• The Korean Journal of Physiology and Pharmacology
• /
• v.21 no.5
• /
• pp.531-546
• /
• 2017

Activation of Toll-like receptor-4 (TLR-4) in articular chondrocytes increases the catabolic compartment and leads to matrix degradation during the development of osteoarthritis. In this study, we determined the proteomic and genomic alterations in human chondrocytes during lipopolysaccharide (LPS)-induced inflammation to elucidate the underlying mechanisms and consequences of TLR-4 activation. Human chondrocytes were cultured with LPS for 12, 24, and 36 h to induce TLR-4 activation. The TLR-4-induced inflammatory response was confirmed by real-time PCR analysis of increased interleukin-1 beta ($IL-1{\beta}$), interleukin-6 (IL-6), and tumor necrosis factor alpha ($TNF-{\alpha}$) expression levels. In TLR-4-activated chondrocytes, proteomic changes were determined by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectroscopy analysis, and genomic changes were determined by microarray and gene ontology analyses. Proteomics analysis identified 26 proteins with significantly altered expression levels; these proteins were related to the cytoskeleton and oxidative stress responses. Gene ontology analysis indicated that LPS treatment altered specific functional pathways including 'chemotaxis', 'hematopoietic organ development', 'positive regulation of cell proliferation', and 'regulation of cytokine biosynthetic process'. Nine of the 26 identified proteins displayed the same increased expression patterns in both proteomics and genomics analyses. Western blot analysis confirmed the LPS-induced increases in expression levels of lamin A/C and annexins 4/5/6. In conclusion, this study identified the time-dependent genomic, proteomic, and functional pathway alterations that occur in chondrocytes during LPS-induced TLR-4 activation. These results provide valuable new insights into the underlying mechanisms that control the development and progression of osteoarthritis.

• Journal of Life Science
• /
• v.21 no.1
• /
• pp.36-43
• /
• 2011

Diabetes mellitus is associated with vascular complications. Diabetic patients exhibit high levels of glycated adducts in serum compared to non-diabetic individuals. The aim of this study was to investigate whether extracellular glycated albumin (GA) predisposes vascular smooth muscle cells (VSMCs) to pro-inflammatory phenotype. Exposure of rat aortic smooth muscle cells (AoSMCs) to GA not only enhanced interleukin-6 (IL-6) release but also activated promoter activity of the IL-6 gene. GA-induced IL-6 promoter activation was suppressed by dominant-negative forms of Toll-like receptor (TLR)-4 and myeloid differentiation factor 88 (MyD88), but not by dominant-negative-forms of TLR-2 and TIR-domain-containing adapter-inducing interferon-$\beta$ (TRIF). Extracellular signal-regulated kinase (ERK) inhibition and diphenyleneiodium (DPI) also attenuated IL-6 induction by GA. Mutation at the nuclear factor-${\kappa}B$ (NF-${\kappa}B$)-binding site in the IL-6 promoter region suppressed promoter activation in response to GA. The present study proposes that GA would contribute to inflammatory reaction in the stressed vasculature by inducing IL-6 in VSMCs, and that TLR-4, EKR, and NF-${\kappa}B$ play active roles in the process.

• Korean Journal of Poultry Science
• /
• v.46 no.3
• /
• pp.185-191
• /
• 2019

Nucleoporin 210 (Nup210) is associated with several physiological processes including muscle and neural cell differentiation, autoimmune diseases, and peripheral T cell homeostasis. Chicken Nup210 (chNup210) gene was originally identified as one of the differentially expressed genes (DEGs) in the kidney tissues of chicken. To elucidate the role of Nup210 in metabolic disease of chicken, we studied the molecular characteristics of chNup210 and analyzed its gene expression under the stimulation of Toll-like receptor 3 (TLR3) ligands. The Nup210 genomic DNA and amino acid sequences of various species including fowls, fishes, and mammals were retrieved from the Ensemble database and subjected to bioinformatics analyses. The expression of Nup210 from several chicken tissues was probed through qRT-PCR, and chicken fibroblast DF-1 cell line was used to determine the change in expression of chNup210 after stimulation with TLR3 ligand, polyinosinic-polycytidylic acid (poly (I:C)). The chNup210 gene was highly expressed in chicken lung and spleen tissues. Although highly conserved among the species, chNup210 was evolutionary clustered in the same clade as that of duck compared to other mammals. Furthermore, this study revealed that chNup210 is expressed in TLR3 signaling pathway and provides fundamental information on Nup210 expression in chicken. Future studies that offer insight into the involvement of chNup210 in the chicken innate immune response against viral infection are recommended.

• The Korean Journal of Physiology and Pharmacology
• /
• v.26 no.5
• /
• pp.335-345
• /
• 2022

Pyroptosis is an inflammatory form of programmed cell death that is linked with invading intracellular pathogens. Cardiac pyroptosis has a significant role in coronary microembolization (CME), thus causing myocardial injury. Tanshinone IIA (Tan IIA) has powerful cardioprotective effects. Hence, this study aimed to identify the effect of Tan IIA on CME and its underlying mechanism. Forty Sprague-Dawley (SD) rats were randomly grouped into sham, CME, CME + low-dose Tan IIA, and CME + high-dose Tan IIA groups. Except for the sham group, polyethylene microspheres (42 ㎛) were injected to establish the CME model. The Tan-L and Tan-H groups received intraperitoneal Tan IIA for 7 days before CME. After CME, cardiac function, myocardial histopathology, and serum myocardial injury markers were assessed. The expression of pyroptosis-associated molecules and TLR4/MyD88/NF-κB/NLRP3 cascade was evaluated by qRT-PCR, Western blotting, ELISA, and IHC. Relative to the sham group, CME group's cardiac functions were significantly reduced, with a high level of serum myocardial injury markers, and microinfarct area. Also, the levels of caspase-1 p20, GSDMD-N, IL-18, IL-1β, TLR4, MyD88, p-NF-κB p65, NLRP3, and ASC expression were increased. Relative to the CME group, the Tan-H and Tan-L groups had considerably improved cardiac functions, with a considerably low level of serum myocardial injury markers and microinfarct area. Tan IIA can reduce the levels of pyroptosis-associated mRNA and protein, which may be caused by inhibiting TLR4/MyD88/NF-κB/NLRP3 cascade. In conclusion, Tanshinone IIA can suppress cardiomyocyte pyroptosis probably through modulating the TLR4/MyD88/NF-κB/NLRP3 cascade, lowering cardiac dysfunction, and myocardial damage.

• Asian-Australasian Journal of Animal Sciences
• /
• v.25 no.7
• /
• pp.1021-1028
• /
• 2012

Peripheral blood mononuclear cells (PBMCs) discriminate microbial pathogens and induce T-cell responses of appropriate effector phenotype accordingly. Toll-like receptors (TLRs), in part, mediate this microbial recognition and differentiation while the development of T-cell effector functions critically depends on the release of Th1- or Th2- type cytokines. In the present study, buffalo PBMCs were stimulated under in vitro culture conditions by Bacillus subtilis cell wall petidoglycan, a TLR2 ligand, in a dose- and time- dependent manner. The expression of TLR2 as well as the subsequent differential induction of the Th1 and Th2 type cytokines was measured. Stimulation was analyzed across five doses of peptidoglycan ($10{\mu}g/ml$, $20{\mu}g/ml$, $30{\mu}g/ml$, $40{\mu}g/ml$ and $50{\mu}g/ml$) for 3 h, 12 h, 24 h and 36 h incubation periods. We observed the induction of TLR2 expression in a dose- and time-dependent manner and the peptidoglycan induced tolerance beyond $30{\mu}g/ml$ dose at all incubation periods. The correlation between peptidoglycan stimulation and TLR2 induction was found positive at all doses and for all incubation periods. Increased production of all the cytokines was observed at low doses for 3 h incubation, but the expression of IL-4 was relatively higher than IL-12 at the higher antigen doses, indicating tailoring towards Th2 response. At 12 h incubation, there was a pronounced decrease in IL-4 and IL-10 expression relative to IL-12 in a dose- dependent manner, indicating skewing to Th1 polarization. The expression of IL-12 was highest for all doses across all the incubation intervals at 24 h incubation, indicating Th1 polarization. The relative expression of TNF-${\alpha}$ and IFN-${\gamma}$ was also higher while that of IL-4 and IL-10 showed a decrease. For 36 h incubation, at low doses, relative increase in the expression of IL-4 and IL-10 was observed which decreased at higher doses, as did the expression of all other cytokines. The exhaustion of cytokine production at 36 h indicated that PBMCs became refractory to further stimulation. It can be concluded from this study that the cytokine response to sPGN initially was of Th2 type which skews, more pronouncedly, to Th1 type with time till the cells become refractory to further stimulation.

• Korean Journal of Plant Resources
• /
• v.34 no.5
• /
• pp.411-419
• /
• 2021

In this study, we investigated in vitro immune-enhancing and anti-obesity activity of Abelmoschus manihot roots (AMR) in mouse macrophage RAW264.7 cells and mouse adipocytes 3T3-L1 cells. AMR increased the production of immunostimulatory factors such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in RAW264.7 cells. The inhibition of toll like receptor (TLR) 2 and 4 blocked AMR-mediated production of immunostimulatory factors in RAW264.7 cells. In addition, the inhibition of mitogen-activated protein kinases (MAPKs) signaling pathway reduced AMR-mediated production of immunostimulatory factors. From these results, AMR is considered to have immune-enhancing activity through TLR2/4-mediated activation of MAPKs signaling pathway. In addition, AMR inhibited lipid accumulation and reduced the protein level such as CCAAT enhancer-binding protein alpha (CEBPα), peroxisome proliferator-activated receptor gamma (PPARγ), perilipin-1, adiponectin and fatty acid binding protein 4 (FABP4) associated with lipid accumulation in 3T3-L1 cells, indicating that AMR may have anti-obesity activity. Based on these results, AMR is expected to be used as a potential functional agent for immune enhancement and anti-obesity.

• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.1
• /
• pp.41-51
• /
• 2016

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activating TLR4-NF-${\kappa}B$ signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-${\kappa}B$ pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPS-induced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.6
• /
• pp.860-873
• /
• 2018

Although ginseng marc is a by-product obtained during manufacturing of various commercial ginseng products and has been routinely discarded as a waste, it still contains considerable amounts of potential bioactive compounds, including saponins and polysaccharides. Previously, we reported that ginseng oligosaccharides derived from ginseng marc polysaccharides by enzymatic hydrolysis exert immunostimulatory activities in macrophages and these activated macrophages are in turn able to inhibit the growth of skin melanoma cells by inducing apoptosis. In the present study, a more detailed investigation of the immunostimulatory activity and underlying action mechanisms of an enzymatic hydrolysate (GEH) containing these oligosaccharides derived from ginseng marc polysaccharides was performed. The levels of proinflammatory cytokines and anti-inflammatory cytokines were measured in GEH-stimulated RAW264.7 macrophages using RT-PCR analysis and ELISA. The expression levels of Toll-like receptor 2 (TLR2) and TLR4, Dectin-1, and MerTK were measured by RT-PCR analysis or western blot analysis, and the phagocytic activities of GEH-challenged bone marrow-derived macrophages toward apoptotic Jurkat cells were assayed using fluorescence microscopy. GEH induced the production of both proinflammatory cytokines $TNF-{\alpha}$ and IL-6, and anti-inflammatory cytokine IL-10 in RAW 264.7 cells. The expression of the TLR2 and MerTK mRNAs was increased upon GEH treatment. Phagocytosis of apoptotic Jurkat cells was enhanced in GEH-treated macrophages. Based on the results, this enzymatic hydrolysate (GEH) containing oligosaccharides exerts immunostimulatory effects by maintaining the balance between M1 and M2 cytokines, facilitating macrophage activation and contributing to the efficient phagocytosis of apoptotic cells. Therefore, the GEH could be developed as value-added, health-beneficial food materials with immunostimulatory effects.

• Molecules and Cells
• /
• v.43 no.3
• /
• pp.251-263
• /
• 2020

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasome-dependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in anti-flagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-β (IFN-β) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo. Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-β, but not for other pro-inflammatory cytokines. In addition, we found that anti-flagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-β produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.

• Biomolecules & Therapeutics
• /
• v.31 no.1
• /
• pp.27-39
• /
• 2023

Extensive research supported the therapeutic potential of curcumin, a naturally occurring compound, as a promising cytokine-suppressive anti-inflammatory drug. This study aimed to investigate the synergistic anti-inflammatory and anti-cytokine activities by combining 6-shogaol and 10-shogaol to curcumin, and associated mechanisms in modulating lipopolysaccharides and interferon-γ-induced proinflammatory signaling pathways. Our results showed that the combination of 6-shogaol-10-shogaolcurcumin synergistically reduced the production of nitric oxide, inducible nitric oxide synthase, tumor necrosis factor and interlukin-6 in lipopolysaccharides and interferon-γ-induced RAW 264.7 and THP-1 cells assessed by the combination index model. 6-shogaol-10-shogaol-curcumin also showed greater inhibition of cytokine profiling compared to that of 6-shogaol-10-shogaol or curcumin alone. The synergistic anti-inflammatory activity was associated with supressed NFκB translocation and downregulated TLR4-TRAF6-MAPK signaling pathway. In addition, SC also inhibited microRNA-155 expression which may be relevant to the inhibited NFκB translocation. Although 6-shogaol-10-shogaol-curcumin synergistically increased Nrf2 activity, the anti-inflammatory mechanism appeared to be independent from the induction of Nrf2. 6-shogaol-10-shogaol-curcumin provides a more potent therapeutic agent than curcumin alone in synergistically inhibiting lipopolysaccharides and interferon-γ induced proinflammatory mediators and cytokine array in macrophages. The action was mediated by the downregulation of TLR4/TRAF6/MAPK pathway and NFκB translocation.