• The Korean Journal of Physiology and Pharmacology
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• v.19 no.1
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• pp.1-7
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• 2015

Our previous study has shown berberine prevents damage to the intestinal mucosal barrier during early phase of sepsis in rat through mechanisms independent of the NOD-like receptors signaling pathway. In this study, we explored the regulatory effects of berberine on Toll-like receptors during the intestinal mucosal damaging process in rats. Male Sprague-Dawlay (SD) rats were treated with berberine for 5 d before undergoing cecal ligation and puncture (CLP) to induce polymicrobial sepsis. The expression of Toll-like receptor 2 (TLR 2), TLR 4, TLR 9, the activity of nuclear factor-kappa B ($NF-{\kappa}B$), the levels of selected cytokines and chemokines, percentage of cell death in intestinal epithelial cells, and mucosal permeability were investigated at 0, 2, 6, 12 and 24 h after CLP. Results showed that the tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and interleukin-6 (IL-6) level were significantly lower in berberine-treated rats compared to the control animals. Conversely, the expression level of tight junction proteins, percentage of cell death in intestinal epithelial cells and the mucosal permeability were significantly higher in berberine-treated rats. The mRNA expression of TLR 2, TLR 4, and TLR 9 were significantly affected by berberine treatment. Our results indicate that pretreatment with berberine attenuates tissue injury and protects the intestinal mucosal barrier in early phase of sepsis and this may possibly have been mediated through the TLRs pathway.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.27.1-27.16
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• 2022

Background: The role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood. Objectives: This study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-β, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes. Methods: CRFK cells and monocytes from feline coronavirus (FCoV)-seronegative cats and FCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours post-infection (hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-β, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence. Results: FIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-β expression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showed lower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfected monocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositive cats downregulated TLR7 and TNF-α expression between 4 and 24 hpi, and 4 and 12 hpi, respectively. IFN-β was upregulated early in FIPV-infected monocytes from FCoV-seropositive cats, with a significant difference observed at 12 hpi compared to FCoV-seronegative cats. The viral load in the CRFK and FIPV-infected monocytes in both cohorts of cats was similar over time.ConclusionTLR7 may be the key TLR involved in evading the innate response against inhibiting TNF-α production. Distinct TLR expression profiles between FCoV-seronegative and FCoV-seropositive cats were observed. The associated TLR that plays a role in the induction of IFN-β needs to be explored further.

• BMB Reports
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• v.46 no.7
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• pp.376-381
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• 2013

It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response against bacterial lipopolysaccharide (LPS) stimulation. LPS-mediated pro-inflammatory cytokine productions on mBMMCs obtained from Toll-like receptor4 (TLR4)-deficient mice, TLR2-defficient mice, and their wildtype, were specifically attenuated by the addition of either mouse MBL-A or ficolin-A in a dose-dependent manner. However, the inhibitory effects by mouse MBL-A or ficolin-A were restored by the addition of mannose or N-acetylglucosamine, respectively. These results suggest that mouse MBL-A and ficolin-A bind to LPS via its carbohydrate-recognition domain and fibrinogen-like domain, respectively, whereby cytokine production by LPS-mediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells.

• Journal of Physiology & Pathology in Korean Medicine
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• v.26 no.4
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• pp.511-518
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• 2012

The mechanisms of Toll-like receptor (TLR) signaling have been the focus of extensive studies because TLRs are the target of therapeutic intervention on multiple diseases. In this study, we investigated the inhibitory potential of Vigna angularis (azuki bean) on the TLR signaling. The effect of Vigna angularis extract (JSD) on TLR activation was investigated by assessing NF-${\kappa}B$ and AP-1 inducible secreted embryonic alkaline phosphatase (SEAP) activity. JSD significantly inhibited SEAP activity induced by poly I:C (TLR3 ligand) and poly I (TLR7 ligand) in a dose-dependent manner at concentration below 100 ${\mu}g/ml$ with no sign of cytotoxicity. Pretreatment of JSD markedly suppressed mRNA expressions of pro-inflammatory cytokines and adhesive molecules such as TNF-${\alpha}$, IL-6, RANTES, MCP-1 and ICAM-1 induced by TLR ligands. It also diminished the phosphorylation of $I{\kappa}B$ kinase and $I{\kappa}B$, and followed by $I{\kappa}B$-mediated nuclear translocation of p50, p65, and phosphorylation of p38, JNK, and IRF signaling pathway. In conclusion, our results suggest that Vigna angularis has inhibitory activity on TLR-3 and -7 signaling and it can be further developed as a remedy in curing TLR-related multiple diseases.

• Journal of Ginseng Research
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• v.46 no.1
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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.

• Biomedical Science Letters
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• v.16 no.2
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• pp.91-95
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• 2010

Meso-dihydroguaiaretic acid (MDGA) is a medicinal herbal product isolated from the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae). It exhibits a neuroprotective effect and also exerts cytotoxicity to certain cancer cells. In the present study, we investigated whether or not MDGA inhibits inflammatory reaction through the inhibition of nitric oxide (NO) generation. The results showed that MDGA (5~$25 {\mu}M$) inhibited 100 ng/ml lipopolysaccharide (LPS)- induced NO generation in macrophage Raw 264.7 cells in a concentration-dependent manner. We also measured the cytotoxic effects of MDGA on Raw 264.7 cells and found no evidence of cytotoxicity. The inhibition of NO generation by MDGA was consistent with the inhibitory effect on the expression of inducible nitric oxide synthase (iNOS). In addition, MDGA inhibited the LPS-induced gene expression of $interleukin-1{\beta}$ $(IL-1{\beta})$ as well as tumor necrosis $factor-{\alpha}$ $(TNF-{\alpha})$. The present results may provide that MDGA has anti-inflammatory properties through inhibition of the toll-like receptors (TLRs) pathway, and suggest that MDGA can be used as an anti-inflammatory agent.

• BMB Reports
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• v.44 no.7
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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.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.1-9
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• 2018

Objective: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. Methods: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1-10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. Results: Fallopian tube epithelial cells expressed TLRs 1-10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. Conclusion: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.4
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• pp.379-389
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• 2018

A nucleobase adenine is a fundamental component of nucleic acids and adenine nucleotides. Various biological roles of adenine have been discovered. It is not produced from degradation of adenine nucleotides in mammals but produced mainly during polyamine synthesis by dividing cells. Anti-inflammatory roles of adenine have been supported in IgE-mediated allergic reactions, immunological functions of lymphocytes and dextran sodium sulfate-induced colitis. However adenine effects on Toll-like receptor 4 (TLR4)-mediated inflammation by lipopolysaccharide (LPS), a cell wall component of Gram negative bacteria, is not examined. Here we investigated anti-inflammatory roles of adenine in LPS-stimulated immune cells, including a macrophage cell line RAW264.7 and bone marrow derived mast cells (BMMCs) and peritoneal cells in mice. In RAW264.7 cells stimulated with LPS, adenine inhibited production of pro-inflammatory cytokines $TNF-{\alpha}$ and IL-6 and inflammatory lipid mediators, prostaglandin $E_2$ and leukotriene $B_4$. Adenine impeded signaling pathways eliciting production of these inflammatory mediators. It suppressed $I{\kappa}B$ phosphorylation, nuclear translocation of nuclear factor ${\kappa}B$ ($NF-{\kappa}B$), phosphorylation of Akt and mitogen activated protein kinases (MAPKs) JNK and ERK. Although adenine raised cellular AMP which could activate AMP-dependent protein kinase (AMPK), the enzyme activity was not enhanced. In BMMCs, adenine inhibited the LPS-induced production of $TNF-{\alpha}$, IL-6 and IL-13 and also hindered phosphorylation of $NF-{\kappa}B$ and Akt. In peritoneal cavity, adenine suppressed the LPS-induced production of $TNF-{\alpha}$ and IL-6 by peritoneal cells in mice. These results show that adenine attenuates the LPS-induced inflammatory reactions.

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