Ganglioside GM3 suppresses lipopolysaccharide‐induced inflammatory responses in rAW 264.7 macrophage cells through NF‐κB,AP‐1, and MAPKs signaling

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase‐2 (COX‐2) protein and mRNA levels in lipopolysaccharide (LPS)‐activated RAW 264.7 cells in a dose‐dependent manner. Moreover, GM3 inhibited the expression and release of pro‐inflammatory cytokines of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS‐induced nuclear translocation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and activator protein (AP)‐1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen‐activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS‐activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS‐induced inflammatory response in RAW 264.7 macrophages by suppression of NF‐κB, AP‐1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.     

Low‐intensity pulsed ultrasound attenuates cardiac inflammation of CVB3‐induced viral myocarditis via regulation of caveolin‐1 and MAPK pathways

The aggressive immunological activity elicited by acute viral myocarditis contributes to a large amount of cardiomyocytes loss and poor prognosis of patients in clinic. Low‐intensity pulsed ultrasound (LIPUS), which is an effective treatment modality for osteoarthropathy, has been recently illustrated regulating the overactive inflammatory response in various diseases. Here, we aimed to investigate whether LIPUS could attenuate coxsackievirus B3 (CVB3) infection‐induced injury by coordinating the inflammatory response. Male BALB/c mice were inoculated intraperitoneally with CVB3 to establish the model of acute viral myocarditis. LIPUS treatment was given on Day 1, Day 1, 3 and Day 1, 3, 5 post‐inoculation, respectively. All mice were followed up for 14 days. Day 1, 3, 5 LIPUS treatment significantly improved the survival rate, attenuated the ventricular dysfunction and ameliorated the cardiac histopathological injury of CVB3‐infected mice. Western blotting analysis showed Day 1, 3, 5 LIPUS treatment decreased pro‐inflammatory cytokines, increased the activation of caveolin‐1 and suppressed p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (ERK) signallings in heart tissue. RAW264.7 cells were treated with lipopolysaccharides (LPS) to simulate the augmented inflammatory response in vivo. LIPUS treatment on RAW264.7 inhibited the expression of pro‐inflammatory cytokines, activated caveolin‐1 and suppressed p38 MAPK and ERK signallings. Transfecting RAW264.7 with caveolin‐1 siRNA blunted the suppression of pro‐inflammatory cytokines and MAPK signallings by LIPUS treatment. Taken together, we demonstrated for the first time that LIPUS treatment attenuated the aggressive inflammatory response during acute viral myocarditis. The underlying mechanism may be activating caveolin‐1 and suppressing MAPK signallings.     

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC₅₀ value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of caveolin‐1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor‐class B type I (SR‐BI) and cluster of differentiation‐36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin‐1. Our aim was to define the contribution of human CD36, SR‐BI, and caveolin‐1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)‐ and heavily (H)‐OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR‐BI, or caveolin‐1, we found that (1) CD36 increases M‐ and H‐OxLDL‐protein uptake; (2) SR‐BI drives M‐OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin‐1 increases M‐ and H‐OxLDL‐protein uptake and decreases CE selective uptake from M‐OxLDL. Also, incubation with M‐ or H‐OxLDL decreases the levels of SR‐BI and LDL‐receptor in control HepG2 cells which can be overcome by caveolin‐1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin‐1 in cells overexpressing this protein. Thus, hepatic caveolin‐1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels. J. Cell. Biochem. 108: 906–915, 2009. © 2009 Wiley‐Liss, Inc.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.     

Neobavaisoflavone inhibits osteoclastogenesis through blocking RANKL signalling‐mediated TRAF6 and c‐Src recruitment and NF‐κB,MAPK and Akt pathways

Psoralea corylifolia (P corylifolia) has been popularly applied in traditional Chinese medicine decoction for treating osteoporosis and promoting fracture healing since centuries ago. However, the bioactive natural components remain unknown. In this study, applying comprehensive two‐dimensional cell membrane chromatographic/C18 column/time‐of‐flight mass spectrometry (2D CMC/C18 column/TOFMS) system, neobavaisoflavone (NBIF), for the first time, was identified for the bioaffinity with RAW 264.7 cells membranes from the extracts of P corylifolia. Here, we revealed that NBIF inhibited RANKL‐mediated osteoclastogenesis in bone marrow monocytes (BMMCs) and RAW264.7 cells dose dependently at the early stage. Moreover, NBIF inhibited osteoclasts function demonstrated by actin ring formation assay and pit‐formation assay. With regard to the underlying molecular mechanism, co‐immunoprecipitation showed that both the interactions of RANK with TRAF6 and with c‐Src were disrupted. In addition, NBIF inhibited the phosphorylation of P50, P65, IκB in NF‐κB pathway, ERK, JNK, P38 in MAPKs pathway, AKT in Akt pathway, accompanied with a blockade of calcium oscillation and inactivation of nuclear translocation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In vivo, NBIF inhibited osteoclastogenesis, promoted osteogenesis and ameliorated bone loss in ovariectomized mice. In summary, P corylifolia‐derived NBIF inhibited RANKL‐mediated osteoclastogenesis by suppressing the recruitment of TRAF6 and c‐Src to RANK, inactivating NF‐κB, MAPKs, and Akt signalling pathways and inhibiting calcium oscillation and NFATc1 translocation. NBIF might serve as a promising candidate for the treatment of osteoclast‐associated osteopenic diseases.     

The effect of oxidized low density lipoprotein (oxLDL)-induced heme oxygenase-1 on LPS-induced inflammation in RAW 264.7 macrophage cells

Macrophages take up oxidized low density lipoprotein (oxLDL) after being exposed to it in the blood vessels. oxLDL transforms macrophages into foam cells, which are a hallmark of atherosclerosis. The effects that oxLDL have on the inflammatory responses of foam cells are not clear. Here, we investigated how oxLDL modulates lipopolysaccharide (LPS)-induced inflammatory mediators in RAW 264.7 murine macrophages. Our results showed that oxLDL dramatically induced HO-1 expression, but did not increase pro-inflammatory mediators such as interleukin-1β, tumor necrosis factor-α, iNOS, and monocyte chemoattractant protein (MCP)-1. In RAW 264.7 macrophages, oxLDL markedly inhibited LPS-induced inflammatory mediators such as inducible nitric oxide synthase (iNOS), IL-1β, IL-6, granulocyte macrophage colony-stimulating factor and stromal cell-derived factor-1. Interestingly, however, the down-regulation of HO-1 by siRNA did not recover the inhibition of LPS-induced expression and/or the secretion of inflammatory mediators. oxLDL blocked LPS-induced NF-κB nuclear translocation by inhibiting inhibitory κB (IκB) degradation. Taken together, our results suggest that oxLDL could modulate LPS-induced inflammatory responses by inhibiting NF-κB signaling independently of HO-1 expression.     

Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

Caffeic acid phenethyl ester,an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley‐Liss, Inc.     

Flagellin‐dependent TLR5/caveolin‐1 as a promising immune activator in immunosenescence

The age‐associated decline of immune responses causes high susceptibility to infections and reduced vaccine efficacy in the elderly. However, the mechanisms underlying age‐related deficits are unclear. Here, we found that the expression and signaling of flagellin (FlaB)‐dependent Toll‐like receptor 5 (TLR5), unlike the other TLRs, were well maintained in old macrophages, similar to young macrophages. The expression and activation of TLR5/MyD88, but not TLR4, were sensitively regulated by the upregulation of caveolin‐1 in old macrophages through direct interaction. This interaction was also confirmed using macrophages from caveolin‐1 or MyD88 knockout mice. Because TLR5 and caveolin‐1 were well expressed in major old tissues including lung, skin, intestine, and spleen, we analyzed in vivo immune responses via a vaccine platform with FlaB as a mucosal adjuvant for the pneumococcal surface protein A (PspA) against Streptococcus pneumoniae infection in young and aged mice. The FlaB‐PspA fusion protein induced a significantly higher level of PspA‐specific IgG and IgA responses and demonstrated a high protective efficacy against a lethal challenge with live S. pneumoniae in aged mice. These results suggest that caveolin‐1/TLR5 signaling plays a key role in age‐associated innate immune responses and that FlaB‐PspA stimulation of TLR5 may be a new strategy for a mucosal vaccine adjuvant against pneumococcal infection in the elderly.     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.     

Enhancement of oxidised low-density lipoprotein uptake by macrophages in response to macrophage migration inhibitory factor

We examined the expression of macrophage migration inhibitory factor (MIF) mRNA in murine macrophage cell line (RAW 264.7 cells) in response to oxidized low-density lipoprotein (oxLDL), and investigated the influence of MIF on the uptake and degradation of oxLDL by RAW 264.7 cells. MIF mRNA expression was markedly upregulated in the presence of oxLDL. Consistent with this, the MIF level of the culture medium was increased by stimulation with oxLDL in dose- and time-dependent manners. Next, we added recombinant rat MIF to the culture medium and examined its effects on the uptake of(125)I-labelled oxLDL. Pretreatment with MIF enhanced both the uptake and degradation of(125)I-oxLDL. Taken together, these results suggest that MIF released from macrophages in response to oxLDL stimulates oxLDL uptake and degradation in an autocrine and paracrine fashion, which potentially results in atherosclerosis.     

Silencing of ATP2B1‐AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA‐mediated NF‐κB signalling pathway

Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca²⁺ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.