LPS-activated RAW264 Research Articles

Bisbenzylisoquinoline alkaloids of lotus (Nelumbo nucifera Gaertn.) seed embryo inhibit lipopolysaccharide-induced macrophage activation via suppression of Ca2+-CaM/CaMKII pathway

ABSTRACTMacrophage hyperactivation is associated with various inflammatory diseases. In this study, the effects of nine bisbenzylisoquinoline alkaloids on NO production by LPS-activated RAW264.7 macrophages were evaluated. It was found that certain bisbenzylisoquinoline alkaloids with single oxygen bridge have more potent effects than those with two oxygen bridges. Further studies revealed that three bisbenzylisoquinoline alkaloids (Neferine, Liensinine and Isoliensinine) of lotus (Nelumbo nucifera Gaertn.) seed embryo, a homology of traditional Chinese medicine and food, can also reduce LPS-induced expression of iNOS and production of IL-1β, IL-6 and TNF-α by blocking IκBα phosphorylation and degradation. And this effect is mediated at least partly by the inhibition of cytosolic Ca2+ level and CaM expression as well as the binding of three alkaloids with CaM, thereby inhibiting the subsequent phosphorylation of CaMKII. These results revealed for the first time that three bisbenzylisoquinoline alkaloids of lotus embryo play an anti-inflammatory role via suppressing Ca2+-CaM/CaMKII pathway.

Thiazolidinedione Derivative Suppresses LPS-induced COX-2 Expression and NO Production in RAW 264.7 Macrophages.

The present study was designed to investigate the inhibitory effect of 2,4 bis-[(4-ethoxyphenyl)azo] 5-(3-hydroxybenzylidene) thiazolidine-2,4-dione (TZD-OCH2CH3) on the cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. The effects of TZD-OCH2CH3 on COX-2 and iNOS mRNA expression in LPS-activated RAW 264.7 cells were detected by real time PCR. Also, to understand structure and substrate specificity, we have utilized molecular docking simulations (AutoDock Vina) and the active residues in the binding pocket were determined from COX-2 and iNOS. The treatment of RAW 264.7 cells with TZD-OCH2CH3 significantly inhibited LPS-induced COX-2 mRNA expression, corresponding to 46.1% and 61.06% at 30 and 60 μg/mL, respectively. The present study revealed that the TZD-OCH2CH3 had a little effect on iNOS mRNA expression. Meanwhile, the TZD-OCH2CH3 also could inhibit the production of NO compared to single LPS-stimulated cell. According to the results obtained, TZD-OCH2CH3 dramatically suppressed lipopolysaccharide (LPS) induced nitric oxide (NO) production after 24 h, in a concentration-dependent manner with an IC50 of 65 μg/mL. Our data suggest that TZD-OCH2CH3, as a functionally novel agent, inhibits the inflammatory pathway via suppression of COX-2 mRNA expression and also by the inhibition of the iNOS activity. Therefore, this compound could be suggested as a novel therapeutic strategy for inflammation-associated disorders.

Terpenoids from the roots of Leontopodium longifolium and their inhibitory activity on NO production in RAW264.7 cells

Phytochemical investigation of the roots of Leontopodium longifolium, led to the isolation of a novel norsesquiterpene, named as longifolactone (1), along with three known diterpenes. The structures of these compounds were elucidated by analysis of HR-ESI-MS, UV, IR and 1D and 2D NMR spectroscopic data. The absolute configuration of the new compound was determined by electronic circular dichroism (ECD) using both experimental and calculated ECD spectra. Furthermore, their anti-inflammatory effects were evaluated in LPS-activated RAW264.7 cells to determine their effects on the release of NO. Longifolactone (1) showed weak cytotoxicity towards two human cancer cell lines.

3‑Bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol inhibits LPS-induced pro-inflammatory responses by preventing ROS production and downregulating NF-κB in vitro and in a zebrafish model

The anti-inflammatory effects of 3‑bromo‑5‑(ethoxymethyl)‑1,2‑benzenediol (BEMB) from Polysiphonia morrowii were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and zebrafish embryo. BEMB showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS), and the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the LPS-activated RAW264.7 cells and zebrafish embryo without cytotoxicity. Moreover, BEMB suppressed the protein and mRNA expression levels of nuclear factor (NF)-κB (p65 and inhibitor of NF-κB [IκB]-A) in RAW264.7 cells and zebrafish embryo, respectively. Collectively, the results of this study indicate that BEMB suppressed the production of pro-inflammatory mediators such as NO, iNOS, and COX-2 as well as their regulation in LPS-induced RAW264.7 cells and zebrafish embryos by inhibiting ROS production and NF-κB expression. Therefore, this study suggests that BEMB could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.

Three new labdane-type diterpenoids from Callicarpa macrophylla Vahl.

Three new labdane-type diterpenoids, callicapene M3-M5 (1–3) were isolated from the Callicarpa macrophylla Vahl. Their structures were identified by spectroscopic method. The isolated compounds were evaluated for inhibitory activity on NO production in LPS-activated RAW 264.7 macrophage cells by using MTT assays. Compounds 1–3 showed potent inhibitory activity, with IC50 value of 48.15, 46.31 and 38.72 μM respectively.

Mycetia cauliflora methanol extract exerts anti-inflammatory activity by directly targeting PDK1 in the NF-κB pathway

Ethnopharmacological relevanceMycetia cauliflora Reinw. (Rubiaceae) has been used as a traditional remedy to ameliorate clinical signs of inflammatory diseases, including pain, inflammation, ulcers, and wounds. Among the Mycetia subfamilies, the molecular and cellular mechanisms of Mycetia longifolia (Rubiaceae) have been studied. However, those of Mycetia cauliflora are not clearly understood. Comprehensive investigation of this plant is necessary to evaluate its potential for ethnopharmacological use. Materialsand methods: The activities of Mycetia cauliflora methanol extract (Mc-ME) on the secretion of inflammatory mediators, the mRNA expression of proinflammatory cytokines, and identification of its molecular targets were elucidated using lipopolysaccharide (LPS)-induced macrophage-like cells. Moreover, the suppressive actions of Mc-ME were examined in an LPS-induced peritonitis mouse model. ResultsAt nontoxic concentrations, Mc-ME downregulated the release of nitric oxide (NO), the mRNA expression of inducible nitric oxide synthase (iNOS), and the mRNA expression of interleukin (IL)-1β from LPS-activated RAW264.7 cells. This extract also inhibited the nuclear translocation of p65 and p50 and the phosphorylation of IκBα, IKK, and AKT. Western blot analysis and in vitro kinase assays confirmed that phosphoinositide-dependent kinase-1 (PDK1) is the direct immunopharmacological target of Mc-ME effect. In addition, Mc-ME significantly reduced inflammatory signs in an animal model of acute peritonitis. These effects were associated with decreased NO production and decreased AKT phosphorylation. ConclusionOur results suggest that Mc-ME displays anti-inflammatory actions in LPS-treated macrophage-like cells and in an animal model of acute inflammatory disease. These actions are preferentially managed by targeting PDK1 in the nuclear factor (NF)-κB signaling pathway.

Gambogic acid induces heme oxygenase-1 through Nrf2 signaling pathway and inhibits NF-κB and MAPK activation to reduce inflammation in LPS-activated RAW264.7 cells

Gambogic acid (GA), a natural product with a xanthone structure, was previously demonstrated to exert anti-inflammatory effects. The aim of this study was to evaluate the anti-inflammatory activity of GA on LPS-stimulated mouse macrophage RAW264.7 and its anti-inflammatory mechanism. Pretreatment with GA inhibited LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) through reducing the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). GA also decreased the expressions of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β. The activation of nuclear factor-κB (NF-κB) and the Mitogen activated phosphokinases (MAPKs) regulates pro-inflammatory factors. Further experiments demonstrated that the nuclear translocation of NF-κB, a promoting regulator in inflammation, was blocked via inhibiting the phosphorylation event of IκBα by GA. Meanwhile, the Mitogen activated phosphokinase (MAPK) signaling pathways were also suppressed. However, activation of nuclear factor erythroid 2-related factor (Nrf2) can inhibit inflammation. GA could activate the nucleus translocation of Nrf2 and up-regulated the expression of heme oxygenase-1 (HO-1). Taken together, GA exhibited its anti-inflammatory activities through Nrf2 activation and NF-κB depression, thus could be a candidate for the prevention and treatment of diseases that involve excessive inflammation.

Compound K, a ginsenoside metabolite, plays an antiinflammatory role in macrophages by targeting the AKT1-mediated signaling pathway

BackgroundCompound K (CK) is an active metabolite of ginseng saponin, ginsenoside Rb1, that has been shown to have ameliorative properties in various diseases. However, its role in inflammation and the underlying mechanisms are poorly understood. In this report, the antiinflammatory role of CK was investigated in macrophage-like cells. MethodsThe CK-mediated antiinflammatory mechanism was explored in RAW264.7 and HEK293 cells that were activated by lipopolysaccharide (LPS) or exhibited overexpression of known activation proteins. The mRNA levels of inflammatory genes and the activation levels of target proteins were identified by quantitative and semiquantitative reverse transcription polymerase chain reaction and Western blot analysis. ResultsCK significantly inhibited the mRNA expression of inducible nitric oxide synthase and tumor necrosis factor-α and morphological changes in LPS-activated RAW264.7 cells under noncytotoxic concentrations. CK downregulated the phosphorylation of AKT1, but not AKT2, in LPS-activated RAW264.7 cells. Similarly, CK reduced the AKT1 overexpression-induced expression of aldehyde oxidase 1, interleukin-1β, interferon-β, and tumor necrosis factor-α in a dose-dependent manner. ConclusionOur results suggest that CK plays an antiinflammatory role during macrophage-mediated inflammatory actions by specifically targeting the AKT1-mediated signaling pathway.

Irpeksins A–E, 1,10-seco-Eburicane-Type Triterpenoids from the Medicinal Fungus Irpex lacteus and Their Anti-NO Activity

Five new triterpenoids, irpeksins A-E (1-5), were isolated from fruiting bodies of the medicinal fungus Irpex lacteus. The structures as well as absolute configurations of the new compounds were established via extensive spectroscopic analysis, computational methods, and Cotton effects. Compounds 1-4 are featured by a scaffold of 1,10- seco- and ring B aromatic eburicane (24-methyllanostane), and compound 5 is characterized by a scaffold of 1,10-9,11- diseco- and ring B aromatic eburicane, which represents unprecedented cleavage patterns in the lanostane family. Compounds 1-5 showed significant inhibitory activity against NO production in LPS-activated RAW 264.7 macrophage cells with IC50 values varying from 2.2 to 19.6 μM.

Activation of Nrf2/HO-1 Pathway by Nardochinoid C Inhibits Inflammation and Oxidative Stress in Lipopolysaccharide-Stimulated Macrophages.

The roots and rhizomes of Nardostachys chinensis have neuroprotection and cardiovascular protection effects. However, the specific mechanism of N. chinensis is not yet clear. Nardochinoid C (DC) is a new compound with new skeleton isolated from N. chinensis and this study for the first time explored the anti-inflammatory and anti-oxidant effect of DC. The results showed that DC significantly reduced the release of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-activated RAW264.7 cells. The expression of pro-inflammatory proteins including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were also obviously inhibited by DC in LPS-activated RAW264.7 cells. Besides, the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also remarkably inhibited by DC in LPS-activated RAW264.7 cells. DC also suppressed inflammation indicators including COX-2, PGE2, TNF-α, and IL-6 in LPS-stimulated THP-1 macrophages. Furthermore, DC inhibited the macrophage M1 phenotype and the production of reactive oxygen species (ROS) in LPS-activated RAW264.7 cells. Mechanism studies showed that DC mainly activated nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, increased the level of anti-oxidant protein heme oxygenase-1 (HO-1) and thus produced the anti-inflammatory and anti-oxidant effects, which were abolished by Nrf2 siRNA and HO-1 inhibitor. These findings suggested that DC could be a new Nrf2 activator for the treatment and prevention of diseases related to inflammation and oxidative stress.

Identification, Quantification, and Anti-inflammatory Activity of 5- n-Alkylresorcinols from 21 Different Wheat Varieties.

The characteristic constituent and anti-inflammatory activity of 5- n-alkylresorcinols (ARs) from 21 wheat bran samples in China were investigated in this study. The amount of ARs ranged from 697 to 1732 μg/g in the tested samples, which were composed of five different homologues. Among these homologues, C19:0 and C21:0 were the most abundant, followed by C17:0, C23:0, and C25:0. Moreover, the mRNA expression of IL-1β, IL-6, and TNF-α in LPS-activated RAW264.7 macrophage cells were significantly inhibited by ARs supplementation. The molecular mechanisms behind its anti-inflammatory activity could result from the suppression of nuclear factor-κB (NF-κB) and JNK/MAPK activation. ARs treatment notably decreased NF-κB p65 nuclear translocation and inhibitor κB (IκBα) kinase and JNK phosphorylation. Additionally, ARs homologues C17:0 had been proven to be the main active constituent. The results from this study could be used to promote the comprehensive utilization of wheat and its byproducts in improving human health.

STANDARDIZED SUPERCRITICAL CO2 EXTRACT OF ACANTHUS ILICIFOLIUS (LINN.) LEAVES INHIBITS THE PRO-INFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR-Α IN LIPOPOLYSACCHARIDE-ACTIVATED MURINE RAW 264.7 MACROPHAGE CELLS

Objective: Acanthus ilicifolius Linn. (Acanthaceae) is a medicinal mangrove plant used in the treatment of inflammation. Previous phytochemical studies have identified 2-benzoxazolinone (BOA) from the leaves of A. ilicifolius. In the present study, we attempted to standardize the supercritical CO2 leaf extract of A. ilicifolius (SCFE-AI) for BOA content and investigate the tumor necrosis factor-α (TNF-α) inhibitory effect of SCFE-AI and BOA on the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. The acute oral toxicity of SCFE-AI and BOA was also established.Methods: SCFE-AI was standardized for BOA content using high-performance thin-layer chromatography (HPTLC) method. The cytotoxicity of SCFE-AI and BOA was evaluated using MTS colorimetric method. The in vitro anti-inflammatory effect of SCFE-AI and BOA on TNF-α production in LPS-activated RAW 264.7 cells was quantified using ELISA method. Acute oral toxicity studies were performed following the Organization for Economic Co-operation and Development test guideline No. 423.Results: The amount of BOA was found 0.8% w/w of SCFE-AI. The RAW 264.7 cell viability was unaffected by SCFE-AI and BOA treatments within a concentration range <1000 mg/ml after 24 h incubation. SCFE-AI decreased the production of TNF-α in a dose-dependent manner compared to BOA. The LD50 value for SCFE-AI was found to be >2000 mg/kg and ranges from 300 to 2000 mg/kg with BOA.Conclusion: The HPTLC chromatogram could serve as an analytical tool for authentication and quantification of BOA content. The anti-inflammatory mechanism of A. ilicifolius might be through the inhibition of TNF-α production.

2'-O-Galloylhyperin Isolated From Pyrola incarnata Fisch. Attenuates LPS-Induced Inflammatory Response by Activation of SIRT1/Nrf2 and Inhibition of the NF-κB Pathways in Vitro and Vivo.

2′-O-galloylhyperin, a major compound of Pyrola incarnata Fisch., possesses a variety of biological and pharmacological activities, including anti-oxidative and anti-inflammatory activities. Nevertheless, the underlying molecular mechanisms of 2′-O-GH in microbial infection and sepsis are not clear. In this study, we investigated the anti-inflammatory effects of 2′-O-GH. We found that 2′-O-GH significantly reduced the production of TNF-α, IL-6, and nitric oxide (NO), suppressed the expression levels of iNOS, blocked the translocation of NF-κB from the cytosol to nucleus, and decreased the MAPK activation in LPS-activated RAW 264.7 cells. 2′-O-GH also enhanced the nuclear translocation of Nrf2 and up-regulated the expression of heme oxygenase-1 (HO-1) and SIRT1. In addition, the administration of 2′-O-GH attenuated the TNF-α and IL-6 production in the serum, infiltration of inflammatory cells, liver tissue damage, and the mortality rate of LPS-challenged mice. Moreover, 2′-O-GH significantly upregulated Nrf2 and SIRT1 expression and inhibited the inflammatory responses in the liver of septic mice. The collective data indicate that 2′-O-GH could potentially be a novel functional food candidate in the treatment of sepsis.

Brain Natriuretic Peptide-Regulated Expression of Inflammatory Cytokines in Lipopolysaccharide (LPS)-Activated Macrophages via NF-κB and Mitogen Activated Protein Kinase (MAPK) Pathways.

BackgroundThis study aimed to investigate the effects of recombinant human brain natriuretic peptide (rhBNP) on IL-6, TNF-α, and IL-10 secretion in LPS-activated RAW 264.7 cells and human peripheral blood mononuclear cells (PBMCs) in vitro and to explore the related signaling pathways of the regulation mechanisms of BNP in systemic inflammatory response syndrome (SIRS).Material/MethodsMTT assay was used to evaluate the effects of rhBNP on cell viabilities. Lipopolysaccharide (LPS) was used to induce inflammation response. The whole study was divided into 8 groups: Control, low, middle, and high concentrations of rhBNP, LPS, LPS with low, middle, and high concentrations of rhBNP. Levels of IL-6, TNF-α, and IL-10 were evaluated using the Cytometric Bead Array Kit and RT-PCR assay. Western blotting was used to test the effects of rhBNP on inflammation-related NF-κB and MAPK pathways.ResultsExcept for the concentrations ≥1.6 ng/mL, all concentrations of rhBNP showed little effect on cell viabilities of RAW264.7 cells and PBMCs after 24 h and 48 h, suggesting a weak cytotoxicity to cells. Expression of IL-6 and TNF-α significantly increased and expression of IL-10 significantly decreased at protein and mRNA levels after LPS treatment, and these effects were strongly inhibited in a dose-dependent manner by pretreatment of rhBNP. Similarly, the LPS-induced increase of NF-κB and MAPK pathway phosphorylation levels were also significantly inhibited by rhBNP.ConclusionsrhBNP can regulate expression of IL-6, TNF-α, and IL-10 in LPS-activated RAW 264.7 cells and PBMCs through inhibiting NF-κB and MAPK pathways. These results may reveal potential causes of the increase of BNP in SIRS and may provide an experimental basis for treatment of SIRS.

Four new diterpenoid alkaloids with anti-inflammatory activities from Aconitum taronense Fletcher et Lauener

Four new C19-diterpenoid alkaloids, named taronenines A-D (1-4), have been isolated from the roots of Aconitum taronense Fletcher et Lauener (Ranunculaceae), which has long been used as a folk medicine to treat rheumatism and arthritis in China. Their structures were established on the basis of extensive spectroscopic analyses. The isolated compounds were evaluated for their potential anti-inflammatory effects using LPS-stimulated RAW 264.7 macrophages. Compounds 1, 2 and 4 showed certain inhibition activities of interleukin-6 in LPS-activated RAW 264.7 cells with IC50 values of 29.60 ± 0.08 μg/mL, 18.87 ± 0.14 μg/mL, and 25.39 ± 0.11 μg/mL, respectively.

Mesenchymal stem cells alleviate acute kidney injury by down-regulating C5a/C5aR pathway activation.

Acute kidney injury (AKI) leads to serious renal damage, and early inhibition of inflammation is necessary for its treatment. C5a/C5aR signaling activation promotes inflammatory response in tissue injury. Anti-inflammatory activity of mesenchymal stem cells (MSCs) makes it possible to alleviate AKI by controlling the C5a/C5aR signaling activation. Ischemia reperfusion (I/R)-induced AKI models in wild-type and C5aR KO mice were used. In addition, human bone marrow MSCs (hBM-MSCs) or C5aR antagonist were injected in this model. All animals were killed at 72h after reperfusion. In vitro, the LPS-activated macrophage line RAW264.7 cells were co-cultured with or without hBM-MSCs in the presence of recombinant C5a or not for indicated time points. After that, C5aR expression, the inflammatory factor production, and NF-κB translocation in RAW264.7 cells were measured. hBM-MSC treatment and C5a/C5aR signaling blockade or C5aR-deficiency exhibited similar attenuated effects on I/R-induced AKI, macrophages infiltration, and the pro-inflammatory cytokines TNF-α and IL-1β expression in renal tissues in mice. Moreover, hBM-MSC administration led to a significant reduction in C5a levels in serum and C5aR expression in the kidney tissues in mice after I/R. In vitro, upon co-culture with hBM-MSCs, both C5aR expression and the secretion of pro-inflammatory factors TNF-α, IL-6, and nitric oxide in LPS-activated macrophages were markedly reduced. Accordingly, recombinant complement C5a accelerated LPS-induced NF-κB translocation and pro-inflammatory factors expression in macrophages, but the addition of hBM-MSCs reversed these C5a-induced effects. The present study indicates that hBM-MSCs alleviate AKI via suppressing C5a/C5aR-NF-κB pathway activation.

Furan fragment isomerized mexicanolide-type Limonoids from the stem barks of Khaya senegalensis

Four new mexicanolide-type limonoids with isomerized furan fragment khaysenelides G–J (1–4) were isolated from the stem barks of Khaya senegalensis. Their structures were elucidated on the basis of (1D and 2D) NMR and HRESIMS, and the normal furan rings in 1-4 were isomerized to hydroxybutenolide moieties. Bioactivities screening of new compounds indicated that 1 showed potent cytotoxicities against MDA-MB-231 and HepG2 human cancer cell lines with IC50 values of 6.02 and 8.98 μM, and significant NO inhibitory activities in LPS-activated RAW 264.7 macrophages with IC50 values of 3.51 μM.

Luteolin activates ERK1/2- and Ca2+-dependent HO-1 induction that reduces LPS-induced HMGB1, iNOS/NO, and COX-2 expression in RAW264.7 cells and mitigates acute lung injury of endotoxin mice.

Although luteolin has shown to have anti-inflammatory action, no report is available whether luteolin inhibits HMGB1 and protects acute lung injury (ALI) in endotoxin rodents. We hypothesized that HO-1 induction by luteolin might play a crucial role for inhibition of pro-inflammatory mediators including HMGB1 through MAPK signaling in LPS-induced RAW264.7 cells, and it ameliorates ALI of endotoxin mice. The effects of luteolin on the production of pro-inflammatory mediators in LPS-activated RAW264.7 cells and LPS-injected mice were evaluated. The mechanisms were investigated using various signal inhibitors. Luteolin significantly increased HO-1 expression through ERK1/2 signaling in a time- and concentration-dependent manner. Indeed, luteolin inhibited pro-inflammatory mediators (HMGB1, iNOS/NO, COX-2, and NF-κB activity) in LPS-activated RAW264.7 cells. In addition, PD98059, an ERK1/2 inhibitor, treatment failed to inhibit production of these pro-inflammatory mediators by luteolin. Interestingly, luteolin augmented HO-1 induction through Ca2+ influx in RAW264.7 cells. Administration of luteolin significantly inhibited plasma HMGB1 level, and iNOS expression in the lung that resulted in a significant reduction of ALI in endotoxin mice that was reversed by a HO-1 inhibitor, ZnPPIX. Therefore, we conclude that luteolin has a great potential for treatment of ALI and related diseases, where HMGB1 is a therapeutic target.

Structural characterization of blackberry wine polysaccharides and immunomodulatory effects on LPS-activated RAW 264.7 macrophages

Three polysaccharide fractions were isolated from blackberry wine. The crude extract BWPs was obtained with ethanol precipitation and freeze-thawing process, it was then submitted to Fehling treatment, giving soluble BWPFs and insoluble BWPFp fractions. These fractions were characterized by Gas Chromatography-Mass Spectrometry (GC–MS) and Nuclear Magnetic Resonance (NMR). Major polysaccharides were identified for each fraction: mannan, type II arabinogalactan and type I rhamnogalacturonan for BWPs, a mannan formed by a major chain of α-Manp(1 → 6)-linked units, O-2 substituted with α-d-Manp(1 → 2)-linked side chains for BWPFp and a AG II formed by a major chain of β-d-Galp(1 → 3)-linked, substituted at O-6 by side chains of the β-d-Galp(1 → 6)-linked, which then are substituted at O-3 by non-reducing units of α-l-Araf and a RG I, formed by [→4)-α-d-GalpA-(1 → 2)-α-l-Rhap-(1→]n for BWPFs. Anti-inflammatory effects of polysaccharide fractions were evaluated in RAW 264.7 cells. Fractions markedly reduced nitric oxide (NO) and pro-inflammatory cytokine production (TNF-α and IL-1β) in LPS-treated cells.

Macrophage migration inhibitory factor (MIF) inhibitor, Z-590 suppresses cartilage destruction in adjuvant-induced arthritis via inhibition of macrophage inflammatory activation

Background: Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory mediator that is involved in the progression of rheumatoid arthritis (RA). Previously, we demonstrated a small molecule compound 3-[(biphenyl-4-ylcarbonyl) carbamothioyl] amino benzoic acid (Z-590) could inhibit MIF activity with docking-based virtual screening and experimental evaluation.Methods: The LPS activated RAW264.7 macrophage cells were used to determine the anti-inflammatory effects of Z-590 in vitro. A rat adjuvant-induced arthritis (AIA) model was used to determine the anti-arthritic effects of Z-590 in vivo.Results: MIF inhibitor Z-590 significantly inhibited the production of NO, TNF-α and IL-6 in LPS-activated RAW 264.7 macrophage cells and markedly inhibited LPS-induced expression of TNF-α, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Z-590 also significantly reduced paw edema, serum level of TNF-α, IL-6 and spleen index in the adjuvant-induced arthritis (AIA) rat model. Furthermore, Z-590 markedly ameliorated joint inflammation and articular cartilage damage in AIA rat model.Conclusion: MIF inhibitor Z-590 possesses potent anti-arthritic activity through suppression of macrophage activation, and could be a potential therapeutic treatment for RA.