• Journal of Life Science
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• 제33권6호
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• pp.463-470
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• 2023

Desmarestia tabacoides Okamura is a brown macroalgae that is found worldwide. Although several genera of Desmarestia have been reported as having anti-tumorigenic, anti-melanogenic, and photoprotective properties, the anti-inflammatory activity of D. tabacoides Okamura has not yet been evaluated. In this study, we analyzed the anti-inflammatory mechanisms of D. tabacoides Okamura ethanol extract (DTEE) via the inhibition of nitric oxide (NO) and prostaglandin (PG) E₂ production and the expression of their corresponding enzymes, inducible NO synthase (iNOS), and cyclooxygenase (COX)-2. In addition, their upstream signaling molecules were evaluated by Western blot analysis, such as nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK), and phosphoinositide-3-kinase (PI3K)/Akt, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The DTEE treatment significantly inhibited LPS-induced NO and PGE₂ production as well as the expression of their corresponding enzymes, iNOS, and COX-2 without cytotoxicity. The stimulated transcription factor NF-κB and upstream signaling molecules extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 were attenuated by the DTEE treatment, which was statistically significant, while Akt did not provide any inhibitory effect. Moreover, the DTEE treatment significantly mitigated the LPS-activated adaptor molecules, toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) in the RAW 264.7 cells. These results suggest that DTEE attenuates TLR4-mediated inflammatory responses by inhibiting NF-κB activation and suppressing MAPK phosphorylation in LPS-stimulated RAW 264.7 cells.

• Journal of Animal Science and Technology
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• 제64권1호
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• pp.123-134
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• 2022

Toll-like receptors (TLRs), as a part of innate immunity, plays an important role in detecting pathogenic molecular patterns (PAMPs) which are structural components or product of pathogens and initiate host defense systems or innate immunity. Precise negative feedback regulations of TLR signaling are important in maintaining homeostasis to prevent tissue damage by uncontrolled inflammation during innate immune responses. In this study, we identified and characterized the function of the pancreatic progenitor cell differentiation and proliferation factor (PPDPF) as a negative regulator for TLR signal-mediated inflammation in chicken. Bioinformatics analysis showed that the structure of chicken PPDPF evolutionarily conserved amino acid sequences with domains, i.e., SH3 binding sites and CDC-like kinase 2 (CLK2) binding sites, suggesting that relevant signaling pathways might contribute to suppression of inflammation. Our results showed that stimulation with polyinosinic:polycytidylic acids (Poly [I:C]), a synthetic agonist for TLR3 signaling, increased the mRNA expression of PPDPF in chicken fibroblasts DF-1 but not in chicken macrophage-like cells HD11. In addition, the expression of pro-inflammatory genes stimulated by Poly(I:C) were reduced in DF-1 cells which overexpress PPDPF. Future studies warrant to reveal the molecular mechanisms responsible for the anti-inflammatory capacity of PPDPF in chicken as well as a potential target for controlling viral resistance.

• Journal of Ginseng Research
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• 제46권1호
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• pp.156-166
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• 2022

Background: Panax ginseng Meyer (P. ginseng), a herb distributed in Korea, China and Japan, exerts benefits on diverse inflammatory conditions. However, the underlying mechanism and active ingredients remains largely unclear. Herein, we aimed to explore the active ingredients of P. ginseng against inflammation and elucidate underlying mechanisms. Methods: Inflammation model was constructed by lipopolysaccharide (LPS) in C57BL/6 mice and RAW264.7 macrophages. Molecular docking, molecular dynamics, surface plasmon resonance imaging (SPRi) and immunofluorescence were utilized to predict active component. Results: P. ginseng significantly inhibited LPS-induced lung injury and the expression of proinflammatory factors, including TNF-α, IL-6 and IL-1β. Additionally, P. ginseng blocked fluorescencelabeled LPS (LPS488) binding to the membranes of RAW264.7 macrophages, the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Furthermore, molecular docking demonstrated that ginsenoside Ro (GRo) docked into the LPS binding site of toll like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex. Molecular dynamic simulations showed that the MD2-GRo binding conformation was stable. SPRi demonstrated an excellent interaction between TLR4/ MD2 complex and GRo (K_D value of 1.16 × 10^-9 M). GRo significantly inhibited LPS488 binding to cell membranes. Further studies showed that GRo markedly suppressed LPS-triggered lung injury, the transcription and secretion levels of TNF-α, IL-6 and IL-1β. Moreover, the phosphorylation of NF-κB and MAPKs as well as the p65 subunit nuclear translocation were inhibited by GRo dose-dependently. Conclusion: Our results suggest that GRo exerts anti-inflammation actions by direct inhibition of TLR4 signaling pathway.

• IMMUNE NETWORK
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• 제8권2호
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• pp.59-65
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• 2008

Background: Toll-like receptors (TLRs) detect microbial infection and can directly induce innate host defense responses, which are thought to play critical roles in protecting the tubotympanum from infection. However, little is known about the relationship between TLRs, which are related to innate immunity, and immunoglobulins, which are related to adaptive immunity, in recurrent otitis media with effusion (OME). We therefore investigated the expression of TLR2 and TLR4 and immunoglobulin in children with OME. Methods: The study population consisted of 72 children with OME, 31 with more than 4 episodes in 12 months or more than 3 episodes in 6 months (otitis-prone group), and 41 with fewer than 3 episodes in 12 months (non-otitis prone group). The expression in middle ear effusion of TLR2 and TLR4 mRNA, as determined by Real time- -polymerase chain reaction (RT-PCR), and the concentrations of IgG, IgA, and IgM, as determined by Enzyme-linked immunosorbent assay(ELISA), were compared between the two groups. Results: Expression of TLR2 and TLR4 mRNA was lower in the otitis prone than in the non-otitis prone group, but the difference was not statistically significant (p>0.05). Between group differences in the concentrations of IgG, IgA and IgM in effusion fluid were not significant (p>0.05), and there were no correlations between immunoglobulin concentration and the expression of TLR2 and TLR4. Conclusion: Although there was a trend toward lower expression of TLR2 and TLR4 in the otitis-prone group, the differences, and those in immunoglobulin concentration, did not differ significantly between the otitis-prone and non-prone groups.

• Clinical and Experimental Pediatrics
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• 제48권1호
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• pp.6-12
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• 2005

In the last few years, a spectrum of dendritic cells(DCs), including toll like receptors(TLRs), might play a critical role in regulating allergy and asthma. DC plays a central role in initiating immune responses, linking innate and adaptive responses to pathogen. Human peripheral blood has three non-overlapping dendritic subset that expressed various 11 TLRs. These dendritic subsets and TLR contribute significant polarizing influences on T helper differentiation, but how this comes about is less clear. A better understanding of DC immunobiology may lead to the comprehension of allergy pathophysiology to prevent early stage allergic march.

• BMB Reports
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• 제44권7호
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• pp.468-472
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• 2011

Toll-like receptors (TLRs) are pattern recognition receptors that recognize molecular structures derived from microbes and initiate innate immunity. TLRs have two downstream signaling pathways, the MyD88- and TRIF-dependent pathways. Dysregulated activation of TLRs is closely linked to increased risk of many chronic diseases. Previously, we synthesized fumaryl pyrrolidinone, (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1-yl)-2-butenoate (IPOP), which contains a fumaric acid isopropyl ester and pyrrolidinone, and demonstrated that it inhibits the activation of nuclear factor kappa B by inhibiting the MyD88-dependent pathway of TLRs. However, the effect of IPOP on the TRIF-dependent pathway remains unknown. Here, we report the effect of IPOP on signal transduction via the TRIF-dependent pathway of TLRs. IPOP inhibited lipopolysaccharide- or polyinosinic-polycytidylic acidinduced interferon regulatory factor 3 activation, as well as interferon-inducible genes such as interferon inducible protein-10. These results suggest that IPOP can modulate the TRIF-dependent signaling pathway of TLRs, leading to decreased inflammatory gene expression.

• The Korean Journal of Physiology and Pharmacology
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• 제19권1호
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• pp.51-57
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• 2015

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of grampositive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.

• BMB Reports
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• 제44권2호
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• pp.129-134
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• 2011

Toll-like receptors (TLRs), which recognize structurally conserved components among pathogens, are mainly expressed by antigen-presenting cells such as dendritic cells (DCs), B cells, and macrophages. Recognition through TLRs triggers innate immune responses and influences antigen-specific adaptive immune responses. Although studies on the expression and functions of TLRs in antigen-presenting cells have been extensively reported, studies in lymphoid tissue inducer (LTi) cells have been limited. In this study, we observed that LTi cells expressed TLR2 and TLR4 mRNA as well as TLR2 protein and upregulated OX40L, CD30L, and TRANCE expression after stimulation with the TLR2 ligand zymosan or TLR4 ligand LPS. The expression of tumor necrosis factor superfamily (TNFSF) members was significantly upregulated when cells were cocultured with DCs, suggesting that upregulated TNFSF expression may contribute to antigen-specific adaptive immune responses.

• Journal of Veterinary Science
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• 제23권3호
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• pp.50.1-50.12
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• 2022

Background: There is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity. Objectives: To develop a reliable and rapid strategy to improve diagnostic tools for bTB. Methods: In this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect. Results: The results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity. It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen. Conclusions: Molecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.

• Clinical and Experimental Pediatrics
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• 제52권6호
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• pp.667-673
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• 2009

Purpose : Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections. The purpose of this study was to analyze TLR2 surface expressions and TLR2-mediated tumor necrosis factor-alpha ($TNF-{\alpha}$) and interleukin-6 (IL-6) production in patients with BCG vaccine-associated suppurative lymphadenitis. Methods : Peripheral monocytes were separated from 16 patients with BCG vaccine-associated suppurative lymphadenitis and 10 healthy controls using a magnet cell isolation kit. Monocytes ($1{\times}10^6$ cells/well) were incubated with a constant amount of $Pam_3CSK_4$ ($100{\mu}g/mL$) for 24 hours. TLR2 surface expression on monocytes was analyzed by FACS analysis and TLR-2 mRNA expression was determined by RT-PCR. TLR2-mediated $TNF-{\alpha}$ and IL-6 production were measured by ELISA. Results : In patients with BCG vaccine-associated suppurative lymphadenitis, low TLR2 expression on monocytes ($3.39{\pm}$1.2% versus $4.64{\pm}2.6%$) together with significantly lower TLR2 mRNA expression than in the healthy controls was seen after $Pam_3CSK_4$ stimulation. TLR2-mediated $TNF-{\alpha}$ and IL-6 production in patients with BCG vaccine-associated suppurative lymphadenitis ($TNF-{\alpha}$, $775.5{\pm}60.8pg/mL$; IL-6, $4,645.8{\pm}583.9pg/mL$) were also lesser than that in healthy controls ($TNF-{\alpha}$, $1,098.5{\pm}94.3pg/mL$; IL-6, $6,696.3{\pm}544.3pg/mL$). Conclusion : These findings suggest that low TLR2 expression on monocytes might be associated with increased susceptibility to BCG vaccine-associated suppurative lymphadenitis.