) analysis showed that 4-1BB-deficient responder cells showed enhanced cellular proliferation over littermate controls. In contrast, IL-2 production was diminished only in 4-1BB knockout cultures. The IFN expression, on the other hand, was comparable between the groups. When female C57BL/6 ($H-2^b$) mice were grafted with the trunk skin of DBA/2 ($H-2^d$) mice, the in vivo tissue destruction of 4-1BB-deficient mice was not distinct from the normal littermates. Conclusion: These data suggest that 4-1BB is critical for the induction of alloreactive responses in vitro but 4-1BB alone could not change the course of skin rejection in vivo.

• 생약학회지
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• 제45권1호
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• pp.28-34
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• 2014

Rhei Rhizoma (RR) and Moutan cortex (MC) have been reported to have anti-inflammatory effects. However, little is known about the effects of RR and MC on endothelial inflammation and insulin resistance (IR). This study aims to investigate whether the water extracts of RR and MC could exert protection against palmitic acid (PA)-induced inflammation and IR in human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated for 6 h with RR or MC, and then exposed to PA for 24 h. The levels of interleukin-6 (IL-6) and tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) were determined by enzyme-linked immunosorbant assay kits. Western blot analysis was performed for activation of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and insulin receptor substrate-1 (IRS-1). In HUVECs stimulated with PA, both RR and MC significantly inhibited the production of TNF-${\alpha}$ and IL-6 and the activation of NF-${\kappa}B$. At the same concentrations, the inhibitory effects of RR were more potent than those of MC. PA reduced insulin-induced phosphorylation of IRS-1, which was reversed by RR and MC. The results suggest that RR and MC are effective in inhibiting PA-associated endothelial inflammation and ameliorating IR by beneficial regulation of NF-${\kappa}B$ and IRS-1 activation.

• Nutrition Research and Practice
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• 제9권4호
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• pp.364-369
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• 2015

BACKGROUND/OBJECTIVES: Inflammation is associated with various types of acute and chronic alcohol liver diseases. In this study, we examined whether umbelliferone (7-hydroxycoumarin, UF) ameliorates chronic alcohol-induced liver damage by modulating inflammatory response and the antioxidant system. METHODS: Rats were fed a Liber-Decarli liquid diet containing 5% alcohol with or without UF (0.05 g/L) for 8 weeks, while normal rats received an isocaloric carbohydrate liquid diet. RESULTS: Chronic alcohol intake significantly increased serum tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and interleukin 6 levels and decreased interleukin 10 level; however, UF supplementation reversed the cytokines related to liver damage. UF significantly suppressed hepatic lipopolysaccharide binding protein, toll-like receptor 4 (TLR4), nuclear factor kappa B, and TNF-${\alpha}$ gene expression increases in response to chronic alcohol intake. Masson's trichrome staining revealed that UF improved mild hepatic fibrosis caused by alcohol, and UF also significantly increased the mRNA expressions and activities of superoxide dismutase and catalase in liver, and thus, decreased lipid peroxide and mitochondrial hydrogen peroxide levels. CONCLUSIONS: The findings of this study indicate that UF protects against alcohol-induced liver damage by inhibiting the TLR4 signaling pathway and activating the antioxidant system.

• Biomolecules & Therapeutics
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• 제22권4호
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• pp.295-300
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• 2014

The actin cytoskeleton plays an important role in macrophage-mediated inflammatory responses by modulating the activation of Src and subsequently inducing nuclear factor (NF)-${\kappa}B$ translocation. In spite of its critical functions, few papers have examined how the actin cytoskeleton can be regulated by the activation of toll-like receptor (TLR). Therefore, in this study, we further characterized the biological value of the actin cytoskeleton in the functional activation of macrophages using an actin cytoskeleton disruptor, cytochalasin B (Cyto B), and explored the actin cytoskeleton's involvement in morphological changes, cellular attachment, and signaling events. Cyto B strongly suppressed the TLR4-mediated mRNA expression of inflammatory genes such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-${\alpha}$, and inducible nitric oxide (iNOS), without altering cell viability. This compound also strongly suppressed the morphological changes induced by lipopolysaccharide (LPS), a TLR4 ligand. Cyto B also remarkably suppressed NO production under non-adherent conditions but not in an adherent environment. Cyto B did not block the co-localization between surface glycoprotein myeloid differentiation protein-2 (MD2), a LPS signaling glycoprotein, and the actin cytoskeleton under LPS conditions. Interestingly, Cyto B and PP2, a Src inhibitor, enhanced the phagocytic uptake of fluorescein isothiocyanate (FITC)-dextran. Finally, it was found that Cyto B blocked the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at 1 min and the phosphorylation of heat shock protein 27 (HSP27) at 5 min. Therefore, our data suggest that the actin cytoskeleton may be one of the key components involved in the control of TLR4-mediated inflammatory responses in macrophages.

• Journal of Nutrition and Health
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• 제49권4호
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• pp.241-246
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• 2016

Purpose: Aloe-emodin (AE), an ingredient of aloe, is known to exhibit anti-inflammatory activities. However, little is known about the underlying molecular mechanisms of its inflammatory modulatory activity in vitro. In the present study, we investigated the anti-inflammatory potential of AE using $Pam_3CSK_4$-stimulated macrophages. Methods: RAW 264.7 macrophages were treated with AE (0~20 mM) for 1 h, followed by treatment with $Pam_3CSK_4$ for 1 h. After incubation, mRNA expression levels of cytokines were measured. The effect of AE on TLR2-related molecules was also investigated in $Pam_3CSK_4$-stimulated RAW 264.7 macrophages. Results: AE attenuated $Pam_3CSK_4$-stimulated expression of proinflammatory cytokines, including tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-6 (IL-6), and interleukin-$1{\beta}$ ($IL-1{\beta}$) in RAW 264.7 macrophages. Two concentrations of AE ($10{\mu}M$ and $20{\mu}M$) effectively reduced mRNA expression of TLR2 by 41.18% and 54.43%, respectively, compared to that in control cells (p < 0.05). AE also decreased nuclear factor-kappa B ($NF-{\kappa}B$) activation and mitogen-activated protein kinase (MAPK) phosphorylation. Phosphorylation levels of ERK1/2, p38, and JNK were markedly reduced by $20{\mu}M$ AE. In particular, AE decreased phosphorylation of ERK in a dose-dependent manner in $Pam_3CSK_4$-stimulated RAW 264.7 macrophages. Conclusion: Our data indicate that AE exerts its anti-inflammatory effect by suppressing TLR2-mediated activation of $NF-{\kappa}B$ and MAPK signaling pathways in macrophages.

• Biomolecules & Therapeutics
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• 제25권6호
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• pp.641-647
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• 2017

Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum. In this study, we investigated the anti-inflammatory effects of galangin under in vitro and in vivo neuroinflammatory conditions caused by polyinosinic-polycytidylic acid (poly(I:C)), a viral mimic dsRNA analog. Galangin suppressed the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in poly(I:C)-stimulated BV2 microglia. On the other hand, galangin enhanced anti-inflammatory interleukin (IL)-10 production. Galangin also suppressed the expression of pro-inflammatory markers in poly(I:C)-injected mouse brains. Further mechanistic studies showed that galangin inhibited poly(I:C)-induced nuclear factor (NF)-${\kappa}B$ activity and phosphorylation of Akt without affecting MAP kinases. Interestingly, galangin increased the expression and transcriptional activity of peroxisome proliferator-activated receptor (PPAR)-${\gamma}$, known to play an anti-inflammatory role. To investigate whether PPAR-${\gamma}$ is involved in the anti-inflammatory function of galangin, BV2 cells were pre-treated with PPAR-${\gamma}$ antagonist before treatment of galangin. We found that PPAR-${\gamma}$ antagonist significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-${\alpha}$ and IL-6 in poly(I:C)-stimulated microglia. In conclusion, our data suggest that PI3K/Akt, NF-${\kappa}B$, and PPAR-${\gamma}$ play a pivotal role in mediating the anti-inflammatory effects of galangin in poly(I:C)-stimulated microglia.

• 한국발생생물학회지:발생과생식
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• 제19권4호
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• pp.227-234
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• 2015

The objective of this study was to investigate the expression of bovine luteum expressed sequence tags (ESTs), vascular endothelial growth factor (VEGF), and tumor necrosis factor receptor 1 (TNFR1) and the presence of functional ESTs in the bovine corpus luteum (CL) during different stages of the estrus cycle. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a difference in the expression of ESTs during the CL stage. Concentration of ESTs in the CL tissue increased significantly from the mid-luteal stage and decreased thereafter. RT-PCR analysis showed higher levels of the EST genes in the CL of the mid-luteal stage than in other stages, and the same level of expression of VEGF. Immunohistochemistry analysis of the tissue from CL formation to regression showed low cytosol and aggregation of the nucleus. And activity caspase 3 (apoptosis detector) was most strongly detected in the CL1 stage of bovine. During the estrous cycle, the cytosol was magnified and differentiation of the nucleus was clearly manifested. The ESTs affected the CL, and the relationship between VEGF and TNFR1 played a pivotal role for CL development and activation, dependent on the stage of CL. These results suggest local production of ESTs, the presence of functional ESTs in the bovine CL, and that ESTs play a role in regulating the function of cell death in bovine CL.

• International Journal of Oral Biology
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• 제40권3호
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• pp.111-116
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• 2015

To determine the effect of diabetes on root resorption in periodontitis, we investigated odontoclast formation and root resorption in diabetic rats with periodontitis. Odontoclast formation was observed in three groups of F344 rats: Controls (C) were normal rats without diabetes or periodontitis; the periodontitis (P) group had mandibular first molars to be ligatured; the periodontitis with diabetes (PD) group was intravenously administered streptozotocin (50 mg/kg) to induce diabetes and had mandibular first molars to be ligatured. On days 3, 10, and 20 after ligature, tumor necrosis factor (TNF)-${\alpha}$ and receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL) expression, odontoclast formation, and root resorption areas were evaluated by immunohistochemistry, tartrate-resistant acid phosphatase staining, and hematoxylin and eosin staining, respectively. The PD group showed frequent urination, weight loss, and hyperglycemia. Numbers of TNF-${\alpha}$- and RANKL-positive cells were higher in the P and PD groups than in the C group. It was more prevalent in PD group on day 3. Odontoclast formation was greater in the P and PD groups than in the C group on days 3 and 10, then decreased to same level as the C group by day 20. Root resorption in the PD and P groups showed increases on days 3 and 10, respectively, compared to the C group. These results suggest that diabetes may transiently increase root resorption on day 3 with high expression of TNF-${\alpha}$ and RANKL after periodontitis induction. This study could aid the understanding of root resorption in diabetic patients with periodontitis.