• BMB Reports
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• v.49 no.5
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• pp.276-281
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• 2016

Corosolic acid (CA), a triterpenoid compound isolated from Lagerstroemia speciosa L. (Banaba) leaves, exerts anti-inflammatory effects by regulating phosphorylation of interleukin receptor- associated kinase (IRAK)-2 via the NF-κB cascade. However, the protective effect of CA against endotoxic shock has not been reported. LPS (200 ng/mL, 30 min) induced phosphorylation of IRAK-1 and treatment with CA (10 μM) significantly attenuated this effect. In addition, CA also reduced protein levels of NLRP3 and ASC which are the main components of the inflammasome in BMDMs. LPS-induced inflammasome assembly through activation of IRAK-1 was down-regulated by CA challenge. Treatment with Bay11-7082, an inhibitor of IκB-α, had no effect on CA-mediated inhibition of IRAK-1 activation, indicating that CA-mediated attenuation of IRAK-1 phosphorylation was independent of NF-κB signaling. These results demonstrate that CA ameliorates acute inflammation in mouse BMDMs and CA may be useful as a pharmacological agent to prevent acute inflammation.

• Molecules and Cells
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• v.41 no.8
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• pp.762-770
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• 2018

Adiponectin, a hormone produced by adipose tissue, is very abundant in plasma, and its anti- and pro-inflammatory effects are reported. However, the mechanisms of these pro- and anti-inflammatory effects are not fully defined. Herein, we evaluated the dual inflammatory response mechanism of adiponectin in macrophages. Short-term globular adiponectin (gAd) treatment induced $I{\kappa}B{\alpha}$ degradation, $NF-{\kappa}B$ nuclear translocation, and $TNF-{\alpha}$ production in RAW 264.7 cells. Polymyxin B pretreatment did not block gAd-induced $I{\kappa}B{\alpha}$ degradation, and heated gAd was unable to degrade $I{\kappa}B{\alpha}$, suggesting that the effects of gAd were not due to endotoxin contamination. gAd activated IKK and Akt, and inhibition of either IKK or Akt by dominant-negative $IKK{\beta}$ ($DN-IKK{\beta}$) or DN-Akt overexpression blocked gAd-induced $I{\kappa}B{\alpha}$ degradation, suggesting that short-term incubation with gAd mediates inflammatory responses by activating the $I{\kappa}B/NF-{\kappa}B$ and PI3K/Akt pathways. Contrastingly, long-term stimulation with gAd induced, upon subsequent stimulation, tolerance to gAd, lipopolysaccharide, and CpG-oligodeoxynucleotide, which is associated with gAd-induced downregulation of IL-receptor-associated kinase-1 (IRAK-1) due to IRAK-1 transcriptional repression. Conclusively, our findings demonstrate that the pro- and anti-inflammatory responses to gAd in innate immune cells are time-dependent, and mediated by the activation of the $I{\kappa}B/NF-{\kappa}B$ pathway, and IRAK-1 downregulation, respectively.

• YAKHAK HOEJI
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• v.47 no.3
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• pp.180-183
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• 2003

Crystal structure of TRAF6 in complex with TRAF6-binding sites from CD40 was previously determined. The structure revealed a distinct TRAF6-binding groove of CD40, the key structural determinant of interaction. The structural information leads to a proposed TRAF6-binding motif. This allows the identification of TRAF6-binding sequences in the hIRAK protein, whose functional requirement in IL-1/Toll-like receptor superfamilies-mediated signal transduction is further demonstrated using site-directed mutagenesis. The mutational effects of hIRAK on the down-stream NF-kB signaling shows the importance of the TRAF6 interface for signaling by IL-1/Toll-like receptor superfamilies.

• BMB Reports
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• v.51 no.6
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• pp.308-313
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• 2018

Small-molecule inhibitors are widely used to treat a variety of inflammatory diseases. In this study, we found a novel anti-inflammatory compound, 1-[(2R,4S)-2-methyl-4-(phenylamino)-1,2,3,4-tetrahydroquinolin-1-yl]prop-2-en-1-one (MPQP). It showed strong anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. These effects were exerted through the inhibition of the production of NO and pro-inflammatory cytokines, such as interleukin (IL)-6, $IL-1{\beta}$, and tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$). Furthermore, MPQP decreased the expression levels of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). Additionally, it mediated the inhibition of the phosphorylation of p38, c-Jun N-terminal kinase (JNK), the inhibitor of ${\kappa}B{\alpha}$ ($I{\kappa}B{\alpha}$), and their upstream kinases, $I{\kappa}B$ kinase (IKK) ${\alpha}/{\beta}$, mitogen-activated protein kinase kinase (MKK) 3/6, and MKK4. Furthermore, the expression of IL-1 receptor-associated kinase 1 (IRAK1) that regulates $NF-{\kappa}B$, p38, and the JNK signaling pathways, was also increased by MPQP. These results indicate that MPQP regulates the IRAK1-mediated inflammatory signaling pathways by targeting IRAK1 or its upstream factors.

• Journal of Ginseng Research
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• v.47 no.1
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• pp.81-88
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• 2023

Background: Air pollution has led to an increased exposure of all living organisms to fine dust. Therefore, research efforts are being made to devise preventive and therapeutic remedies against fine dust-induced chronic diseases. Methods: Research of the respiratory protective effects of KRG extract in a particulate matter (PM; aerodynamic diameter of <4 ㎛) plus diesel exhaust particle (DEP) (PM4+D)-induced airway inflammation model. Nitric oxide production, expression of pro-inflammatory mediators and cytokines, and IRAK-1, TAK-1, and MAPK pathways were examined in PM4-stimulated MH-S cells. BALB/c mice exposed to PM4+D mixture by intranasal tracheal injection three times a day for 12 days at 3 day intervals and KRGE were administered orally for 12 days. Histological of lung and trachea, and immune cell subtype analyses were performed. Expression of pro-inflammatory mediators and cytokines in bronchoalveolar lavage fluid (BALF) and lung were measured. Immunohistofluorescence staining for IRAK-1 localization in lung were also evaluated. Results: KRGE inhibited the production of nitric oxide, the expression of pro-inflammatory mediators and cytokines, and expression and phosphorylation of all downstream factors of NF-κB, including IRAK-1 and MAPK/AP1 pathway in PM4-stimulated MH-S cells. KRGE suppressed inflammatory cell infiltration and number of immune cells, histopathologic damage, and inflammatory symptoms in the BALF and lungs induced by PM4+D; these included increased alveolar wall thickness, accumulation of collagen fibers, and TNF-α, MIP2, CXCL-1, IL-1α, and IL-17 cytokine release. Moreover, PM4 participates induce alveolar macrophage death and interleukin-1α release by associating with IRAK-1 localization was also potently inhibited by KRGE in the lungs of PM4+D-induced airway inflammation model. KRGE suppresses airway inflammatory responses, including granulocyte infiltration into the airway, by regulating the expression of chemokines and inflammatory cytokines via inhibition of IRAK-1 and MAPK pathway. Conclusion: Our results indicate the potential of KRGE to serve as an effective therapeutic agent against airway inflammation and respiratory diseases.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.1
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• pp.44-52
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• 2023

Objective: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. Methods: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. Results: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1β, IL-6, IL-12, interferon α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. Conclusion: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.

• Natural Product Sciences
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• v.28 no.3
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• pp.121-129
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• 2022

Despite considerable efforts, cancer remains an aggressive killer worldwide. Chemotherapeutic drugs that are currently in use lead to destructive side effects and have not succeeded in fulfilling expectations. For centuries, medicinal plants are used for treating various diseases and are also known to have anticancer activity. The main aim of this research was to evaluate antiproliferative activity of saffron, clove, fenugreek, and green tea on Vero and MDA-MB-231 cell lines and to subsequently analyze the effect of these extracts on IRAK-4, TAK1, IKK-alpha, IKK-beta, NF-Kappa B, IRF3, IRF7 genes in Toll Like Receptors (TLRs) pathway. Antiproliferative assay was done by Neutral Red Dye uptake assay. Methanolic extract of green tea was found to be most effective against both cell lines as IC₅₀ was achieved at least concentration of the extract. For molecular studies, MDAMB-231 cells were sensitized with methanolic extract of green tea at same IC₅₀, and RT-PCR was performed to determine the relative expression of genes. Expression of IRAK-4, TAK1, IKK-beta, NF-Kappa B, IRF3 genes was down regulated and IRF7 and IKKalpha was upregulated. Green tea has a potential cytotoxic effect on both cell lines which was demonstrated by its effect on the expression of (TLRs) pathway genes.

• Biomolecules & Therapeutics
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• v.30 no.2
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• pp.126-136
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• 2022

Liver fibrosis is part of the wound healing process to help the liver recover from the injuries caused by various liver-damaging insults. However, liver fibrosis often progresses to life-threatening cirrhosis and hepatocellular carcinoma. To overcome the limitations of current in vivo liver fibrosis models for studying the pathophysiology of liver fibrosis and establishing effective treatment strategies, we developed a new mouse model of liver fibrosis using polyhexamethylene guanidine phosphate (PHMG-p), a humidifier sterilizer known to induce lung fibrosis in humans. Male C57/BL6 mice were intraperitoneally injected with PHMG-p (0.03% and 0.1%) twice a week for 5 weeks. Subsequently, liver tissues were examined histologically and RNA-sequencing was performed to evaluate the expression of key genes and pathways affected by PHMG-p. PHMG-p injection resulted in body weight loss of ~15% and worsening of physical condition. Necropsy revealed diffuse fibrotic lesions in the liver with no effect on the lungs. Histology, collagen staining, immunohistochemistry for smooth muscle actin and collagen, and polymerase chain reaction analysis of fibrotic genes revealed that PHMG-p induced liver fibrosis in the peri-central, peri-portal, and capsule regions. RNA-sequencing revealed that PHMG-p affected several pathways associated with human liver fibrosis, especially with upregulation of lumican and IRAK3, and downregulation of GSTp1 and GSTp2, which are closely involved in liver fibrosis pathogenesis. Collectively we demonstrated that the PHMG-p-induced liver fibrosis model can be employed to study human liver fibrosis.

• Korean Journal of Food Science and Technology
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• v.39 no.2
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• pp.175-180
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• 2007

Toll-like receptors induce innate immune responses recognizing conserved microbial structural molecules that are known as pathogen-associated molecular patterns (PAMPs). Ligand-induced homotypic oligomerization was found to proceed in LPS-induced activation of TLR4 signaling pathways. TLR2 is known to heterodimerize with TLR1 or TLR6 and recognize diacyl- or triacyl-lipopeptide, respectively. These results suggest that ligand-induced receptor dimerization of TLR4 and TLR2 is required for the activation of downstream signaling pathways. Therefore, receptor dimerization may be one of the first lines of regulation in the activation of TLR-mediated signaling pathways and induction of subsequent innate and adaptive immune responses. Here, we report biochemical evidence that curcumin from the plant Curcuma longa inhibits activation of $NF-{\kappa}B$, expression of COX-2, and dimerization of TLRs induced by TLR2, TLR3 and TLR4 agonists. These results imply that curcumin can modulate the activation of TLRs and subsequent immune/inflammatory responses induced by microbial pathogens.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.10a
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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.