LPS-activated Murine Macrophages Research Articles

BP5 regulated B cell development promoting anti-oxidant defence

Bursa of Fabricius is the humoral immune system for B cell differentiation and antibody production. Bursopentine (BP5) is a novel immunomodulatory peptide and significantly stimulated an antigen-specific immune response in mice. BP5 was also found to protect LPS-activated murine peritoneal macrophages from oxidative stress. In this study, the effects of BP5 on B cell development were examined. The results suggested that BP5 markedly promoted B cell development by increasing CFU-pre B, and affected the redox homeostasis regulation of B cells. To study the molecular mechanism of effect of bursal-derived BP5, this research utilized 2D-E and MALDI-TOF/TOF to analyze the differentially expressed proteins of BP5-treated WEHI-231 cells. The results showed that BP5 affected the redox homeostasis regulation of WEHI-231 cells and induced alterations in the protein expression profiles related to the oxidoreduction coenzyme metabolic process, precursor metabolites and energy, proteolysis, RNA splicing and translation and cellular process, respectively. BP5 also induced glucose-6-phosphate dehydrogenase (G6PD) activity, an essential anti-oxidant cofactor. We found that the redox homeostasis regulation effect of BP5 was reduced in G6PD-deficient cells. These data suggested that BP5 affected the redox balance toward reducing conditions by promoting the expression of G6PD, which in turn regulated the glutathione redox cycle and other processes.

Unsaponifiable fraction from extra virgin olive oil inhibits the inflammatory response in LPS-activated murine macrophages

Extra virgin olive oil (EVOO) has demonstrated great anti-inflammatory properties. Nowadays, it is clear that its minor components have a key role in these beneficial effects. However, the contribution of the unsaponifiable fraction (UF) to these healthy effects remains unknown. The present study was designed to evaluate UF in LPS stimulated peritoneal macrophages isolated from mice. NO production was analysed by the Griess method and intracellular ROS by fluorescence analysis. In addition, MAPK family activation, IKBα degradation, NFκB-p65, iNOS and COX-2 expression were determined by Western blot. UF exerted anti-inflammatory and anti-oxidant effects inhibiting LPS-induced intracellular ROS and nitrite production. Additionally, UF decreased COX-2 and iNOS protein expression. These effects were related with a down-regulation in NFκB signal signalling pathways and in MAPK phosphorylation. UF of EVOO compounds could play an important role in the anti-inflammatory effect of virgin olive oils and probably provide an attractive complement in management of inflammatory diseases.

The Suppressive Effects of Bursopentine (BP5) on Oxidative Stress and NF-ĸB Activation in Lipopolysaccharide-activated Murine Peritoneal Macrophages

Background/Aim: Bursopentine (BP5) is a novel thiol-containing pentapeptide isolated from chicken bursa of Fabricius, and is reported to exert immunomodulatory effects on B and T lymphocytes. It has been found that some thiol compounds, such as glutathione (GSH) and N-acetylcysteine (NAC) protect living cells from oxidative stress. This led us to investigate whether BP5 had any ability to protect macrophages from oxidative stress as well as any mechanism that might underlie this process. Methods: Murine peritoneal macrophages activated by lipopolysaccharide (LPS) (2 µg/ml) were treated with single bouts (0, 25, 50, and 100 µM) of BP5. Results: BP5 potently suppressed the markers for oxidative stress, including nitric oxide (NO), reactive oxygen species (ROS), lipid peroxidation, and protein oxidation. It also decreased the expression and activity of inducible nitric oxide synthase (iNOS) and promoted a protective antioxidant state by elevating GSH content and by activating the expression and activity of certain key antioxidant and redox enzymes, including glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT). This suppressive effect on oxidative stress was accompanied by down-regulated expression and activity of nuclear factor kappa B (NF-ĸB). Conclusion: These findings demonstrate that BP5 can protect LPS-activated murine peritoneal macrophages from oxidative stress. BP5 may have applications as an anti-oxidative stress reagent.

Potential Antioxidant, Antiinflammatory, and Proapoptotic Anticancer Activities of Kakadu Plum and Illawarra Plum Polyphenolic Fractions

Kakadu plum (Terminalia ferdinandiana Exell, Combretaceae) and Illawarra plum (Podocarpus elatus Endl., Podocarpaceae) extracts were fractionated, using a bioassay-guided approach and screened for antioxidant activity [oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays] and antiinflammatory activity (nitrite concentration and prostaglandin E2 release in lipopolysaccharide (LPS)-activated murine macrophages). Among 8 fractions obtained from KP and 5 fractions obtained from IP, fraction KPF5 from KP exhibited superior activity in all assays, with an ORAC value of 3,776 ± 603 μmol Trolox/g DW and a CAA value of 52.2 ± 8.6 μmol quercetin equivalents/g DW. In addition, KPF5 further demonstrated an upregulation of the Nrf2/Keap1 ratio in Hep G2 cells. KPF5 also inhibited the expression of COX-2 and iNOS in LPS-activated murine macrophages, potentially through the NF-κB, p44/42 mitogen activated protein kinase and Akt pathways. KPF5 also induced apoptosis and DNA damage in HT-29 cells, as determined by the cytokinesis block micronucleus cytome assay.

Andrographolide inhibits extracellular signal-regulated kinase 1/2 signaling pathway in activatedmacrophages

To investigate the effects of andrographolide on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and tumor necrosis factor-α (TNF-α) expression in lipopolysaccharide (LPS)-activated macrophages. LPS-activated mouse peritoneal macrophages were cultured in media with different concentrations of andrographolide. Cytotoxicity of andrographolide was detected by cell counting kit-8. The macrophages were lysed, and then expressions of phosphorylated ERK1/2, JNK and p38 and nuclear factor-κB inhibitor (IκBα) protein were detected by Western blotting and TNF-α mRNA expression was detected by reverse transcription-polymerase chain reaction. Supernatants of the macrophages were used to detect content of TNF-α protein by enzyme-linked immunosorbent assay. Andrographolide at 1-100 μg/mL showed no cytotoxicity on LPS-activated mouse peritoneal macrophages. Andrographolide inhibited ERK1/2 phosphorylation in LPS-activated murine peritoneal macrophages, which was concentration-dependent (P<0.01). Andrographolide at 1-25 μg/mL had no effects on phosphorylation levels of JNK and p38 and IκBα degradation in LPS-stimulated mouse peritoneal macrophages. In activated macrophages, TNF-α expression was inhibited by 12 μg/mL andrographolide and 20 μmol/L PD98059 (inhibitor of ERK1/2 signaling pathway) at both mRNA expression and protein secretion levels. In LPS-activated macrophages, andrographolide may inhibit the expression of TNF-α by inhibiting ERK1/2 signaling pathway.

Biological activity of water-soluble inclusion complexes of 1′-acetoxychavicol acetate with cyclodextrins

1′-Acetoxychavicol acetate (ACA), isolated from the rhizomes and the seeds of the Zingiberaceae plant, has a variety of biological activities such as antitumor, antiallergic and repellent effects. However, ACA seems to have some disadvantages which may limit for future possible clinical applications, for example, its poor water solubility. Furthermore, ACA is not stable in aqueous solutions and undergoes hydrolysis and/or isomerization. To improve the solubility and stability of ACA in water, we prepared the inclusion complexes with various β-cyclodextrins (β-CDs).In aqueous solution, the association constants of ACA with various CDs were estimated at 662 ± 95 (β-CD), 336 ± 70 (methyl-β-CD, Meβ-CD), and 322 ± 44 M −1 (hydroxypropyl-β-CD, HPβ-CD), respectively, by a spectrofluorometric displacement method based on competition between a guest and a fluorescent probe for CDs. It was revealed that almost all ACAs existed as a free molecule in the CD-containing aqueous solution. However, in the case of preparing the inclusion complexes of CDs with ACA by a solid phase ‘high-speed vibration milling’ technique, the average inclusion rates of the obtained water-soluble complexes were calculated as 88 ± 13% (β-CD), 70 ± 1% (Meβ-CD), and 63 ± 2% (HPβ-CD), respectively, by 1H NMR analysis. To characterize the structures of the CD·ACA complexes, 2,3,6-trimethyl-β-CD (TMeβ-CD)·ACA complex was prepared as a model compound (inclusion rate: 40%). As a result of 2D ROESY experiments, it was considered that the aromatic ring of ACA is located in the narrow side of the hydrophobic cavity of the TMeβ-CD and both 1′- and 4-acetoxy groups of ACA positioned in the vicinity of the secondary and primary methoxy groups of TMeβ-CD, respectively. Furthermore, we examined the apoptogenic activity of CD·ACA complexes to evaluate whether or not the bioactivities of ACA were affected by their inclusion. Although the cytotoxicity of all CD·ACA complexes in human epithelial carcinoma HeLa cells and murine adenocarcinoma colon26 cells were diminished as compared with the ACA alone, only HPβ-CD·ACA maintained high levels of activity. In addition, HPβ-CD·ACA, and Meβ-CD·ACA showed suppressive effect for the transcription factor NF-κB activation on LPS-activated murine macrophage RAW264.7 cells and the former was more active complex. Furthermore, HPβ-CD·ACA inhibited the in vivo tumor growth of tumor-bearing mice, although the activity was slightly weak compared with that of free ACA. These results indicate that HPβ-CD is the best host molecule for ACA to form a water-soluble complex with the similar biological activity of free ACA.

Synthesis and biological evaluation of unique stereodimers of sinomenine analogues as potential inhibitors of NO production

Inhibition of the excessive NO production has been recognized as a potential means for the treatment of rheumatoid arthritis (RA). In order to discover more potent inhibitors and explore the preliminary structure activity relationship, a series of unique stereodimers of sinomenine analogues were designed and synthesized. Their inhibitory activity on NO production and cytotoxicity were evaluated using LPS-activated murine macrophages RAW264.7 assay and MTT method, respectively. Among these compounds, 1a, 2, 2a, 2b, and 4 showed potent inhibitory activity on NO production without obvious cytotoxicity. Furthermore, 2, 2a, and 2b significantly suppressed mRNA expression of iNOS. Interestingly, ( S)-dimers displayed a better bioactivity than ( R)-dimers. These compounds may sever as lead candidates in the development of novel therapeutic drugs for RA treatment.

Native Australian fruit polyphenols inhibit COX-2 and iNOS expression in LPS-activated murine macrophages

Purified polyphenolic-rich extracts from four native Australian fruits, Illawarra Plum (Podocarpus elatus Endl., Podocarpaceae), Kakadu Plum (Terminalia ferdinandiana Exell, Combretaceae), Muntries (Kunzea pomifera F. Muell., Myrtaceae) and Native Currant (Acrotriche depressa R.Br., Epacridaceae), were screened for their ability to modulate anti-inflammatory activity in LPS-activated murine macrophages (RAW 264.7). The Kakadu Plum extract inhibited the expression of inducible nitric oxide synthase (iNOS), and importantly was the only fruit in this study that displayed differential inhibition of the expression of cyclooxygenase (COX)-2 but not COX-1. Illawarra Plum (COX-2 and iNOS) and Native Currant (iNOS only) also inhibited inflammatory enzymes, while Muntries exhibited none of these activities under the same conditions. All evaluated extracts inhibited the production of prostaglandin E2 and nitric oxide, suggesting the involvement of alternative pathways in their regulation for the Muntries extract. Further molecular investigations, showed that Kakadu Plum inhibited the NF-кB pathway, but not the p44/42 mitogen activated protein kinase (MAPK) pathway. Collectively, these results demonstrate potential anti-inflammatory activities of native Australian fruits, in particular Kakadu Plum, in LPS-activated murine macrophages, thus confirming the potential biological activities of these fruits.

Anti-inflammatory and antinociceptive properties of the leaves of Eriobotrya japonica

The leaves of Eriobotrya japonica Lindl. have been widely used as a traditional medicine for the treatment of many diseases including coughs and asthma. The present study was designed to validate the anti-inflammatory and antinociceptive properties of the n-BuOH fraction of E. japonica (LEJ) leaves. The anti-inflammatory properties of LEJ were studied using IFN-γ/LPS activated murine peritoneal macrophage model. The antinociceptive effects of LEJ were assessed using experimental models of pain, including thermal nociception methods, such as the tail immersion test and the hotplate test, and chemical nociception induced by intraperitoneal acetic acid and subplantar formalin in mice. To examine the possible connection of the opioid receptor to the antinociceptive activity of LEJ, we performed a combination test with naloxone, a nonselective opioid receptor antagonist. In the IFN-γ and LPS-activated murine peritoneal macrophage model, LEJ suppressed NO production and iNOS expression via down-regulation of NF-κB activation. It also attenuated the expression of COX-2 and the secretion of pro-inflammatory cytokines like TNF-α and IL-6. Moreover, LEJ also demonstrated strong and dose-dependent antinociceptive activity compared to tramadol and indomethacin in various experimental pain models. In a combination test using naloxone, diminished analgesic activities of LEJ were observed, indicating that the antinociceptive activity of LEJ is connected with the opioid receptor. The results indicate that LEJ had potent inhibitory effects on the inflammatory mediators including nitric oxide, iNOS, COX-2, TNF-α and IL-6 via the attenuation of NF-κB translocation to the nucleus. LEJ also showed excellent antinociceptive activity in both central and peripheral mechanism as a weak opioid agonist. Based on these results, LEJ may possibly be used as an anti-inflammatory and an analgesic agent for the treatment of pains and inflammatory diseases.

Synthesis of a novel series of diphenolic chromone derivatives as inhibitors of NO production in LPS-activated RAW264.7 macrophages

A novel series of diphenolic chromone derivatives were synthesized and their inhibitory activity on nitric oxide (NO) production and cytotoxicity were evaluated using LPS-activated murine macrophages RAW264.7 assay and MTT method, respectively. Among these compounds, (5,7-dihydroxy-4-oxo-4 H-chromen-3-yl) methyl esters ( 6b, 6c, 6f, 6g, and 6h) showed quite potent inhibitory activities with IC 50 values of 2.20, 3.48, 0.35, 0.80, and 0.61 μM, respectively. The MTT results showed that all of the active compounds exhibited no cytotoxicity at the effective concentrations. The preliminary mechanism of the most potent compounds ( 6b, 6c, 6f, 6g, and 6h) was further examined based on the RT-PCR results and the compounds 6f, 6g, and 6h inhibited NO production by suppressing the expression of iNOS mRNA in a dose dependent manner. Furthermore, a computational analysis of physicochemical parameters revealed that the most of the compounds possessed drug-like properties.

Ethyl pyruvate decreases HMGB1 release and ameliorates murine colitis

Signals from stressed cells and the enteric microbiota activate macrophages and dendritic cells and mediate intestinal inflammation. HMGB1 serves as an immunogenic stimuli causing release of inflammatory cytokines by myeloid cells. Ethyl pyruvate inhibits secretion of HMGB1 and improves survival in models of endotoxemia and hemorrhagic shock. We reasoned that ethyl pyruvate may be protective in colitis, which involves similar inflammatory pathways. In IL-10(-/-) mice with established chronic colitis, ethyl pyruvate administration ameliorated colitis and reduced intestinal cytokine production. IL-10(-/-) mice demonstrated increased intestinal HMGB1 expression and decreased expression of RAGE compared with wild-type mice. Fecal HMGB1 levels were decreased in ethyl pyruvate-treated mice. Furthermore, ethyl pyruvate induced HO-1 expression in intestinal tissue. In TNBS-induced colitis, intrarectal administration of ethyl pyruvate resulted in amelioration of colitis and reduced intestinal cytokine production. In LPS-activated murine macrophages, ethyl pyruvate decreased expression of IL-12 p40 and NO production but did not affect IL-10 levels. Ethyl pyruvate did not inhibit nuclear translocation of NF-kappaB family members but attenuated NF-kappaB DNA binding. Additionally, ethyl pyruvate induced HO-1 mRNA and protein expression and HO-1 promoter activation. Moreover, ethyl pyruvate prevented nuclear-to-cytoplasmic translocation of HMGB1. In conclusion, the HMGB1/RAGE pathway has pathophysiologic and diagnostic significance in experimental colitis. Ethyl pyruvate and other strategies to inhibit HMGB1 release and function represent promising interventions in chronic inflammatory diseases.

IL-13 pre-treatment of murine peritoneal macrophages increases their anti-Toxoplasma gondii activity induced by lipopolysaccharides

Th1 cytokines and microbial lipopolysaccharides (LPS) activate macrophages to produce inflammatory mediators and effector molecules. Althrough Th2 cytokines often have an opposite action to Th1 cytokines and down-modulate the inflammatory response of macrophages, they can induce a distinct alternative activation that is beneficial in host defence. In this study, we report that IL-13 enhances the anti-Toxoplasma activity of LPS-activated murine macrophages. The inhibition of parasite proliferation was not related to reduced Toxoplasma gondii penetration into the cells, nor to the conversion of tachyzoites into bradyzoites. Used alone, IL-13 triggers the polarisation of macrophages towards type 2. However, in LPS-activated macrophages, we show the priming capacity of this cytokine to enhance the expression of inducible nitric oxide synthase (iNOS), a major marker of type 1 macrophages. This effect of IL-13 was not dependent on the activation state of macrophages (resident versus thioglycolate-elicited) or the timing of pre-treatment. We demonstrate a correlation between the enhancement of NO production and upgrading of the microbicidal effectiveness of the macrophages. Thus, both Th2 and Th1 cytokines could activate macrophages to control infections.

Evaluation of nitric oxide scavenging activity, In Vitro and Ex Vivo, of selected medicinal plants traditionally used in inflammatory diseases

Steroidal and non-steroidal antiinflammatory drugs, despite their various side effects, are in great demand worldwide. Alternatively, herbal formulations provide relief to a large percentage of the population suffering from inflammatory diseases. Therefore, such practices need to be rationalized through a mechanistic approach. Thus, four traditional medicinal plants, namely Ventilago madraspatana Gaertn., Rubia cordifolia Linn., Lantana camara Linn. and Morinda citrifolia Linn. were selected for a study on the inhibition of nitric oxide (NO*), a key mediator in the phenomenon of inflammation, signifying the presence of effective antiinflammatory constituents therein. Plant samples were extracted with different solvents for evaluation of their inhibitory activity on NO* produced in vitro from sodium nitroprusside, and in LPS-activated murine peritoneal macrophages, ex vivo. Further, the inhibition of NO* synthesis was correlated with the reduction of iNOS protein expression through Western blot. Notable NO* scavenging activity was exhibited in vitro by some extracts of V. madraspatana, R. cordifolia and L. camara (IC(50) < 0.2 mg/mL). Most of them showed marked inhibition (60%-80%), ex vivo, at a dose of 80 microg/mL without appreciable cytotoxic effect on the cultured macrophages. Immunoblot analysis confirmed that the modulatory effect of the samples had occurred through suppression of iNOS protein.

Cancer chemoprevention by tea polyphenols through modulating signal transduction pathways.

The action mechanisms of several chemopreventive agents derived from herbal medicine and edible plants have become attractive issues in cancer research. Tea is the most widely consumed beverage worldwide. Recently, the cancer chemopreventive actions of tea have been intensively investigated. It have been demonstrated that the active principles of tea were attributed to their tea polyphenols. Recently, tremendous progress has been made in elucidating the molecular mechanisms of cancer chemoprevention by tea and tea polyphenols. The suppression of various tumor biomarkers including growth factor receptor tyrosine kinases, cytokine receptor kinases, PI3K, phosphatases, ras, raf, MAPK cascades, N x FB, I x B kinase, PKA, PKB, PKC, c-jun, c-fos, c-myc, cdks, cyclins, and related transducing proteins by tea polyphenols has been studied in our laboratory and others. The I x B kinase (IKK) activity in LPS-activated murine macrophages (RAW 264.7 cells) was found to be inhibited by various tea polyphenols including (-) epigallocatechin-3-gallate (EGCG), theaflavin (TF-1), theaflavin-3-gallate (TF-2) and theaflavin-3,3'-digallate (TF-3). TF-3 inhibited IKK activity in activated macrophages more strongly than did the other tea polyphenols. TF-3 inhibited both IKK1 and IKK2 activity and prevented the degradation of I x B x and I x B x in activated macrophage cells. The results suggested that the inhibition of IKK activity by TF-3 and other tea polyphenols could occur by a direct effect on IKKs or on upstream events in the signal transduction pathway. TF-3 and other tea polyphenols blocked phosphorylation of IB from the cytosolic fraction, inhibited NFB activity and inhibited increases in inducible nitric oxide synthase levels in activated macrophage. TF-3 and other tea polyphenols also inhibited strongly the activities of xanthine oxidase, cyclooxygenase, EGF-receptor tyrosine kinase and protein kinase C. These results suggest that TF-3 and other tea polyphenols may exert their cancer chemoprevention through suppressing tumor promotion and inflammation by blocking signal transduction. The mechanisms of this inhibition may be due to the blockade of the mitogenic and differentiating signals through modulating EGFR function, MAPK cascades, NFkappaB activation as well as c-myc, c-jun and c-fos expression.

Yomogin Inhibits the Degranulation of Mast Cells and the Production of the Nitric Oxide in Activated RAW 264.7 Cells

Yomogin (1), an eudesmane sesquiterpene isolated from Artemisia princeps, was tested for the effects on the degranulation process of cultured mast cells and on the nitric oxide production in LPS-activated murine macrophages. It demonstrated a significant inhibition on the release of beta-hexosaminidase from the cultured RBL-2H3 cells in a dose-dependent manner (IC50 value, 50 microM) and also exhibited a potent inhibition on the nitric oxide production from the activated RAW264.7 cells (IC50 value, 3 microM).

Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.

Sensitivity of Bacteria to NaNO2 and to L‐Arginine‐Dependent System in Murine Macrophages

Sensitivity of bacteria to NO2-/NO and to L-arginine-dependent system in murine macrophages was tested. The growth of all bacteria tested, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus epidermidis, was significantly inhibited by NaNO2 at the concentration of 5 to 10 mM. However, L-arginine-dependent effector mechanism of LPS-activated murine macrophages was ineffective in killing these bacteria. It seems that the concentration of NO2-/NO in phagolysosome in the activated macrophages is insufficient to kill the bacteria in situ. We concluded that L-arginine-dependent effector mechanism can hardly work on bacteria killing in activated macrophages.