LPS-induced Nitric Oxide Production In RAW Research Articles

Salvimulticanol from Salvia multicaulis suppresses LPS-induced inflammation in RAW264.7 macrophages: in vitro and in silico studies.

Sustained inflammatory responses can badly affect several vital organs and lead to chronic inflammation-related disorders, such as atherosclerosis, pneumonia, rheumatoid arthritis, obesity, diabetes, Alzheimer's disease, and cancers. Salvia multicaulis is one of the widely distributed plants that contains several biologically active phytochemicals and diterpenoids with anti-inflammatory effects. Therefore, finding alternative and safer natural plant-extracted compounds with good curative anti-inflammatory efficiencies is an urgent need for the clinical treatment of inflammation-related diseases. In the current study, S. multicaulis Vahl was used to extract and isolate two compounds identified as salvimulticanol and candesalvone B methyl ester to examine their effects against inflammation in murine macrophage RAW264.7 cells that were induced by lipopolysaccharide (LPS). Accordingly, after culturing RAW264.7 cells and induction of inflammation by LPS (100ng/ml), cells were exposed to different concentrations (9, 18, 37.5, 75, and 150µM) of each compound. Then, Griess assay for detection of nitric oxide (NO) levels and western blotting for the determination of inducible nitric oxide synthase (iNOS) expression were performed. Molecular docking and molecular dynamics (MD) simulation studies were employed to investigate the anti-inflammatory mechanism. Our obtained results validated that the level of NO was significantly decreased in the macrophage cell suspensions as a response to salvimulticanol treatment in a dose-dependent manner (IC50: 25.1 ± 1.2µM) as compared to the methyl ester of candesalvone B which exerted a weaker inhibition (IC50: 69.2 ± 3.0µM). This decline in NO percentage was comparable with a down-regulation of iNOS expression by western blotting. Salvimulticanol strongly interacted with both the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex and the inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKβ) to disrupt their inflammatory activation due to the significant hydrogen bonds and effective interactions with amino acid residues present in the target proteins' active sites. S.multicaulis is a rich natural source of the aromatic abietane diterpenoid, salvimulticanol, which exerted a strong anti-inflammatory effect through targeting iNOS and diminishing NO production in LPS-induced RAW264.7 cells in a mechanism that is dependent on the inhibition of TLR4-MD-2 and IKKβ as activators of the classical NF-κB-mediated inflammatory pathway.

ANTIOXIDANT AND INHIBITORY EFFECT OF SELECTED GHANAIAN VEGETABLES ON NITRIC OXIDE EXPRESSION IN LIPOPOLYSACCHARIDE-INDUCED MACROPHAGE CELLS

Objective: Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation. Inhibitors of NO may be useful candidates for the treatment of inflammatory diseases. The study aimed to determine the antioxidant and inhibitory effect of commonly used Ghanaian vegetables, namely Corchorus olitorius (CO), Solanum melongena (SM), Solanum torvum (ST), Xanthosoma sagittifolia (XS) and Abelmoschus esculentus (AE) on NO expression in a Lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cell line. Methods: The cytotoxic effects of the vegetables on the cell line were determined using a tetrazolium-based colorimetric assay. The inflammatory activity was determined by measuring the inhibition of NO production in LPS-induced RAW 264.7 macrophage cells. Total antioxidant activity, total phenolic, flavonoid, and reduced glutathione contents were evaluated using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay, Folin-Ciocalteu, aluminium chloride, and O-Phthalaldehyde methods, respectively. Results: Our results showed that CO and ST significantly inhibited NO production in a concentration-dependent manner with good cell viability. Solanum torvum also exhibited strong antioxidant activity (IC50= 0.466±0.23 mg/ml) with total phenolic content of 230.73±1.84 mg/g GAE, while CO showed high flavonoid content (291.45±2.14 mg/g QUE). Abelmoschus esculentus recorded the highest glutathione content (58.6 µg/g GSH. Saponins, alkaloids, tannins, terpenoids, and cardiac glycosides were present in all the samples except SM and AE, which lacked terpenoids. Conclusion: These findings suggest that CO and ST possess anti-inflammatory and antioxidant activities that could be explored as potential therapeutic remedies for inflammatory disorders.

Compounds isolated from the pericarp of Zanthoxylum bungeanum and inhibitory activity against LPS-induced NO production in RAW264.7

Seventeen compounds were isolated and identified from the ethyl acetate part of Zanthoxylum bungeanum Maxim., including one new compound 18-acetyloxyneocryptotanshinone (1) and 16 known compounds (2–17). Their structures were elucidated by extensive spectroscopy. The absolute configuration of 1 was confirmed by electronic circular dichroism (ECD). All compounds were evaluated for the inhibition of LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, of which 1 and 10 exhibited the most significant inhibitory effect, with IC50 of 17.29 and 10.27 μM, respectively.

Essential Oil from Glossogyne tenuifolia Inhibits Lipopolysaccharide-Induced Inflammation-Associated Genes in Macro-Phage Cells via Suppression of NF-κB Signaling Pathway.

Glossogyne tenuifolia Cassini (Hsiang-Ju in Chinese) is a perennial herb native to Taiwan. It was used in traditional Chinese medicine (TCM) as an antipyretic, anti-inflammatory, and hepatoprotective agent. Recent studies have shown that extracts of G. tenuifolia possess various bioactivities, including anti-oxidant, anti-inflammatory, immunomodulation, and anti-cancer properties. However, the pharmacological activities of G. tenuifolia essential oils have not been studied. In this study, we extracted essential oil from air-dried G. tenuifolia plants, then investigated the anti-inflammatory potential of G. tenuifolia essential oil (GTEO) on lipopolysaccharide (LPS)-induced inflammation in murine macrophage cells (RAW 264.7) in vitro. Treatment with GTEO (25, 50, and 100 μg/mL) significantly as well as dose-dependently inhibited LPS-induced pro-inflammatory molecules, such as nitric oxide (NO) and prostaglandin E2 (PGE2) production, without causing cytotoxicity. Q-PCR and immunoblotting analysis revealed that the inhibition of NO and PGE2 was caused by downregulation of their corresponding mediator genes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), respectively. Immunofluorescence and luciferase reporter assays revealed that the inhibition of iNOS and COX-2 genes by GTEO was associated with the suppression of nuclear export and transcriptional activation of the redox-sensitive transcription factor, nuclear factor -κB (NF-κB). In addition, GTEO treatment significantly inhibited phosphorylation and proteosomal degradation of the inhibitor of NF-κB (I-κBα), an endogenous repressor of NF-κB. Moreover, treatment with GTEO significantly blocked the LPS-mediated activation of inhibitory κB kinase α (IKKα), an upstream kinase of the I-κBα. Furthermore, p-cymene, β-myrcene, β-cedrene, cis-β-ocimene, α-pinene, and D-limonene were represented as major components of GTEO. We found that treatment with p-cymene, α-pinene, and D-limonene were significantly inhibiting LPS-induced NO production in RAW 264.7 cells. Taken together, these results strongly suggest that GTEO inhibits inflammation through the downregulation of NF-κB-mediated inflammatory genes and pro-inflammatory molecules in macrophage cells.

Semi-Synthesis of Flavonoid Glycosides and Their Anti-Inflammatory and Antitumor Activities towards Triple Negative Breast Cancer.

Flavonoid glycosides are known to possess diverse bioactivities including antitumor and anti-inflammatory properties. Hesperetin is abundant in nature and can be used to synthesize bioactive flavonoids. This has the advantages of low cost, short synthetic steps, simple operation, and good yields. In this study, we aimed to synthesize bioactive flavonoids and flavonoid glycosides from hesperetin and evaluate the antitumor and anti-inflammatory activities of these compounds. A series of flavonoids and their derivatives were synthesized by methoxylation, oxidative dehydrogenation, benzylation, debenzylation, and deacetylation as well as using a modified peroxyacetone method and a glycoside condensation reaction. Their anti-inflammatory activities were evaluated for their inhibitory effects on nitric oxide (NO), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) production in LPS-induced RAW264.7 mouse macrophages. Their structures were characterized by HRMS, 1 H-NMR, and 13 C-NMR, and their cytotoxicity on the human triple-negative breast cancer cell (TNBC) line, SUM 149, was tested by using the MST assay. Most of the compounds markedly reduced NO production in LPS-stimulated murine macrophages at the tested concentrations in a dose-dependent manner. Among these, compounds 1, 7, 9, and 17 showed significant anti-inflammatory activities against NO production in LPS-induced RAW264.7 mouse macrophages. In addition, they could also reduce the release of TNF-α and IL-6 in a concentration-dependent manner. Most of the tested compounds showed remarkable anti-human TNBC activities. Compounds 1b-1m, 1, and 3 showed a certain degree of growth inhibition effect on the human TNBC cell lines and their IC50 values were all below 16.61 μM. In addition, compound 1l was the most cytotoxic with IC50 values of 1.38±0.31 μM, while the other compounds were inactive with inhibition rates <50 % at the highest concentration tested (20 μM). A novel series of flavonoids were synthesized from the natural flavonoid, hesperetin, including 17 new compounds. Screening tests indicated that most of these compounds reduced NO production in LPS-stimulated murine macrophages at concentrations of 15 to 60 μM, and the inhibition generally increased in a dose-dependent manner. Some compounds showed different degrees of cytotoxicity on the human TBNC cell lines, SUM 149.

Glycoside constituents from Miliusa sinensis leaves and their anti-inflammatory and acetylcholine protective effects

Repeated column chromatography resulted in the isolation of two new glycosides, miliusides A–B (1 and 7), along with six known metabolites (2–6, and 8) from the leaves of Miliusa sinensis Finet and Gagnep. The structures of the purified phytochemicals were elucidated by interpreting their spectroscopic data (NMR, HRMS), as well as comparison with the previous literature. The biological evaluation of acetylcholinesterase (AChE) inhibitory effects and anti-inflammatory activity by measuring nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophage cells, were also conducted. Among them, compounds 5 and 7 exhibited significant AChE inhibitory activities (IC50 = 53.36 ± 4.20 and 88.50 ± 8.79 µM, respectively), compared with the positive control (Galanthamine, IC50 = 1.65 ± 0.15 µM). Only the MeOH extract showed suppression effects on NO production in LPS-induced RAW264.7 cells (IC50 = 38.18 ± 3.25 μg/mL) comparable to that of the positive control, l-NMMA (IC50 = 2.21 ± 0.56 μg/mL).

A new phenylethyl glycoside and a new dibenzocyclooctadiene lignan from the leaves of Kadsura coccinea (Lem.) A. C. Smith

• Two new and seven known compounds were isolated from K. coccinea (Lem.) A. C. Smith leaves. • Chemical structures were established by spectroscopic, MS data, or analytical HPLC experiment. • Isolates were evaluated for their inhibition of NO production in LPS-induced RAW264.7 cells. A new phenylethyl glycoside ( 1 ), ( S )-1-phenylethyl-6- α -L-arabinopyranosyl- β -D-glucopyranoside), and a new dibenzocyclooctadiene lignan ( 7 ), Kasuracin A, together with seven known compounds, were isolated and identified from the leaves of Kadsura coccinea . Their structures were determined by analysis of multinuclear and multidimensional nuclear magnetic resonance (NMR), circular dichroism (CD), mass spectrometry, hydrolysis analysis, or comparison to those reported in the literature. Then, all the isolates 1 ‒ 9 were evaluated for their inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Among them, compounds 2 and 3 were isolated for the first time from this plant and compounds 3 , 7 , 8 , and 9 significantly inhibited NO production with IC 50 values of 36.4, 10.2, 12.3, and 21.7 μM, respectively.

Polyacetylene glucosides from the florets of Carthamus tinctorius and their anti-inflammatory activity

Five previously undescribed polyacetylene glucosides, namely, four C10- and one C14-acetylenes, together with three known analogues, were isolated from the florets of Carthamus tinctorius L. The structures of these novel compounds were elucidated to be (5R)-5-acetoxy-8,10,12-tetradecatriyne-1-O-β-D-glucopyranoside, (2Z)-decaene-4,6,8-triyne-1-O-β-D-glucopyranoside, (8Z)-1-[(3-O-β-D-glucosyl)-isovaleroyloxy]-8-decaene-4,6-diyne, (8Z)-decaene-1-isovaleroyloxy-4,6-diyne-10-O-β-D-glucopyranoside, and (2E,8E)-decadiene-4,6-diyne-1-O-β-D-glucopyranoside via spectroscopic and chemical methods. All of the isolated compounds were tested for cytotoxicity against cancer cell lines, antibacterial activity, and inhibitory effects on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production. The results showed that (5R)-5-acetoxy-8,10,12-tetradecatriyne-1-O-β-D-glucopyranoside significantly inhibited LPS-induced NO production in RAW264.7 cells in a dose-dependent manner.

Anti-inflammatory effect of neo-lignan isoamericanin A via suppression of NF-κB in liposaccharide-stimulated RAW 264.7 cells

Purpose: To investigate the potential anti-inflammatory effects of the seeds of Opuntina humifusa and its active constituents.Methods: The extract of O. humifusa seeds was tested for the inhibition of nitric oxide (NO) production in liposaccharide (LPS)-stimulated RAW 264.7 cells using Griess reagent. The active constituents were isolated using bioassay-guided isolation methods. The effects of the active constituent on NO, proinflammatory cytokines, nuclear factor-kappa B (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot analysis.Results: The seed extract of O. humifusa significantly attenuated LPS-induced NO production in RAW 264.7 cells (p &lt; 0.05). Bioassay-guided fractionation resulted in the isolation of isoamericanin A as an active constituent. Isoamericanin A reduced LPS-induced production of NO, iNOS, and proinflammatory cytokines (TNF-α and IL-6) in a concentration-dependent manner (p &lt; 0.05). Furthermore, the effect was accompanied by decreased translocation of NF-κB from the cytosol to the nucleus and the decreased phosphorylation of IκB in the cytosol induced by LPS (p &lt; 0.05).Conclusion: The seed extract of O. humifusa and its active constituent, isoamericanin A, have antiinflammatory effects in LPS-stimulated RAW 264.7 cells, suggesting that they have potentials as antiinflammatory agents.&#x0D; Keywords: Opuntia humifusa seeds, Isoamericanin A, Nitric oxide, RAW 264.7 cells, NF-kappa B

Lactobacillus paraplantarum THG-G10 as a potential anti-acne agent with anti-bacterial and anti-inflammatory activities

Cutibacterium acnes (formerly Propionibacterium acnes) is the main bacterium targeted for the prevention and medical treatment of acne vulgaris. Lactic acid bacteria (LAB) are a group of microorganisms classified by their ability to produce lactic acid through fermentation. Although the activities of LAB have been studied, their potential anti-acne effects are not well known. Here, Lactobacillus paraplantarum THG-G10, which has anti-bacterial activity against C. acnes, was isolated from traditional Kimchi in Republic of Korea. The anti-acne effects of dried cell-free supernatant of L. paraplantarum THG-G10 (DC-G10) were evaluated by determining its anti-microbial and anti-inflammatory activities. Anti-microbial activity was examined by a broth dilution assay: 25 mg/ml of DC-G10 inhibited the growth of C. acnes KCTC 5012 and KACC 1194; salicylic acid and benzoyl peroxide for acne treatment inhibited the growth of C. acnes KCTC 5012 and KACC 11946 at concentrations of 1.25 and 7.5 mg/ml, respectively; and tea tree oil inhibited the growth of C. acnes KCTC 5012 but not the growth of C. acnes KACC 11946 at 50 mg/ml. Anti-inflammatory activity was evaluated by a nitric oxide (NO) assay: only DC-G10 and ascorbic acid reduced LPS-induced NO production in RAW 264.7 cells in a dose-dependent manner. In addition, the toxicities of erythromycin, salicylic acid, benzoyl peroxide, tea tree oil, and DC-G10 were examined in HaCaT cells and normal human dermal fibroblasts (NHDFs). In these cells, the cytotoxic effects of DC-G10 were weaker than the effects of erythromycin, benzoyl peroxide, and ascorbic acid. Furthermore, scanning electron microscopy revealed that DC-G10 induces deleterious morphological changes in the bacterial cell membrane. These results demonstrate that DC-G10 may be an effective and safe treatment for acne vulgaris.

Anti-inflammatory xanthone derivatives from Garcinia delpyana

Two new xanthones delpyxanthone A (1) and delpyxanthone B (3), together with four known ones, gerontoxanthone I (2), α-mangostin (4), cowanin (5) and cowanol (6) were isolated from the stem bark of Garcinia delpyana. The chemical structures of 1–6 were established mainly using nuclear magnetic resonance (NMR) and mass spectrometry (MS). The anti-inflammatory activity of the isolated compounds was evaluated against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells in vitro. Compounds 1–4 showed significant inhibitory activity against the LPS-induced NO production in RAW264.7 cells with IC50 values ranging from 14.5 to 28.2 μM, but the others were inactive. The results suggested that G. delpyana and its constituents might be potential anti-inflammatory agents on RAW 264.7 cells.

Anti-arthritic and anti-inflammatory effects of (−)-Epicatechin-3-O-β-d-allopyranoside, a constituent of Davallia formosana

Background(−)-Epicatechin-3-O-β-d-allopyranoside (ECAP) is isolated from the popular Chinese herbal medicine Davallia formosana, which has been used to treat bone diseases including bone fracture, arthritis, and osteoporosis. PurposeTo investigate the antiarthritic and the anti-inflammatory effect of ECAP on a mouse model of collagen-induced arthritis (CIA) and in vitro. MethodsMale DBA/1 J mice were immunized by administering an intradermal injection of 100 µg of type II collagen in Freund's complete adjuvant. The control groups (vehicle) and ECAP were administered orally at doses of 1 ml/kg (H2O), 50 and 100 mg/ml/kg once a day from Day 22 to Day 42 after primary immunization. Paw swelling, arthritis severity score, and histological changes were examined. Enzyme-linked immunosorbent assay was used to measure the levels of cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-10, IL-17, IL-4, and interferon-γ (IFN-γ), in splenocytes. Furthermore, the anti-inflammatory activities of ECAP were investigated in vitro by measuring nitric oxide (NO) levels in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. ResultsIn the CIA model, the oral administration of ECAP ameliorated paw edema and reduced the arthritis severity score and disease incidence. Histopathological examination demonstrated that ECAP treatment effectively protected the bone and cartilage of knee joints from erosion, lesion formation, and deformation compared with the vehicle treatment. ECAP also reduced IL-1β and MMP-9 expression in inflamed joints. Compared with the vehicle-treated mice with CIA, the reduced severity of the disease in ECAP-treated mice was associated with decreased levels of TNF-α and IL-17 and increased levels of IL-10 and IL-4 in the supernatants of splenocyte cultures. Flow cytometry analysis demonstrated that ECAP increased the population of CD4+CD25+ regulatory T cells, thereby inhibiting the B cell population. Anticollagen IgG1 and IgG2a levels decreased in the serum of ECAP-treated mice. ECAP suppressed LPS-induced NO production in RAW264.7 macrophages. ConclusionThe administration of ECAP effectively suppressed inflammation and inflammatory pain and adjuvant-induced arthritis, indicating its therapeutic potential in the treatment of rheumatoid arthritis.

Ginsenoyne C, a polyacetylene isolated from Panax Ginseng inhibit inflammatory mediators via regulating extracellular regulated kinases signaling

Background: Panax ginseng has been used as a traditional medicine for various pathological conditions. Ginsenoyne C (GSC) is a polyacetylene, minor constituent. Objective: The objective of the present study was to determine the anti-inflammatory activities of GSC for the protection of inflammation. Materials and Methods: The effects of GSC on cell viability and nitric oxide (NO) production in lipopolysaccharide (LPS)–induced RAW 264.7 cells were investigated through MTT assay and the Griess reaction, respectively. The levels of inflammatory cytokines such as interleukin (IL-1β) and IL-6 and inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and pERK were measured using enzyme-linked immunosorbent assay and Western blot analysis, respectively. Results: GSC suppressed NO production in LPS-induced RAW 264.7 cells. GSC suppressed the production of inflammatory mediators through downregulating phosphorylation of extracellular regulated kinases signaling in LPS-induced RAW 264.7. Conclusion: GSC has a potential therapeutic agent for protection of inflammation. Abbreviation used: CCK: Cell counting kit; COX-2: Cyclooxygenase-2; ELISA: Enzyme linked-immunosorbent assay; ERK: Extracellular regulated kinases; FBS: Fetal bovine serum; FEG: Fermented ginseng extract; GSC: Ginsenoyne C; IL: Interleukin; iNOS: Inducible nitric oxide synthase; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein kinases; NO: Nitric oxide; PVDF: Polyvinylidene difluoride; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

2-Benzyl-benzofurans from the tubers of Ophiopogon japonicus

BackgroundThe overproduction of nitric oxide (NO) is known to involve in various inflammatory processes. A methanol extract of the tubers of Ophiopogon japonicus was found to strongly inhibit NO production. The present paper deals with the isolation, structural identification and NO inhibitory effect of five compounds isolated from the MeOH extract of O. japonicus tubers.ResultsThree new compounds were elucidated to be (2R)-(4-methoxybenzyl)-5,7-dimethyl-6-hydroxyl-2,3-dihydrobenzofuran (1), 2-(2-hydroxyl-4-methoxy-benzyl)-5-methyl-6-methoxyl-2,3-dihydrobenzofuran (2), and 2-(4-hydroxy-benzyl)-5,6-dihydroxybenzofuran (3). In addition, two known compounds were isolated from a natural source for the first time including 2-(4-methoxy-benzyl)-6,7-dimethoxyl-2,3-dihydrobenzofuran (4), and 2-(4-methoxy-benzyl)-6,7-methylenedioxy-2,3-dihydrobenzofuran (5). The absolute configuration of compound 1 was determined by experimental and calculated circular dichroism spectra. The effects of the isolated compounds on LPS-induced NO production in RAW264.7 cells were evaluated. Compound 1 and 2 showed the inhibitory activity with IC50 values of 11.4 and 29.1 μM, respectively.ConclusionsThe class of 2-benzyl-2,3-dihydrobenzofuran is uncommon in nature. In this work, three such compounds were isolated from O. japonicus. Two of them showed promising anti-inflammatory activity by inhibition of NO production.

Pleurotus giganteus (Berk. Karun & Hyde), the giant oyster mushroom inhibits NO production in LPS/H2O2 stimulated RAW 264.7 cells via STAT 3 and COX-2 pathways

BackgroundPleurotus giganteus (Berk. Karunarathna and K.D. Hyde), has been used as a culinary mushroom and is known to have medicinal properties but its potential as an anti-inflammatory agent to mitigate inflammation triggered diseases is untapped. In this study, the molecular mechanism underlying the protective effect of ethanol extract of P. giganteus (EPG) against lipopolysaccharide (LPS) and combination of LPS and hydrogen peroxide (H2O2)-induced inflammation on RAW 264.7 macrophages was investigated.MethodThe effect of EPG on nitric oxide (NO) production as an indicator of inflammation in RAW 264.7 macrophages was estimated based on Griess reaction that measures nitrite level. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), NF-kB activating protein (NKAP), signal transducer and activator of transcription 3 protein (STAT 3) and glutathione peroxidase (GPx) genes were assessed using real time reverse transcription polymerase chain reaction (RT-PCR) approach.ResultsEPG (10 μg/ml) showed the highest reduction in the LPS-induced NO production in RAW 264.7 macrophages and significantly suppressed (p < 0.05) the expression iNOS, STAT 3 and COX-2. There was a significant increase (p < 0.05) in combination of LPS and H2O2- induced iNOS production when compared to the LPS-induced iNOS production in RAW 264.7 macrophages and this concurred with the NO production which was attenuated by EPG at 10 μg/ml. A significant (p < 0.05) down regulation was observed in the combination of LPS and H2O2-induced iNOS and GPx expression by EPG.ConclusionsOur data suggest that the anti-inflammatory activity of EPG is mediated via the suppression of the STAT 3 and COX-2 pathways and can serve as potential endogenous antioxidant stimulant.

Hispidulin-7-O-Neohesperidoside from Cirsium japonicum var. ussuriense Attenuates the Production of Inflammatory Mediators in LPS-Induced Raw 264.7 Cells and HT-29 Cells.

Background:Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract and involves secretion of inflammatory mediators. The flavone diglycoside hispidulin-7-O-neohesperidoside (HN) isolated from the methanolic extract of aerial parts of Cirsium japonicum var. ussuriense, but its pharmacologic activities, with the exception of alleviation of alcohol toxicity, have not been investigated to date.Objective:The aim of the present study was to investigate the anti-inflammatory activities of HN for the treatment of chronic inflammatory illnesses, including IBD.Materials and Methods:In lipopolysaccharide (LPS)-induced RAW264.7 cells and HT-29 cells, the effects of HN on cell viability and nitric oxide (NO) production were examined via MTT assay and the Griess reaction, respectively. The expression levels of interleukin (IL)-1α, IL-8, and tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) protein levels were measured by enzyme-linked immunosorbent assay and Western blotting, respectively.Results:HN concentration-dependently inhibited NO production in LPS-induced RAW 264.7 cells. Treatment with HN considerably downregulated the levels of the pro-inflammatory cytokines, IL-1β and TNF-α and the iNOS protein level in LPS-induced RAW 264.7 cells. Furthermore, HN inhibited the production of the chemotactic cytokine, IL-8, in LPS-induced HT-29 cells.Conclusion:HN has potential as an anti-inflammatory agent to prevent and/or treat IBD.SUMMARY Hispidulin-7-O-neohesperidoside (HN) is flavone diglycoside isolated from the methanolic extract of aerial parts of Cirsium japonicum var. ussuriense.HN concentration-dependently inhibited NO production and considerably downregulated the levels of the proinflammatory cytokines, IL-1β and TNF-α, and the iNOS protein level in LPS-induced RAW 264.7 cells.HN inhibited the production of the chemotactic cytokine, IL-8, in LPS-induced HT-29 cells.HN has potential as an anti-inflammatory agent to prevent and/or treat IBD. Abbreviations used: IBD: Inflammatory bowel disease, HN: hispidulin-7-O-neohesperidoside, LPS: lipopolysaccharide, NO: nitric oxide, IL: interleukin, TNF: tumor necrosis factor, iNOS: inducible nitric oxide synthase, CD: Crohn's disease, UC: ulcerative colitis, RT: room temperature, DMEM: Dulbecco's modified Eagle's medium, FBS: fetal bovine serum, PBS: phosphate buffered saline, SDS: sodium dodecyl sulfate, PVDF: polyvinylidene difluoride, SD: standard deviation

Alkaloids and phenolic glycosides from Clematis mandshurica and their inhibitory effects against NO production in LPS-induced RAW 246.7 macrophages

Two new alkaloids (1 and 2), one new phenolic glycoside (3), and five known structures (4⿿8) were isolated from the roots and rhizomes of Clematis mandshurica. Their structures were elucidated on the basis of spectroscopic evidence and hydrolysis products. All isolates were evaluated for inhibitory activity against LPS-induced nitric oxide (NO) production in RAW 246.7 macrophages. Feruloyl and isoferuloyl derivates showed moderate inhibitory effects with the inhibition ratios (%) from 29.3 to 38.7 at 50μM, whereas 3-carboxy-indole derivates showed potent inhibitory activities with IC50 from 7.7 to 12.4μM.

선학초(仙鶴草)추출물의 대식세포에서의 LPS-유도 염증반응에 대한 효능 연구

Objectives : The aerial parts of Agrimonia pilosa Ledeb (Agrimoniae Herba; AH) has been traditionally used as a Korean medicine to treatment of abdominal pain, sore throat, headaches, bloody discharge, parasitic infections and eczema. In this study, we investigated the effect of AH ethanol extract on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells. Methods : AH was extracted by 30% ethanol (AH-E). Raw264.7 cells were treated with AH-E extract at different concentrations for 30 min and then stimulated with LPS (1μg/ml) or without for indicated times. Cell viability was measured by MTT assay, and nitric oxide (NO) production was measured by Griess assay. The expression of inflammatory mediators, iNOS and COX-2 and inflammatory cytokines, TNF-α, IL-1β, and IL-6 was detected by RT-PCR, and the phosphorylation of ERK1/2, p38 and JNK MAP kinases (MAPKs) was analyzed by Western blot. Also, the expression of NF-κB in nuclear and cytosol was detected by Western blot. Results : AH-E extract significantly decreased LPS-induced NO production in RAW264.7 cells. AH-E extract inhibited the mRNA expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6 in LPS-stimulated cells with a dose-dependent manner. In addition, the phosphorylation of ERK, p38 and JNK MAPKs was also inhibited by AH-E extract. AP-E extract inhibited the nuclear translocation of NF-κB in LPS-stimulated cells. Conclusions : Our results suggest that AH-E extract has an anti-inflammatory activity in macrophages-mediated inflammation.

Effects of Agrimoniae Herba 30% ethanol extract on LPS-induced inflammatory responses in RAW264.7 macrophage cells

Objectives : The aerial parts of Agrimonia pilosa Ledeb (Agrimoniae Herba; AH) has been traditionally used as a Korean medicine to treatment of abdominal pain, sore throat, headaches, bloody discharge, parasitic infections and eczema. In this study, we investigated the effect of AH ethanol extract on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells. Methods : AH was extracted by 30% ethanol (AH-E). Raw264.7 cells were treated with AH-E extract at different concentrations for 30 min and then stimulated with LPS (1μg/ml) or without for indicated times. Cell viability was measured by MTT assay, and nitric oxide (NO) production was measured by Griess assay. The expression of inflammatory mediators, iNOS and COX-2 and inflammatory cytokines, TNF-α, IL-1β, and IL-6 was detected by RT-PCR, and the phosphorylation of ERK1/2, p38 and JNK MAP kinases (MAPKs) was analyzed by Western blot. Also, the expression of NF-κB in nuclear and cytosol was detected by Western blot. Results : AH-E extract significantly decreased LPS-induced NO production in RAW264.7 cells. AH-E extract inhibited the mRNA expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6 in LPS-stimulated cells with a dose-dependent manner. In addition, the phosphorylation of ERK, p38 and JNK MAPKs was also inhibited by AH-E extract. AP-E extract inhibited the nuclear translocation of NF-κB in LPS-stimulated cells. Conclusions : Our results suggest that AH-E extract has an anti-inflammatory activity in macrophages-mediated inflammation.

IVSE, isolated from Inula japonica,suppresses LPS-induced NO production via NF-κB and MAPK inactivation in RAW264.7 cells

AimsOur previous study showed that the extract of Inula japonica Thunb. (I. japonica) has anti-inflammatory and anti-asthmatic activities. In an attempt to find anti-inflammatory compounds from I. japonica, we recently isolated 1,6α-dihydroxy-4αH-1,10-secoeudesma-5(10),11(13)-dien-12,8β-olide (SE), 6α-isobutyryloxy-1-hydroxy-4αH-1,10-secoeudesma-5(10),11(13)-dien-12,8β-olide (IBSE), and 6α-isovaleryloxy-1-hydroxy-4αH-1,10-secoeudesma-5(10),11(13)-dien-12,8β-olide (IVSE) from the extract of I. japonica, and investigated their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Main methodsThe inhibitory effect of IVSE, SE and IBSE on NO production in LPS-induced RAW264.7 cells was examined using Griess reagent, and the effects of IVSE on the expressions of inducible nitric oxide synthase (iNOS) and its upstream signal proteins including IκB kinase (IKK)/inhibitor kappa B (IκB)-α/nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) were investigated by Western blot. Key findingsAmong the 3 compounds isolated, SE, IBSE, and IVSE inhibited NO production at 2.5μM with 5.1%, 40.4%, and 52.8%, respectively. IVSE displayed the most potent inhibition of NO production. Mechanism analysis indicated that IVSE dramatically decreased the expression of iNOS, reduced the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation and degradation of IκB-α, and inhibited the activation of the upstream mediator IKK α/β. Furthermore, our results showed that IVSE inhibited the phosphorylation of MAPKs including extracellular regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK) and p38. SignificanceIVSE exhibited anti-inflammatory activity by inhibiting NO production, in which inactivation of NF-κB and MAPKs might be involved. Our results suggest that IVSE might become an anti-inflammatory drug candidate.