Effects of Echinococcus multilocularis miR-71 mimics on murine macrophage RAW264.7 cells

The microRNAs (miRNAs) are a class of small regulatory non-coding RNA that contributes to the activation of host-pathogen cross-talk during infection. In helminthes, miR-71 is highly conserved and it has recently been detected in nematode exosomes, as well as in the sera and/or fluids of infected humans and mice. However, the role of miR-71 during infection remains poorly characterized. Herein, we show that Ago1 and Ago4, which encode key components of the small RNA-induced silencing complex (RISC), were up-regulated in murine macrophage RAW264.7 cells transfected by Echinococcus multilocularis miR-71 (emu-miR-71) mimics. Using a miRNA PCR array, none of the 84 miRNAs involved in inflammation or autoimmunity were significantly up- or down-regulated in the transfected cells (p > 0.05). Although it did not influence IL-10 production by the treated cells (p > 0.05), the mimics significantly repressed the production of NO 12 h after treatment with LPS and IFN-γ (p < 0.01), identifying another potential mechanism whereby parasites can carefully regulate host levels of NO. These findings indicate that the release of parasite-derived miR-71 into hosts can affect the functions of macrophages, and possibly represents an exciting direction for studies of the interplay between parasites and hosts.     

Tectorigenin inhibits IFN-γ/LPS-induced inflammatory responses in murine macrophage RAW 264.7 cells

Tectorigenin (Tg) and tectoridin (Td) are the major compounds isolated from the rhizomes of iridaceous plant Belamcanda chinensis which is well known as a chinese traditional medicine for the treatment of inflammatory diseases. In this study we investigated whether tectorigenin and tectoridin can be applied to the suppression of interferon-γ and lipopolysaccharide (IFN-γ/LPS)-induced inflammatory responses in macrophages. Anti-inflammatory activities of tectorigenin and tectoridin were compared with genistein (Ge), well known isoflavonoid as a phytoestrogen and regarded as an emerging anti-inflammatory agent. Both compounds showed low cytotoxic effect. In Raw 264.7 cells activated with IFN-γ/LPS, pre-treated tectorigenin was found to inhibit the expression of inducible nitric oxide synthase (iNOS), the production of nitric oxide (NO) and the secretion of interleukin (IL)-1β dose-dependently. Tectorigenin also decreased the expression of cyclooxigenase (COX)-2 and the production of prostaglandin E₂ (PGE₂) in dose-dependent manner. These inhibitory effects of tectorigenin were found to be caused by the blocking of nuclear factor kappa-B (NF-κB) activation. Compared with genistein and tectoridin, tectorigenin showed significant inhibitory effect for almost anti-inflammatory tests in this study. All these results clearly demonstrated that tectorigenin appears to have the potential to prevent inflammation.     

Activation of murine macrophage cell line RAW 264.7 by Korean propolis

Monocytes and macrophages play a major role in defense mechanism of the host response to tumor, in part through the secretion of several potent products and macrophage cytokines. Monocytes and tissue macrophages produce at least two groups of protein mediators of inflammation, interleukin 1 (IL-1) and tumor necrosis factor (TNF). Recent studies emphasizes that TNF and IL-1 modulate the inflammatory function of endothelial cells, leukocytes, and fibroblasts. In this study, our work is directed toward studying the in vitro effects of Korean propolis on the ability to induce cellular and secretory responses in murine macrophage cell line, RAW 264.7. It was found that Water Extract of Korean Propolis (WEP) could activate macrophages by producing cytokines. The production of the macrophage cytokines, IL-1 and TNF-alpha, by RAW 264.7 treated with WEP was examined from 2.5 microg/ml up to 25 microg/ml with dose dependent manner. Nitric oxide (NO) production was also increased when cells were exposed to combination of LPS and WEP from 2.5 microg/ml up to 25 microg/ml. At high dose of WEP (50 to 100 microg/ml) used to prescribe for anti-inflammatory and analgesic medicine showed inhibition of NO production in LPS-stimulated macrophage. Besides cytokine production, NO release, surface molecule expression and cell morphologic antigen expression were increased in response to the stimulation by WEP. These results suggested WEP may function through macrophage activation.     

Chemical constituents from Bletilla striata and their NO production suppression in RAW 264.7 macrophage cells

A novel glucoside bletilloside A (1) was isolated from the tubers of Bletilla striata, together with seven known compounds (2–8). Their structures were determined on the basis of extensive spectroscopic analyses. All compounds were evaluated for the inhibition on NO production effects in RAW 264.7 macrophage cells, while militarine (4) and dactylorhin A (5) exhibited moderate inhibitory effects.     

Anti-inflammatory activity of Ternstroemia gymnanthera stem bark extracts in bacterial lipopolysaccharide-stimulated RAW264.7 murine macrophage cells

Context: Ternstroemia gymnanthera Sprague (Theaceae) possesses various known pharmacological properties. However, its anti-inflammatory activity has not been reported.

Objective: The anti-inflammatory activity of Ternstroemia gymnanthera stem bark aqueous extract (TGSBE) was evaluated using LPS-stimulated RAW264.7 macrophages.

Materials and methods: Cytotoxicity was assessed by MTT assay after 24?h with TGSBE (25–200?μg/mL). Further testing used TGSBE at 100 and 200?μg/mL. Griess and ELISA methods after 24?h with TGSBE determined NO and cytokine levels, respectively; then, mRNA levels (iNOS &; cytokines) were analyzed by Quantitative-PCR after 12?h. NF-κB and MAPK were assessed by immunoblotting after TGSBE treatment for 12?h, followed by LPS for 30?min. Immunofluorescence assay was also performed for NF-κB. ROS and MMP, after 12?h with TGSBE, were determined by flow cytometry. The antioxidant potential of TGSBE was analyzed by ABTS assay. The Folin–Ciocalteu method determined the total phenolic content of TGSBE. LPS concentration was 0.5?μg/mL.

Results: TGSBE at 200?μg/mL showed about 96.2% viability while suppressing the production of NO (88.99%), TNFα (24.38%), IL-6 (61.70%) and IL-1β (55.12%) and gene expression by 67.88, 45.24, 65.84, and 70.48%, respectively. TGSBE decreased ROS (79.26%) and improved MMP (48.01%); it inhibited translocation of NF-κB and MAPK activation. Radical scavenging activity was 50% at 402.17?μg/mL (ascorbic acid standard: 88.8?μg/mL). Total phenolic content was 240.9?mg GAE/g.

Discussion and conclusion: TGSBE suppresses the inflammatory response by inhibiting the NF-κB and MAPK cascades exhibiting therapeutic potential to treat inflammatory diseases associated with increased activation of macrophages.     

Multidrug resistance related protein (ABCC1) and its role on nitrite production by the murine macrophage cell line RAW 264.7

Multidrug resistance related protein 1 (MRP1/ABCC1) is an ABC transporter protein related to the extrusion of reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH-conjugates, as well as leukotriene C(4) and cyclopentane prostaglandins. Inhibition of ABCC1 activity impairs lymphocyte activation. The present work studied ABCC1 expression and activity on a murine macrophage cell line, RAW 267.4 and the effects of ABCC1 classical inhibitors, as well as GSH metabolism modulators, on LPS induced activation. Approximately, 75% of resting cells were positive for ABCC1 and the classical ABCC1 reversors (indomethacin, 0.1-2mM; probenecid, 0.1-10mM and MK571, 0.01-1mM) were able to enhance intracellular CFDA accumulation in a concentration-dependent manner, suggesting ABCC1 inhibition. After LPS (100ng/ml) activation 50% of the population was positive for ABCC1, and this protein was still active. In LPS-activated cells, ABCC1 activity was also impaired by BSO (1mM), an inhibitor of GSH synthesis. Conversely, GSH (5mM) reversed the BSO effect. ABCC1 inhibition by indomethacin, probenecid or MK571 decreased LPS induced nitrite production in a concentration-dependent manner, the same result was observed with BSO and again GSH reversed its effect. The ABCC1 reversors were also able to inhibit iNOS expression. In conclusion, LPS modulated the expression and activity of ABCC1 transporters in RAW macrophages and inhibitors of these transporters were capable of inhibiting nitrite production suggesting a role for ABCC1 transporters in the inflammatory process.     

In vitro antiinflammatory activity of kalopanaxsaponin A isolated from Kalopanax pictus in murine macrophage RAW 264.7 cells

In the present study, effects of various hederagenin monodesmosides isolated from the stem bark of Kalopanax pictus Nakai, such as hederagenin, 5-hederin, kalopanaxsaponin A, kalopanaxsaponin 1, and sapindoside C, have been evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) release by the macrophage cell line RAW 264.7. Among the tested monodesmosides, kalopanxsaponin A was the most potent inhibitor of NO production, and it also significantly decreased PGE2 and TNF-alpha release. Consistent with these observations, the expression level of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 enzyme was inhibited by kalopanxsaponin A in a concentration-dependent manner. Thus, this study suggests that kalopanaxsaponin A-mediated inhibition of iNOS, COX-2 expression, and TNF-alpha release may be one of the mechanisms responsible for the anti-inflammatory effects of the stem bark of Kalopanax pictus Nakai.     

Methyl galbanate,a novel inhibitor of nitric oxide production in mouse macrophage RAW264.7 cells

It is well known that inflammation is associated with various neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. An inflammatory mediator, nitric oxide (NO), is produced by inducible NO synthase (iNOS) in microglia and seems to be one of the possible causes of neurodegeneration. Several natural and synthetic compounds which exert anti-inflammatory effects by inhibiting NO production have been reported to date. The aim of this work was to investigate whether any of the 6 terpenoid coumarins (methyl galbanate, galbanic acid, farnesiferol A, badrakemone, umbelliprenin, and aurapten) isolated from Ferula szowitsiana DC. have inhibitory activity against NO production in RAW264.7 mouse macrophage cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Of the 6 terpenoid coumarins tested, methyl galbanate significantly decreased NO production in LPS/IFN-γ-stimulated RAW264.7 cells. In the presence of methyl galbanate, LPS/IFN-γ-induced iNOS mRNA expression was significantly decreased to 52% of the level found with LPS/IFN-γ stimulation alone. Methyl galbanate slightly attenuated COX-2 mRNA expression. Using the RAW264.7-tsAM5NE co-culture system, we showed that methyl galbanate protected neuronally differentiated tsAM5NE cells from NO-induced cell death by inhibiting the production of NO. Our finding suggests that methyl galbanate may be useful for developing a new drug against neurodegenerative diseases.     

In vitro anti-inflammatory activity of 23-hydroxyursolic acid isolated from Cussonia bancoensis in murine macrophage RAW 264.7 cells

In the present study, the effects of various triterpenoids isolated from the stem bark of Cussonia bancoensis, namely, ursolic acid ( 1), 23-hydroxyursolic acid ( 2), 3-O-alpha- L-arabinopyranosyl-23-hydroxyursolic acid (3), and 3-O-beta-D-glucopyranosyl-23-hydroxyursolic acid ( 4) were evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) release by the macrophage cell line RAW 264.7. Of the tested triterpenoids, 23-hydroxyursolic acid ( 2) was found to be the most potent inhibitor of NO production, and also significantly reduced PGE (2) release. Consistent with these observations, the protein and mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 enzymes were inhibited by 23-hydroxyursolic acid ( 2) in a concentration-dependent manner. Furthermore, 23-hydroxyursolic acid ( 2) inhibited the LPS-induced DNA binding activity of nuclear factor- kappaB (NF- kappaB), which was associated with a decrease of p65 protein levels in the nucleus. These results suggest that the 23-hydroxyursolic acid-mediated inhibition of iNOS and COX-2 expression, via blocking NF- kappaB activation, may mechanistically responsible for the anti-inflammatory effects of Cussonia bancoensis stem bark in vitro.     

Participation of various kinases in staurosporine induced apoptosis of RAW 264.7 cells

Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage-like cell line, as determined by DNA fragmentation, the increase of annexin V-stained cells, and the cleavage of poly(ADP-ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub-G(1) cells revealed that the DNA fragmentation occurred in a time- and concentration-dependent manner at 0.021-2.1 microM of staurosporine. Staurosporine induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal-regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 potentiated the staurosporine-induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H-89 potentiated the staurosporine-induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro-31-8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine-induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and PI3K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation.     

Secretory phospholipase A2 induces apoptosis through TNF-alpha and cytochrome c-mediated caspase cascade in murine macrophage RAW 264.7 cells

Phospholipase A2 (PLA2) is an esterase that cleaves the sn-2 ester bond in glycerophospholipids, thereby releasing free fatty acids and lysophospholipids. In addition to the apoptotic activity of cytosolic PLA2 and Ca2+-independent PLA2, recent studies showed that secretory PLA2 (sPLA2) also play a role in apoptosis. However, the details of molecular mechanism have not been fully elucidated. Our data demonstrated that group IB PLA (IB PLA2)-exposed murine macrophage 264.7 cells showed characteristic features of apoptosis such as morphological changes, DNA laddering, staining positive for propidium iodide (PI) as well as Annexin V and activation of caspases and subsequent cleavage of poly (ADP-ribose) polymerase (PARP) in dose- and time-dependent manner. Moreover, IB PLA2 was found to elicit tumor necrosis factor (TNF)-alpha production and release of cytochrome c, suggesting that IB PLA2 exerts its apoptotic activity via the induction of TNF-alpha production and cytochrome c release, which results in triggering the activation of caspase cascade and PARP cleavage.     

Chemical constituents from Belamcanda chinensis and their inhibitory effects on nitric oxide production in RAW 264.7 macrophage cells

Archives of Pharmacal Research - Belamcanda chinensis (L.) DC., which belongs to the family of Iridaceae, has been used as a folk medicine for the treatment of inflammation, asthma, tonsillitis,...     

Anti-inflammatory effect of Lycium Fruit water extract in lipopolysaccharide-stimulated RAW 264.7 macrophage cells

Lycium Fruit has been used as a traditional drug for low back pain and chronic cough in east-Asian countries. However, inhibitory effects of Lycium Fruit water extract (LFWE) on inflammation remain unknown. In this study, we investigated the inhibitory effects of LFWE on pro-inflammatory mediator production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. LFWE inhibited LPS-induced nitric oxide (NO), prostaglandin (PG) E?, tumor necrosis factor (TNF)-α and interleukin (IL)-6 production as well as their synthesizing enzyme inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 gene expression. Furthermore, LFWE inhibited phosphorylations of extracellular signal-regulated kinase (ERK), p38 and c-Jun NH?-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) as well as suppression of IκBα degradation and nuclear translocation of nuclear factor (NF)-κB upon LPS stimulation. In addition, LFWE suppressed NO, PGE?, TNF-α and IL-6 production in LPS-stimulated peritoneal macrophage cells. Taken together, our results suggest that LFWE inhibits the production of various inflammatory mediators via blockade on the MAPKs and NF-κB pathways. This finding first explains the mechanism of anti-inflammatory effect by LFWE in LPS-stimulated macrophage cells.     

Toxic response of HIPCO single-walled carbon nanotubes in mice and RAW264.7 macrophage cells

In this study, we identified the toxic response of pristine single-walled carbon nanotubes (P-SWCNTs) synthesized by HIPCO method in mice and RAW264.7 cells, a murine peritoneal macrophage cell line. P-SWCNT contained a large amount of Fe ion (36 wt%). In the lungs of mice 24 h after intratracheal administration, P-SWCNTs increased the secretion of IL-6 and MCP-1, and the number of total cells, the portion of neutrophils, lymphocytes, and eosinophils, also significantly increased at a 100 μg/mL of concentration. In RAW264.7 cells, cell viability and ATP production decreased in a dose-dependent manner at 24 h after exposure, whereas the generations of ROS and NO were enhanced at all concentrations together with the activation of the MAP kinase pathway. Moreover, the levels of both apoptosis- and autophagy-related proteins and ER stress-related proteins clearly increased, and the concentrations of Fe, Cu, and Zn ions, but not of Mn ions, increased in a dose-dependent manner. TEM images also revealed that P-SWCNTs induced the formation of autophagosome-like vacuoles, the dilatation of the ER, the generation of mitochondrial flocculent densities, and the separation of organelle by disappearance of the cell membrane. Taken together, we suggest that P-SWCNTs cause acute inflammatory response in the lungs of mice, and induce autophagy accompanied with apoptosis through mitochondrial dysfunction and ER stress in RAW264.7 cells. Furthermore, further study is required to elucidate how the physicochemical properties of SWCNTs determine the cell death pathway and an immune response.     

Chlorophyllin suppression of lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells

Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, functions as an anticarcinogen and antioxidant. In the present study, we investigated the effect of CHL on nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Treatment with CHL inhibited nitric oxide production in the LPS-stimulated RAW 264. 7 cells in a dose-related manner. Competitive RT-PCR analysis, using a DNA competitor as an internal standard, demonstrated that the treatment with 1, 10, and 50 microM CHL decreased LPS-induced iNOS mRNA expression in a concentration-dependent manner. Since the expression of the iNOS gene is mainly regulated by NF-kappaB, we then examined the effects of CHL on the NF-kappaB DNA binding activity, using an electrophoretic mobility shift assay. CHL down-regulated the NF-kappaB DNA binding on its cognate recognition site at the concentrations just noted. Employing a transfection and reporter gene expression system with p(NF-kappaB)(3)-chloramphenicol acetyl transferase (CAT), the treatment of CHL produced a dose-dependent inhibition of CAT activity in RAW 264.7 cells. Furthermore, CHL partially restored LPS-decreased IkappaBalpha, an inhibitory protein against NF-kappaB activation, in the cytosolic extract from the LPS-treated cells determined by immunoblot analysis. CHL also protected the hydroxyl radical-induced cytotoxicity in RAW 264.7 cells, indicating its antioxidant effect. These results suggest that CHL suppresses the nitric oxide production and iNOS mRNA expression mediated by the inhibition of NF-kappaB activation, and its action mechanism may be based on its antioxidant effect.     

Inhibition of methanol extract from the aerial parts of Saururus chinensis on lipopolysaccharide-induced nitric oxide and prostagladin E2 production from murine macrophage RAW 264.7 cells

As an attempt to search for bioactive natural products exerting antiinflammatory activity, we have evaluated the effects of the methanol extract from the aerial parts of Saururus chinensis (LOUR.) BAILL (Saururaceae) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release by the macrophage cell line RAW 264.7. Our data indicate that this extract is a potent inhibitor of NO production and it also significantly decreased PGE(2) release. Consistent with these observations, the protein and mRNA expression level of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 was inhibited by MeOH extracts of the aerial part of S. chinensis (SCM) in a dose-dependent manner. Furthermore, SCM inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB), which was associated with decreased p65 protein levels in the nucleus. These results suggest that SCM inhibits LPS-induced iNOS and COX-2 expression by blocking NF-kappaB activation.     

Activation of mitogen-activated protein kinases and AP-1 by polysaccharide isolated from the radix of Platycodon grandiflorum in RAW 264.7 cells

The root of Platycodon grandiflorum has been widely used for the treatment of various diseases in oriental medicine. Our previous study showed that the PG, a polysaccharide isolated from P. grandiflorum, activates macrophages via Toll-like receptor 4 (TLR4). However, the associated biological mechanisms are not fully understood. To elucidate the molecular mechanism responsible for the macrophage activation, we investigated the effect of PG on the activity of mitogen-activated protein kinases (MAPKs) and activator protein-1 (AP-1) in RAW 264.7 cells, a murine macrophage cell line. Treatment of RAW 264.7 cells with PG produced a marked induction of AP-1 DNA binding activity. Moreover, all three MAPKs were activated by PG, and PG-induced activation of MAPKs was abrogated by the treatment of PD98059, curcumin, and SB203580, specific inhibitors of MEK-1/2, stress-activated protein kinases/jun N-terminal kinase (SAPK/JNK), and p38 MAP kianse, respectively. The induction of AP-1 DNA binding activity by PG was also inhibited by these MAPK inhibitors. Moreover, supershift analysis identified that JunB and Fra-1 are major components involved in the PG-mediated induction of AP-1 DNA binding. Additionally, curcumin and SB203580 suppressed PG-induced production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), whereas PD98059 showed an inhibitory effect only on the TNF-alpha production. Taken together, these results suggest that macrophage activation by PG is mediated, at least in part, by MAPKs and AP-1.