Inhibition of Nitric Oxide (NO) Production in Lipopolysaccharide (LPS)-Activated Murine Macrophage RAW 264.7 Cells by the Norsesterterpene Peroxide, Epimuqubilin A

Excess nitric oxide (NO) induces apoptosis of some cell types, including macrophages. As NO is synthesized by NO synthase (NOS) from arginine, a common substrate of arginase, these two enzymes compete for arginine. There are two known isoforms of arginase, types I and II. Using murine macrophage-like RAW 264.7 cells, we asked if the induction of arginase II would downregulate NO production and hence prevent apoptosis. When cells were exposed to lipopolysaccharide (LPS) and interferon-γ (IFN-γ), the inducible form of NOS (iNOS) was induced, production of NO was elevated, and apoptosis followed. When dexamethasone and cAMP were further added, both iNOS and arginase II were induced, NO production was much decreased, and apoptosis was prevented. When the cells were transfected with an arginase II expression plasmid and treated with LPS/IFN-γ, some cells were rescued from apoptosis. An arginase I expression plasmid was also effective. On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added. These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.

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Comparing the protective effects of resveratrol, curcumin and sulforaphane against LPS/IFN-γ-mediated inflammation in doxorubicin-treated macrophages

Scientific Reports ◽

10.1038/s41598-020-80804-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Haidy A. Saleh ◽

Eman Ramdan ◽

Mohey M. Elmazar ◽

Hassan M. E. Azzazy ◽

Anwar Abdelnaser

Keyword(s):

Nitric Oxide ◽

Inflammatory Response ◽

Raw 264.7 Cells ◽

Inos Mrna ◽

No Production ◽

Raw 264.7 ◽

Mrna Levels ◽

Protective Effects ◽

Tnf Α ◽

Ifn Γ

AbstractDoxorubicin (DOX) chemotherapy is associated with the release of inflammatory cytokines from macrophages. This has been suggested to be, in part, due to DOX-mediated leakage of endotoxins from gut microflora, which activate Toll-like receptor 4 (TLR4) signaling in macrophages, causing severe inflammation. However, the direct function of DOX on macrophages is still unknown. In the present study, we tested the hypothesis that DOX alone is incapable of stimulating inflammatory response in macrophages. Then, we compared the anti-inflammatory effects of curcumin (CUR), resveratrol (RES) and sulforaphane (SFN) against lipopolysaccharide/interferon-gamma (LPS/IFN-γ)-mediated inflammation in the absence or presence of DOX. For this purpose, RAW 264.7 cells were stimulated with LPS/IFN-γ (10 ng/mL/10 U/mL) in the absence or presence of DOX (0.1 µM). Our results showed that DOX alone is incapable of stimulating an inflammatory response in RAW 264.7 macrophages. Furthermore, after 24 h of incubation with LPS/IFN-γ, a significant increase in tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) mRNA levels was observed. Similarly, nitric oxide (NO) production and TNF-α and IL-6 protein levels were significantly upregulated. Moreover, in LPS/IFN-γ-treated macrophages, the microRNAs (miRNAs) miR-146a, miR-155, and miR-21 were significantly overexpressed. Interestingly, upon testing CUR, RES, and SFN against LPS/IFN-γ-mediated inflammation, only SFN was able to significantly reverse the LPS/IFN-γ-mediated induction of iNOS, TNF-α and IL-6 and attenuate miR-146a and miR-155 levels. In conclusion, SFN, at the transcriptional and posttranscriptional levels, exhibits potent immunomodulatory action against LPS/IFN-γ-stimulated macrophages, which may indicate SFN as a potential treatment for DOX-associated inflammation.

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Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages

Foods ◽

10.3390/foods9030269 ◽

2020 ◽

Vol 9 (3) ◽

pp. 269 ◽

Cited By ~ 1

Author(s):

Su Cheol Baek ◽

Dahae Lee ◽

Mun Seok Jo ◽

Kwang Ho Lee ◽

Yong Hoon Lee ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Sea Buckthorn ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Tnf Α ◽

Mouse Macrophages ◽

Cox 2 ◽

Oxide Synthase

Hippophae rhamnoides L. (Elaeagnaceae; commonly known as “sea buckthorn” and “vitamin tree”), is a spiny deciduous shrub whose fruit is used in foods and traditional medicines. The H. rhamnoides fruit (berry) is rich in vitamin C, with a level exceeding that found in lemons and oranges. H. rhamnoides berries are usually washed and pressed to create pomace and juice. Today, the powder of the aqueous extract of H. rhamnoides berries are sold as a functional food in many countries. As part of our ongoing effort to identify bioactive constituents from natural resources, we aimed to isolate and identify those from the fruits of H. rhamnoides. Phytochemical analysis of the extract of H. rhamnoides fruits led to the isolation and identification of six compounds, namely, a citric acid derivative (1), a phenolic (2), flavonoids (3 and 4), and megastigmane compounds (5 and 6). Treatment with compounds 1–6 did not have any impact on the cell viability of RAW 264.7 mouse macrophages. However, pretreatment with these compounds suppressed lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse macrophages in a concentration-dependent manner. Among the isolated compounds, compound 1 was identified as the most active, with an IC50 of 39.76 ± 0.16 μM. This value was comparable to that of the NG-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor with an IC50 of 28.48 ± 0.05 μM. Western blot analysis demonstrated that compound 1 inhibited the LPS-induced expression of IKKα/β (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), nuclear factor kappa-B (NF-κB) p65, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2) in RAW 264.7 cells. Furthermore, LPS-stimulated cytokine production was detected using a sandwich enzyme-linked immunosorbent assay. Compound 1 decreased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) production in LPS-stimulated RAW 264.7 cells. In summary, the mechanism of action of 1 included the suppression of LPS-induced NO production in RAW 264.7 cells by inhibiting IKKα/β, I-κBα, NF-κB p65, iNOS, and COX-2, and the activities of IL-6 and TNF-α.

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Naphthoquinone Derivatives with Anti-Inflammatory Activity from Mangrove-Derived Endophytic Fungus Talaromyces sp. SK-S009

Molecules ◽

10.3390/molecules25030576 ◽

2020 ◽

Vol 25 (3) ◽

pp. 576 ◽

Cited By ~ 1

Author(s):

Hongju Liu ◽

Chong Yan ◽

Changqun Li ◽

Tingting You ◽

Zhigang She

Keyword(s):

Nitric Oxide ◽

Endophytic Fungus ◽

Raw 264.7 Cells ◽

Inflammatory Factors ◽

No Production ◽

Raw 264.7 ◽

Mrna Levels ◽

Tnf Α ◽

Ic50 Values ◽

Oxide Synthase

Twelve 1, 4-naphthoquinone derivatives, including two new (1 and 2) and 10 known (3–12), were obtained from endophytic fungus Talaromyces sp. SK-S009 isolated from the fruit of Kandelia obovata. All structures were identified through extensive analysis of the nuclear magnetic resonance (NMR), mass spectrometry (MS) and circular dichroism (CD), as well as by comparison with literature data. These compounds significantly inhibited the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in the murine macrophage cell line (RAW 264.7 cells). The half maximal inhibitory concentration (IC50) values, except for compound 2, were lower than that of indomethacin (26.3 μM). Compound 9 inhibited the LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions in RAW 264.7 macrophages. Additionally, compound 9 reduced the mRNA levels of pro-inflammatory factors interleukin (IL)1β, IL-6, and tumor necrosis factor (TNF)-α. The results of this study demonstrated that these 1, 4-naphthoquinone derivatives can inhibit LPS-induced inflammation.

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In vitro and in vivo anti-inflammatory activities of a sterol-enriched fraction from freshwater green alga, Spirogyra sp.

Fisheries and Aquatic Sciences ◽

10.1186/s41240-020-00172-9 ◽

2020 ◽

Vol 23 (1) ◽

Author(s):

Lei Wang ◽

You-Jin Jeon ◽

Jae-Il Kim

Keyword(s):

Nitric Oxide ◽

Green Alga ◽

Raw 264.7 Cells ◽

Ros Generation ◽

Prostaglandin E ◽

No Production ◽

Raw 264.7 ◽

Anti Inflammatory

Abstract Background Inflammation plays a crucial role in the pathogenesis of many diseases such as arthritis and atherosclerosis. In the present study, we evaluated anti-inflammatory activity of sterol-rich fraction prepared from Spirogyra sp., a freshwater green alga, in an effort to find bioactive extracts derived from natural sources. Methods The sterol content of ethanol extract of Spirogyra sp. (SPE) was enriched by fractionation with hexane (SPEH), resulting 6.7 times higher than SPE. Using this fraction, the in vitro and in vivo anti-inflammatory activities were evaluated in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells and zebrafish. Results SPEH effectively and dose-dependently decreased the production of nitric oxide (NO) and prostaglandin E2 (PGE2). SPEH suppressed the production of pro-inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β through downregulating nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW 264.7 cells without cytotoxicity. The in vivo test results indicated that SPEH significantly and dose-dependently reduced reactive oxygen species (ROS) generation, cell death, and NO production in LPS-stimulated zebrafish. Conclusions These results demonstrate that SPEH possesses strong in vitro and in vivo anti-inflammatory activities and has the potential to be used as healthcare or pharmaceutical material for the treatment of inflammatory diseases.

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Rotenoid Derivatives from Derris Trifoliata with Nitric Oxide Production Inhibitory Activity

Natural Product Communications ◽

10.1177/1934578x1200701117 ◽

2012 ◽

Vol 7 (11) ◽

pp. 1934578X1200701

Author(s):

Chihiro Ito ◽

Tomiyasu Murata ◽

Hugh T.-W. Tan ◽

Norio Kaneda ◽

Hiroshi Furukawa ◽

...

Keyword(s):

Nitric Oxide ◽

Inhibitory Activity ◽

Chemical Constituents ◽

Interferon Γ ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Nitric Oxide Production ◽

Murine Macrophage ◽

Isolation And Identification ◽

Oxide Production

Study of the chemical constituents of the stems of Derris trifoliata Lour. (Leguminosae) collected in Singapore led to the isolation and identification of three known and two new rotenoid derivatives. The new derivatives, named derrisfolin A (1) and B (2), inhibited nitric oxide production in murine macrophage-like RAW 264.7 cells stimulated with interferon-γ and lipopolysaccharide.

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ANTI-INFLAMMATORY ACTIVITY OF TINOCRISPOSIDE BY INHIBITING NITRIC OXIDE PRODUCTION IN LIPOPOLYSACCHARIDES-STIMULATED RAW 264.7 CELLS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2018.v11i4.23739 ◽

2018 ◽

Vol 11 (4) ◽

pp. 149 ◽

Cited By ~ 1

Author(s):

Adek Zamrud Adnan ◽

Muhammad Taher ◽

Tika Afriani ◽

Annisa Fauzana ◽

Dewi Imelda Roesma ◽

...

Keyword(s):

Nitric Oxide ◽

Inflammatory Diseases ◽

Positive Control ◽

Inflammatory Activity ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Dependent Manner ◽

Anti Inflammatory

Objective: The aim of this study was to investigate in vitro anti-inflammatory activity of tinocrisposide using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cells. Tinocrisposide is a furano diterpene glycoside that was isolated in our previous study from Tinospora crispa.Methods: Anti-inflammatory effect was quantified spectrometrically using Griess method by measuring nitric oxide (NO) production after the addition of Griess reagent.Results: The sample concentrations of 1, 5, 25, 50, and 100 μM and 100 μM of dexamethasone (positive control) have been tested against the LPS-stimulated RAW 264.7 cells, and the results showed NO level production of 39.23, 34.00, 28.9, 20.25, 16.3, and 13.68 μM, respectively, and the inhibition level of 22.67, 33.00, 43.03, 60.10, 68.00, and 73%, respectively.Conclusions: From the study, it could be concluded that tinocrisposide was able to inhibit the formation of NO in the LPS-stimulated RAW 264.7 cells in concentration activity-dependent manner, with half-maximal inhibition concentration 46.92 μM. It can be developed as anti-inflammatory candidate drug because NO is a reactive nitrogen species which is produced by NO synthase. The production of NO has been established as a mediator in inflammatory diseases.

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Sesquiterpenes from Inula japonica with Inhibitory Effects on Nitric Oxide Production in Murine Macrophage RAW 264.7 Cells

Journal of Natural Products ◽

10.1021/acs.jnatprod.5b01106 ◽

2016 ◽

Vol 79 (6) ◽

pp. 1548-1553 ◽

Cited By ~ 9

Author(s):

Qinghao Jin ◽

Jin Woo Lee ◽

Hari Jang ◽

Ji Eun Choi ◽

Dongho Lee ◽

...

Keyword(s):

Nitric Oxide ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Nitric Oxide Production ◽

Murine Macrophage ◽

Inhibitory Effects ◽

Oxide Production

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IN VITRO ANTI-INFLAMMATORY ACTIVITY OF THE COMPONENTS OF AMOMUM TSAO-KO IN MURINE MACROPHAGE RAW 264.7 CELLS

African Journal of Traditional Complementary and Alternative Medicines ◽

10.21010/ajtcamv15i2.4 ◽

2018 ◽

Vol 15 (2) ◽

pp. 26

Author(s):

Chun Whan Choi ◽

Ju Young Shin ◽

Changon Seo ◽

Seong Su Hong ◽

Eun-Kyung Ahn ◽

...

Keyword(s):

Nitric Oxide ◽

Benzoic Acid ◽

Inflammatory Activity ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Dependent Manner ◽

Murine Macrophage ◽

Etoh Extract ◽

Anti Inflammatory

Background: Plants still remain the prime source of drugs for the treatment of inflammation and can provide leads for the development of novel anti-inflammatory agents. Material and methods: An in vitro bioassay guide revealed that the 80% ethanol (EtOH) extract of the whole plant, Amomum tsao-ko (Zingiberaceae), displayed anti-inflammatory activity after assessing its effects on murine macrophage RAW 264.7 cells. Result: Phytochemical study of the 80% EtOH extract of Amomum tsao-ko led to the isolation of eight compounds: 4-hydroxy-3-methoxy-benzoic acid (1), meso-hannokinol (2), (+)-hannokinol (3), coumaric acid (4), 4-hydroxy-benzoic acid (5), (+)-epicatechin (6), (-)-catechin (7), and myrciaphenone A (8). The results indicated that two of the isolated components, (+)-epicatechin (6) and (-)-catechin (7), inhibited the production of nitric oxide (NO) significantly in lipopolysaccharide treated RAW 264.7 cells. Conclusion: LPS-induced interleukin tumor necrosis factor-alpha (TNF-), IL-1β and IL-10 production was also decreased in a dose-dependent manner. In addition, western blot analysis revealed that (+)-epicatechin (6) and (-)-catechin (7) reduced the expression of inducible nitric oxide synthase and inhibited nuclear localization of nuclear factor kappa-B (NF-κB).

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