Effects of compounds from Kaempferia parviflora on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha productions in RAW264.7 macrophage cells

Kaempferia parviflora Wall. ex Baker, is one of the plants in the Zingiberaceae family, locally known in Thai as kra-chai-dam. The rhizome of this plant has been used for treatment of gout, apthous ulcer and abscesses. Since K. parviflora rhizomes have long been used for treatment of inflammation and possessed marked nitric oxide (NO) inhibitory activity (IC(50)=7.8microg/ml), we thus investigated the inhibitory activity of compounds isolated from this plant against lipopolysaccharide (LPS)-induced NO release in RAW264.7 cells. From bioassay-guided fractionation of K. parviflora, seven methoxyflavones were isolated from the hexane fraction and were tested for their anti-inflammatory effects. Among the isolated compounds, compound 5 (5-hydroxy-3,7,3',4'-tetramethoxyflavone) exhibited the highest activity against NO release with an IC(50) value of 16.1microM, followed by 4 (IC(50)=24.5microM) and 3 (IC(50)=30.6microM). Compound 5 was also tested on LPS-induced prostaglandin E(2) (PGE(2)) and tumor necrosis factor-alpha (TNF-alpha) releases from RAW264.7 cells. It was revealed that 5 showed appreciable inhibitory effect on PGE(2) release (IC(50)=16.3microM), but inactive on TNF-alpha (IC(50)>100microM). These findings may support the use in Thai traditional medicine of K. parviflora for treatment of inflammatory-related diseases through the inhibition of NO and PGE(2) releases but partly due to that of TNF-alpha.     

Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells

The rhizomes of Cyperus rotundus (C. rotundus) have been used in oriental traditional medicines for the treatment of stomach and bowel disorders, and inflammatory diseases. Nitric oxide (NO) and superoxide (O2-) are important mediators in the pathogenesis of inflammatory diseases. This study was undertaken to address whether the metanol (MeOH) extract of rhizomes of C. rotundus could modulate NO and O2- productions by murine macrophage cell line, RAW 264.7 cells. The MeOH extract of rhizomes of C. rotundus showed the inhibition of NO production in a dose-dependent manner by RAW 264.7 cells stimulated with interferon-gamma plus lipopolysaccharide. The inhibition of NO production by the extract was due to the suppression of iNOS protein, as well as iNOS mRNA expression, determined by Western and Northern blotting analyses, respectively. In addition, the MeOH extract suppressed the production of O2- by phorbol ester-stimulated RAW 264.7 cells in dose- and time-dependent manners. Collectively, these results suggest that the MeOH extract of rhizomes of C. rotundus could be developed as anti-inflammatory candidate for the treatment of inflammatory diseases mediated by overproduction of NO and O2-.     

Potent cytotoxic lignans from Justicia procumbens and their effects on nitric oxide and tumor necrosis factor-alpha production in mouse macrophages

A new lignan glycoside, 4-O-alpha-L-arabinopyranosyl-(1' "-->2' ')-beta-D-apiofuranosyldiphyllin (2), named procumbenoside A, and 11 known compounds were isolated from the whole plant of Justicia procumbens. The structure of 2 was established by spectral analysis and chemical methods. The known compounds justicidin A (1), diphyllin (3), and tuberculatin (4) showed potent cytotoxic effects against a number of cancer cells in vitro. Compounds 1 and 4 also strongly enhanced tumor-necrosis factor-alpha (TNF-alpha) generation from mouse macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS).     

Inhibitory effects of butanol fraction of the aqueous extract of Forsythia koreana on the nitric oxide production by murine macrophage-like RAW 264.7 cells

The aim of this study was to investigate the effect of butanol fraction of the aqueous extract of Forsythia koreana fruits on the nitric oxide (NO) production and inducible nitric oxide synthesis (iNOS) gene expression in murine macrophage-like RAW 264.7 cells. Butanol fraction alone affected neither NO production nor iNOS gene expression in macrophage-like RAW 264.7 cells. However, the butanol fraction inhibited NO production and iNOS gene expression in RAW 264. 7 cells stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). These findings suggest that inhibition of NO production by this butanol fraction in RAW 264.7 cells stimulated with IFN-gamma plus LPS was due to the suppression of iNOS gene expression.     

Inhibitory phenolic amides on lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells from Beta vulgaris var. cicla seeds

Four phenolic compounds were isolated from Beta vulgaris L. var. cicla L. (Chenopodiaceae). These isolated compounds were identified as N-cis-feruloyl 3-O-methyldopamine (1), N-cis-feruloyl tyramine (2), N-trans-feruloyl 3-O-methyldopamine (3), N-trans-feruloyl tyramine (4), respectively, by spectroscopic analysis. The phenolic amides 1-4 exhibited modest inhibitory activity on LPS-activated nitric oxide production dose-dependently in RAW 264.7 cells.     

Acidic polysaccharide isolated from Phellinus linteus enhances through the up-regulation of nitric oxide and tumor necrosis factor-alpha from peritoneal macrophages

Medicinal mushrooms are increasingly used to treat a wide variety of disease processes. Aqueous extract from the fruiting body or mycelia of Phellinus linteus has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro. However, the mechanisms underlying its tumoricidal effects are poorly understood. The tumoricidal activity of peritoneal macrophages (PM) cultured with acidic polysaccharide (PL) isolated from Phellinus linteus against B16 melanoma cells was enhanced in a dose-dependent manner; growth inhibition increased 4-fold with 200 microg/ml of PL. To further characterize the mechanisms of PL, we investigated the effects of PL on phagocytosis and the release of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and reactive oxygen intermediates (ROI). To investigate the phagocytosis of PM, the uptake of Dextran (Dex)-FITC between PL-untreated and PL-treated PM was compared. We found some augment in phagocytosis of PL-treated PM compared untreated group. PL stimulated a dose-dependent increase in NO and TNF-alpha, but not in ROI production in PM. We suggested that PL has cytotoxicity against Yac-1 cells through the up-regulation of NO and TNF-alpha production. Also, PL enhanced the expression of costimulatory molecules, CD80 and CD86, and major histocompatibility complex (MHC) molecules II in PM. The ability of PL upon the up-regulation of these surface molecules involved in antigen-presenting processes may, by inference, activate T-cell-mediated immunity against malignant cells in vivo. Taken together, these results suggest that PL act as an effective immunomodulator and enhances the anti-tumoral activity of PM.     

Suppressive effects of methoxyflavonoids isolated from Kaempferia parviflora on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been traditionally used in Thailand to treat abscesses, gout, and peptic ulcers.

Aim

Previously, we reported that the chloroform fraction of a Kaempferia parviflora extract had an inhibitory effect on rat paw-edema. In the present study, we isolated the constituents of this fraction and investigated the anti-inflammatory mechanism against nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) and the expression of inducible nitric oxide synthase (iNOS) as well as phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK). In addition, effects of trimethylapigenin (4) on the enzyme activities of protein kinases possibly leading to iNOS expression were examined to clarify the targets.

Materials and methods

The chloroform fraction was isolated using silica gel column chromatography and HPLC. Isolated compounds were tested against NO and TNF-α using RAW264.7 cells. Cytotoxicity and iNOS, p-ERK and p-JNK expression were also examined.

Results

Three active components, 5,7-dimethoxyflavone (2), trimethylapigenin (4), and tetramethylluteolin (5), markedly inhibited the production of NO in lipopolysaccharide (LPS)-activated RAW264.7 cells. Compounds 2, 4, and 5 moderately inhibited production of TNF-α. Compounds 2, 4, and 5 strongly inhibited expression of iNOS mRNA and iNOS protein in a dose-dependent manner, but did not inhibit p-ERK or p-JNK protein expression. The most active compound, 4, did not inhibit the enzyme activity of inhibitor of κB kinases or mitogen-activated protein kinases, but inhibited that of spleen tyrosine kinase (SYK).

Conclusion

The mechanism responsible for the anti-inflammatory activity of methoxyflavonoids from the chloroform fraction of the rhizomes of Kaempferia parviflora is mainly the inhibition of iNOS expression, and the inhibition of SYK by 4 may be involved in the suppression of LPS-induced signaling in macrophages.     

Inhibition of lipopolysaccharide and interferon-gamma-induced expression of inducible nitric oxide synthase and tumor necrosis factor-alpha by Lithospermi radix in mouse peritoneal macrophages

Lithospermi radix (LR, root of Lithospermum erythrorhizon Siebold. et Zuccarinii) has been used to treat various conditions, such as septic shock, eczema and burns. In this study, the effect of LR on lipopolysaccharide (LPS) and recombinant interferon-gamma (rIFN-gamma)-induced production of nitric oxide (NO) and tumor necrosis factor (TNF)-alpha were examined using mouse peritoneal macrophages. At 0.01-1 mg/ml, LR inhibited the LPS/rIFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and TNF-alpha release. To clarify the mechanism involved, the effect of LR on the activation of nuclear factor (NF)-kappaB was examined. The LPS/rIFN-gamma-induced activation of NF-kappaB was almost completely blocked by LR at 1mg/ml without cytotoxicity. These findings demonstrate that the inhibition of the LPS/rIFN-gamma-induced production of NO and TNF-alpha by LR involves the inhibition of NF-kappaB activation.     

Effect of honey bee venom on microglial cells nitric oxide and tumor necrosis factor-alpha production stimulated by LPS

Abnormal activation of microglial cells has been implicated in various neurodegenerative diseases. Results showed that venom (KBV) produced and purified in Korea regulated lipopolysaccharides (LPS)-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in the murine microglia, BV-2 cell line. The production of proinflammatory cytokines, NO, and TNF-alpha was examined by LPS in BV-2 cell. The effect of KBV on the expression of inducible nitric oxide synthase (iNOS) and TNF-alpha was investigated by Western blot and RT-PCR in LPS-stimulated BV-2 cells. KBV suppressed the NO, iNOS, and TNF-alpha production, and decreased the levels of iNOS and TNF-alpha mRNA. These results suggest that KBV has anti-inflammatory properties that inhibit iNOS and TNF-alpha expression. KBV could be useful in inhibiting the production of inflammatory cytokine and NO production in neurodegenerative diseases. Further studies on the pharmacological aspects of the individual components of KBV are recommended.     

两种哒嗪酮衍生物对小鼠腹腔巨噬细胞体外释放肿瘤坏死因子的影响

 目的研究两种哒嗪酮衍生物即：6-(a,a-二苯基乙酰哌嗪基苯基)-4,5-二氢-5-甲基-3-(2H)-哒嗪酮(简称DMDP)和6-(a-苯基乙酰哌嗪基苯基)-4,5-二氢-5-甲基-3-(2H)-哒嗪酮(简称PMDP)及对照药物维拉帕米(verapamil,Ver)对小鼠腹腔巨噬细胞体外释放肿瘤坏死因子(tumor necrosis factor,TNF)的影响。方法体外诱生TNF,以L929细胞为靶细胞的细胞毒结晶紫染色法进行TNF的生物定量测定。结果在10^-6～10^-4mol·L^-1浓度范围内,DMDP的抑制作用呈剂量依赖性;浓度达10^-4mol·L^-1时,可完全抑制TNF的释放。IC50<10-6mol·L^-1。PMDP的抑制作用更强：10^{-8～10-6mol.L-1时,该抑制作用呈剂量依赖性;10-6mol·L-1时,即可完全抑制TNF的释放。IC50<1O-8mol·L-1。结论DMDP,PMDP及Ver对小鼠腹腔巨噬细胞体外释放TNF具有显著的抑制作用。}     

Inhibitory effects of several flavonoids on E-selectin expression on human umbilical vein endothelial cells stimulated by tumor necrosis factor-alpha

The present study investigates the suppressive effect of flavonoids on TNF-alpha-stimulated E-selectin expression on HUVECs by carrying out a comparative examination of the 37 flavonoids. Several flavonoids: fisetin, luteolin and apigenin (subclass of flavone), kaempferol and quercetin (flavonols), eriodictyol (flavanones), genistein (isoflavones) and butein (chalcone) exhibit the inhibitory effects. Considerations to the structure of flavonoids, the C2-C3 double bond of C-ring and 4-oxo functional group are essential for their inhibition activities. These results help to explain the pharmacological efficacy of flavonoids as anti-inflammatory compounds.     

Involvement of heme oxygenase-1 in the anti-inflammatory activity of Chrysanthemum boreale Makino extracts on the expression of inducible nitric oxide synthase in RAW264.7 macrophages

Aim of the study

This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Materials and methods

Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed.

Results

The CB extract at the highest concentration (100 μg/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 μg/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract.

Conclusion

These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.     

Inhibitory effects of Euterpe oleracea Mart. on nitric oxide production and iNOS expression

The palm Euterpe oleracea is a plant of great economic value in Brazil. Although the heart of palm extracted from its trunk is considered a delicacy the world over, its fruits are popular only among Brazilians. In some poor regions of Brazil, there are reports on the popular use of its juice in the treatment of several disorders, mainly those of oxidative onset as cardiovascular ones. Because of its wide utilization; because there are very few scientific studies of this species, and to discover if its use in folk medicine for problems related with oxidation is in fact justifiable, we decided, in this study, to evaluate the effects of Euterpe oleracea flowers, fruits and spikes fractions on: nitric oxide (NO) production, NO scavenger capacity, and on the expression of inducible nitric oxide synthase enzyme, as well. Results showed that the fractions obtained from fruits were the most potent in inhibiting NO production, followed by those from flowers and spikes. Only in high doses, did some fractions reduce cell viability. Reduction on NO production was not due to NO scavenger activity. These results were accompanied by inhibition of iNOS expression. The more pronounced effect was observed in the fractions in which the concentration of cyanidin-3-O-glucoside and cyanidin-3-O-rhamnoside were higher. To sum up, our results indicate that fractions from Euterpe oleracea inhibits NO production by reducing the levels of inducible nitric oxide synthase expression.     

Suppression of lipopolysaccharide-induced inflammatory responses in RAW 264.7 murine macrophages by aqueous extract of Clinopodium vulgare L. (Lamiaceae)

Ethnopharmacological relevance

The wild basil Clinopodium vulgare L. is commonly used in Bulgarian folk medicine for treatment of irritated skin, mastitis- and prostatitis-related swelling, as well as for some disorders accompanied with significant degree of inflammation (e.g. gastric ulcers, diabetes, and cancer).

Aim of study

To determine the effect of aqueous extract of Clinopodium vulgare L. on LPS-induced inflammatory responses of murine RAW 264.7 macrophages.

Materials and methods

Cell cytotoxicity was evaluated by MTT assay. Protein expression levels were monitored by Western blot analysis. Production of NO and PGE₂ was measured by the Griess colorimetric method and enzyme immunoassay, respectively. Activation of MMP-9 was visualized by gelatin zymography. Cytokine levels were determined by BioPlex assay. Intracellular ROS and free radical scavenging potential were measured by DCFH-DA and DPPH method, respectively. Xanthine oxidase activity was evaluated spectrophotometrically.

Results

The extract suppresses NF-κB activation by preventing Iκ-B phosphorylation and inhibits the phosphorylation of p38 and SAPK/JNK MAPKs. It down-regulates iNOS expression which manifests as a drastic decrease of NO production, inhibits MMP-9 activation, but does not affect COX-2 protein levels and reduces only slightly the released PGE₂. Secretion of IL-1β and Il-10 is greatly reduced, whereas suppression of TNF-α and GM-CSF production is less dramatic. The extract has strong free radical scavenging properties and exerts inhibitory effect on xanthine oxidase activity, which lowers the levels of intracellular ROS.

Conclusion

The study provides evidence for the anti-inflammatory potential of Clinopodium vulgare L. aqueous extract.     

The methanol extract of Spiraea prunifolia var. simpliciflora root inhibits the generation of nitric oxide and superoxide in RAW 264.7 cells

It is well established that nitric oxide (NO) and superoxide radicals play pivotal roles in the pathogenesis of inflammatory diseases and fever. This study is undertaken to address whether the methanol extract of Spiraea prunifolia var. simpliciflora root, a traditional medicine as an antipyretic, modulates the generation of NO and superoxide in IFN-gamma primed or polymyristic acetate (PMA) stimulated RAW 264.7 cells, respectively. The generation of NO as well as the expression of inducible nitric oxide synthase (iNOS) protein from IFN-gamma primed RAW 264.7 cells is markedly decreased by the methanol extract in a dose dependent manner. However, the methanol extract does not affect the viability of RAW 264.7 cells, as assessed by methylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay. In addition, the methanol extract suppresses the generation of superoxide in PMA-stimulated RAW 264.7 cells in a dose and a time dependent manner. Taken together, anti-pyretic effects of Spiraea prunifolia var. simpliciflora root extract could result from direct suppression of NO and decreased superoxide generation.     

Production of nitric oxide in mouse peritoneal macrophages after priming with interferon-gamma by the stem of Sinomenium acutum.

The present study demonstrates that the aqueous extract of Sinomenium acutum stem (SSAE) produces nitric oxide (NO) upon treatment with recombinant interferon gamma (rIFN-gamma) in mouse peritoneal macrophages. Apparently SSAE has no effect on NO production by itself. This production is dependent on L-arginine and can be inhibited by the L-arginine analogue N(G)-monomethyl-L-arginine. The increased production of NO from rIFN-gamma plus SSAE-stimulated cells was decreased by the treatment of protein kinase C inhibitor. Tumor necrosis factor-alpha (TNF-alpha) has been shown to stimulate the oxidative metabolism of L-arginine to produce NO. Mouse peritoneal macrophages secrete high levels of TNF-alpha after incubation with rIFN-gamma plus SSAE. In addition, SSAE-induced NO production is progressively inhibited by anti-murine TNF-alpha neutralizing antibody. These results show that the capacity of SSAE to increase NO production from rIFN-gamma-primed mouse peritoneal macrophages is the result of SSAE-induced TNF-alpha secretion.     

槲皮素对LPS诱导小鼠RAW264.7细胞炎症的保护作用

目的研究槲皮素对脂多糖(LPS)诱导小鼠RAW264.7细胞炎症的保护作用。方法 CCK-8法检测细胞活力,Griess法检测NO释放,RT-PCR法检测TNF-α、IL-6、IL-1β、iNOS、COX-2、MCP-1、TLR2、TLR4、MyD88 mRNA表达,Western blot法检测iNOS、COX-2、TLR4、IκBα、p-IκBα、p-NF-κB p65蛋白表达。结果不同浓度(0～50μmol/L)槲皮素对细胞活力无明显影响(P0.05)。与LPS组比较,槲皮素组(25、50μmol/L)显著抑制NO释放(P0.05);槲皮素组(5、15、25μmol/L)显著降低TNF-α、IL-6、IL-1β、iNOS、COX-2、MCP-1、TLR4、MyD88 mRNA表达和iNOS、COX-2蛋白表达(P0.05,P0.01),并呈浓度依赖性;槲皮素组(25μmol/L)显著下调TLR4、p-IκBα、p-NF-κB p65蛋白表达(P0.05),显著上调IκBα蛋白表达(P0.05)。结论槲皮素可抑制LPS诱导小鼠RAW264.7细胞炎症,其机制可能与调节TLR4/NF-κB信号通路有关。     

喜炎平注射液对巨噬细胞分泌炎性因子的影响

目的：研究喜炎平注射液对LPS诱导的小鼠单核巨噬细释放炎性因子的抑制作用。方法：用LPS刺激体外培养的小鼠单核巨噬细胞RAW264．7释放TNF—α、IL-6、NO等前炎性因子，检测不同浓度的药物对巨噬细胞释放以上炎性细胞因子的抑制作用。通过ELISA的方法检测TNF—α、IL-6的含量，采用Griess法检测培养液上清中NO的含量，以MTT法评价细胞毒性。结果：喜炎平注射液在3—200μg／ml的浓度范围内可明显抑制LPS诱导的巨噬细胞RAW264．7释放炎性因子TNF—α、IL-6及NO，并呈现良好的剂量依赖关系。结论：喜炎平能明显抑制巨噬细胞释放炎性细胞因子TNF—α、IL-6和NO。