Cryptotanshinone Suppressed Inflammatory Cytokines Secretion in RAW264.7 Macrophages through Inhibition of the NF-κB and MAPK Signaling Pathways

Cryptotanshinone (CTS), a major constituent extracted from the medicinal herb Salvia miltiorrhiza Bunge, has well-documented antioxidative and anti-inflammatory effects. In the present study, the pharmacological effects and underlying molecular mechanisms of CTS on lipopolysaccharide (LPS)-induced inflammatory responses were investigated. By enzyme-linked immunosorbent assay, we observed that CTS reduced significantly the production of proinflammatory mediators (tumor necrosis factor-α and interleukin-6) induced by LPS in murine macrophage-like RAW264.7 cells. Mechanistically, CTS inhibited markedly the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, p38MAPK, and JNK, which are crucially involved in regulation of proinflammatory mediator secretion. Moreover, immunofluorescence and western blot analysis indicated that CTS abolished completely LPS-triggered nuclear factor-κB (NF-κB) activation. Taken together, these data implied that NF-κB and MAPKs might be the potential molecular targets for clarifying the protective effects of CTS on LPS-induced inflammatory cytokine production in macrophages.     

Thymol Inhibits LPS-Stimulated Inflammatory Response via Down-Regulation of NF-κB and MAPK Signaling Pathways in Mouse Mammary Epithelial Cells

Thymol is a natural monoterpene phenol primarily found in thyme, oregano, and tangerine peel. It has been shown to possess anti-inflammatory property both in vivo and in vitro. In the present paper, we studied the anti-inflammatory effect of thymol in lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (mMECs). The mMECs were stimulated with LPS in the presence or absence of thymol (10, 20, 40 μg/mL). The concentrations of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and IL-1β in the supernatants of culture were determined using enzyme-linked immunosorbent assay. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB), and inhibitor protein of NF-κB (IκBα) were measured using western blot. The results showed that thymol markedly inhibited the production of TNF-α and IL-6 in LPS-stimulated mMECs. The expression of iNOS and COX-2 was also suppressed by thymol in a dose-dependent manner. Furthermore, thymol blocked the phosphorylation of IκBα, NF-κB p65, ERK, JNK, and p38 mitogen-activated protein kinases (MAPKs) in LPS-stimulated mMECs. These results indicate that thymol exerted anti-inflammatory property in LPS-stimulated mMECs by interfering the activation of NF-κB and MAPK signaling pathways. Thereby, thymol may be a potential therapeutic agent against mastitis.     

Benzoylaconine Modulates LPS-Induced Responses Through Inhibition of Toll-Like Receptor-Mediated NF-κB and MAPK Signaling in RAW264.7 Cells

Inflammation - Previous studies have shown that benzoylaconine (BAC), a representative monoester alkaloid, has a potential anti-inflammatory effect. This study investigated the underlying molecular...     

Thrombospondin-1 Production Regulates the Inflammatory Cytokine Secretion in THP-1 Cells Through NF-κB Signaling Pathway

Thrombospondin-1 (TSP-1) is upregulated in several inflammatory diseases. Recent data have shown that macrophages from TSP-1-deficient mice have a reduced inflammatory phenotype, suggesting that TSP-1 plays a part in macrophage activation. DNA microarray approach revealed that Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS) may induce the enhanced TSP-1 expression in human monocytes, suggesting a role of TSP-1-mediated pathogenesis in periodontitis. Until recently, the function of TSP-1 has been a matter of debate. In this study, we explored the role of TSP-1 in inflammatory cytokine secretions and its putative mechanism in pathogenesis of periodontitis. We demonstrated that TSP-1 expression was significantly upregulated in gingival tissues with periodontitis and in P. gingivalis LPS-stimulated THP-1 cells. Deficiency of TSP-1 by transfecting siRNAs decreased IL-6, IL-1β, and TNF-α secretions in THP-1 cells, whereas overexpression of TSP-1 resulted in an upregulation of IL-6, IL-1β, and TNF-α productions. Additional experiments showed that Pyrrolidine dithiocarbamate (PDTC) inhibited IL-6, IL-1β, and TNF-α expression induced by overexpression of TSP-1, accompanying with downregulation of phosphorylated p65 and IκBα protein levels in response to P. gingivalis LPS. These results indicated that TSP-1 played a significant role in P. gingivalis LPS-initiated inflammatory cytokines (IL-6, IL-1β, and TNF-α) secretions of THP-1 cells, and the NF-κB signaling is involved in its induction of expression. Thus, TSP-1 effectively elevated P. gingivalis LPS-induced inflammation mediated by the NF-κB pathway and may be critical for pathology of periodontitis.     

Hesperetin Suppresses Inflammatory Responses in Lipopolysaccharide-Induced RAW 264.7 Cells via the Inhibition of NF-κB and Activation of Nrf2/HO-1 Pathways

Hesperetin (Hesp), a common flavanone glycoside, was extracted from the fruit peel of Citrus aurantium L. (Rutaceae). Hesp has been shown to possess various biological properties, including antioxidant, neuroprotective, and anti-inflammatory properties. In this study, we investigated the protective effect of Hesp on inflammatory responses in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Our results indicated that Hesp treatment dramatically suppressed secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β; reduced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression; inhibited NF-κB (p65) phosphorylation; and blocked IκBα phosphorylation and degradation. Further studies revealed Hesp markedly enhanced the heme oxygenase (HO)-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression, which were involved with inducing Nrf2 nuclear translocation and decreasing Keap1 protein expression. Together, these results indicated that the anti-inflammatory effect of Hesp may be associated with NF-κB inhibition and Nrf2/HO-1 activation.     

Suppression of MAPK and NF-κB Pathways by Limonene Contributes to Attenuation of Lipopolysaccharide-Induced Inflammatory Responses in Acute Lung Injury

The present study aimed to investigate the protective role of limonene in lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS (0.5 mg/kg), and limonene (25, 50, and 75 mg/kg) was injected intraperitoneally 1 h prior to LPS administration. After 12 h, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Limonene pretreatment at doses of 25, 50, and 75 mg/kg decreased LPS-induced evident lung histopathological changes, lung wet-to-dry weight ratio, and lung myeloperoxidase activity. In addition, pretreatment with limonene inhibited inflammatory cells and proinflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in BALF. Furthermore, we demonstrated that limonene blocked the phosphorylation of IκBα, nuclear factor-κB (NF-κB) p65, p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase in LPS-induced ALI. The results presented here suggest that the protective mechanism of limonene may be attributed partly to decreased production of proinflammatory cytokines through the inhibition of NF-κB and MAPK activation.     

Selenium Inhibits LPS-Induced Pro-inflammatory Gene Expression by Modulating MAPK and NF-κB Signaling Pathways in Mouse Mammary Epithelial Cells in Primary Culture

Mastitis is characterized by an inflammation of the mammary gland of dairy animals and humans; this condition is one of the major causes of economic losses in dairy industries. Selenium (Se), a biological trace element, modulates the functions of many regulatory proteins in signal transduction and provides advantages for animals with inflammatory diseases, including mastitis. The current study aimed to assess the protective effects and the active mechanism of Na₂SeO₃ against lipopolysaccharide (LPS)-induced inflammation in mouse mammary epithelial cells (MMECs). Our results showed that LPS-induced expressions of cyclooxygenase-2 and tumor necrosis factor-α significantly decreased after Se was supplemented to Se-deficient MMECs. Na₂SeO₃ also suppressed LPS-induced nuclear factor-κB activation, inhibitory kappa B degradation, and ERK, JNK, and P38 phosphorylation in a dose-dependent manner. These results suggested that Se functions as an anti-inflammatory agent in mastitis.     

Gamma-Linolenic Acid Inhibits Inflammatory Responses by Regulating NF-κB and AP-1 Activation in Lipopolysaccharide-Induced RAW 264.7 Macrophages

Gamma linolenic acid (GLA) is a member of the n-6 family of polyunsaturated fatty acids and can be synthesized from linoleic acid (LA) by the enzyme delta-6-desaturase. The therapeutic values of GLA supplementation have been documented, but the molecular mechanism behind the action of GLA in health benefits is not clear. In this study, we assessed the effect of GLA with that of LA on lipopolysaccharide (LPS)-induced inflammatory responses and further explored the molecular mechanism underlying the pharmacological properties of GLA in mouse RAW 264.7 macrophages. GLA significantly inhibited LPS-induced protein expression of inducible nitric oxide synthase, pro-interleukin-1β, and cyclooxygenase-2 as well as nitric oxide production and the intracellular glutathione level. LA was less potent than GLA in inhibiting LPS-induced inflammatory mediators. Both GLA and LA treatments dramatically inhibited LPS-induced IκB-α degradation, IκB-α phosphorylation, and nuclear p65 protein expression. Moreover, LPS-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) nuclear protein–DNA binding affinity and reporter gene activity were significantly decreased by LA and GLA. Exogenous addition of GLA but not LA significantly reduced LPS-induced expression of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK)-1. Our data suggest that GLA inhibits inflammatory responses through inactivation of NF-κB and AP-1 by suppressed oxidative stress and signal transduction pathway of ERK and JNK in LPS-induced RAW 264.7 macrophages.     

Stevioside Plays an Anti-inflammatory Role by Regulating the NF-κB and MAPK Pathways in S. aureus-infected Mouse Mammary Glands

Mastitis is an inflammatory disease caused by microbial infection. Staphylococcus aureus is one of the primary bacteria responsible for mastitis. Stevioside is isolated from Stevia rebaudiana and is known to have therapeutic functions. This study was designed to evaluate the effects of stevioside in a mouse model of S. aureus-induced mastitis. In this study, the mouse mammary gland was infected with S. aureus to induce the mastitis model. The stevioside was administered intraperitoneally after the S. aureus infection was established. Hematoxylin–eosin (HE) staining, ELISA, Western blot, and q-PCR methods were used. The results show that stevioside significantly reduced the inflammatory cell infiltration and the levels of TNF-α, IL-1β, and IL-6 and the respective expression of their messenger RNAs (mRNAs). Further studies revealed that stevioside downregulated the TLR2, NF-κB, and (mitogen-activated protein kinase) MAPK signaling pathways in the S. aureus-infected mouse mammary gland. Our results demonstrate that stevioside reduced the expression of TNF-α, IL-1β, and IL-6 by inhibiting the phosphorylation of proteins in the NF-κB and MAPK signaling pathways dose-dependently, but that their mRNA expression was not obviously changed.     

A Leaf Methanolic Extract of Wercklea insignis Attenuates the Lipopolysaccharide-Induced Inflammatory Response by Blocking the NF-κB Signaling Pathway in RAW 264.7 Macrophages

The biological activity of Wercklea insignis (WI) in inflammation and the underlying mechanisms of action of extracts of this plant are largely unknown. In the present study, we investigated the effects of a WI methanolic extract on lipopolysaccharide-stimulated inflammation in the mouse macrophage cell line, RAW 264.7. A WI methanolic extract significantly inhibited NO, PGE₂, IL-6, IL-1β, and TNF-α production in LPS-stimulated RAW 264.7 cells. Expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α were suppressed by the extract at both the mRNA and protein levels in lipopolysaccharide (LPS)-stimulated cells. Additionally, the attenuation of inflammatory responses in RAW 264.7 cells by the WI extract was closely associated with suppression of phosphorylation of mitogen-activated protein kinase (MAPK) molecules, including ERK, JNK1/2, and p38 MAPK and translocation of the nuclear factor (NF)-κB p65 subunit into the nucleus. The effect of WI extract was investigated against carrageenan-induced paw edema in female (20–25 g). Our results collectively indicate that the WI extract inhibits LPS-induced inflammatory responses by blocking the NF-κB signaling pathway in macrophages, supporting use of the extract as a therapeutic anti-inflammatory treatment.     

Oxymatrine Lightened the Inflammatory Response of LPS-Induced Mastitis in Mice Through Affecting NF-κB and MAPKs Signaling Pathways

Mastitis, an inflammatory reaction of the mammary gland, is recognized as one of the most costly diseases in dairy cattle. Oxymatrine, one of the alkaloids extracted from Chinese herb Sophora flavescens Ait, has been reported to have many biological activities, such as anti-inflammatory, anti-virus, and anti-hepatic fibrosis properties. The aim of this study was to investigate the protective effect and the anti-inflammatory mechanism of oxymatrine on lipopolysaccharide (LPS)-induced mastitis in mice. The mouse mastitis was induced by 10 μg of LPS for 24 h. Oxymatrine was intraperitoneally administered with the dose of 30, 60, and 120 mg/kg 1 h before and 12 h after LPS induction. The results showed that oxymatrine significantly attenuated the damage of the mammary gland induced by LPS. Oxymatrine inhibited the phosphorylation of NF-κB p65 and IκB in NF-κB signal pathway and reduced the phosphorylation of p38, ERK, and JNK in mitogen-activated protein kinase (MAPKs) signal pathway. The results showed that oxymatrine had a protective effect on LPS-induced mastitis, and the anti-inflammatory mechanism of oxymatrine was related to the inhibition of NF-κB and MAPKs signal pathways.     

Lipoxin A4 Inhibits NF-κB Activation and Cell Cycle Progression in RAW264.7 Cells

Lipoxins (LXs), including lipoxin A₄ (LXA₄), etc., have been approved for potent anti-inflammatory and immunomodulatory properties. Based on the important roles of macrophages in inflammation and immunomodulation, we investigate the effects of LXA₄ on lipopolysaccharide (LPS)-induced proliferation and the possible signal transduction pathways in RAW264.7 macrophages. RAW264.7 cells were treated in vitro with or without LPS in the absence or presence of LXA₄. [³H]-TdR incorporation assay and flow cytometry were used for detecting cell proliferation and cycle, respectively. Moreover, Western blot was applied to evaluate the protein expression levels of Cyclin E, IκBα, nuclear factor-κB (NF-κB), and IκB kinase (IKK). Our research showed that LXA₄ suppressed LPS-induced proliferation, increased the proportion of the G₀/G₁ phase, decreased the proportion of the S phase, and downregulated the expression of Cyclin E. Besides these, LXA₄ suppressed LPS-induced IκBα degradation, NF-κB translocation, and the expression of IKK. The data suggested that LXA₄ inhibited LPS-induced proliferation through the G₀/G₁ phase arrest in RAW264.7 macrophages, and the inhibitory effect might depend on NF-κB signaling transduction pathway.     

Defective NF-κB Signaling in Dedifferentiated Hepatoma Cells

Dedifferentiated rat hepatoma cells contain defects that result in the loss of hepatic gene expression, including the liver-enriched HNF4/HNF1 pathway. We examined induction of NF-B, a key mediator of the inflammatory response, in hepatoma and dedifferentiated hepatoma cells. We show that exposure of dedifferentiated hepatoma cells, but not rat and human hepatoma cell lines, to proinflammatory cytokines or lipopolysaccharide resulted in rapid and sustained NF-B induction. IB- levels, but not NF-B subunit p65 or IB- levels, were elevated compared with those for parental hepatoma cells. Interestingly, LPS-mediated activation of NF-B was found to be independent of degradation of IB- or IB-. Thus, these results suggest that loci responsible for maintaining hepatic gene expression also influence cellular responses to inflammatory agents.     

7,8-Dihydroxyflavone exhibits anti-inflammatory properties by downregulating the NF-κB and MAPK signaling pathways in lipopolysaccharide-treated RAW264.7 cells

7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, has received considerable attention as a selective tyrosine kinase receptor B agonist. Several studies have indicated that 7,8-DHF has neurotrophic and antioxidant activities. However, little is known about the cellular and molecular mechanisms underlying the anti-inflammatory activity of 7,8-DHF. Therefore, we investigated whether 7,8-DHF affects the expression of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Our results indicated that 7,8-DHF significantly attenuated secretion of LPS-induced inflammatory mediators nitric oxide (NO), prostaglandin E? (PGE?) and interleukin-1β (IL-1β) in RAW264.7 cells. Additionally, LPS-induced expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2 and IL-1β was decreased by pre-treatment with 7,8-DHF. Our results also showed that 7,8-DHF reduces LPS-induced nuclear factor-κB (NF-κB) activity via the suppression of the nuclear translocation of NF-κB p65 and the degradation of inhibitor κB (lκB). In addition, 7,8-DHF inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) such as extracellular-signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). These results suggest that the anti-inflammatory property of 7,8-DHF is related to the downregulation of iNOS, COX-2 and IL-1β, due to NF-κB inhibition as well as to the negative regulation of MAPK activation in RAW264.7 cells. Thus, 7,8-DHF may be a novel therapeutic agent for the prevention of various inflammatory diseases.     

Schisantherin A Exhibits Anti-inflammatory Properties by Down-Regulating NF<Emphasis Type="Italic">-</Emphasis>κB and MAPK Signaling Pathways in Lipopolysaccharide-Treated RAW 264.7 Cells

Schisantherin A, a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, has been used as an antitussive, tonic, and sedative agent under the name of Wuweizi in Chinese traditional medicine. In the present study, we carry out a screening program to identify the anti-inflammatory potentials of schisantherin A. We found that schisantherin A reduced lipopolysaccharide (LPS (1 mg/L))-induced levels of TNF-α, IL-6, NO, and PGE2 (p < 0.01 or p < 0.05), and also reduced levels of iNOS and COX-2 in RAW 264.7 macrophages in a concentration-dependent manner. We further investigated signal transduction mechanisms to determine how schisantherin A affects. RAW264.7 cells were pretreated with 0.5, 2.5, or 25 mg/L of schisantherin A 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation and IκBα was measured by Western blot. Alternatively, cells were fixed and nuclear factor-κB (NF-κB) activation was measured using immunocytochemical analysis. Signal transduction studies showed that schisantherin A significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH2-terminal kinase (JNK) phosphorylation protein expression. Schisantherin A also inhibited p65-NF-κB translocation into the nucleus by IκBα degradation. By using specific inhibitors of ERK, JNK and p38, we found that schisantherin A may inhibit TNF-α mostly through ERK pathway. Therefore, schisantherin A may inhibit LPS-induced production of inflammatory cytokines by blocking NF-κB and MAPKs signaling in RAW264.7 cells.     

Signaling Pathways for Fcγ Receptor-Stimulated Tumor Necrosis Factor-α Secretion and Respiratory Burst in RAW 264.7 Macrophages

Fc gamma receptor (Fc gammaR) signaling mediates several important macrophage functions including cytokine secretion and respiratory burst. The present study describes the development of a model using the macrophage cell line, RAW 264.7 for studying Fc gammaR-stimulated tumor necrosis factor-alpha (TNF-alpha) secretion and hydrogen peroxide (H2O2) production. In unprimed cells these functions were low but pretreatment with interferon-gamma augmented Fc gammaR-stimulated TNF-alpha secretion and H2O2 production to levels that were about half that caused by lipopolysaccharide (LPS) and zymosan, respectively. Studies on the signaling pathways found that TNF-alpha secretion stimulated by either Fc gammaR or LPS was decreased by inhibitors of PKC, MAPK p42/p44, and MAPK p38. TNF-alpha secretion was also reduced by the combination of PLC and PLD inhibitors but not by the individual inhibitors alone. H2O2 production stimulated by either Fc gammaR or zymosan was blocked by inhibitors of PKC, PLC, PLD, and MAPK p42/44 but not by MAPK p38. Thus, interferon-gamma treated RAW 264.7 cells are a model of inflammatory macrophages and are well suited for further study of these signaling pathways.