芦荟大黄素对LPS诱导的RAW264.7细胞NO生成及iNOS表达的影响

目的观察芦荟大黄素(aloe-emodin)对脂多糖(LPS)诱导的RAW264.7细胞一氧化氮(NO)生成及诱生型一氧化氮合酶(iNOS)mRNA表达的作用。方法采用LPS诱导的RAW264.7细胞株建立细胞炎症反应模型。采用Griess试剂法测定NO释放量;采用硝普钠释放NO法测定NO自由基含量的变化;采用反转录聚合酶链反应(RT-PCR)分析iNOS mRNA表达改变。结果芦荟大黄素在0.69～2.50mg·L-1剂量范围内可抑制LPS诱导的RAW264.7细胞NO的释放,并呈剂量和时间依赖关系;芦荟大黄素在0.63～5.00mg·L-1剂量范围内可下调LPS诱导的RAW264.7细胞iNOS mRNA含量;而此范围内芦荟大黄素无直接清除NO自由基作用,不影响iNOS活性。结论芦荟大黄素可明显降低LPS诱导的RAW264.7细胞NO释放,呈时间和剂量依赖关系,此作用并非通过捕捉NO或抑制iNOS活性来实现,而是通过抑制iNOS mRNA表达发挥作用的。     

Inhibitory effects of ivermectin on nitric oxide and prostaglandin E2 production in LPS-stimulated RAW 264.7 macrophages

We have previously shown that ivermectin inhibits LPS-induced production of inflammatory cytokines. In the present study, we investigated the effect of ivermectin on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 macrophages. Ivermectin inhibited LPS-induced NO and PGE₂ production. Consistent with these observations, the protein and mRNA expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes were inhibited by ivermectin in a concentration-dependent manner. Furthermore, the phosphorylation of p38, ERK1/2, and JNK in LPS-stimulated RAW 264.7 cells was suppressed by ivermectin in a dose-dependent manner. These results suggest that ivermectin suppresses NO and PGE₂ production, as well as iNOS and COX-2 expression, by inhibiting phosphorylation of mitogen-activated protein kinases (MAPK) (p38, ERK1/2, and JNK) in LPS-stimulated RAW 264.7 cells.     

Inhibitory effect of CDP, a polysaccharide extracted from the mushroom Collybia dryophila, on nitric oxide synthase expression and nitric oxide production in macrophages

The effect of Collybia dryophila polysaccharide (CDP), a (1-->3), (1-->4)-beta-D-glucan extracted from the mushroom C. dryophila, was evaluated on nitric oxide (NO) production induced by lipopolysaccharide (LPS) and gamma interferon (IFNgamma) or by LPS alone in RAW 264.7 cells. CDP significantly inhibited NO production in a dose-dependent manner without affecting cell viability. The inhibition of NO by CDP was consistent with decreases in both inducible nitric oxide synthase (iNOS) protein and mRNA expression suggesting that CDP exerts its effect by inhibiting iNOS gene expression. In addition, CDP at concentrations of 400 and 800 microg/ml was shown to significantly increase prostaglandin E2 (PGE2) production in LPS- and IFNgamma-induced macrophages when compared to the control.     

Tanshinone IIA from Salvia miltiorrhiza inhibits inducible nitric oxide synthase expression and production of TNF-alpha, IL-1beta and IL-6 in activated RAW 264.7 cells

The inhibitory effects of tanshinone IIA, a diterpene isolated from Salvia miltiorrhiza root, on the production of nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and the expression of inducible nitric oxide synthase (iNOS) were investigated in activated RAW 264.7 cells. This compound markedly inhibited the production of NO, IL-1beta and TNF-alpha, and suppressed the expression of iNOS in a dose-dependent manner. These results suggest that the traditional use of S. miltiorrhiza as an anti-inflammatory herbal medicine may be explained, in part, by the inhibition of NO, IL-1beta, IL-6 and TNF-alpha production, and expression of iNOS.     

NF-kappaB signaling pathway, not IFN-beta/STAT1, is responsible for the selenium suppression of LPS-induced nitric oxide production

Upon stimulation of macrophages with lipopolysaccharide (LPS), Toll-like receptor 4 recognizes LPS, leading to expression of inducible nitric oxide synthase (iNOS), via MyD88/NF-kappaB and TRIF/IFN-beta/STAT pathways. Although selenium (Se) was reported to inhibit nitric oxide (NO) production, it is unclear which signaling pathway is inhibited by Se. Here, we investigated how Se inhibits NO production in LPS-stimulated RAW 264.7 cells. When the cells were pretreated with Se for 1 h followed by LPS treatment, iNOS mRNA expression and subsequent NO production declined significantly in a dose-dependent manner. Se inhibited IkappaBalpha degradation in the cytosol and NF-kappaB binding to its recognition site in the nucleus of the LPS-stimulated cells. Meanwhile, Se did not inhibit IFN-beta mRNA induction or STAT1 phosphorylation in the LPS-stimulated cells. These results suggest that Se down-regulates iNOS gene expression and NO production in the LPS-stimulated macrophages through inhibition of the NF-kappaB activation pathway but not the IFN-beta/STAT1 signaling pathway.     

In vitro inducible nitric oxide synthesis inhibitors from Alismatis Rhizoma.

Bioassay-guided fractionation of an aqueous extract of Alismatis Rhizoma has furnished two inducible nitric oxide synthase (iNOS) inhibitory compounds, alismol (1) and alisol B monoacetate (2), together with an inactive triterpene, alisol C monoacetate (3). Compounds 1 and 2 inhibited nitric oxide (NO) synthesis in a dose-dependent manner in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibitory effects of 1 and 2 on NO synthesis were partly due to suppression of iNOS mRNA expression as determined by Northern blotting.     

Inhibitory effects of a fermented ginseng extract, BST204, on the expression of inducible nitric oxide synthase and nitric oxide production in lipopolysaccharide-activated murine macrophages

In this study, the effects of BST204, a fermented ginseng extract, on the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production are looked into. Crude ginseng extract was incubated with ginsenoside-beta-glucosidase to prepare BST204. BST204, unlike lipopolysaccharide (LPS) and crude ginseng extract, did not affect the level of iNOS protein and NO production in unstimulated RAW 264.7 cells. However, it suppressed the level of iNOS protein and NO production in LPS-stimulated RAW 264.7 cells but did not manifest the same effect on the iNOS mRNA level. An investigation of the activating phosphorylation of p70 S6 kinase and 4E-BP1, which are important for translation, was conducted to investigate the suppressive mechanism of iNOS protein. LPS increased the phosphorylation of p70 S6 kinase, but not 4E-BP1, in a time-dependent manner, and BST204 inhibited it in a dose-dependent manner. The expression of iNOS protein, however, was partially suppressed by rapamycin, an upstream inhibitor of p70 S6 kinase. Therefore, this paper suggests that the suppression of iNOS protein by BST204 was partially correlated with the inhibition of p70 S6 kinase activation.     

Equol inhibits nitric oxide production and inducible nitric oxide synthase gene expression through down-regulating the activation of Akt

In the present study, we report the inhibitory effect of equol on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in murine macrophages. In vivo administration of equol (i.p.) attenuated NO production by peritoneal adherent cells isolated from lipopolysaccharide (LPS)-treated mice. Equol dose-dependently inhibited the LPS-induced production of NO in isolated peritoneal adherent cells and RAW 264.7 cells. The mRNA expression of iNOS was also blocked by equol in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced activation of Akt was suppressed by equol in RAW 264.7 cells while the activation of ERK, SAPK/JNK and p38 MAP kinase was not affected. Equol also blocked LPS-induced NF-kappaB activation. Moreover, the LPS-induced NO production and NF-kappaB activation was inhibited by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase/Akt pathway, in RAW 264.7 cells. These results suggest that equol might inhibit NO production and iNOS gene expression, at least in part, by blocking Akt activation and subsequent down-regulation of NF-kappaB activity.     

Saucerneol D, a naturally occurring sesquilignan, inhibits LPS-induced iNOS expression in RAW264.7 cells by blocking NF-kappaB and MAPK activation

We evaluated the ability of saucerneol D (SD), a tetrahydrofuran-type sesquilignan isolated from Saururus chinensis, to regulate the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells. SD consistently inhibited nitric oxide (NO) production in a dose-dependent manner, with an IC(50) of 2.62 microM, and also blocked LPS-induced iNOS expression. SD potently suppressed both the reporter gene expression and DNA-binding activity of nuclear factor-kappaB (NF-kappaB). In addition, SD inhibited IkappaB-alpha degradation in a concentration- and time-dependent manner. SD also inhibited LPS-induced activation of various mitogen-activated protein kinases, including extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These findings suggest that SD may inhibit LPS-induced iNOS expression by blocking NF-kappaB and MAPK activation.     

Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1

The role of heme oxygenase-1 (HO-1) played in the inhibitory mechanism of flavonoids in lipopolysaccharide (LPS)-induced responses remained unresolved. In the present study, flavonoids, including 3-OH flavone, baicalein, kaempferol, and quercetin, induced HO-1 gene expression at the protein and mRNA levels in the presence or absence of LPS in RAW264.7 macrophages. This effect was associated with suppression of LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein expression. Hemin induced HO-1 protein expression and this was associated with the suppression of LPS-induced NO production and iNOS protein expression in a dose-dependent manner. In addition, an increase in bilirubin production was found in flavonoid- and hemin-treated cells. Hemin, at the doses of 10, 20, and 50 microM, dose-dependently stimulated the flavonoid (50 microM)-induced HO-1 protein expression, and enhanced their inhibitory effects on LPS-induced NO production and iNOS protein expression. Pretreatment of the HO-1 inhibitor, tin protoporphyrin (10 microM), attenuated the inhibitory activities of the indicated flavonoids on LPS-induced NO production. Morphologic analysis showed that 3-OH flavone, baicalein, kaempferol, quercetin, hemin, and tin protoporphyrin did not cause any change in cell viability in the presence or absence of LPS. In contrast, only 3-OH flavone showed a significant inhibition of cell growth using the MTT assay. Transfection of an HO-1 vector in macrophages (HO-1/RAW264.7) resulted in a 3-fold increase in HO-1 protein compared with that the parental RAW264.7 cells. NO production mediated by LPS in HO-1 over-expressed RAW264.7 cells (HO-1/RAW264.7) was significant less than that in parental RAW264.7 cells. 3-OH Flavone, baicalein, kaempferol, and quercetin showed a more significant inhibition on LPS-induced NO production in HO-1/RAW264.7 cells than in parental RAW264.7 cells. These results provide evidence on the role of HO-1 in the inhibition of LPS-induced NO production by flavonoids. A combination of HO-1 inducers (i.e. hemin) and flavonoids might be an effective strategy for the suppression of LPS-induced NO production.     

Protective effect of pine (Pinus morrisonicola Hay.) needle on LDL oxidation and its anti-inflammatory action by modulation of iNOS and COX-2 expression in LPS-stimulated RAW 264.7 macrophages.

The protective effects of pine (Pinus morrisonicola Hay.) needle on low-density lipoprotein (LDL) oxidation and nitric oxide production in macrophages as well as its bioactive compounds were investigated. Of the four solvent extracts, the ethyl acetate extract of pine needle (EAE-PN) exhibited the strongest scavenging action on free radicals. EAE-PN significantly inhibited copper-induced LDL oxidation through prolonging the lag phase of conjugated dienes formation and decreasing the relative electrophoretic mobility of LDL. Lipid accumulation and foam cell formation were significantly reduced when EAE-PN (75 microg/mL) was added to the medium co-incubated with macrophages cells and copper-induced LDL. EAE-PN also markedly inhibited reactive oxygen species production in RAW 264.7 cells stimulated with lipopolysaccharide (LPS). As regards NO production in cells, EAE-PN showed dose-dependent inhibitory effect on nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells. The inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions in LPS-stimulated RAW 264.7 cells were inhibited by EAE-PN. RT-PCR analysis indicated that the iNOS and COX-2 mRNA expression were suppressed by EAE-PN. The major phenolic compounds in EAE-PN were epicatechin and p-coumaric acid by HPLC analysis. The presence of epicatechin and p-coumaric acid in EAE-PN may be partially responsible for the biological action of EAE-PN. Taken together, these results suggest that EAE-PN may provide potential protective effects against LDL oxidation and attenuating excessive NO generation at inflammatory sites; consequently, this may contribute to anti-atherosclerotic and anti-inflammatory effects of EAE-PN.     

Nicotine enhancement of lipopolysaccharide/interferon-gamma-induced cytotoxicity with elevating nitric oxide production

Nicotine has been shown to induce relaxation via nitric oxide (NO) production with activation of endothelium nitric oxide synthase (eNOS), however the effect of nicotine on lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production and inducible NOS (iNOS) gene expression is still undefined. Here, nicotine alone did not affect the NO and PGE2 production in RAW264.7 and primary peritoneal macrophages. Interestingly, nicotine showed the dose-dependent stimulatory effect on LPS (20 ng/ml)/IFN-gamma (10 ng/ml)-induced NO but not PGE2 production in both cells. Although nicotine stimulates NO production in the presence of LPS/IFN-gamma, LPS at the dose of 20 ng/ml, nicotine showed no obvious inductive effect on the expression of iNOS protein by Western blotting in both cells. However, nicotine significantly stimulates LPS (2.5, 5 ng/ml)/IFN-gamma (10 ng/ml)-induced iNOS expression and NO production in RAW264.7 cells. Cytotoxicity assay showed that nicotine enhanced LPS (20 ng/ml) and IFN-gamma (10 ng/ml)-induced cytotoxicity, which was inhibited by an NOS inhibitor N-nitro-L-arginine (NLA) in RAW264.7 cells. Direct and indirect NOS activity assays indicated that nicotine did not affect NOS activity. And, iNOS protein stability was not changed by nicotine after LPS/IFN-gamma treatment. These data indicates that nicotine may potentiate LPS/IFN-gamma-induced cytotoxic effects by enhancing NO production; enhancing iNOS gene expression induced by LPS/IFN-gamma is involved. A cross-talk between inflammation and smoking was proposed in the present study.     

Scopoletin: an inducible nitric oxide synthesis inhibitory active constituent from Artemisia feddei.

Bioassay-guided fractionation of an H2O extract of Artemisia feddei has furnished an inducible nitric oxide synthase (iNOS) inhibitory coumarin, scopoletin (1) and one of the inactive sesquiterpenes, achillin (2). Compound 1 showed inhibition of nitric oxide (NO) synthesis in a dose-dependent manner in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibition of NO synthesis of 1 was due to suppression of iNOS mRNA and iNOS protein, as determined by Northern and Western blotting, respectively.     

Fulvic acid promotes extracellular anti-cancer mediators from RAW 264.7 cells,causing to cancer cell death in vitro

Fulvic acid (FA) is known to promote electrochemical balance as a donor or a receptor possessing many biomedical functions. Nevertheless, the effect of FA on the anti-cancer activity has not been elucidated. In the current study, we first isolated FA from humus and investigated whether FA regulates immune-stimulating functions, such as production of nitric oxide (NO), in RAW 264.7 cells. Our data showed that FA slightly enhances cell viability in a dose-dependent manner and secretion of NO from RAW 264.7 cells. It upregulated the protein and mRNA expression of inducible NO synthesis (iNOS). In addition, FA enhanced the DNA-binding activity of nuclear factor-κB (NF-κB) in RAW 264.7 cells; the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) effectively attenuated the expression of FA-stimulated iNOS, suggesting that FA stimulates NF-κB to promote iNOS and NO production. Finally, FA-stimulated culture media (FA-CM) from RAW 264.7 cells were collected and MCA-102 fibrosarcoma cells were cultured in this media. The FA-CM augmented MCA-102 fibrosarcoma cell apoptosis; however, an NO inhibitor N^G-monomethyl-l-arginine (NMMA) slightly inhibited the FA-CM-mediated MCA-102 fibrosarcoma cell apoptosis, which was accompanied by low levels of NO. In the present study, we found that FA induces the generation of NO and iNOS in RAW 264.7 cells by inducing NF-κB activation; however, NO did not significantly stimulate MCA-102 fibrosarcoma cell apoptosis in the current study. In addition, FA-CM enhanced cell death in various human cancer cells such as Hep3B, LNCaP, and HL60. Taken together, FA most likely stimulates immune-modulating molecules such as NO and induces cancer cell apoptosis.     

Chitooligosaccharides in combination with interferon-gamma increase nitric oxide production via nuclear factor-kappaB activation in murine RAW264.7 macrophages.

A low-molecular weight chitosan (LMWC) with a molecular mass of 20 kDa and a chitooligosaccharide mixture (oligomixture) which is composed of sugars with a degree of polymerization (DP) of 1-6 were isolated from the chitosan hydrolysate. The effects of the chitosan hydrolysate, LMWC and oligomixture on the production of nitric oxide (NO) in RAW 264.7 macrophages were evaluated, and their effects on NF-kappaB activation and the gene expression of inducible NO synthase (iNOS) were further investigated. None of the tested 3 samples of hydrolysate, LMWC and oligomixture alone affected the NO production in RAW 264.7 macrophages. However, treatment of macrophages with a combination of hydrolysate/oligomixture and interferon-gamma (IFN-gamma) significantly induced NO production in a dose-dependent manner, whereas a combination of LMWC and IFN-gamma inhibited NO production. These effects on NO synthesis were evidenced via regulating the iNOS gene expression. Both hydrolysate and oligomixture promoted the migration of NF-kappaB into the nucleus and enhanced its DNA binding activity. MG132, a specific inhibitor of NF-kappaB, eliminated the NO synthesis in IFN-gamma plus hydrolysate/oligomixture-induced RAW264.7 macrophages. The treatment of RAW264.7 macrophages with anti-CD14, anti-TLR4, and anti-CR3 antibodies significantly blocked NO production induced by IFN-gamma plus hydrolysate/oligomixture. These results demonstrated that the oligomixture, which is the main functional component in the chitosan hydrolysate, in combination with IFN-gamma, synergistically induced NF-kappaB activation and NO production through binding with the receptors of CD14, TLR4 and CR3 in RAW264.7 macrophages.     

In vitro inducible nitric oxide synthesis inhibitory active constituents from Fraxinus rhynchophylla

Bioassay-guided fractionation of an H2O extract of the barks of Fraxinus rhynchophylla has furnished two inducible nitric oxide synthase (iNOS) inhibitory compounds, ferulaldehyde (1) and scopoletin (3) together with a coumarin, fraxidin (2). Compounds 1 and 3 showed inhibition of nitric oxide (NO) synthesis in a dose-dependent manner by murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS). The inhibition of NO synthesis of 1 was reflected in the decreased amount of iNOS protein, as determined by Western blotting.     

Gigantol isolated from the whole plants of Cymbidium goeringii inhibits the LPS-induced iNOS and COX-2 expression via NF-kappaB inactivation in RAW 264.7 macrophages cells

During our efforts to find bioactive natural products with anti-inflammatory activity, we isolated gigantol from the whole plants of Cymbidium goeringii (Orchidaceae) by activity-guided chromatographic fractionation. Gigantol was found to have potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E (2) (PGE (2)) production in RAW 264.7 cells. Consistent with these findings, gigantol suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in RAW 264.7 cells in a concentration-dependent manner. Our data also indicate that gigantol is a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) release and influenced the mRNA expression levels of these cytokines in a dose-dependent manner. Furthermore, a reporter gene assay for nuclear factor kappa B (NF-kappaB) and an electromobility shift assay (EMSA) demonstrated that gigantol effectively inhibited the activation of NF-kappaB, which is necessary for the expression of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6. Thus, our studies suggest that gigantol inhibits LPS-induced iNOS and COX-2 expression by blocking NF- kappaB activation.