Glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing TLR4 recruitment into lipid rafts in RAW264.7 cells

Background

The aim of this study was to investigate the effect of glycyrrhizin on LPS-induced endotoxemia in mice and clarify the possible mechanism.

Methods

An LPS-induced endotoxemia mouse model was used to confirm the anti-inflammatory activity of glycyrrhizin in vivo. In vitro, RAW264.7 cells were stimulated with LPS in the presence or absence of glycyrrhizin. The expression of cytokines was determined by ELISA. Toll-like receptor 4 (TLR4) was determined by Western blot analysis. Nuclear factor-kB (NF-κB) and Interferon regulatory factor 3 (IRF3) activation were detected by Western blotting and luciferase assay. Lipid raft staining was detected by immunocytochemistry.

Results

In vivo, the results showed that glycyrrhizin can improve survival during lethal endotoxemia. In vitro, glycyrrhizin dose-dependently inhibited the expression of TNF-α, IL-6, IL-1β and RANTES in LPS-stimulated RAW264.7 cells. Western blot analysis showed that glycyrrhizin suppressed LPS-induced NF-κB and IRF3 activation. However, glycyrrhizin did not inhibit NF-κB and IRF3 activation induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. Moreover, glycyrrhizin did not affect the expression of TLR4 and CD14 induced by LPS. Significantly, we found that glycyrrhizin decreased the levels of cholesterol of lipid rafts and inhibited translocation of TLR4 to lipid rafts. Moreover, glycyrrhizin activated ABCA1, which could induce cholesterol efflux from lipid rafts.

Conclusion

Glycyrrhizin exerts an anti-inflammatory property by disrupting lipid rafts and inhibiting translocation of TLR4 to lipid rafts, thereby attenuating LPS-mediated inflammatory response.

General significance

Learning the anti-inflammatory mechanism of glycyrrhizin is crucial for the anti-inflammatory drug development.     

Ursolic acid inhibits nuclear factor-κB signaling in intestinal epithelial cells and macrophages,and attenuates experimental colitis in mice

Aims

Ursolic acid (UA), a natural pentacyclic triterpenoid acid, has been reported to show immunomodulatory activity. This study investigated the effects of UA on nuclear factor-kappa B (NF-κB) signaling in cells and experimental murine colitis.

Main methods

Human intestinal epithelial cells (IECs) COLO 205 and peritoneal macrophages from IL-10-deficient (IL-10^−/−) mice were pretreated with UA and then stimulated with tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS), respectively. The expression of pro-inflammatory cytokines was determined by real-time RT-PCR and ELISA. The effect of UA on NF-κB signaling was examined by immunoblot analysis to detect IκBα phosphorylation/degradation and electrophoretic mobility shift assay to assess the DNA binding activity of NF-κB. For in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in C57BL/6 wild-type mice and chronic colitis in IL-10^−/− mice were treated with or without UA. Colitis was quantified by histopathologic evaluation. Immunohistochemical staining for phosphorylated IκBα was performed in the colonic tissue.

Key findings

UA significantly inhibited the production of pro-inflammatory cytokines, IκBα phosphorylation/degradation and NF-κB DNA binding activity in both IEC and IL-10^−/− peritoneal macrophages stimulated with TNF-α and LPS, respectively. UA significantly reduced the severity of DSS-induced murine colitis, as assessed by the disease activity index, colon length, and histopathology. UA also significantly ameliorated the severity of colitis in IL-10^−/− mice. Furthermore, UA suppressed IκBα phosphorylation in the colonic tissue.

Significance

UA inhibits NF-κB activation in both IECs and macrophages, and attenuates experimental murine colitis. These results suggest that UA is a potential therapeutic agent for inflammatory bowel disease.     

Degraded λ-carrageenan activates NF-κB and AP-1 pathways in macrophages and enhances LPS-induced TNF-α secretion through AP-1

Background

Carrageenan (CGN), a high molecular weight sulfated polysaccharide, is a traditional ingredient used in food industry. Its degraded forms have been identified as potential carcinogens, although the mechanism remains unclear.

Methods

The effects of degraded λ-carrageenan (λ-dCGN) on murine RAW264.7 cells and human THP-1-derived macrophage cells were investigated by studying its actions on tumor necrosis factor alpha (TNF-α) secretion, Toll-like receptor 4 (TLR4) expression, and activation of nuclear factor-κb (NF-κB) and activation protein-1 (AP-1) pathways.

Results

We found that λ-dCGN was much stronger than native λ-CGN in the activation of macrophages to secrete TNF-α. Treatment of RAW264.7 cells with λ-dCGN resulted in the upregulation of TLR4, CD14 and MD-2 expressions, but it did not increase the binding of lipopolysacchride (LPS) with macrophages. Meanwhile, λ-dCGN treatment activated NF-κB via B-cell lymphoma/leukemia 10 (Bcl10) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation. In addition, λ-dCGN induced extracellular signal-regulated kinases/1/2/mitogen-activated protein kinases (ERK1/2/MAPK) and AP-1 activation. Interestingly, pretreatment of RAW264.7 cells with λ-dCGN markedly enhanced LPS-stimulated TNF-α secretion. This pretreatment resulted in the enhanced phosphorylation of ERK1/2 and c-Jun N-terminal kinase (JNK) and intensified activation of AP-1.

Conclusions

λ-dCGN induced an inflammatory reaction via both NF-κB and AP-1, and enhanced the inflammatory effect of LPS through AP-1 activation.

General significance

The study demonstrated the role of λ-dCGN to induce the inflammatory reaction and to aggravate the effect of LPS on macrophages, suggesting that λ-dCGN produced during food processing and gastric digestion may be a safety concern.     

NF-κB is involved in the LPS-mediated proliferation and apoptosis of MAC-T epithelial cells as part of the subacute ruminal acidosis response in cows

Objectives

To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T.

Results

MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells.

Conclusions

LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.

     

Renoprotective effect of paricalcitol via a modulation of the TLR4-NF-κB pathway in ischemia/reperfusion-induced acute kidney injury

Background

The pathophysiology of ischemic acute kidney injury (AKI) is thought to include a complex interplay between vascular endothelial cell dysfunction, inflammation, and tubular cell damage. Several lines of evidence suggest a potential anti-inflammatory effect of vitamin D in various kidney injury models. In this study, we investigated the effect of paricalcitol, a synthetic vitamin D analog, on renal inflammation in a mouse model of ischemia/reperfusion (I/R) induced acute kidney injury (AKI).

Methods

Paricalcitol was administered via intraperitoneal (IP) injection at 24 h before ischemia, and then I/R was performed through bilateral clamping of the renal pedicles. Twenty-four hours after I/R, mice were sacrificed for the evaluation of injury and inflammation. Additionally, an in vitro experiment using HK-2 cells was also performed to examine the direct effect of paricalcitol on tubular cells.

Results

Pre-treatment with paricalcitol attenuated functional deterioration and histological damage in I/R induced AKI, and significantly decreased tissue neutrophil and macrophage infiltration and the levels of chemokines, the pro-inflammatory cytokine interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). It also decreased IR-induced upregulation of Toll-like receptor 4 (TLR4), and nuclear translocation of p65 subunit of NF-κB. Results from the in vitro study showed pre-treatment with paricalcitol suppressed the TNF-α-induced depletion of cytosolic IκB in HK-2 cells.

Conclusion

These results demonstrate that pre-treatment with paricalcitol has a renoprotective effect in ischemic AKI, possibly by suppressing TLR4-NF-κB mediated inflammation.     

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with pro-inflammatory,oxidative-glycation markers and sRAGE in diabetic retinopathy

Background

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with diabetic retinopathy (DR). However, the mechanism on how it affects the disease development is still unclear.

Aim

This study aims to investigate the relationship between 2245G/A RAGE gene polymorphism and selected pro-inflammatory, oxidative-glycation markers in DR patients.

Methods

A total of 371 unrelated type 2 diabetic patients [200 with retinopathy, 171 without retinopathy (DNR)] and 235 healthy subjects were recruited. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method followed by DNA sequencing. The nuclear and cytosolic extracts from peripheral blood mononuclear cells were used for nuclear factor kappa B (NF-κB) p65 and superoxide dismutase activity measurement respectively. Plasma was used for glutathione peroxidase activity, advanced oxidation protein product (AOPP), monocyte chemoattractant protein (MCP)-1, pentosidine and soluble RAGE (sRAGE) measurements.

Results

DR patients with 2245GA genotype had significantly elevated levels of activated NF-κB p65, plasma MCP-1, AOPP and pentosidine but lower level of sRAGE when compared to DR patients with wild-type 2245GG.

Conclusion

The RAGE gene polymorphism 2245G/A is associated with pro-inflammatory, oxidative-glycation markers and circulating sRAGE in DR patients. Patients with 2245GA RAGE genotype could aggravate DR possibly via NF-κB mediated inflammatory pathway.     

Resveratrol enhances TNF-α production in human monocytes upon bacterial stimulation

Background

Resveratrol is a key component of red wine that has been reported to have anti-carcinogenic and anti-aging properties. Additional studies conducted in vitro and in animal models suggested anti-inflammatory properties. However, data from primary human immune cells and in vivo studies are limited.

Methods

A pilot study was performed including 10 healthy volunteers. Plasma cytokine levels were measured over 48 h after oral application of 5 g resveratrol.To verify the in vivo findings, cytokine release and gene expression in human peripheral blood mononuclear cells (PBMC) and/or monocytes was assessed after treatment with resveratrol or its metabolites and stimulation with several toll-like receptor (TLR)-agonists. Additionally, the impact on intracellular signaling pathways was analyzed using a reporter cell line and Western blotting.

Results

Resveratrol treated individuals showed a significant increase in tumor necrosis factor-α (TNF-α) levels 24 h after treatment compared to baseline. Studies using human PBMC or isolated monocytes confirmed potentiation of TNF-α production with different TLR agonists, while interleukin (IL)-10 was inhibited. Moreover, we observed significantly enhanced nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB) activation using a reporter cell line and found increased phosphorylation of p105, which is indicative of alternative NF-κB pathway activation.

General significance

By administering resveratrol to healthy humans and utilizing primary immune cells we were able to detect TNF-α enhancing properties of the agent. In parallel, we found enhanced alternative NF-κB activation. We report on a novel pro-inflammatory property of resveratrol which has to be considered in concepts of its biologic activity.     

Titanium and zirconia particle-induced pro-inflammatory gene expression in cultured macrophages and osteolysis,inflammatory hyperalgesia and edema in vivo

Aims

The biological reaction to wear debris is critical to the osteolysis underlying aseptic loosening of joint prosthetic implants. In an attempt to reduce aseptic loosening, ceramics have been introduced. This study was designed to evaluate, compare and correlate the expression of Toll-like receptors (TLRs), their intracellular adaptors and proinflammatory cytokines in cultured macrophages challenged with titanium or zirconia particles, as well as particle-induced osteolysis in calvaria and hyperalgesia and edema in hind paw.

Main methods

TLRs and their adaptors were evaluated at the mRNA level by RT-PCR, and cytokine expression was evaluated at the mRNA and protein levels. Osteolysis and hyperalgesia and edema were evaluated in vivo, in calvaria and hind paw, respectively.

Key findings

Cultured macrophages challenged with zirconia or titanium particles expressed increased mRNA for TLRs 2, 3, 4 and 9, and their adaptors MyD88, TRIF and NF-κB and cytokines TNF-α, IL-1β and IL-6, which were also increased at protein level. Quantitative differences are evident and, in general, zirconia particle-induced pro-inflammatory gene expression was lower than that induced by titanium particles. In in vivo experiments, exposition to titanium or zirconia particles induced osteolysis in calvaria and hyperalgesia and edema in hind paw; however those induced by zirconia particles were significantly lower. There is a strong and positive correlation between the expressions of mRNA for TLR4, NF-κB, TNF-α, IL-1β and IL-6.

Significance

Collectively, our data suggest that zirconia ceramic particles are less bioactive than titanium particles.     

Tiliroside,a dietary glycosidic flavonoid,inhibits TRAF-6/NF-κB/p38-mediated neuroinflammation in activated BV2 microglia

Background

Tiliroside is a dietary glycosidic flavonoid which has shown in vivo anti-inflammatory activity. This study is aimed at evaluating the effect of tiliroside on neuroinflammation in BV2 microglia, and to identify its molecular targets of anti-neuroinflammatory action.

Methods

BV2 cells were stimulated with LPS + IFNγ in the presence or absence of tiliroside. TNFα, IL-6, nitrite and PGE₂ production was determined with ELISA, Griess assay and enzyme immunoassay, respectively. iNOS, COX-2, phospho-p65, phospho-IκBα, phospho-IKKα, phospho-p38, phospho-MK2, phosopho-MKK3/6 and TRAF-6 were determined by western blot analysis. NF-κB activity was also investigated using a reporter gene assay in HEK293 cells. LPS-induced microglia ROS production was tested using the DCFDA method, while HO-1 and Nrf2 activation was determined with western blot.

Results

Tiliroside significantly suppressed TNFα, IL-6, nitrite and PGE₂ production, as well as iNOS and COX-2 protein expression from LPS + IFNγ-activated BV2 microglia. Further mechanistic studies showed that tiliroside inhibited neuroinflammation by targeting important steps in the NF-κB and p38 signalling in LPS + IFNγ-activated BV2 cells. This compound also inhibited LPS-induced TRAF-6 protein expression in BV2 cells. Antioxidant activity of tiliroside in BV2 cells was demonstrated through attenuation of LPS + IFNγ-induced ROS production and activation of HO-1/Nrf2 antioxidant system.

Conclusions

Tiliroside inhibits neuroinflammation in BV2 microglia through a mechanism involving TRAF-6-mediated activation of NF-κB and p38 MAPK signalling pathways. These activities are possibly due, in part, to the antioxidant property of this compound.

General Significance

Tiliroside is a potential novel natural compound for inhibiting neuroinflammation in neurodegenerative disorders.     

Enalapril inhibits nuclear factor-κB signaling in intestinal epithelial cells and peritoneal macrophages and attenuates experimental colitis in mice

Aims

Enalapril, an angiotensin-converting enzyme (ACE) inhibitor, has pleiotropic effects such as anti-inflammatory effects. This study investigated the effect of enalapril on the nuclear factor-kappa B (NF-κB) pathway and on experimental colitis.

Main methods

The human intestinal epithelial cell (IEC) line COLO 205 and peritoneal macrophages from C57BL/6 wild-type mice and IL-10-deficient (IL-10^−/−) mice were prepared and subsequently stimulated with lipopolysaccharide (LPS) alone or LPS plus enalapril. The effect of enalapril on NF-κB signaling was examined by western blotting to detect IκBα phosphorylation/degradation; an electrophoretic mobility shift assay (EMSA) to assess the DNA binding activity of NF-κB; and ELISAs to qualify IL-8, TNF-α, IL-6, and IL-12 production. In in vivo studies, dextran sulfate sodium (DSS)-induced acute colitis in wild-type mice and chronic colitis in IL-10^−/− mice were treated with or without enalapril. Colitis was quantified by histologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry.

Key findings

Enalapril significantly inhibited LPS-induced IκBα phosphorylation/degradation, NF-κB binding activity, and pro-inflammatory cytokine production in both IEC and peritoneal macrophages. The administration of enalapril significantly reduced the severity of colitis, as assessed based on histology in both murine colitis models. Furthermore, in colon tissue, the up-regulation of IκBα phosphorylation with colitis induction was attenuated in enalapril-treated mice.

Significance

Enalapril may block the NF-κB signaling pathway, inhibit the activation of IECs and macrophages, and attenuate experimental murine colitis by down-regulating IκBα phosphorylation. These findings suggest that enalapril is a potential therapeutic agent for inflammatory bowel disease.     

S-nitrosylation of surfactant protein D as a modulator of pulmonary inflammation

Background

Surfactant protein D (SP-D) is a member of the family of proteins termed collagen-like lectins or “collectins” that play a role in non-antibody-mediated innate immune responses [1]. The primary function of SP-D is the modulation of host defense and inflammation [2].

Scope of review

This review will discuss recent findings on the physiological importance of SP-D S-nitrosylation in biological systems and potential mechanisms that govern SP-D mediated signaling.

Major conclusions

SP-D appears to have both pro- and anti-inflammatory signaling functions.SP-D multimerization is a critical feature of its function and plays an important role in efficient innate host defense. Under baseline conditions, SP-D forms a multimer in which the N-termini are hidden in the center and the C-termini are on the surface. This multimeric form of SP-D is limited in its ability to activate inflammation. However, NO can modify key cysteine residues in the hydrophobic tail domain of SP-D resulting in a dissociation of SP-D multimers into trimers, exposing the S-nitrosylated N-termini. The exposed S-nitrosylated tail domain binds to the calreticulin/CD91 receptor complex and initiates a pro-inflammatory response through phosphorylation of p38 and NF-κB activation [3,4]. In addition, the disassembled SP-D loses its ability to block TLR4, which also results in activation of NF-κB.

General significance

Recent studies have highlighted the capability of NO to modify SP-D through S-nitrosylation, causing the activation of a pro-inflammatory role for SP-D [3]. This represents a novel mechanism both for the regulation of SP-D function and NO's role in innate immunity, but also demonstrates that the S-nitrosylation can control protein function by regulating quaternary structure. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation.     

Sodium butyrate inhibits interferon-gamma induced indoleamine 2,3-dioxygenase expression via STAT1 in nasopharyngeal carcinoma cells

Aims

Indoleamine 2,3-dioxygenase (IDO) inhibits T-cell proliferation by catalyzing the conversion of l-tryptophan to l-kynurenine. IDO-induced immune tolerance weakens the clinical outcomes of immunotherapies. Sodium butyrate (NaB), one of the histone deacetylase inhibitors (HDACIs), has potential anti-tumor effects. Our previous studies revealed that NaB could inhibit IFN-γ induced IDO expression in nasopharyngeal carcinoma cells, CNE2. In the present study, we aim to investigate to the mechanism of NaB interfering with the interferon-gamma (IFN-γ)-mediated IDO expression signaling transduction.

Main methods

IDO expression and STAT1 phosphorylation in CNE2 cells were analyzed by western blotting and STAT1 acetylation was evaluated by immunoprecipitation. STAT1 nuclear translocation and NF-κB activity were detected by transient transfection and reporter gene assay.

Key findings

We found that NaB inhibited IFN-γ-induced IDO expression in CNE2 cells via decreasing phosphorylation and nuclear translocation of STAT1, but not via down-regulation of IFN-γ-receptor (IFNGR). Immunoprecipitation assays revealed that NaB increased STAT1 acetylation. Furthermore, NaB elevated the activity of NF-κB in CNE2 cells, and blocking the NF-κB activity had no effect on the IFN-γ-induced IDO expression.

Significance

These results suggest that NaB inhibited IFN-γ-induced IDO expression via STAT1 increased acetylation, decreased phosphorylation, and reduced nuclear translocation. These provided new evidence for the anti-tumor action of NaB and potential drug targets to reduce the IDO-induced immune tolerance.