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.     

Comparative analysis of the anti-inflammatory activity of Huang-lian extracts in lipopolysaccharide-stimulated RAW264.7 murine macrophage-like cells using oligonucleotide microarrays

In this study, we attempted to determine the anti-inflammatory activity of a medicinal plant huang-lian using gene expression profiles as an index. Huang-line extracts (CEXs) were prepared from seven different plant origins and compared for their chemical composition and biological activity. In order to achieve this, RAW264.7 cells were treated with CEXs in the absence or presence of LPS for 6 h, and the differential gene expression profiles were analyzed using oligonucleotide microarrays. The alkaloid content of CEXs was determined by high performance liquid chromatography analysis. Evaluation of anti-inflammatory activity of CEXs was by measuring a decrease in cytokines and nitric oxide production in LPS-stimulated RAW264.7 cells. Hierarchical clustering analysis revealed that three CEXs from Coptis chinensis formed a cluster separate from the other four CEXs in LPS-stimulated cells, and were the most effective anti-inflammatoryagents. The extract prepared from Picrorrhiza kurrooa neither induced any changes in gene expression profiles nor possessed any anti-inflammatory activity. The extract from Jeffersonia dubia, which exhibited the highest cytotoxicity among the CEXs tested, was most effective in suppressing LPS-induced nitric oxide production but was not able to inhibit proinflammatory cytokine production. The results obtained in this study demonstrate that overall gene expression profiles of the extracts correlated well with their biological activity and that CEXs prepared from plants of diverse origins vary in their biological activity. These data also suggest that gene expression profiles may serve as a good indicator for the pharmacological activities of medicinal plants arising from diverse origins.     

Anti-inflammatory effect of fucoxanthin derivatives isolated from Sargassum siliquastrum in lipopolysaccharide-stimulated RAW 264.7 macrophage

In this study, the anti-inflammatory effect of fucoxanthin (FX) derivatives, which was isolated from Sargassum siliquastrum were evaluated by examining their inhibitory effects on pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. The FX derivatives were isolated from activity-guided chloroform fraction using inhibition of nitric oxide (NO) production and identified as 9′-cis-(6′R) fucoxnathin (FXA), and 13-cis and 13′-cis-(6′R) fucoxanthin complex (FXB) on the basis of a comparison of NMR spectroscopic data. Both FXA and FXB significantly inhibited the NO production and showed slightly reduce the PGE2 production. However, FXB exhibited cytotoxicity at the whole tested concentration, therefore, the results of FXA was only illustrate for further experiments. FXA induced dose-dependent reduction in the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) proteins as well as mRNA expression. In addition, FXA reduced the LPS-stimulated production and mRNA expressions of TNF-α and IL-6 in a dose-dependent manner whereas IL-1β production do not inhibit by addition of FXA. Taken together, these findings indicate that the anti-inflammatory properties of FXA may be due to the inhibition of iNOS/NO pathway which associated with the attenuation of TNF-α and IL-6 formation. Thus FXA may provide a potential therapeutic approach for inflammation related diseases.     

Bioassay guided fractionation and identification of active anti-inflammatory constituent from Delonix elata flowers using RAW 264.7 cells

Abstract

Context: Delonix elata (L.) Gamble (Fabaceae) has been used in the Indian traditional medicine system to treat rheumatism and inflammation.

Aim: To assess the anti-inflammatory effect of Delonix elata flowers and to isolate the active principle.

Materials and methods: The prompt anti-inflammatory constituent was isolated from Delonix elata flower extracts using bioassay guided fractionation in liposaccharide (LPS) stimulated RAW 264.7 macrophage cell line. The anti-inflammatory activity of extracts/fractions/sub-fractions/compounds (10, 25, and 50?µg/ml) was evaluated by estimating the levels of nitric oxide (NO), TNF-α, and IL-1β after 24?h of LPS induction (1?μg/ml). The isolated active compound was subjected to NMR, IR, and UV analyses for structure determination.

Results: In an attempt to search for anti-inflammatory constituents, the active pure principle was isolated and crystallized as a white compound from Delonix elata flowers methanol extract. This active compound (50?µg/ml) decreased the release of inflammatory mediators levels such as NO (0.263?±?0.03?µM), TNFα (160.20?±?17.57?pg/ml), and IL-1β (285.79?±?15.16?pg/ml) significantly (p?<?0.05); when compared to the levels of NO (0.774?±?0.08?µM), TNFα (501.71?±?25.14?pg/ml), and IL-1β (712.68?±?52.25?pg/ml) from LPS-stimulated macrophage cells. The active compound was confirmed as hesperidin with NMR, IR, and UV spectroscopy data. This is the first report of this compound from Delonix elata flowers.

Conclusion: The findings of the study support the traditional use of Delonix elata flowers to treat inflammation.     

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.     

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.     

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.     

Anti-inflammatory activity of aucubin by inhibition of tumor necrosis factor-alpha production in RAW 264.7 cells

To elucidate a possible mechanism for the anti-inflammatory action of iridoid glycosides, the effects of both aucubin (AU) and its hydrolyzed product (H-AU) by beta-glucosidase treatment were studied on the production of TNF-alpha in RAW 264.7 cells. H-AU suppressed the production of both mRNA for TNF-alpha and subsequent TNF-alpha protein in the culture, but AU did not. The production of TNF-alpha protein was inhibited in a dose-dependent manner with an IC (50) of 9.2 microM. In addition, treatment with H-AU blocked both the I-kappa B alpha degradation and the translocation of NF-kappa B from the cytosol fraction to the nuclear fraction (55 % inhibition) in the culture. However, treatment with H-AU did not affect the intracellular level of cAMP formed by forskolin treatment in human monocytes U937 culture, implying that there is no influence on the cAMP level in other cell systems. The present study indicates a possible justification for those medicinal plants containing iridoid glycoside that have been used for the treatment of inflammation.     

Effect of T-2 toxin-injected shrimp muscle extracts on mouse macrophage cells (RAW264.7)

Following intramuscular injections of 0.1?mL, 3?mg?kg^?1?BW^?1(1/10 LD₅₀) T-2 toxin (T-2), the tissue concentration of T-2 in shrimp was quantitatively detected using LC-MS/MS. The biological half-time (t_1/2) of T-2 in blood was 40.47?±?0.24?min. The highest number of intramuscular T-2 shrimp could tolerate when given at blood t_1/2 intervals was 4. The shrimps which were injected 5 T-2 died. The T-2 toxin highest accumulation was 0.471?±?0.012?ng?g^?1?BW^?1. The effect of toxic shrimp muscle subjected to different processing conditions (high pressure, trifluoroacetic acid, acid and alkali digestions, artificial digestive juice [to simulate exposure to gastric and intestinal juices]) on mouse macrophage cells (RAW267.4) were evaluated by the MTT assay. The inhibition ratio of 2% muscle extract on RAW267.4 was 85.70?±?2.63%. The immunocytotoxicity of muscle extracts to RAW264.7 was highest in muscle extracts subjected to physical and chemical digestion (high pressure >?NaOH?> trifluoroacetic acid >?0.02 M HCl?>?0.2 M HCl?>?controls), and also artificial digestion (artificial intestinal juice >?artificial gastric juice >?N type intestinal juice >?N type gastric liquid >?controls). Results showed that high-pressure and artificial intestinal juice were most effective in the release of modified T-2 to free T-2 thus enhancing toxicity. These results can be interpreted as measurement of T-2 in food being of little value because of enhanced toxicity of T-2-contaminated food as they pass through the gastrointestinal tract.     

Participation of mitogen-activated protein kinase in thapsigargin- and TPA-induced histamine production in murine macrophage RAW 264.7 cells

1. Stimulation of the murine macrophage cell line RAW 264.7 with thapsigargin, an endomembrane Ca(2+)-ATPase inhibitor, induced histamine production in a time- and concentration-dependent manner. 2. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), also enhanced histamine production. 3. alpha-Fluoromethylhistidine, a suicide substrate of L-histidine decarboxylase (HDC), suppressed the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production. 4. Both thapsigargin (30 nM) and TPA (30 nM) induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. 5. PD98059, a specific inhibitor of MEK-1 which phosphorylates p44/p42 MAP kinase, strongly suppressed both the thapsigargin (30 nM)- and TPA (30 nM)-induced histamine production, whereas SB203580, a specific inhibitor of p38 MAP kinase, inhibited them only partially. 6. The other MEK-1 inhibitor, U-0126, also inhibited both the thapsigargin- and TPA-induced histamine production in a concentration-dependent manner. 7. Thapsigargin (30 nM) and TPA (30 nM) increased the levels of HDC mRNA at 4 h, but PD98059 suppressed both the thapsigargin- and TPA-induced increases in the HDC mRNA level. 8. These findings indicate that thapsigargin and TPA induce histamine production in RAW 264.7 cells by increasing the level of HDC mRNA, and that both the thapsigargin- and TPA-induced histamine production are regulated largely by p44/p42 MAP kinase and partially by p38 MAP kinase.     

Immunomodulatory activities of Gelidium amansii gel extracts on murine RAW 264.7 macrophages

The objective of this study was to investigate the effects of Gelidium amansii, a red algae cultivated in the northeastern coast of Taiwan, on immune regulatory activities in RAW 264.7 macrophages. G. amansii gel was prepared by boiling and filtering the dried G. amansii algae. Phosphate-buffered saline (PBS) and ethanol extracts of the freeze-dried gel were then used to treat the cells. The results showed that the PBS extracts of G. amansii gel activated the macrophage by increasing cell proliferation and by enhancing the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6; the ethanol extracts did not show such effects. By contrast, neither PBS nor ethanol extracts of G. amansii gel affected NO production in lipopolysaccharide (LPS)-stimulated macrophages, although the ethanol extracts suppressed LPS-stimulated TNF-α, IL-1β, and IL-6 production. In conclusion, the PBS extracts of G. amansii gel activated the macrophage by enhancing the production of immune mediators, whereas the ethanol extracts showed anti-inflammatory activities by suppressing the cytokine production in LPS-stimulated RAW 264.7 macrophages.     

Anti-inflammatory activity of hydroxycinnamic acid derivatives isolated from corn bran in lipopolysaccharide-stimulated Raw 264.7 macrophages

In this study, the effect of the 80% ethanolic extract of corn bran (EECB) on inhibition of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells was investigated. The EECB inhibited LPS-induced NO production and iNOS expression in a dose-dependent manner. Four hydroxycinnamic acid derivatives (HADs), including two free cinnamic acids, p-coumaric acid (CA) and ferulic acid (FA), and their conjugate phenolic amides, p-dicoumaroyl-putrescine (DCP) and diferuloylputrescine (DFP), were found to be present in the EECB by LC–MS analysis, and DFP (378.66 μg/g) was the predominant phenolic compound, followed by DCP (7.83 μg/g) > CA (5.58 μg/g) > FA (1.84 μg/g). The four HADs significantly inhibited NO production and iNOS expression in a dose-dependent manner. Among the four HADs tested, DFP showed the most potent inhibition on NO production and iNOS mRNA and protein expression, followed by DCP > FA ? CA. DFP also exhibited the strongest inhibition on LPS-induced iNOS and NF-κB luciferase activity, which was followed by DCP ? FA (CA) > CA (FA). Thus, these results suggest that phenolic amides in the corn bran may be a potential source of natural anti-inflammatory agents.     

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 &...     

Anti-inflammatory activity of 20(S)-protopanaxadiol: enhanced heme oxygenase 1 expression in RAW 264.7 cells

20( S)-Protopanaxadiol (PPD) is one of the metabolites of ginsenosides from Panax ginseng. In this study, we demonstrate that PPD inhibits the increase in lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression through inactivation of nuclear factor-kappaB by preventing degradation of inhibitory factor-kappaBalpha. PPD also induces heme oxygenase 1 (HO-1) expression in RAW 264.7 cells, at the mRNA and protein levels, in the presence and absence of LPS. This effect is associated with suppression of LPS-induced nitric oxide (NO) production and iNOS expression. The HO-1 inducer hemin is associated with the suppression of LPS-induced NO production in a dose-dependent manner, and the HO-1 inhibitor tin protoporphyrin attenuates the inhibitory activity of PPD on LPS-induced NO production. These results provide evidence for the role of HO-1 in the inhibition of LPS-induced NO production by PPD.     

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.     

Thymus peptides regulate activity of RAW 264.7 macrophage cells: inhibitory analysis and a role of signal cascades

Objectives: The aim of this study was to reveal T-lymphocyte-independent mechanisms of thymic peptide-mediated immunomodulation.

Methods: The effects of two thymic peptides— thymulin and thymopentin were studied in cultured RAW 264.7 macrophages (lipopolysaccharide-stimulated or unstimulated) by measuring cytokine production and signal protein levels.

Results: Both peptides increased proinflammatory cytokine secretion by unstimulated RAW 264.7 macrophages and these effects were blocked by the NF-κB cascade inhibitor, stress-activated protein kinase (SAPK)/JNK cascade inhibitor and, to a lesser extent, Toll-like 4 receptor activity inhibitor. In macrophages stimulated by bacterial lipopolysaccharide, peptides alone did not affect cytokine secretion, but significantly enhanced effects of each of the inhibitors. Thymopentin increased activation of both NF-κB and SAPK/JNK cascades in unstimulated macrophages, while thymulin significantly decreased activation of the SAPK/JNK but not NF-κB cascade in LPS-stimulated macrophages. Thymulin and thymopentin increased production of the heat shock protein HSP72 both in LPS-stimulated and unstimulated cells.

Conclusions: Thymulin and thymopentin are effective anti-inflammatory modulators with direct actions on innate immune cells; the effects involve multiple signal cascades, including NF-κB and SAPK/JNK pathways. Since signaling cascades are now considered to be targets for new therapies, thymic peptides may be prospective modulators of signaling cascades, acting alone or in combination with other agents.     

Lead alters intracellular protein signaling and suppresses pro-inflammatory activation in TLR4 and IFNR-stimulated murine RAW 264.7 cells,in vitro

Lead (Pb) is a persistent environmental pollutant that has a structure and charge similar to many ions, such as calcium, that are essential for normal cellular function. Pb may compete with calcium for protein binding sites and inhibit signaling pathways within the cell affecting many organ systems including the immune system. The aim of the current study was to assess whether the calcium/calmodulin pathway is a principal target of environmentally relevant Pb during pro-inflammatory activation in a RAW 264.7 macrophage cell line. RAW 264.7 cells were cultured with 5 μM Pb(NO₃)₂, LPS, rIFNγ, or LPS+rIFNγ for 12, 24, or 48 hr. Intracellular protein signaling and multiple functional endpoints were investigated to determine Pb-mediated effects on macrophage function. Western blot analysis revealed that Pb initially modulated nuclear localization of NFκB p65 and cytoplasmic phosphorylation of CaMKIV accompanied by increased phosphorylation of STAT1β at 24 hr. Macrophage proliferation was significantly decreased at 12 hr in the presence of Pb, while nitric oxide (NO) was significantly reduced at 12 and 24 hr. Cells cultured with Pb for 12, 24, or 48 hr exhibited altered cytokine levels after specific stimuli activation. Our findings are in agreement with previous reports suggesting that macrophage pro-inflammatory responses are significantly modulated by Pb. Further, Pb-induced phosphorylation of CaMKIV (pCaMKIV), observed in the present study, may be a contributing factor in metal-induced autophagy noted in our previous study with this same cell line.