TAK-242 selectively suppresses Toll-like receptor 4-signaling mediated by the intracellular domain

TAK-242, a small-molecule antisepsis agent, has shown to suppress lipopolysaccharide (LPS)-induced inflammation. In this study, we demonstrate that TAK-242 is a selective inhibitor of Toll-like receptor (TLR)-4 signaling. TAK-242 almost completely suppressed production of nitric oxide (NO) or tumor necrosis factor (TNF)-alpha induced by a TLR4-specific ligand, ultra-pure LPS, in mouse RAW264.7, human U-937 and P31/FUJ cells, whereas this agent showed little effect on other TLR ligands, Pam(3)CSK(4) (TLR1/2), peptidoglycan (TLR2/6), double strand RNA (TLR3), R-848 (TLR7) and CpG oligonucleotide (TLR9). Furthermore, TAK-242 potently inhibited nuclear factor (NF)-kappaB activation induced by ultra-pure LPS in HEK293 cells transiently expressing TLR4 and co-receptors, myeloid differentiation protein-2 (MD2) and CD14, whereas this agent showed little effect on other TLRs, TLR1/2, TLR2/6, TLR3, TLR5, TLR7 and TLR9. TAK-242 also inhibited ligand-independent NF-kappaB activation resulting from over-expression of TLR4. Although chimera receptors, which are consist of the extracellular domain of CD4 and the intracellular domain of human or mouse TLR4, showed constitutive NF-kappaB activation, TAK-242 potently inhibited the signaling from CD4-TLR4 chimera receptors. In contrast, the NF-kappaB activation mediated by TLR4 adaptors, myeloid differentiation factor 88 (MyD88), TIR-associated protein (TIRAP), Toll/IL-1R homology (TIR)-domain-containing adaptor protein-inducing interferon-beta (TRIF) or TRIF-related adaptor molecule (TRAM) was not affected by TAK-242. TAK-242 is therefore a selective inhibitor of signaling from the intracellular domain of TLR4 and represents a novel therapeutic approach to the treatment of TLR4-mediated diseases.     

Selenium suppresses the activation of transcription factor NF-kappa B and IRF3 induced by TLR3 or TLR4 agonists

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induce innate immune responses by recognizing invading microbial pathogens leading to the activation of the adaptive immune responses. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and TRIF-dependent pathways leading to the expression of pro-inflammatory cytokines and type I interferons (IFNs). The MyD88- and TRIF-dependent pathways lead to the activation of NF-kappa B and IRF3 through the activation of IKK-beta and TBK1, respectively. Selenium is an essential trace element nutrient possessing anticarcinogenic properties. Here, we attempted to identify the molecular targets of selenium in TLR signaling pathways. Selenium inhibited NF-kappaB activation induced by poly[I:C] (TLR3 agonist), LPS (TLR4 agonist) or overexpression of MyD88 or IKK-beta which is the key kinase of MyD88-dependent signaling pathway. Selenium inhibited IRF3 activation induced by poly[I:C], LPS or the overexpression of TRIF or TBK1. Selenium also suppressed the expression of COX-2 and iNOS and the endogenous IFN beta mRNA induced by poly[I:C] or LPS. Therefore, our results suggest that selenium can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression.     

Xiao-Xu-Ming decoction extract alleviates LPS-induced neuroinflammation associated with down-regulating TLR4/MyD88 signaling pathway in vitro and in vivo

In the present study, we aimed to investigate the effects of Xiao-Xu-Ming decoction extract (XXM) on lipopolysaccaride (LPS)-induced neuroinflammation invitro and invivo. Invitro, the microglia BV2 cells were treated with 200 ng/mL LPS for 24 h to induce inflammatory responses. Invivo, mice were treated with 5 mg/kg LPS to induce inflammatory responses. The NO level was determined by Griess Reagents. The levels of IL-1β, IL-6, TNF-α and MCP-1 were determined by ELISA. The expressions of Iba-1, TLR4 and MyD88 at the protein levels were determined by Western blotting analysis. The mRNA levels of TLR4 and MyD88 were determined by real-time PCR. Invitro, XXMsignificantly reduced the levels of various pro-inflammatory factors, including NO, IL-1β, IL-6 and TNF-α, induced by LPS in the supernatant of BV2 cells and suppressedexpressions of inflammatory proteins TLR4 and MyD88 induced by LPS in BV2 cells. Invivo, XXM significantly inhibited microglia activation, attenuated LPS-induced inflammatory factors and chemokine production, such as IL-1β, IL-6, TNF-α and MCP-1, andinhibited the expressions of inflammatory proteins including TLR4 and MyD88, in the cortex of LPS-induced mice. Our findings suggested that XXM could attenuate LPS-induced neuroinflammation via down-regulating TLR4/MyD88 signaling pathway.     

Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization

Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFκB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFκB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFκB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNβ in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFκB or IRF3 induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFκB activation. Further, cinnamaldehyde inhibited ligand-independent NFκB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.     

Suppression of MyD88- and TRIF-dependent signaling pathways of Toll-like receptor by (-)-epigallocatechin-3-gallate, a polyphenol component of green tea

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-kappaB. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-kappaB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKbeta which is the key kinase in the canonical pathway for NF-kappaB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.     

Inhibition of homodimerization of Toll-like receptor 4 by curcumin

Toll-like receptors play a key role in sensing microbial components and inducing innate immune responses. Ligand-induced dimerization of TLR4 is required for the activation of downstream signaling pathways. Thus, the receptor dimerization may be one of the first lines of regulation in activating TLR-mediated signaling pathways and induction of subsequent immune responses. LPS induces the activation of NF-kappaB and IRF3 through MyD88- or TRIF-dependent pathways. Curcumin, a polyphenol found in the plant Curcuma longa, has been shown to suppress the activation of NF-kappaB induced by various pro-inflammatory stimuli by inhibiting IKKbeta kinase activity in MyD88-dependent pathway. Curcumin also inhibited LPS-induced IRF3 activation. These results imply that curcumin inhibits both MyD88- and TRIF-dependent pathways in LPS-induced TLR4 signaling. However, in TRIF-dependent pathway, curcumin did not inhibit IRF3 activation induced by overexpression of TRIF in 293T cells. These results suggest that TLR4 receptor complex is the molecular target of curcumin in addition to IKKbeta. Here, we report biochemical evidence that phytochemicals (curcumin and sesquiterpene lactone) inhibit both ligand-induced and ligand-independent dimerization of TLR4. Furthermore, these results demonstrate that small molecules with non-microbial origin can directly inhibit TLRs-mediated signaling pathways at the receptor level. These results imply that the activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain dietary phytochemicals we consume daily.     

The regulation of the expression of inducible nitric oxide synthase by Src-family tyrosine kinases mediated through MyD88-independent signaling pathways of Toll-like receptor 4

Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to the expression of inflammatory gene products. Src-family tyrosine kinases (STKs) are known to be activated by LPS in monocytes/macrophages. Therefore, we determined the role of STKs in TLR4 signaling pathways and target gene expression in macrophages. The activation of NFkappaB, and p38 MAPK, and the expression of inducible nitric oxide synthase (iNOS) induced by LPS were not affected in macrophages deficient in three STKs (Lyn, Hck, and Fgr). These results suggest that the deletion of the three STKs among possibly nine STKs is not sufficient to abolish total activity of STKs possibly due to the functional redundancy of other STKs present in macrophages. However, two structurally unrelated pan-inhibitors of STKs, PP1 and SU6656, suppressed LPS-induced iNOS expression in MyD88-knockout as well as wild-type macrophages. The suppression of iNOS expression by the inhibitors was correlated with the downregulation of IFNbeta (a MyD88-independent gene) expression and subsequent decrease in STAT1 phosphorylation. Moreover, PP1 suppressed the expression of IFNbeta and iNOS induced by TRIF, a MyD88-independent adaptor of TLR4. PP1 suppressed STAT1 phosphorylation induced by LPS, but not by IFNbeta suggesting that STKs are involved in the primary downstream signaling pathways of TLR4, but not the secondary signaling pathways downstream of IFNbeta receptor. Together, these results demonstrate that STKs play a positive regulatory role in TLR4-mediated iNOS expression in a MyD88-independent (TRIF-dependent) manner. These results provide new insight in understanding the role of STKs in TLR4 signaling pathways and inflammatory target gene expression.     

Novel anti-inflammatory function of NSC95397 by the suppression of multiple kinases

NSC95397 (2,3-bis-[(2-hydroxyethyl)thio]-1,4-naphthoquinone) is a CDC25 inhibitor with anti-cancer properties. Since the anti-inflammatory activity of this compound has not yet been explored, the aim of this study was to examine whether this compound is able to modulate the inflammatory process. Toll like receptor (TLR)-mediated inflammatory responses were induced by lipopolysaccharide (LPS), a TLR4 ligand, and pam3CSK, a TLR2 ligand, in peritoneal macrophages and RAW264.7. The molecular mechanism of NSC95397's anti-inflammatory activity was studied using immunoblotting analysis, nuclear fractionation, immunoprecipitation, overexpression strategies, luciferase reporter gene assays, and kinase assays. NSC95397 dose-dependently suppressed the production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and prostaglandin (PG)E₂, and diminished the mRNA expression of inflammatory genes such as inducible NO synthase (iNOS), cyclooxygenase (COX)-2, interferon (IFN)-β, and TNF-α in peritoneal macrophages and RAW264.7 cells that were stimulated by LPS and pam3CSK. This compound also clearly blocked the activation of NF-κB (p65), AP-1 (c-Fos/c-Jun), and IRF-3 in LPS-treated RAW264.7 cells and TRIF- and MyD88-overexpressing HEK293 cells. In addition, biochemical and molecular approaches revealed that this compound targeted AKT, IKKα/β, MKK7, and TBK1. Therefore, these results suggest that the anti-inflammatory function of NSC95397 can be attributed to its inhibition of multiple targets such as AKT, IKKα/β, MKK7, and TBK1.     

Inhibition of homodimerization of toll-like receptor 4 by 4-oxo-4-(2-oxo-oxazolidin-3-yl)-but-2-enoic acid ethyl ester

Toll-like receptors (TLRs) recognize molecular structures derived from microbes and initiate innate immunity. The stimulation of TLR4 by lipopolysaccharide (LPS) triggers the activation of the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent major downstream signaling pathways. Previously, we synthesized a fumaryl oxazolidinone derivative, 4-oxo-4-(2-oxo-oxazolidin-3-yl)-but-2-enoic acid ethyl ester (OSL07) and demonstrated that it inhibits activation of nuclear factor kappa B (NF-κB) by inhibiting the MyD88-dependent pathway of TLRs. TLR4 and the downstream signaling components are good therapeutic targets for many chronic inflammatory diseases. Here, it is investigated whether OSL07 modulates TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by OSL07. OSL07 inhibited LPS-induced NF-κB and interferon regulatory factor 3 activation by targeting TLR4 dimerization. These results suggest that OSL07 can modulate TLR4 signaling pathway leading to decreased inflammatory gene expression.     

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.     

Guggulsterone suppresses the activation of transcription factor IRF3 induced by TLR3 or TLR4 agonists

Toll-like receptors (TLRs) are vital in the induction of innate immune responses. The microbial components trigger the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent downstream TLR signaling pathways. Guggulsterone, which has been used for centuries to treat many chronic diseases, inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-κB kinase. However, it is not known whether guggulsterone inhibits the TRIF-dependent pathway. Presently, we sought to identify the molecular targets of guggulsterone in this pathway. Guggulsterone inhibited nuclear factor-κB and IRF3 activation induced by lipopolysaccharide or poly[I:C] and activation of IRF3 induced by the overexpression of TRIF, TBK1 or constitutively active IRF3. Guggulsterone also suppressed the lipopolysaccharide-induced phosphorylation of IRF3. These results suggest that guggulsterone can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression.     

Costunolide inhibits cyclooxygenase-2 expression induced by toll-like receptor 3 or 4 agonist

Toll-like receptors (TLRs) play an important role in the induction of innate immune responses recognizing conserved microbial structural molecules. The microbial pathogens trigger the activation of two downstream signaling pathways of TLRs; myeloid differential factor 88 (MyD88)- and toll interleukin-1 receptor domain containing adapter inducing interferon-β (TRIF)-dependent pathways leading to the activation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3). Saussureae Radix has been used for centuries to treat a variety of diseases. Costunolide, one of the active ingredients in Saussureae Radix, has been used to treat many chronic diseases. However, the mechanism of costunolide’s beneficial effects is largely unknown. Here, we report biochemical evidence that costunolide inhibits NF-κB activation and cyclooxygenase-2 expression induced by the TLR3 agonist polyriboinosinic polyribocytidylic acid (poly[I : C]) or the TLR4 agonist lipopolysaccharide (LPS). These results suggest that costunolide can modulate the immune responses regulated by TLR signaling pathways.     

Euxanthone inhibits lipopolysaccharide-induced injury,inflammatory response,and MUC5AC hypersecretion in human airway epithelial cells by the TLR4/MyD88 pathway

Asthma progression is involved in airway epithelial dysfunction, airway inflammatory response, and mucus hypersecretion. Euxanthone has been found to exhibit cytotoxic activity on several human diseases, such as neurological disorders and cancers. Our study aimed to explore the influence of euxanthone on lipopolysaccharide (LPS)-induced injury, inflammatory response, and mucin 5AC (MUC5AC) hypersecretion in human airway epithelial cells (AECs). Network pharmacology analysis was carried out to analyze the drug targets and key pathways of euxanthone against asthma. Cell injury was evaluated by CCK-8, Lactate dehydrogenase (LDH) release assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The production of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and MUC5AC was measured using enzyme-linked immunosorbent assay (ELISA). MUC5AC mRNA expression was detected by qRT-PCR. Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) protein expression was examined by western blot analysis. Venn diagram showed 14 overlapping targets between euxanthone and asthma. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we focused on TLR signaling pathway. LPS exposure evoked viability reduction, increased LDH release and apoptosis, and induced production of inflammatory cytokines (IL-6, IL-8, and MCP-1) and MUC5AC hypersecretion in human AECs, which were alleviated by euxanthone. Mechanistically, we validated that euxanthone attenuated LPS-induced activation of TLR4/MyD88 pathway in AECs. Moreover, inhibition of the TLR4/MyD88 pathway enhanced the inhibitory effect of euxanthone on LPS-induced cell injury, inflammatory response and MUC5AC expression. In conclusion, euxanthone attenuated LPS-induced cell injury, inflammatory response, and MUC5AC expression in AECs by inhibiting the activation of TLR4/MyD88 pathway.     

Baicalin prevents LPS-induced activation of TLR4/NF-κB p65 pathway and inflammation in mice via inhibiting the expression of CD14

Previous studies have shown that baicalin,an active ingredient of the Chinese traditional medicine Huangqin,attenuates LPS-induced inflammation by inhibiting the activation of TLR4/NF-κBp65 pathway,but how it affects this pathway is unknown.It has been shown that CD14 binds directly to LPS and plays an important role in sensitizing the cells to minute quantities of LPS via chaperoning LPS molecules to the TLR4/MD-2 signaling complex.In the present study we investigated the role of CD14 in the anti-inflammatory effects of baicalin in vitro and in vivo.Exposure to LPS(1μg/mL)induced inflammatory responses in RAW264.7 cells,evidenced by marked increases in the expression of MHC II molecules and the secretion of NO and IL-6,and by activation of MyD88/NF-κB p65 signaling pathway,as well as the expression of CD14 and TLR4.These changes were dose-dependently attenuated by pretreatment baicalin(12.5–50μM),but not by baicalin post-treatment.In RAW264.7 cells without LPS stimulation,baicalin dose-dependently inhibit the protein and mRNA expression of CD14,but not TLR4.In RAW264.7 cells with CD14 knockdown,baicalin pretreatment did not prevent inflammatory responses and activation of MyD88/NF-κB p65 pathway induced by high concentrations(1000μg/mL)of LPS.Furthermore,baicalin pretreatment also inhibited the expression of CD14 and activation of MyD88/NF-κB p65 pathway in LPS-induced hepatocyte-derived HepG2 cells and intestinal epithelial-derived HT-29 cells.In mice with intraperitoneal injection of LPS and in DSS-induced UC mice,oral administration of baicalin exerted protective effects by inhibition of CD14 expression and inflammation.Taken together,we demonstrate that baicalin pretreatment prevents LPS-induced inflammation in RAW264.7 cells in CD14-dependent manner.This study supports the therapeutic use of baicalin in preventing the progression of LPS-induced inflammatory diseases.     

5-O-methyldihydroquercetin and cilicicone B isolated from Spina Gleditsiae ameliorate lipopolysaccharide‐induced acute kidney injury in mice by inhibiting inflammation and oxidative stress via the TLR4/MyD88/TRIF/NLRP3 signaling pathway

ObjectiveInflammation and oxidative stress are the major mechanisms implicated in lipopolysaccharide (LPS)-induced AKI. Spina Gleditsiae is a traditional Chinese anti-inflammatory medicine, from which a large number of flavonoids, such as 5-O-methyldihydroquercetin (GS1) and cilicicone B (GS2), were isolated in the present study. Here, we examined the reno-protective effects and potential underlying mechanisms of GS1 and GS2 in mice with LPS-induced AKI.MethodsWe analyzed renal function; the serum metabolic profile, inflammatory cytokine levels, peripheral white blood cell count, renal cell apoptosis, renal oxidant and antioxidant levels, and renal expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), TIR-domain-containing adapter-inducing interferon-β (TRIF), nuclear factor-ĸB (NF-ĸB), interferon regulatory factor 3 (IRF3), NOD-, LRR- and pyrin domain-containing 3 (NLRP3, inflammasome), cleaved caspase-1, and interleukin 1 receptor type I (IL-1R1) in mice with LPS-induced AKI.ResultsGS1 and GS2 improved renal function and significantly reduced the levels of inflammatory cytokines and oxidative stress markers. In addition, PCA score scatter plots suggest that the GS1 and GS2 groups were clustered with the control group, indicating that these compounds contributed to the recovery of mice with AKI toward the normal condition. Moreover, GS1 and GS2 inhibited the expression of TLR4, MyD88, TRIF, p-NF-ĸB, p-IRF3, NLRP3, cleaved caspase-1, and IL-1R1.ConclusionThe reno-protective effects of GS1 and GS2 are mediated via the MyD88/TRIF and NLRP3 pathways to reduce inflammation and oxidative stress through TLR4 signaling.     

Inhibition of homodimerization of toll-like receptor 4 by (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1-yl)-2-butenoate

Toll-like receptors (TLRs) recognize molecular structures derived from microbes and initiate innate immunity. The stimulation of TLR4 by lipopolysaccharide triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain -containing adapter inducing interferon-β (TRIF)-dependent signaling pathways. Previously, we synthesized a fumaryl pyrrolidinone, (E)-isopropyl 4-oxo-4-(2-oxopyrrolidin-1-yl)-2-butenoate (IPOP) and demonstrated that it inhibits activation of nuclear factor kappa B (NF-κB) by inhibiting the MyD88-dependent pathway of TLRs. TLR4 and the downstream signaling components are good therapeutic targets for many chronic inflammatory diseases. Here, it is investigated whether IPOP modulates TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by IPOP. IPOP inhibited LPSinduced NF-κB and interferon regulatory factor 3 (IRF3) activation by targeting TLR4 dimerization. These results suggest that IPOP can modulate TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.     

Suppression of TLRs signaling pathways by 1-[5-methoxy-2-(2-nitrovinyl)phenyl]pyrrolidine

Toll-like receptors (TLRs) play significant roles in recognizing the pathogen-associated molecular patterns that induce innate immunity, and subsequently, acquired immunity. In general, TLRs have two downstream signaling pathways, the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways, which lead to the activation of nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). 1-[5-methoxy-2-(2-nitrovinyl)phenyl]pyrrolidine (MNP) has been previously synthesized in our laboratory. To evaluate the therapeutic potential of MNP, its effect on signal transduction via the TLR signaling pathways was examined. MNP was shown to inhibit the activation of NF-κB and IRF3 induced by TLR agonists, as well as to inhibit the expression of cyclooxygenase-2, inducible nitric oxide synthase, and interferon inducible protein-10. MNP also inhibited the activation of NF-κB and IRF3 induced by the overexpression of downstream signaling components of the MyD88- or TRIF-dependent signaling pathways. These results suggest that MNP can modulate MyD88- and TRIF-dependent signaling pathways of TLRs, leading to decreased inflammatory gene expression.     

脂多糖对类风湿关节炎外周血单核细胞Toll样受体4、髓样分化因子88蛋白表达的影响

目的 观察脂多糖(LPS)对类风湿关节炎(RA)患者外周血单核细胞Toll样受体4(TLR4)、髓样分化因子88(MyD88)蛋白表达的影响。方法 采用活动期RA患者外周血进行单核细胞的分离培养,并与健康志愿者做对照。分为以下5组观察:健康对照组、健康+LPS组、RA对照组、RA+ LPS组、RA+LPS+TAK-242组,并采用Western blot法检测各组TLR4、 MyD88蛋白的表达。结果 健康+LPS组或RA+LPS组TLR4、MyD88蛋白表达与健康对照组或RA对照组比较均明显升高(均P<0.01);RA对照组或RA+LPS组TLR4、MyD88蛋白表达与健康对照组或健康+LPS组比较均明显升高(均P<0.01);RA+LPS+TAK-242组TLR4、MyD88蛋白表达与RA+LPS组比较明显降低(均P<0.01)。结论 RA活动期单核细胞可能存在TLR/MyD88信号通路的激活,且对LPS的刺激反应性增强。     

脂多糖对类风湿关节炎外周血单核细胞TLR4、Myd88蛋白表达的影响

目的 观察脂多糖(LPS)对类风湿关节炎(RA)患者外周血单核细胞TLR4、MyD88蛋白表达的影响。方法 采用活动期RA患者外周血进行单核细胞的分离培养,并与健康志愿者做对照。分为以下5组观察：健康对照组、健康 LPS组、RA对照组、RA LPS组、RA LPS TAK-242组,并采用western blot 法检测各组TLR4、 MyD88蛋白的表达。 结果 健康 LPS组或RA LPS组TLR4、MyD88蛋白表达与健康对照组或RA对照组比较均明显升高(均P<0.01);RA对照组或RA LPS组TLR4、MyD88蛋白表达与健康对照组或健康 LPS组比较均明显升高(均P<0.01); RA LPS TAK-242组TLR4、MyD88蛋白表达与RA LPS组比较明显降低(均P<0.01)。结论RA活动期单核细胞可能存在TLR/MyD88信号通路的激活,且对LPS的刺激反应性增强。