• IMMUNE NETWORK
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• 제9권5호
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• pp.192-202
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• 2009

Background: Little information is available the role of Nitric Oxide (NO) in host defenses during human tuberculosis (TB) infection. We investigated the modulating factor(s) affecting NO synthase (iNOS) induction in human macrophages. Methods: Both iNOS mRNA and protein that regulate the growth of mycobacteria were determined using reverase transcriptase-polymerase chain reaction and western blot analysis. The upstream signaling pathways were further investigated using iNOS specific inhibitors. Results: Here we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-${\gamma}$ synergistically enhanced NO synthesis and iNOS expression induced by Mycobacterium tuberculosis (MTB) or by its purified protein derivatives in human monocyte-derived macrophages. Both the nuclear factor-${\kappa}B$ and MEK1-ERK1/2 pathways were indispensable in the induction of iNOS expression, as shown in toll like receptor 2 stimulation. Further, the combined treatment with 1,25-D3 and IFN-${\gamma}$ was more potent than either agent alone in the inhibition of intracellular MTB growth. Notably, this enhanced effect was not explained by increased expression of cathelicidin, a known antimycobacterial effector of 1,25-D3. Conclusion: These data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.

• Nutrition Research and Practice
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• 제15권6호
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• pp.798-806
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• 2021

BACKGROUND/OBJECTIVES: Obesity is associated with chronic inflammation. The spleen is the largest organ of the lymphatic system and has an important role in immunity. Obesity-induced inflammatory responses are triggered by Toll-like receptor (TLR)-myeloid differentiation primary response 88 (MyD88) pathway signaling. Phenethyl isothiocyanate (PEITC) and 3,3'-diindolylmethane (DIM), major dietary glucosinolates present in cruciferous vegetables, have been reported to produce anti-inflammatory effects on various diseases. However, the effects of PEITC and DIM on the obesity-induced inflammatory response in the spleen are unclear. The purpose of this study was to examine the anti-inflammatory effects of PEITC and DIM on the spleen and their mechanism in high fat/cholesterol diet (HFCD)-fed C57BL/6 mice. MATERIALS/METHODS: We established an animal model of HFCD-induced obesity using C57BL/6 mice. The mice were divided into six groups: normal diet with AIN-93G diet (CON), high fat diet (60% calories from fat) with 1% cholesterol (HFCD), HFCD with PEITC 30 mg/kg/day or 75 mg/kg/day (HFCD+P30, HFCD+P75), and HFCD with DIM 1.5 mg/kg/day or 7.5 mg/kg/day (HFCD+D1.5, HFCD+D7.5). Enzyme-linked immunosorbent assay was used to evaluate pro-inflammatory cytokine secretion. Western blot and quantitative polymerase chain reaction were used to analyze protein and mRNA levels of nuclear factor kappa B (NF-κB) p65, interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), TLR2, TLR4, and MyD88 in spleen tissue. RESULTS: Serum IL-6 levels were significantly higher in the HFCD group than in groups fed a HFCD with PEITC or DIM. Levels of NF-κB p65 protein and TLR2/4, MyD88, NF-κB p65, IL-6, and COX-2 mRNA were significantly higher in the HFCD group than in the CON group and were reduced by the PEITC and DIM supplements. CONCLUSIONS: PEITC- and DIM-supplemented diets improved splenic inflammation by modulating the TLR2/4-MyD88 pathway in HFCD-fed mice. We suggest that dietary glucosinolates may at least partially improve obesity-induced inflammation of the spleen.

• Nutrition Research and Practice
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• 제7권6호
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• pp.460-465
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• 2013

The hepatoprotective activity of Acanthopanax koreanum Nakai extract (AE) was investigated against D-Galactosamine/Lipopolysaccharide (D-GalN/LPS)-induced liver failure rats compared with that of acanthoic acid (AA) isolated from AE. Although D-GalN/LPS (250 mg/kg body weight/$10{\mu}g/kg$ body weight, i.p.) induced hepatic damage, pretreatments with AE (1 and 3% AE/g day) and AA (0.037% AA, equivalent to 3% AE/g day) alleviated the hepatic damage. This effect was the result of a significant decrease in the activity of alanine transaminase. Concomitantly, both the nitric oxide and IL-6 levels in the plasma were significantly decreased by high-dose AE (AE3) treatment compared to the GalN/LPS control (AE0). This response resulted from the regulation of pro-inflammatory signaling via a decrease in TLR4 and CD14 mRNA levels in the liver. While a high degree of necrosis and hemorrhage were observed in the AE0, pretreatment with AE3 and AA reduced the extent of hepatocyte degeneration, necrosis, hemorrhage and inflammatory cell infiltrates compared to the AE0. In conclusion, these results suggest that especially high-dose AE are capable of alleviating D-GalN/LPS-induced hepatic injury by decreasing hepatic toxicity, thereby mitigating the TLR 4-dependent cytokine release. The anti-inflammatory effect of AE could be contributing to that of AA and AE is better than AA.

• BMB Reports
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• 제52권5호
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• pp.336-341
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• 2019

The cGAS-STING pathway plays an important role in pathogen-induced activation of the innate immune response. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) found predominantly in the synovial fluid of osteoarthritis (OA) patients increases the expression of catabolic factors via the toll-like receptor-2 (TLR-2) signaling pathway. In this study, we investigated whether 29-kDa FN-f induces inflammatory responses via the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) pathway in human primary chondrocytes. The levels of cGAS and STING were elevated in OA cartilage compared with normal cartilage. Long-term treatment of chondrocytes with 29-kDa FN-f activated the cGAS/STING pathway together with the increased level of gamma-H2AX, a marker of DNA breaks. In addition, the expression of pro-inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF-2), granulocyte colony-stimulating factor (G-CSF/CSF-3), and type I interferon ($IFN-{\alpha}$), was increased more than 100-fold in 29-kDa FN-f-treated chondrocytes. However, knockdown of cGAS and STING suppressed 29-kDa FN-f-induced expression of GM-CSF, G-CSF, and $IFN-{\alpha}$ together with the decreased activation of TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), and inhibitor protein ${\kappa}B{\alpha}$ ($I{\kappa}B{\alpha}$). Furthermore, NOD2 or TLR-2 knockdown suppressed the expression of GM-CSF, G-CSF, and $IFN-{\alpha}$ as well as decreased the activation of the cGAS/STING pathway in 29-kDa FN-f-treated chondrocytes. These data demonstrate that the cGAS/STING/TBK1/IRF3 pathway plays a critical role in 29-kDa FN-f-induced expression of pro-inflammatory cytokines.

• Animal Bioscience
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• 제37권2호
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• pp.303-314
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• 2024

Objective: Rutin, also called vitamin P, is a flavonoids from plants. Previous studies have indicated that rutin can alleviate the injury of tissues and cells by inhibiting oxidative stress and ameliorating inflammation. There is no report on the protective effects of rutin on goat rumen epithelial cells (GRECs) at present. Hence, we investigated whether rutin can alleviate lipopolysaccharide (LPS)-induced damage in GRECs. Methods: GRECs were cultured in basal medium or basal medium containing 1 ㎍/mL LPS, or 1 ㎍/mL LPS and 20 ㎍/mL rutin. Six replicates were performed for each group. After 3-h culture, the GRECs were harvested to detect the relevant parameters. Results: Rutin significantly enhanced the cell activity (p<0.05) and transepithelial electrical resistance (TEER) (p<0.01) and significantly reduced the apoptosis rate (p<0.05) of LPS-induced GRECs. Rutin significantly increased superoxide dismutase, glutathione peroxidase, and catalase activity (p<0.01) and significantly decreased lactate dehydrogenase activity and reactive oxygen species and malondialdehyde (MDA) levels in LPS-induced GRECs (p<0.01). The mRNA and protein levels of interleukin 6 (IL-6), IL-1β, and C-X-C motif chemokine ligand 8 (CXCL8) and the mRNA level of tumor necrosis factor-α (TNF-α) and chemokine C-C motif ligand 5 (CCL5) were significantly increased in LPS-induced GRECs (p<0.05 or p<0.01), while rutin supplementation significantly decreased the mRNA and protein levels of IL-6, TNF-α, and CXCL8 in LPS-induced GRECs (p<0.05 or p<0.01). The mRNA level of toll-like receptor 2 (TLR2), and the mRNA and protein levels of TLR4 and nuclear factor κB (NF-κB) was significantly improved in LPS-induced GRECs (p<0.05 or p<0.01), whereas rutin supplementation could significantly reduce the mRNA and protein levels of TLR4 (p<0.05 or p<0.01). In addition, rutin had a tendency of decreasing the protein levels of CXCL6, NF-κB, and inhibitor of nuclear factor kappa-B alpha (0.05

• BMB Reports
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• 제46권12호
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• pp.594-599
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• 2013

The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-${\alpha}$, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signal-regulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, $8.79{\times}10^5M^{-1}$. Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK.

• The Korean Journal of Physiology and Pharmacology
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• 제16권6호
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• pp.447-453
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• 2012

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-$1{\beta}$ and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-$1{\beta}$ in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-$1{\beta}$, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-$1{\beta}$. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-$1{\beta}$. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-$1{\beta}$ via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.

• IMMUNE NETWORK
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• 제22권2호
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• pp.20.1-20.9
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• 2022

Despite the high prevalence of chronic dermatitis and the accompanied intractable itch, therapeutics that specifically target itching have low efficacy. Increasing evidence suggests that TLRs contribute to immune activation and neural sensitization; however, their roles in chronic itch remain elusive. Here, we show that the RBL-2H3 mast cell line expresses TLR4 and that treatment with a TLR4 antagonist opposes the LPS dependent increase in mRNA levels of Th2 and innate cytokines. The pathological role of TLR4 activation in itching was studied in neonate rats that developed chronic itch due to neuronal damage after receiving subcutaneous capsaicin injections. Treatment with a TLR4 antagonist protected these rats with chronic itch against scratching behavior and chronic dermatitis. TLR4 antagonist treatment also restored the density of cutaneous nerve fibers and inhibited the histopathological changes that are associated with mast cell activation after capsaicin injection. Additionally, the expression of IL-1β, IL-4, IL-5, IL-10, and IL-13 mRNA in the lesional skin decreased after TLR4 antagonist treatment. Based on these data, we propose that inhibiting TLR4 alleviated itch in a rat model of chronic relapsing itch, and the reduction in the itch was associated with TLR4 signaling in mast cells and nerve fibers.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.686-694
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• 2008

The inhibitory effects of 5,6,3',5'-tetramethoxy 7,4'-hydroxyflavone (labeled as p7F) were elucidated on the productions of proinflammatory cytokines as well as inflammatory mediators in human synovial fibroblasts and macrophage cells. p7F inhibited IL-1${\beta}$ or TNF-${\alpha}$ induced expressions of inflammatory mediators (ICAM-1, COX-2, and iNOS). p7F also inhibited LPS-induced productions of nitric oxide and prostaglandin $E_2$ in RAW 264.7 cells. In order to investigate whether p7F would inhibit IL-1 signaling, p7F was added to the D10S Th2 cell line (which is responsive to only IL-1${\beta}$ and thus proliferates), revealing that p7F inhibited IL-1${\beta}$-induced proliferation of D10S Th2 cells in a dose-response manner. A flow cytometric analysis revealed that p7F reduced the intracellular level of free radical oxygen species in RAW 264.7 cells treated with hydrogen peroxide. p7F inhibited IkB degradation and NF-${\kappa}$B activation in macrophage cells treated with LPS, supporting that p7F could inhibit signaling mediated via toll-like receptor. Taken together, p7F has inhibitory effects on LPS-induced productions of inflammatory mediators on human synovial fibroblasts and macrophage cells and thus has the potential to be an anti-inflammatory agent for inhibiting inflammatory responses.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 추계학술대회
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• pp.92-92
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• 2019

Excessive or chronic inflammation contributes to the pathogenesis of many inflammatory diseases such as sepsis, rheumatoid arthritis, and ulcerative colitis. Hibiscus syriacus L. has been used as a medicinal plant in many Asian countries, even though its anti-inflammatory activity has been unclear. Therefore, we investigated the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. varieties Pulsae (PS) on the lipopolysaccharide (LPS)-induced expression of proinflammatory mediators and cytokines in RAW264.7 macrophages. PS suppressed LPS-induced nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) secretion concomitant with downregulation of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as tumor necrosis factor-alpha ($TNF-{\alpha}$), interleukin-6 (IL-6), and IL-12 in LPS-stimulated RAW264.7 macrophages. Further study showed that PS significantly decreased LPS-induced nuclear translocation of the nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) subunits, p65 and p50. Molecular docking data showed that many anthocyanins from PS fit into the hydrophobic pocket of MD2 and bound to Toll-like receptor 4 (TLR4), indicating that PS inhibits the TLR4-MD2-mediated inflammatory signaling pathway. Especially, apigenin-7-O-glucoside most powerfully bound to MD2 and TLR4 through LYS122, LYS122, and SER127 at a distance of $2.205{\AA}$, $3.098{\AA}$, and $2.844{\AA}$ and SER441 at a distance of $2.873{\AA}$ (docking score: -8.4) through hydrogen bonding, respectively. Additionally, PS inhibited LPS-induced TLR4 dimerization/expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation. PS completely blocked LPS-mediated mortality in zebrafish larvae by diminishing the recruitment of neutrophil and macrophages accompanied by low levels of proinflammatory cytokines. Taken together, our results indicate that PS attenuates LPS-mediated inflammation in both in vitro and in vivo by blocking the TLR4/MD2-MyD88/IRAK4-$NF-{\kappa}B$ axis. Therefore, PS might be used as a novel modulatory candidate for effective treatment of LPS-mediated inflammatory diseases.