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.     

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.     

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,...     

Triptolide suppresses lipopolysaccharide-induced activity of toll-like receptor 4 in mouse macrophage cell line RAW 264.7

Toll-like receptor 4 (TLR4) has attracted a great deal of attention in ischemia–reperfusion injury in recent years. Triptolide has potent anti-inflammatory and immunosuppressive effects; however, the mechanism has not been fully delineated. The objective of this study was to evaluate the effects of triptolide on TLR4 expression in the mouse macrophage cell line RAW 264.7. In the first part of this study, the mouse macrophage cell line RAW 264.7 was treated with different concentrations of lipopolysaccharide (LPS) for 6?h to find the concentration of LPS to be used in the second part of this study. In the second part of the study, mouse macrophage cell line RAW 264.7 was pre-treated with triptolide at different concentrations for 1?h, and then exposed to LPS. The expression of the TLR4 and HSP70 mRNA and protein, as well as the levels of tumour necrosis factor (TNF)-α and interleukin (IL)-6, were assessed. In the first part of this study, the expression level of TLR4 mRNA was increased significantly in the macrophage cell line treated with LPS, and reached a plateau at 100?ng/ml. In the second part of this study, the mouse macrophage cell line RAW 264.7 pre-treated with different concentrations of triptolide, showed a dose-dependent inhibition of the levels of TLR4 mRNA and protein, TNF-α and IL-6. Triptolide can suppress LPS-induced TLR4 expression at both the mRNA and protein levels in the mouse macrophage cell line RAW 264.7.     

甘草次酸及其衍生物TY501对小鼠巨噬细胞RAW264.7增殖的影响

目的探究甘草次酸及其衍生物TY501对小鼠巨噬细胞RAW264.7增殖的影响。方法以小鼠巨噬细胞RAW264.7为靶细胞,设置空白组,脂多糖(LPS)1μg/mL处理组(模型组),LPS1μg/mL加80、40、20、10μg/mL药物处理组(受试药物TY501,对照药物泼尼松龙、甘草次酸、吡罗昔康),每组设置4个复孔在给药刺激24h后按照CCK-8试剂盒说明测定各药物对细胞增殖抑制率。结果TY501半数抑制浓度(IC50)为233μg/mL,泼尼松龙IC50为1571μg/mL,甘草次酸IC50为31μg/mL,吡罗昔康IC50为14187μg/mL。结论甘草次酸对RAW264.7巨噬细胞的增殖具有明显的抑制作用,甘草次酸衍生物TY501也可抑制小鼠巨噬细胞RAW264.7的增殖,在同等质量浓度条件下其对小鼠巨噬细胞增殖的抑制程度强于泼尼松龙,弱于甘草次酸,表明甘草次酸及其衍生物TY501对于小鼠巨噬细胞RAW264.7增殖的抑制作用可能是其发挥抗炎作用的机制之一。     

M-CSF促进RAW 264.7细胞MMP-9的分泌及其可能机制

目的探讨巨噬细胞集落刺激因子(MCSF)致动脉粥样硬化的作用是否与其影响基质金属蛋白酶(MMP)表达和活性改变有关及其可能机制。方法应用明胶酶图分析方法观察MCSF和(或)PD98059对体外培养的RAW264.7细胞MMP9活性的影响;Westerblot检测MCSF和(或)PD98059对体外培养的RAW264.7细胞pERK1/2表达的影响。结果MCSF能增强RAW264.7细胞MMP9的活性,并呈一定的剂量依赖性;同时MCSF也呈时间、浓度依赖性促进pERK1/2的表达;PD98059不仅阻断了ERK1/2的磷酸化,而且也降低了MMP9的活性。结论MCSF可诱导RAW264.7细胞MMP9的活性增加,其机制可能与MCSF激活MAPKERK1/2通路有关。     

血红铆钉菇多糖对RAW264.7巨噬细胞免疫调节作用研究

目的 研究血红铆钉菇多糖对RAW264.7小鼠单核巨噬细胞的免疫调节作用.方法 将小鼠的巨噬细胞进行复苏培养,制备细胞悬液,设置空白对照组以及不同质量浓度的血红铆钉菇多糖(CRPS25-Ⅱ)组(1、20、40、80和160μg/ml).采用MTT法测定血红铆钉多糖对小鼠巨噬细胞RAW264.7的细胞毒性;采用RT-PC...     

LC2W胞外多糖对大肠杆菌脂多糖诱导RAW264.7细胞因子表达的影响

<正>乳杆菌对维持肠道内微生态平衡、提高机体抗感染、抗病毒能力,发挥机体局部或全身的防御功能具有重要作用。乳杆菌的生理功能可能与其所分泌的胞外多糖(Exopolysac-charide,EPS)有密切关系[1-2]。药理研究表明,多糖能激活     

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.     

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.     

Effects of prenylated flavonoids and biflavonoids on lipopolysaccharide-induced nitric oxide production from the mouse macrophage cell line RAW 264.7

Certain flavonoid derivatives possess anti-inflammatory activity in vitro and in vivo. Besides their antioxidative properties and effects on the arachidonic acid metabolism including cyclooxygenase/lipoxygenase inhibition, some flavones and flavonols were previously found to show inhibitory activity on nitric oxide production by inducible nitric oxide synthase (iNOS; NOS type 2) through suppression of iNOS induction. As part of our continuing investigations, the effects of unique and minor flavonoids (prenylated flavonoids and biflavonoids) on nitric oxide production from lipopolysaccharide-induced macrophage cell line (RAW 264.7) were evaluated in order to establish their inhibitory activity on NO production and correlate this action with their in vivo anti-inflammatory potential. Among the derivatives tested, prenylated compounds including morusin, kuwanon C, and sanggenon D and biflavonoids such as bilobetin and ginkgetin were found to inhibit NO production from lipopolysaccharide (LPS)-induced RAW 264.7 cells at > 10 microM. Inhibition of nitric oxide production was mediated by suppression of iNOS enzyme induction but not by direct inhibition of iNOS enzyme activity. An exception was echinoisoflavanone that inhibited iNOS enzyme activity (IC50 = 83 microM) and suppressed iNOS enzyme induction as well. While most prenylated derivatives showed cytotoxicity to RAW cells at 10-100 microM, all biflavonoids tested were not cytotoxic. Since nitric oxide (NO) produced by inducible NO synthase (iNOS) plays an important role in inflammatory disorders, inhibition of NO production by these flavonoids may contribute, at least in part, to their anti-inflammatory and immunoregulating potential in vivo.     

The effect of linarin on LPS-induced cytokine production and nitric oxide inhibition in murine macrophages cell line RAW264.7

The herb, Chrysanthemum zawadskii var, latilobum commomly known as Gu-Jul-Cho in Korea, used in traditional medicine to treat pneumonia, bronchitis, cough, common cold, pharyngitis, bladder-related disorders, gastroenteric disorders, and hypertension. Linarin is the main active compound and the biological mechanisms of its activity are unclear. It is believed that effects of this herb may be exerted through the pluripotent effectors of linarin due to its ability to treat a variety of afflictions. In this study, the effects of linarin on the mouse macrophages cell line, RAW 264.7, were investigated. It was found that linarin could activate macrophages by producing cytokines. Monocytes and tissue macrophages produce at least two groups of protein mediators of inflammation, interleukin 1 (IL-1) and the tumor necrosis factor (TNF). Recent studies have shown that TNF and IL-1 modulate the inflammatory function of endothelial cells, leukocytes, and fibroblasts. TNF-alpha production by macrophages treated with linarin occured in a dose dependent manner. However, IL-1 production was largely unaffected by this natural product. This study demonstrated the ability of linarin to activate macrophages both directly and indirectly. Linarin also affect both cytokine production and nitric oxide inhibition, in addition to the expression of some surface molecules. Nitric oxide (NO), derived from L-argin-ine, is produced by two forms(constitutive and inducible) of nitric oxide synthase (NOS). The NO produced in large amounts by inducible NOS is known to be responsible for the vasodilation and hypotension observed in septic shock. Linarin was found to inhibit NO production in the LPS-activated RAW 264.7 cells. Linarin may be a useful candidate as a new drug for treating endotoxemia and the inflammation accompanied by NO overproduction. The linarin-treated total lymphocytes exhibited cytotoxicity in a dose dependent manner between 20 microg/ml and 40 microg/ml. These results suggest that linarin may function through macrophage activation.     

甘草次酸及其衍生物TY501对小鼠巨噬细胞RAW264.7增殖的影响

植物中的活性成分是植物药发挥疗效的物质基础,植物活性成分研究是阐释植物药的生物活性、临床疗效和毒性的必要手段,也是新药发现和创制的可行途径,更是中药药效物质基础研究、质量控制以及配伍合理性及作用规律研究的前提和基础。近些年来,随着国际上     

Effects of solubility of urban air fine and coarse particles on cytotoxic and inflammatory responses in RAW 264.7 macrophage cell line

We investigated the inflammatory and cytotoxic activities of the water-soluble and -insoluble as well as organic-solvent-soluble and -insoluble fractions of urban air fine (PM_2.5-0.2) and coarse (PM_10-2.5) particulate samples. The samples were collected with a high volume cascade impactor (HVCI) in 7-week sampling campaigns of selected seasons in six European cities. Mouse macrophage cells (RAW 264.7) were exposed to the samples for 24 h. The production of nitric oxide (NO) and proinflammatory cytokines (TNFα, IL-6), and cytotoxicity (MTT-test, apoptosis, cell cycle) were measured. The inflammatory and cytotoxic responses in both size ranges were mostly associated with the insoluble particulate fractions. However, both the water- and organic-solvent-soluble particulate fractions induced TNFα production and apoptosis and had some other cytotoxic effects. Soil-derived water-soluble and -insoluble components of the chemical PM_2.5-0.2 mass closure had consistent positive correlations with the responses, while the correlations were negative with the secondary inorganic anions (NO₃⁻, NH₄⁺, non-sea-salt SO₄²⁻) and particulate organic matter (POM). With the PM_10-2.5 samples, sea salt and soluble soil components correlated positively with the induced toxic responses. In this size range, a possible underestimation of the insoluble, soil-related compounds containing Si and Ca, and biological components of POM, increased uncertainties in the evaluation of associations of the mass closure components with the responses. It is concluded that insoluble components of the complex urban air particulate mixture exert the highest inflammatory and cytotoxic activities in the macrophage cell line but, at the same time, they may operate as carriers for active water- and lipid-soluble components.     

梓醇对AGEs刺激巨噬细胞介导肾系膜细胞损伤的影响

目的探讨梓醇对晚期糖基化终末产物(AGEs)刺激巨噬细胞(RAW264.7)介导肾系膜细胞(MMCs)损伤的影响。方法将MMCs接种于Transwell小室,RAW264.7接种于下层孔板共培养,设置空白对照组、模型组、梓醇组(0.1、1.0、10.0μmol·L^-1),设置氨基胍组(10.0μmol·L^-1)作为阳性对照。各组加入药物孵育1 h后,用AGEs(100 mg·L^-1)刺激RAW264.7,继续孵育23 h。采用MTT法检测MMCs增殖;免疫荧光法检测MMCs中COL-Ⅳ的表达; Western blot法检测MMCs中FN、COL-Ⅳ、TGF-β的表达; ELISA法检测RAW264.7上清液中IL-6、IL-12、TNF-α水平。结果梓醇可抑制AGEs刺激巨噬细胞介导系膜细胞的增殖(P<0.05,P <0.01),下调系膜细胞FN、COL-Ⅳ、TGF-β蛋白表达(P <0.05,P <0.01),降低巨噬细胞上清中IL-6、IL-12、TNF-α水平(P <0.05,P &...     

Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships.

Flavonoids affect the inflammatory process of the mammalian system and possess anti-inflammatory as well as immunomodulatory activities in vitro and in vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of various naturally occurring flavonoids on NO production in LPS-activated RAW 264.7 cells were evaluated in vitro. Flavonoids such as apigenin, wogonin, luteolin, tectorigenin, and quercetin inhibited NO production, as measured by nitrite formation at 10-100 microM. The most active among 26 flavonoid derivatives tested were apigenin, wogonin, and luteolin, having IC50 values of 23, 17, and 27 microM, respectively, while AMT, a synthetic selective iNOS inhibitor, had an IC50 value of 0.09 microM. In contrast, flavanones, such as naringenin, and flavonoid glycosides, such as apiin, did not demonstrate significant inhibition up to 100 microM. These results clearly indicated that a C-2,3 double bond might be important, and that the potency of inhibition depended upon the substitution patterns of the flavonoid molecules. The inhibitory activity of flavonoids was not due to direct inhibition of iNOS enzyme activity because they did not reasonably inhibit iNOS activity, as measured by [3H]citrulline formation from [3H]arginine, up to 100 microM. In contrast, wogonin and luteolin concentration-dependently reduced iNOS enzyme expression, when measured by western blotting, at 10-100 microM. All these results clearly demonstrated that certain flavonoids inhibit NO production in lipopolysaccharide-activated RAW 264.7 cells, and their inhibitory activity might be due to reduction of iNOS enzyme expression.     

Exposure of the murine RAW 264.7 macrophage cell line to hydroxyapatite dispersions of various composition and morphology: Assessment of cytotoxicity,activation and stress response

Cellular stress responses leading to the release of cytotoxic mediators are discussed as indicators of the hazard presented by particles, and in particular ultrafine particles or nanomaterials. The present study was designed to investigate effects of the following materials on RAW 264.7 macrophages: three hydroxyapatite materials of various morphologies, i.e., nano-sized with rod-like (HA-NR), plate-like (HA-NP) or needle-shaped (HA-NN) morphology, and an irregularly shaped composite of hydroxyapatite and protein (HPC) in the low micrometer range. Concentrations of 50, 100, 500, 1000 and 5000 μg/ml were applied and cells were analyzed for viability (XTT-test), cytokine production (TNF-α) and induction of nitric oxide (NO) after 18 and 42 h. DQ12 quartz and lipopolysaccharide (LPS) served as positive controls. Up to concentrations of 500 μg/ml, cell viability was not considerably impaired by the test samples at both timepoints. Overall, viability was about one order of magnitude higher than with comparable concentrations of quartz. TNF-α release was induced in all samples after 18 h, with HA-NR showing the most pronounced induction at 100 μg/ml, still clearly below the LPS signal. No or little induction was observed after 42 h. NO production was low after 18 and 42 h. The results support the conclusion that the tested materials exhibit good biocompatibility and are safe to use.     

Proanthocyanidins and related compounds: antileishmanial activity and modulatory effects on nitric oxide and tumor necrosis factor-alpha-release in the murine macrophage-like cell line RAW 264.7

A series of 17 proanthocyanidins and structurally related compounds was tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Most of the polyphenols significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.8-10.6 nM) when compared with the antileishmanial drug Pentostam (EC50 10.6 nM), but all were inactive against the extracellular form (EC50 7.8 to >86 nM). Noteworthy is that all compounds exhibited only moderate or no cytotoxicity against the murine host cells (EC50 7.8 to >56 nM; >25 microg/ml). These polyphenols were further evaluated for immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumor necrosis factor-alpha (TNF) and interferon (IFN)-like properties using several functional assays. The results showed that all compounds induced murine RAW 264.7 cells only moderately to release NO (7-26 microM) relative to the reference stimulus IFN-gamma/LPS (119 microM). The TNF-inducing potential of the polyphenols producing 50% lysis in murine L929 cells ranged from absent to 138 U/ml at the host cell subtoxic concentration of 50 microg/ml. The highest TNF-inducing activity was associated with those flavan-3-ols with galloyl groups (98-127 U/ml). For proanthocyanidins, it appeared that an increase in the flavanyl chain length did not enhance the induction of TNF-release (32-86 U/ml and below detection limits for oligomers and polymers, respectively). With interferon-like activities, phylloflavan and a prodelphinidin polymer showed appreciable cytoprotective effects, as reflected by the inhibition of the cytopathic effect of encephalomyocarditis virus on L929 fibroblast cells (38 and 36 U/ml, respectively). All remaining compounds displayed only negligible or moderate protective effects at subtoxic concentrations up to 25 microg/ml (<5 to 12 U/ml). These results indicate that proanthocyanidins and related compounds have favorable antileishmanial activity in vitro and might be considered as beneficial immunological response modifiers provided there are no bioavailability problems.     

清开灵含药血清对小鼠巨噬细胞Toll样受体及其下游信号转导通路的影响

清开灵具有清热解毒、化痰开窍和镇静安神等作用。现代药理研究表明其主要有解热[1]、抗炎免疫、保肝等作用。聚肌胞(POLY I:C)是TLR3的特异性配体,能诱导NF-κB和MAPK的活化。TLR3介导的信号途径中存在着MyD88非依赖途径[2,3]。细菌脂多糖(Lipopolysaccharide,LPS)是G-菌细胞成分之一,是TLR4的特异性配体。本实验观察了清开灵含药血清对LPS和POLY I:C刺激的RAW264·7细胞分泌炎症因子及其TLR3、TLR4和下游信号转导通路的影响。1材料与方法1.1药品及试剂清开灵口服液,北京中医药大学药厂;LPS、POLY I:C,美国Sigma公…