LPS-induced Inflammation In RAW Research Articles

Emodin suppresses LPS-induced inflammation in RAW264.7 cells through a PPARγ-dependent pathway

Inflammation is a defense and protective response to multiple harmful stimuli. Over and uncontrolled inflammation can lead to local tissues or even systemic damages and injuries. Actually, uncontrolled and self-amplified inflammation is the fundament of the pathogenesis of a variety of inflammatory diseases, including sepsis shock, acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Our recent study showed that emodin, the main active component of Radix rhizoma Rhei, could significantly ameliorate LPS-induced ALI/ARDS in mice. However, its underlying signal pathway was not still very clear. Then, the aim of current study was to explore whether emodin could attenuate LPS-induced inflammation in RAW264.7 cells, and its involved potential mechanism. The mRNA and protein expression of ICAM-1, MCP-1 and PPARγ were measured by qRCR and western blotting, the production of TNF-α was evaluated by ELISA. Then, the phosphorylation of NF-κB p65 was also detected by western blotting. And NF-κB p65 DNA binding activity was analyzed by ELISA as well. Meanwhile, siRNA-PPARγ transfection was performed to knockdown PPARγ expression in cells. Our data revealed that LPS-induced the up-regulation of ICAM-1, MCP-1 and TNF-α, LPS-induced the down-regulation of PPARγ, and LPS-enhanced NF-κB p65 activation and DNA binding activity were substantially suppressed by emdoin in RAW264.7 cells. Furthermore, our data also figured out that these effects of emdoin were largely abrogated by siRNA-PPARγ transfection. Taken together, our results indicated that LPS-induced inflammation were potently compromised by emodin very likely through the PPARγ-dependent inactivation of NF-κB in RAW264.7 cells.

Ganglioside GD1a suppresses LPS-induced pro-inflammatory cytokines in RAW264.7 macrophages by reducing MAPKs and NF-κB signaling pathways through TLR4

Gangliosides, sialic acid-containing glycosphingolipids, have been considered to be involved in the development, differentiation, and function of nervous systems in vertebrates. However, the mechanisms for anti-inflammation caused by gangliosides are not clear. In this paper, we investigated the anti-inflammation effects of ganglioside GD1a by using RAW264.7 macrophages. Our data demonstrated that treatment of macrophages with lipopolysaccharide significantly increased the production of NO and pro-inflammatory cytokines. GD1a suppressed the induction of iNOS and COX-2 mRNA and protein expression and secretory pro-inflammatory cytokines in culture medium, such as TNFα, IL-1α and IL-1β. In addition, LPS-induced phosphorylation of mitogen-activating protein kinases and IκBα degradation followed by translocation of the NF-κB from the cytoplasm to the nucleus were attenuated after GD1a treatment. Furthermore, GD1a probably inhibited LPS binding to macrophages and LPS-induced accumulation between TLR4 and MyD88. Taken together, the results demonstrated that ganglioside GD1a inhibited LPS-induced inflammation in RAW 264.7 macrophages by suppressing phosphorylation of mitogen-activating protein kinases and activation of NF-κB through repressing the Toll-like receptor 4 signaling pathway.

Ketamine promotes inflammation through increasing TLR4 expression in RAW264.7 cells.

Ketamine (KTM), a N-methyl-D-aspartate (NMDA) receptor antagonist, was found to has an anti-inflammatory effect, but some patients suffered from exacerbated pro-inflammatory reactions after anesthesia with KTM. The present study was aimed to examine the underlying mechanism of pro-inflammatory effects of KTM. In this study, RAW264.7 cells were exposed to KTM and NMDA alone or combined for 30 min before lipopolysaccharide (LPS) stimulation. The expression levels of IL-6 and TNF-α were detected by RT-PCR and ELISA, and those of NMDA receptors by RT-PCR in RAW264.7 cells. Additionally, the TLR4 expression was determined by RT-PCR and flow cytometry, respectively. The results showed that in RAW264.7 cells, KTM alone promoted the TLR4 expression, but did not increase the expression of IL-6 or TNF-α. In the presence of LPS, KTM caused a significantly higher expression of IL-6 and TNF-α than LPS alone. NMDA could neither alter the IL-6 and TNF-α mRNA expression, nor reverse the enhanced expression of IL-6 and TNF-α mRNA by KTM in LPS-challenged cells. After TLR4-siRNA transfection, RAW264.7 cells pretreated with KTM no longer promoted the IL-6 and TNF-α expression in the presence of LPS. In conclusion, KTM accelerated LPS-induced inflammation in RAW264.7 cells by promoting TLR4 expression, independent of NMDA receptor.

D(-)-Salicin inhibits the LPS-induced inflammation in RAW264.7 cells and mouse models.

D(-)-Salicin is a traditional medicine which has been known to exhibit anti-inflammation and other therapeutic activities. The present study aimed to investigate whether D(-)-Salicin inhibited the LPS-induced inflammation in vivo and in vitro. We evaluated the effect of D(-)-Salicin on cytokines (TNF-α, IL-1β, IL-6 and IL-10) in vivo and in vitro by enzyme-linked immunosorbent assay and signaling pathways (MAPKs and NF-κB) in vivo by Western blot. The results showed that D(-)-Salicin markedly decreased TNF-α, IL-1β and IL-6 concentrations and increased IL-10 concentration. In addition, western blot analysis indicated that D(-)-Salicin suppressed the activation of MAPKs and NF-κB signaling pathways stimulated by LPS. To examine whether D(-)-Salicin ameliorated LPS-induced lung inflammation, inhibitors of MAPKs and NF-κB signaling pathways were administrated intraperitoneally to mice. Interference with specific inhibitors revealed that D(-)-Salicin-mediated cytokine suppression was through MAPKs and NF-κB pathways. In the mouse model of acute lung injury, histopathologic examination indicted that D(-)-Salicin suppressed edema induced by LPS. So it is suggest that D(-)-Salicin might be a potential therapeutic agent against inflammatory diseases.

Rosmarinic acid와 luteolin의 항염증에 대한 상승효과

본 연구에서는 들깨 유래 기능성 물질인 rosmarinic acid (RA)와 luteolin이 RAW264.7 세포에서 항염증작용에 대한 상승 효과가 있는지 알아보고자 하였다. 그 결과 RAW264.7 세포에 RA(<TEX>$50{\mu}M$</TEX>)와 luteolin (<TEX>$1{\mu}M$</TEX>)을 동시에 처리하였을 경우 염증 매개인자인 NO, iNOS, <TEX>$PGE_2$</TEX>, COX-2의 생성을 RA (<TEX>$100{\mu}M$</TEX>)와 luteolin (<TEX>$2{\mu}M$</TEX>)을 각각 처리하였을 때 보다 더 뛰어나게 억제하였다. 또한 RA (<TEX>$50{\mu}M$</TEX>)와 luteolin (<TEX>$1{\mu}M$</TEX>)을 동시에 처리하였을 경우 TNF-<TEX>${\alpha}$</TEX>, IL-6, IL-<TEX>$1{\beta}$</TEX> 같은 염증성 사이토카인의 생성량을 RA (<TEX>$100{\mu}M$</TEX>)와 luteolin (<TEX>$2{\mu}M$</TEX>)을 각각 처리하였을 때 보다 더 뛰어나게 억제하는 것을 확인하였다. 그리고 RA (<TEX>$50{\mu}M$</TEX>)와 luteolin (<TEX>$1{\mu}M$</TEX>)을 동시에 처리하였을 경우 RA (<TEX>$100{\mu}M$</TEX>)와 luteolin (<TEX>$2{\mu}M$</TEX>)을 각각 처리하였을 때 보다 NF-<TEX>${\kappa}B$</TEX>의 subunit인 p65의 translocation과 <TEX>$I{\kappa}B$</TEX>-<TEX>${\alpha}$</TEX>의 degradation을 더 뛰어나게 억제하는 것을 볼 수 있어 두 화합물 간의 상승작용이 뚜렷함을 확인 할 수 있었고, RA와 luteolin 두 화합물을 동시에 처리할 경우 염증관련 질환 치료에 유용하게 활용될 수 있을 것으로 판단된다. The aim of this study was to investigate the synergistic anti-inflammatory effect of rosmarinic acid (RA) and luteolin from perilla (Perilla frutescens L.) leaves in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. A combination of RA and luteolin more strongly inhibited the production of nitric oxide (NO), inducible NOS (iNOS), prostaglandin <TEX>$E_2$</TEX> (<TEX>$PGE_2$</TEX>), and COX-2 than higher concentrations of RA or luteolin alone in LPS-stimulated RAW264.7 macrophages. The combined RA and luteolin synergistically inhibited the production of pro-inflammatory cytokines, such as tumor necrosis factor-<TEX>${\alpha}$</TEX> (TNF-<TEX>${\alpha}$</TEX>), interleukin-6 (IL-6), and interleukin-<TEX>$1{\beta}$</TEX> (IL-<TEX>$1{\beta}$</TEX>), in LPS-stimulated RAW264.7 macrophages. Furthermore, combined RA and luteolin more strongly suppressed NF-<TEX>${\kappa}B$</TEX> activation than RA or luteolin alone, by inhibiting the degradation of inhibitor of NF-<TEX>${\kappa}B(I{\kappa}B)$</TEX>-<TEX>${\alpha}$</TEX> and nuclear translocation of the p65 subunit of NF-<TEX>${\kappa}B$</TEX> in LPS-stimulated RAW264.7 macrophages. Collectively, these results suggest that RA and luteolin in combination exhibit synergistic effects in suppression of LPS-induced inflammation in RAW264.7 macrophages.

Glucosylceramide attenuates the inflammatory mediator expression in lipopolysaccharide-stimulated RAW264.7 cells

The positive effect of glucosylceramide (GlcCer) on skin conditions is well known. Recently, there has been increasing interest in the potential antiinflammatory effects of GlcCer due to its efficacy in relieving atopic skin symptoms. However, the role of GlcCer in inflammation has not been investigated completely. Thus, we hypothesized that GlcCer might exhibit the antiinflammatory effects through the inhibition of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. To test this hypothesis, the antiinflammatory effects and signaling mechanisms of GlcCer were investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. We report that GlcCer inhibited messenger RNA and protein expression of tissue necrosis factor α and interleukin 1β without cytotoxicity. However, it did not affect interleukin 6 production in LPS-stimulated RAW 264.7 macrophages. Glucosylceramide also suppressed prostaglandin E2 but not nitric oxide production, consistent with its inhibition of cyclooxygenase 2 but not of inducible nitric oxide synthase expression. The molecular mechanism of GlcCer-mediated inhibition of LPS-induced inflammation in RAW 264.7 cells is closely related to suppression of NF-κB p65 subunit nuclear translocation as well as to phosphorylation of extracellular signal-regulated kinase and, in particular, p38 MAPK. In addition, GlcCer did not affect c-Jun N-terminal kinase phosphorylation. In conclusion, GlcCer inhibits LPS-induced inflammation by blocking the nuclear translocation of NF-κB and inhibiting the phosphorylation of extracellular signal-regulated kinase/p38 MAPK pathways in macrophages, suggesting that it might be a promising potential drug candidate for various inflammatory diseases.

N-acetylmuramic acid triggers anti-inflammatory capacity in LPS-induced RAW 264.7 cells and mice

Lactic acid bacteria (LAB) have long been used in the manufacture of yogurt and cheese. LAB inhabit the human gastrointestinal tract alongside dozens of varieties of gut bacteria, which play an essential role in modulating the innate immune response to gastrointestinal disorders. This research sought to provide a basis for the investigation of peptidoglycan (PGN) from Lactobacillus acidophilus (L. acidophilus) by macrophages phagocytosis related to the anti-inflammatory effect both in vitro and vivo. Results show that PGN, PGN hydrolysate and PGN monomer N-acetylmuramic acid (NAM) from L. acidophilus were found to have an anti-inflammatory effect on LPS-induced inflammation in RAW 264.7 cells, the profiling of iNOS mRNA levels was also inhibited in NAM-treated (100–300 µg/mL) group. The activation of the Nuclear factor κB (NF-κB) pathway is regulated by the cellular kinase p38 in mitogen-activated protein kinases (MAPK) upon NAM treatment (300 µg/mL). These findings were further supported in Escherichia coli (E. coli)-stimulated ICR mice, in which we found that oral administration of free L. acidophilus PGN and NAM from L. acidophilus exerted a potential anti-inflammatory response. NAM, as the main components of the LAB cell wall, will be a candidate for pharmaceutical application of anti-inflammatory drugs.

Nardostachys jatamansi (D. Don) DC prevents LPS-induced inflammation in RAW 264.7 macrophages by preventing ROS production and down-regulating inflammatory gene expression

Methanol extracts of Nardostachys jatamansi (D. Don) DC (NJ) roots were studied for anti-inflammatory activities in RAW 264.7 macrophages. NJ extracts protected macrophages against lipopolysaccharide (LPS)-induced apoptosis. The cell viability of LPS-stimulated macrophages was increased from 60.5% to 98.9% by a 100 μg/mL NJ extract. A decrease in the reactive oxygen species level contributed to the protective effect of NJ extracts on LPSstimulated macrophages. The anti-inflammatory effect of NJ extracts was assessed via an inhibitory effect on nitric oxide (NO) production. The NO level decreased from 100% to 5.8% after incubation with a 100 μg/mL NJ extract. Inducible iNOS and COX-2 expression was determined using reverse transcription-polymerase chain reaction and Western blot analyses. NJ extracts downregulated both iNOS and COX-2 expression in a dose-dependant manner. mRNA expression of the pro-inflammatory cytokines tumor necrosis TNF-α, IL-1β, and IL-6 decreased in response to NJ extracts.

미나리 발효 식초의 지방세포 분화억제 및 항염증 효과

비만은 감염원 없이 진행되는 낮은 수준의 만성적인 전신성 염증상태로 간주되며, 만성대사질환의 원인이 된다. 본 연구에서는 미나리를 발효하여 만든 식초가 3T3-L1 지방전구세포의 분화 및 RAW 264.7 세포에서의 항염증 효과에 미치는 영향을 확인하였다. MDI (IBMX, dexamethasone, insulin)에 의해 분화가 유발된 3T3-L1 지방전구세포에 대한 미나리 식초의 분화 억제능을 Oil-red-O staining을 통해 확인하였으며, western blot법을 통해 지방세포 형성 시 중요한 전사인자인 peroxisome proliferator-activated receptor-<TEX>${\gamma}$</TEX> (PPAR-<TEX>${\gamma}$</TEX>)와 CCAAT-enhancer-binding protein <TEX>${\alpha}$</TEX> (<TEX>$C/EBP{\alpha}$</TEX>)의 발현을 감소시키는 것으로 나타났다. 또한, 미나리 식초가 lipopolysaccharide (LPS)에 의해 염증이 유발 된 RAW 264.7 세포의 nitric oxide (NO) 생성을 억제시켰으며, inducible NO synthase (iNOS)와 cyclooxygenase-2 (COX-2)의 단백질 발현을 감소시키는 것을 확인하였다. 이러한 결과들을 통해 미나리 발효 식초는 지방세포 분화와 염증 억제 효과를 가지고 있음을 확인하였다. 따라서 미나리 발효 식초는 대사성 질환을 예방할 수 있는 천연물 소재로 이용 가능할 것으로 생각된다. Obesity, which is characterized by a state of mild chronic inflammation, is known to cause metabolic diseases. This study was carried out to investigate the effect of lyophilized dropwort vinegar powder (DVP) on adipocyte differentiation and inflammation in T3-L1 preadipocyte and RAW 264.7 macrophage cell lines. DVP inhibited the differentiation of 3T3-L1 preadipocytes induced by a mixture of IBMX, dexamethasone, and insulin (MDI). Western blot analysis of cell lysates showed that DVP decreased the levels of two major transcription factors involved in adipogenesis, peroxisome proliferator- activated receptor-<TEX>${\gamma}$</TEX> (PPAR-<TEX>${\gamma}$</TEX>) and CCAAT-enhancer-binding protein <TEX>${\alpha}$</TEX> (<TEX>$C/EBP{\alpha}$</TEX>). DVP also significantly suppressed lipopolysaccharide (LPS)-induced production of nitric oxide (NO), and this was accompanied by a decrease in inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. These results demonstrate that DVP inhibits MDI-induced adipocyte differentiation of 3T3-L1 cells and LPS-induced inflammation in RAW 264.7 macrophage cells. The findings indicate that this natural product may be a good candidate as to prevent metabolic diseases.

Punicalagin Inhibits Inflammation in LPS-Induced RAW264.7 Macrophages via the Suppression of TLR4-Mediated MAPKs and NF-κB Activation

Punicalagin (2,3,hexahydroxydiphenoyl-gallagyl-D-glucose and referred to as PUN) is a bioactive ellagitannin isolated from pomegranate, which is widely used for the treatment of inflammatory bowel disease (IBD), diarrhea, and ulcers in Chinese traditional medicine. In this study, we detected the anti-inflammation potentials of PUN in lipopolysaccharide (LPS)-induced macrophages and tried to uncover the underlying mechanism. Results demonstrated that PUN (25, 50, or 100μM) treatment could significantly decrease the LPS-induced production of nitric oxide), prostaglandin E2 (PGE2), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in RAW264.7 cells. Molecular research showed that PUN inhibited the activation of upstream mediator nuclear factor-κB by suppressing the phosphorylation of IκBα and p65. Results also indicated that PUN could suppress the phosphorylation of mitogen-activated protein kinase including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase. In conclusion, we observed that PUN could inhibit LPS-induced inflammation, and it may be a potential choice for the treatment of inflammation diseases.

3,4,5-Trihydroxycinnamic acid inhibits lipopolysaccharide (LPS)-induced inflammation by Nrf2 activation in vitro and improves survival of mice in LPS-induced endotoxemia model in vivo

NF-E2-related factor 2 (Nrf2) has been demonstrated to be a key transcription factor regulating the anti-inflammatory genes including heme oxygenase-1 (HO-1) in experimental sepsis models. Based on the fact that 3,4,5-trihydorxycinnamic acid (THC) has been reported to possess anti-inflammatory properties in BV2 microglial cells, the possible effects of THC and its underlying mechanism was examined against lipopolysaccharide (LPS)-induced RAW 264.7 cell culture and septic mouse models. Pretreatment of RAW 264.7 cells with THC significantly attenuated LPS-induced NO, PGE2 production, and expression of iNOS and COX-2. THC also significantly suppressed LPS-induced release of pro-inflammatory cytokines and degradation of IκB-α. Increased phosphorylation of Nrf2 and nuclear translocation of Nrf2 were observed with THC treatment with consequent expression of HO-1. The data demonstrated that multiple signaling pathways including Akt, p38, and PKC are involved in the THC-induced activation of Nrf2/HO-1 pathway. Treatment of THC resulted in significantly increased survival of LPS-induced septic mice. THC also significantly ameliorated LPS-induced septic features such as hypothermia and increased vascular leakage. In accordance with the data from cell culture model, THC exhibited increased expression of HO-1 in kidney and decreased serum level of pro-inflammatory mediators such as TNF-α, IL-1β, and NO. Taken together, the present study for the first time demonstrates that THC inhibits inflammation in LPS-induced RAW264.7 cells by Nrf2 activation and improves survival of mice in LPS-induced endotoxemia model.

Extracellular polysaccharide from Bacillus sp. strain LBP32 prevents LPS-induced inflammation in RAW 264.7 macrophages by inhibiting NF-κB and MAPKs activation and ROS production

Extracellular polysaccharides (EPSs) are high-molecular weight sugar-based polymers that are synthesized and secreted by many microorganisms. Recently, EPSs have attracted particular attention due to their multiple biological functions including anti-inflammation. However, studies rarely reported the molecular mechanisms underlying their functions. We previously purified an EPS from an oligotrophic bacteria (Bacillus sp. LBP32) found in Lop Nur Desert, which possesses a potent antioxidant activity, while the anti-inflammatory effects of EPS and signaling mechanisms underlying its action have not been clarified. In this study, we demonstrated that EPS significantly inhibited the LPS-induced release of pro-inflammatory mediators, such as nitric oxide (NO), IL-6 and TNF-α, without any significant cytotoxicity. EPS also downregulated the expression of nitric oxide synthase (iNOS) induced by LPS. Furthermore, activation of nuclear factor κB (NF-κB) was abrogated by EPS through inhibited the phosphorylation of IκB kinase (IKK). Activations of Mitogen-activated protein kinases (MAPKs), including p38 MAPK and c-Jun N-terminal kinase (JNK), were also found to be inhibited by EPS. In addition, the level of intracellular reactive oxygen species (ROS) was also significantly decreased with the treatment of EPS. In vivo experiments were conducted and showed that EPS could greatly improve the outcome of mice with LPS-induced endotoxic shock. Taken together, our data indicate that EPS prevents LPS-induced inflammatory response by inhibiting NF-κB and MAPKs activation and ROS production.

Tubeimoside-1 attenuates LPS-induced inflammation in RAW 264.7 macrophages and mouse models

Context: Acute lung injury (ALI), characterized by severe hypoxemia, pulmonary edema and neutrophil accumulation in the lung, is a common clinical problem associated with significant morbidity and mortality in shock, sepsis, ischemia reperfusion, etc.Objective: In this study, we aimed at investigating the protective effect of tubeimoside-1 (TBMS1) on inflammation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and a LPS-induced in vivo lung injury model.Materials and methods: We evaluated the effect of TBMS1 on LPS-induced production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in the culture supernatants of RAW 264.7 cells by enzyme-linked immunosorbent assay. LPS (0.5 mg/kg) was instilled intranasally in phosphate-buffered saline to induce ALI, and the severity of pulmonary injury was evaluated 6 h after LPS challenge.Results: TBMS1 significantly inhibited the production of the pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β in vitro and in vivo. Pretreatment with TBMS1 markedly attenuated the development of pulmonary edema, histological severities and inflammatory cells infiltration in mice with ALI. In addition, we further demonstrated that TBMS1 exerts an anti-inflammatory effect in vivo model of ALI through suppression of IκB activation and p38/extracellular signal-regulated kinase mitogen-activated protein kinases signaling in a dose-dependent manner.Discussion and conclusion: Overall, our data suggest that TBMS1 inhibits inflammation both in vitro and in vivo, and may be a potential therapeutic candidate for the prevention of inflammatory diseases.

Inhibitory effect of a formulated extract from multiple citrus peels on LPS-induced inflammation in RAW 246.7 macrophages

Background: Formulated Citrus Peel Extract (GL) made from the peels of six citrus fruits available in Japan, namely navel oranges, citrus hassaku, citrus limon, citrus natsudaidai, citrus miyauchi and satsuma, was initially developed as a cosmetic product to protect skin from UV irradiation. Anecdotal evidences of anti-cancer property of GL have been reported by consumers based on the cases such as topical application for melanoma, and oral ingestion for prostate, lung and liver cancers. Those anecdotal reports stimulated us to investigate anti-tumorigenesis activity of GL. In the previous study, we reported that the topical application of GL inhibited DMBA/TPA-induced skin tumor formation by decreasing inflammatory gene parameters.Objective: In this study, we mainly investigated the effect of GL on translocation of NF-kB together with production of nitric-oxide and TNF-α induced by LPS in RAW 264.7 cells.Results: This investigation showed that GL decreased the release of TNF-α and nitric oxide from macrophage RAW264.7 cells stimulated by LPS in a dose-dependent manner. In addition, GL suppressed the expression of iNOS and nuclear translocation of NF-kB in RAW264.7 cells, inhibited the degradation of IκB-α, and scavenged hydroxyl radicals (DMPO/OH adduct) in vitro.Conclusions: Our findings suggest that GL suppresses the inflammation in vitro, and exerts chemopreventive activity through the inhibition of production of TNF-α and iNOS proteins due to the inhibition of nuclear translocation of NF-kB and oxidative stress. GL appears to be a novel functional natural product capable of preventing inflammation and inflammation-associated tumorigenesis. Keywords: GL, Citrus peel extract, anti-inflammation, Nitric oxide, iNOS, NF-kB, TNF-α

Low molecular weight fucoidan from the sporophyll of Undaria pinnatifida suppresses inflammation by promoting the inhibition of mitogen-activated protein kinases and oxidative stress in RAW264.7 cells

The suppression of MAPK and oxidative stress could be an important anti-inflammatory mechanism. Fucoidan regulates MAPK activity in several cell lines. However, the mechanism for the anti-inflammatory and oxidative stress effect of low molecular weight fucoidan (LMWF) is poorly understood in RAW264.7 cells. The objective of this study was to examine the critical role of LMWF during LPS-induced inflammation in RAW264.7 cells. To determine the potential role of LMWF, we analysed pro-inflammatory cytokine, transcription factor, inflammation-related and oxidative stress related protein expression in vitro during LPS-induced inflammation in RAW264.7 cells. In this study, we demonstrated the anti-inflammatory effects of LMWF on LPS-induced inflammation in macrophages through the regulation of signalling pathways, including its effect on the attenuation of inflammatory cytokines, such as IL-1β, IL-1 and TNF-α, and the degradation of phosphorylated p38 MAPK, ERK1/2 and JNK. This study also demonstrates that LMWF might block NO as well as the expression of reactive oxygen species (ROS), which subsequently inhibits the iNOS and COX-2 expression induced by LPS. Based on these findings, we suggest that LMWF might have great potential as an external pathogen prevention and intervention agent for inflammatory diseases.

Synergistic Anti-inflammatory Effects of Nobiletin and Sulforaphane in Lipopolysaccharide-Stimulated RAW 264.7 Cells

Inflammation plays important roles in the initiation and progress of many diseases including cancers in multiple organ sites. Herein, we investigated the anti-inflammatory effects of two dietary compounds, nobiletin (NBN) and sulforaphane (SFN), in combination. Noncytotoxic concentrations of NBN, SFN, and their combinations were studied in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that combined NBN and SFN treatments produced much stronger inhibitory effects on the production of nitric oxide (NO) than NBN or SFN alone at higher concentrations. These enhanced inhibitory effects were synergistic based on the isobologram analysis. Western blot analysis showed that combined NBN and SFN treatments synergistically decreased iNOS and COX-2 protein expression levels and induced heme oxygenase-1 (HO-1) protein expression. Real-time polymerase chain reaction analysis indicated that low doses of NBN and SFN in combination significantly suppressed LPS-induced upregulation of IL-1 mRNA levels and synergistically increased HO-1 mRNA levels. Overall, our results demonstrated that NBN and SFN in combination produced synergistic effects in inhibiting LPS-induced inflammation in RAW 264.7 cells.

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.

Abstract 4243: Potential protective role of phytoalexins derived from soybean by biotic elicitor on inflammatory mechanism

Abstract Given the preventive effect of soy intake against several chronic diseases, this study was conducted to investigate the inhibitory activity of phytoalexins glyceollins derived from soybean isoflavones by treating with biotic elicitor against LPS-induced inflammation in RAW 264.7 cell culture system. Our data showed that glyceollins effectively inhibited NO production, IL-6 release, and COX-2 expression induced by LPS. In particular, glyceollins suppressed the LPS-induced phosphorylation of NF-B p65, suggesting that the compounds inhibit the production of NO and transcriptional activation of COX-2 by regulating NF-B activity. In another experiment we found that glyceollins enhanced the expression of heme oxygenase 1 and repressed the phosphorylation of ERK1/2 and p38 but not JNK in LPS-treated RAW 264.7 cells. Glyceollins also reduced TPA-induced skin inflammation in mouse model, confirming the anti-inflammatory activity of glyceollins in vivo system as well as cell culture system. Taken together, glyceollins merit further study as potential therapeutic agents for inflammatory disorders. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4243. doi:10.1158/1538-7445.AM2011-4243

Proteomic response of LAP-activated RAW 264.7 macrophages to the anti-inflammatory property of fungal ergosterol

Ergosterol is the principal fungal sterol in edible mushrooms and yeast lees in wine. In this study, the immunoregulatory action of ergosterol was evaluated by a proteomic approach using two-dimensional gel electrophoresis to identify proteins with modified expression in LPS-challenged RAW 264.7 macrophages treated with ergosterol. By comparing the proteome signatures, alterations of multiple proteins were observed, including NF-κB cascade-related proteins, Rho family GTPase protein, and cytoskeleton-related proteins. The down-regulation of NF-κB cascade-associated proteins coincided with the inhibition of TNF-α production and COX-2 expression in the ergosterol-treated RAW 264.7 cells. Western blotting analysis indicated that ergosterol inhibits LPS-induced inflammation in RAW 264.7 macrophages by suppressing the NF-κB signalling pathway.

Inhibition of Inducible Nitric Oxide Synthase by Agaricus bisporus Extract in RAW 264.7 Macrophages

Agaricus bisporus, also known as white button mushroom, is one of the most popular mushrooms consumed in Korea. This mushroom contains high concentrations of flavanoids and exhibits antioxidant activity. In this study, we examined the effects of Agaricus bisporus ethanol extract (ABE) on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells. Nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein levels were assessed in cells treated with 100 μM LPS in the presence or absence of ABE. 0.5 ㎎/mL of ABE suppressed NO production significantly. Moreover, ABE inhibited levels of iNOS protein. Taken together, these results suggest that ABE exerts anti-inflammatory activity in LPS-induced inflammation in RAW 264.7 cells.