Previously, we reported that vitamin K3 (VK3), but not VK1 or VK2 (=MK-4), inhibits the activity of human DNA polymerase γ (pol γ). In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF)-α induced by lipopolysaccharide (LPS). Moreover, MK-2 was found to inhibit the action of nuclear factor (NF)-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.
vitamin K; MK-2; DNA polymerase λ; enzyme inhibitor; anti-inflammation
Our previous studies found that 4 compounds, namely pseudohypericin, amentoflavone, quercetin, and chlorogenic acid in Hypericum perforatum ethanol extract synergistically inhibited lipopolysaccharide (LPS)-induced macrophage production of prostaglandin E2 (PGE2). Microarray studies led us to hypothesize that these compounds inhibited PGE2 production by activating suppressor of cytokine signaling 3 (SOCS3). In the current study we used siRNA to knockdown the expression of SOCS3 in RAW 264.7 macrophages and investigated the impact of H. perforatum extract and the 4 compounds on inflammatory mediators and cytokines. We found SOCS3 knockdown significantly compromised the inhibition of PGE2 and nitric oxide (NO) by the 4 compounds, but not by the extract. The 4 compounds, but not the extract decreased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), while both of them lowered interleukine-1β. SOCS3 knockdown further decreased IL-6 and TNF-α. Pseudohypericin was the major contributor to the PGE2 and NO inhibition in cells treated with the 4 compounds and its activity was lost with SOCS3 knockdown. Cyclooxygenase-2 (COX-2) and inducible NO synthase protein expression were not altered by the treatments, while COX-2 activity was decreased by the extract and the 4 compounds and increased by SOCS3 knockdown. In summary, we demonstrated that the 4 compounds inhibited LPS-induced PGE2 and NO through SOCS3 activation. The reduction of PGE2 can be partially attributed to COX-2 enzyme activity, which was significantly elevated with SOCS3 knockdown. At the same time, our results also suggest that constituents in H. perforatum extract were alleviating LPS-induced macrophage response through SOCS3 independent mechanisms.
Hypericum perforatum; Inflammation; Lipopolysaccharide; Macrophage; Nitric oxide; Prostaglandin E2; St. John’s wort; Suppressor of cytokine signaling 3
The present study examined the inhibitory effect of licorice compounds glycyrrhizin and a metabolite 18β-glycyrrhetinic acid on the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse and on the 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in differentiated PC12 cells. MPTP treatment increased the activities of total superoxide dismutase, catalase and glutathione peroxidase and the levels of malondialdehyde and carbonyls in the brain compared to control mouse brain. Co-administration of glycyrrhizin (16.8 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. In vitro assay, licorice compounds attenuated the MPP+-induced cell death and caspase-3 activation in PC12 cells. Glycyrrhizin up to 100µM significantly attenuated the toxicity of MPP+. Meanwhile, 18β-glycyrrhetinic acid showed a maximum inhibitory effect at 10µM; beyond this concentration the inhibitory effect declined. Glycyrrhizin and 18β-glycyrrhetinic acid attenuated the hydrogen peroxide- or nitrogen species-induced cell death. Results from this study indicate that glycyrrhizin may attenuate brain tissue damage in mice treated with MPTP through inhibitory effect on oxidative tissue damage. Glycyrrhizin and 18β-glycyrrhetinic acid may reduce the MPP+ toxicity in PC12 cells by suppressing caspase-3 activation. The effect seems to be ascribed to the antioxidant effect.
Glycyrrhizin; MPTP; MPP+; Brain tissue damage; Cell death; Inhibitory effect
This study was aimed to examine the effects of indole-3-carbinol (I3C) and β-phenylethyl isothiocyanate (PEITC), bioactive components present in cruciferous vegetable, on the production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10), in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. Possible mechanisms of the NO-inhibitory effects were also explored. The results indicated that I3C and PEITC inhibited NO production, and this suppression was associated with decreased production of TNF-α and IL-10 by activated macrophages. In addition, I3C suppressed NO production even after the inducible nitric oxide synthase (iNOS) protein had been produced, but such an inhibitory effect was not observed in cells treated with PEITC. Furthermore, both compounds reduced the NO contents generated from an NO donor in a cell-free condition, suggesting that the increased NO clearance may have contributed to the NO-inhibitory effects. In summary, both I3C and PEITC possessed antiinflammatory effects by inhibiting the productions of NO, TNF-α, and IL-10, although the NO-inhibitory effects may have involved in different mechanisms.
Acetogenins from the Annonaceous plant are a fatty acid-derived natural product. Chemically synthesized natural acetogenins, such as mucocin (compound 1), jimenezin (compound 2), muconin (compound 4), pyranicin (compound 5) and pyragonicin (compound 6) were investigated. Concomitantly, 19-epi jimenezin (compound 3), 10-epi pyragonicin (compound 7) and a γ-lactone (compound 8), which is estimated to be a biosynthetic precursor of acetogenins, were synthesized and investigated. Compounds 5 and 6 strongly inhibited, and compound 7 moderately inhibited the activities of mammalian DNA polymerases (pols), such as replicative pol α and repair/recombination-related pol β and λ, and also inhibited human DNA topoisomerase (topos) I and II activities. On the other hand, compounds 1–4 and 8 did not influence the activities of any pols and topos. Compound 5 was the strongest inhibitor of the pols and topos tested, and the IC50 values were 5.0–9.6 μM, respectively. These compounds also suppressed human cancer cell growth with almost the same tendency as the inhibition of pols and topos. Compound 5 was the strongest suppressor of the proliferation of the promyelocytic leukemia cell line, HL-60, in human cancer cell lines tested with an LD50 value of 9.4 μM, and arrested the cells at G1 phases, indicating that it blocks DNA replication by inhibiting the activity of pols rather than topos. This compound also induced cell apoptosis. The relationship between the three-dimensional molecular structure of acetogenins and these inhibitory activities is discussed. The results suggested that compound 5 is a lead compound of potentially useful cancer chemotherapy agents.
acetogenins; pyranicin; enzyme inhibitor; DNA polymerase; DNA topoisomerase; cell cycle arrest; apoptosis; anti-cancer agent; computer simulation
This study discovered that glycyrrhetinic acid inhibited the human 20S proteasome at 22.3 µM. Esterification of the C-3 hydroxyl group on glycyrrhetinic acid with various carboxylic acid reagents yielded a series of analogs with marked improved potency. Among the derivatives, glycyrrhetinic acid 3-O-isophthalate (17) was the most potent compound with IC50 of 0.22 µM, which was approximately 100-fold more potent than glycyrrhetinic acid.
Glycyrrhetinic acid; proteasome inhibitor; triterpene
Bacterial lipopolysaccharides (LPS) are potent inducers of macrophage activation, leading to the production of a number of proinflammatory mediators. Although several cytokines that prime macrophages for enhanced LPS-triggered responses have been identified, far less is known regarding the role that cytokines play in down-regulating macrophage responses to LPS. This study was designed to determine the effects of recombinant transforming growth factor beta 1 (rTGF-beta 1) on macrophage activation by LPS. Pretreatment of either mouse peritoneal macrophages or cells of the RAW 264.7 macrophage-like cell line with rTGF-beta 1 inhibited their ability to produce both tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) in response to LPS. These inhibitory effects were reversed by increasing the concentration of LPS or by priming cells with optimal concentrations of recombinant gamma interferon (rIFN-gamma). Pretreatment of cells with rTGF-beta 1 had only a modest inhibitory effect on the expression of TNF-alpha mRNA. By contrast, the expression of mRNA for the inducible form of nitric oxide synthase (iNOS), which is responsible for NO production in activated macrophages, was significantly inhibited by rTGF-beta 1 pretreatment. Thus, rTGF-beta 1-dependent suppression of macrophage TNF-alpha biosynthesis was manifest at a posttranscriptional level, whereas the inhibition of NO production correlated with a direct effect on iNOS gene expression. Importantly, both of these suppressive effects of rTGF-beta 1 were reversed by exposing the cells to priming concentrations of rIFN-gamma. As with NO production, immunocytochemical analysis of iNOS expression in LPS-stimulated macrophages revealed that rIFN-gamma and rTGF-beta 1 had antagonistic effects, with the former increasing, and the latter reducing, the number of iNOS-expressing cells induced by LPS. These data suggest that a balance between the priming effects of IFN-gamma and the inhibitory effects of TGF-beta 1 can determine the overall level of macrophage activation induced by LPS.
We reported that ailanthoidol, a neolignan from Zanthoxylum ailanthoides and Salvia miltiorrhiza Bunge, inhibited inflammatory reactions by macrophages and protected mice from endotoxin shock. We examined the anti-inflammatory activity of six synthetic ailanthoidol derivatives (compounds 1-6). Among them, compound 4, 2-(4-hydroxyphenyl)-5-(3-hydroxypropenyl)-7-methoxybenzofuran, had the lowest IC50 value concerning nitric oxide (NO) release from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Compound 4 suppressed the generation of prostaglandin (PG) E2 and the expression of inducible NO synthase and cyclooxygenase (COX)-2 induced by LPS, and inhibited the release of LPS-induced pro-inflammatory cytokines from RAW264.7 cells. The underlying mechanism of compound 4 on anti-inflammatory action was correlated with the down-regulation of mitogen-activated protein kinase and activator protein-1 activation. Compound 4 is potentially an effective functional chemical candidate for the prevention of inflammatory diseases.
Ailanthoidol derivatives; AP-1; Cytokines; Inflammation; Macrophage
N,N-Didesmethylgrossularine-1 (DDMG-1), a compound with a rare α-carboline structure, was isolated from an Indonesian ascidian Polycarpa aurata as responsible for the observed inhibitory activity against TNF-α production in lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells. DDMG-1 inhibited the mRNA level of mTNF-α, IκB-α degradation, and binding of NF-κB to the target DNA site in LPS-stimulated RAW 264.7 cells. Moreover, DDMG-1 had an inhibitory effect on the production of IL-8, which is produced in CD14+-THP-1 cells stimulated by LPS. DDMG-1 is thus a promising drug candidate lead compound for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis.
LPS; RAW 264.7 cells; NF-κB; TNF-α; N,N-didesmethylgrossularine-1; natural product
Cudrania tricuspidata extract is an important traditional herbal remedy for tumors, inflammation, gastritis, and liver damage and is predominantly used in Korea, China, and Japan. However, the anti-inflammatory effects of the extract have not yet been conclusively proved.
In this study, we investigated the effects of the CHCl3 fraction (CTC) of a methanol extract of C. tricuspidata on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells and mouse peritoneal macrophages, and the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in RAW 264.7 macrophage cells.
We observed that the protein expression levels of inducible NO synthase and COX-2 enzymes were markedly inhibited by CTC in a concentration-dependent manner. In addition, CTC reduced the production of TNF-α, IL-1β, and IL-6 in the LPS-stimulated RAW 264.7 macrophage cells.
Our results show that the C. tricuspidata extract could modulate macrophage-mediated inflammatory functions such as the overproduction of cytokines, NO, and PGE2. The CTC was found to be the active fraction in this context.
Cudrania tricuspidata; Nitric oxide; Prostaglandin E2; Lipopolysaccharide CHCl3 partitioned methanol extract of Cudrania tricuspidata; CTC, Lipopolysaccharide; LPS, prostaglandin E2; PGE2, nitric oxide; NO
The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria. Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-γ) and other signals, including tumor necrosis factor (TNF). Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF(70-80), was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to kill Plasmodia spp. Therefore, we investigated the capacity of TNF(70-80) to activate the murine macrophage cell line RAW264.7 infected with the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG). When RAW264.7 cells were pretreated with human TNF or TNF(70-80) in the presence of IFN-γ, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of 3H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants. TNF- or TNF(70-80)-induced macrophage activation was dependent on IFN-γ and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-γ antisera. Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors of l-arginine, which blocked the activation of inducible nitric oxide synthase. A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF(70-80), demonstrating that the effect of TNF(70-80) is dependent on signaling through TNF receptor I. The mimetic effects of TNF(70-80) on macrophage activation in vitro suggest that treatment with TNF(70-80) may modulate mycobacterial infections in vivo.
The tumor necrosis factor (TNF-alpha or TNF) gene is activated by both lipopolysaccharide (LPS) and cycloheximide in RAW 264.7 macrophages, whereas neither stimulus activates the gene in 3T3 fibroblasts. Moreover, the pattern of CG methylation within the TNF gene is readily distinguishable in DNA derived from cells of these two types. These findings would suggest that the TNF gene has been rendered inaccessible to transcription in the 3T3 cell environment. When RAW 264.7 cells are fused with 3T3 cells, an immortal pentaploid hybrid results. In the hybrid cell, all three TNF genes contributed by the RAW 264.7 cell parent become highly methylated according to the pattern observed in the 3T3 cell parent. Permanently transfected chloramphenicol acetyl transferase (CAT) reporter constructs, bearing 2.2 kb of upstream sequence (including the entire TNF promoter and 5'-untranslated region [UTR]) as well as 1.0 kb of downstream sequence (including the entire TNF 3'-UTR and termination sequence), are accessible in both RAW 264.7 cells and 3T3 cells, but are silenced in transition from the RAW 264.7 cell to the hybrid cell environment. Moreover, the endotoxin signaling pathway is abrogated, as assessed by transient transfection of hybrid cells with LPS-responsive CAT reporter constructs. It would therefore appear that the fusion of 3T3 cells and RAW 264.7 cells activates a system that silences the TNF gene, as well as the LPS signaling pathway. This system may operate to determine TNF gene accessibility and LPS responsiveness in the course of cell differentiation. The DNA sequences targeted within the TNF gene are included in the CAT reporter construct; therefore, the silencing element has been circumscribed to a region of DNA 3.2 kb in length.
High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. Advances in targeting HMGB1 represent a major challenge to improve the treatment of acute/chronic inflammation.
This study is aimed at verifying whether the inhibition of HMGB1 through dipotassium glycyrrhizate (DPG) is a good strategy to reduce intestinal inflammation.
Human colon adenocarcinoma cell line, HT29, human epithelial colorectal adenocarcinoma, Caco2, and murine macrophage cell line, RAW 264.7, were cultured to investigate the effect of DPG on the secretion of HMGB1. Acute colitis was induced in C57BL/6 mice through administration of 3% dextran sodium sulphate (DSS); a combined treatment with DSS and 3 or 8 mg/kg/day DPG was used to investigate the effects of DPG on intestinal inflammation. Animals were euthanized at seventh day and colonic samples underwent molecular and histological analyses.
DPG significantly reduces in vitro the release of HMGB1 in the extracellular matrix as well as expression levels of pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6, by inhibiting HMGB1. Moreover, DPG significantly decreases the severity of DSS-induced colitis in mice. Murine colonic samples show decreased mRNA levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6, as well as HMGB1 receptors, RAGE and TLR4. Finally, HMGB1, abundantly present in the feces of mice with DSS-induced colitis, is strongly reduced by DPG.
HMGB1 is an early pro-inflammatory cytokine and an active protagonist of mucosal gut inflammation. DPG exerts inhibitory effects against HMGB1 activity, significantly reducing intestinal inflammation. Thus, we reason that DPG could represent an innovative tool for the management of human intestinal inflammation.
Edema Factor toxin (EF) of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3′,5′-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-Oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15 μg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin-(ET) catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure-activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.
Adenylyl cyclase toxin inhibitor; non-nucleotide inhibitors; toxicity profiling; computer aided design; cell based assay
Macrophage-like RAW264.7 cells are killed by the combination of gamma interferon (IFN-gamma) treatment and infection with Rickettsia prowazekii. The roles of tumor necrosis factor alpha (TNF-alpha), the nitric oxide synthase pathway, and lipopolysaccharide (LPS) in this killing were investigated. R. prowazekii, both the Breinl and Madrid E strains, induced RAW264.7 cells to produce TNF-alpha. However, dead rickettsiae (which cannot kill the IFN-gamma-treated RAW264.7 cells) induced the production of as much TNF-alpha as viable rickettsiae. Inhibition of the production of TNF-alpha (by the addition of actinomycin D or emetine during the rickettsial infection) or neutralization of TNF-alpha (by the addition of polyclonal rabbit anti-mouse TNF-alpha serum both during the IFN-gamma treatment and during the rickettsial infection) did not inhibit the killing of the RAW264.7 cells. Addition of polymyxin B (which inhibits many effects of LPS) during the IFN-gamma treatment did not inhibit the ability of IFN-gamma to prepare the RAW264.7 cells to be killed by R. prowazekii. Suppression of nitrite production by addition of the nitric oxide synthase inhibitor aminoguanidine both during the IFN-gamma treatment and during the rickettsial infection also did not inhibit the killing of the RAW264.7 cells. R. prowazekii-mediated killing of the RAW264.7 cells was dramatically suppressed in cultures treated with IFN-gamma plus LPS compared with that in cultures treated with IFN-gamma alone, and inhibition of nitric oxide synthase restored the rickettsia-induced killing of the RAW264.7 cells in cultures treated with IFN-gamma plus LPS. These data indicate that (i) TNF-alpha, LPS, and the nitric oxide synthase pathway are not required in order for IFN-gamma to prepare RAW264.7 cells to be killed by R. prowazekii; (ii) neither TNF-alpha nor the nitric oxide synthase pathway is responsible for the killing of the IFN-gamma-treated RAW264.7 cells by R. prowazekii; and (iii) in cultures treated with IFN-gamma plus LPS and then incubated with rickettsiae, a nitric oxide synthase pathway-dependent mechanism inhibits the killing of the RAW264.7 cells.
We have investigated the anti-inflammatory effects of Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) and carrageenan (Carr)-induced mouse paw edema model. When RAW264.7 macrophages were treated with cinnamic aldehyde together with LPS, a significant concentration-dependent inhibition of nitric oxide (NO), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) levels productions were detected. Western blotting revealed that cinnamic aldehyde blocked protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear transcription factor kappa B (NF-κB), and IκBα, significantly. In the anti-inflammatory test, cinnamic aldehyde decreased the paw edema after Carr administration, and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw tissue. We also demonstrated cinnamic aldehyde attenuated the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity in the edema paw after Carr injection. Cinnamic aldehyde decreased the NO, TNF-α, and PGE2 levels on the serum level after Carr injection. Western blotting revealed that cinnamic aldehyde decreased Carr-induced iNOS, COX-2, and NF-κB expressions in the edema paw. These findings demonstrated that cinnamic aldehyde has excellent anti-inflammatory activities and thus has great potential to be used as a source for natural health products.
Carnosic acid (CA) is a diterpene compound exhibiting antioxidative, anticancer, anti-angiogenic, anti-inflammatory, anti-metabolic disorder, and hepatoprotective and neuroprotective activities. In this study, the effect of CA on various skin inflammatory responses and its inhibitory mechanism were examined. CA strongly suppressed the production of IL-6, IL-8, and MCP-1 from keratinocyte HaCaT cells stimulated with sodium lauryl sulfate (SLS) and retinoic acid (RA). In addition, CA blocked the release of nitric oxide (NO), tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2) from RAW264.7 cells activated by the toll-like receptor (TLR)-2 ligands, Gram-positive bacterium-derived peptidoglycan (PGN) and pam3CSK, and the TLR4 ligand, Gram-negative bacterium-derived lipopolysaccharide (LPS). CA arrested the growth of dermatitis-inducing Gram-positive and Gram-negative microorganisms such Propionibacterium acnes, Pseudomonas aeruginosa, and Staphylococcus aureus. CA also blocked the nuclear translocation of nuclear factor (NF)-κB and its upstream signaling including Syk/Src, phosphoinositide 3-kinase (PI3K), Akt, inhibitor of κBα (IκBα) kinase (IKK), and IκBα for NF-κB activation. Kinase assays revealed that Syk could be direct enzymatic target of CA in its anti-inflammatory action. Therefore, our data strongly suggest the potential of CA as an anti-inflammatory drug against skin inflammatory responses with Src/NF-κB inhibitory properties.
Corticotropin-releasing hormone (CRH) exerts an anti-inflammatory effect indirectly, via cortisole production, and a proinflammatory effect directly on immune cells. The aim of the present work was to examine the effect of CRH on macrophage-derived cytokines both in vitro and in vivo. For the in vitro experiments we used two types of macrophages: (i) the RAW264.7 monocyte/macrophage cell line and (ii) thioglycolate-elicited peritoneal macrophages from BALB/c mice. We have found that CRH enhanced lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 production. For the in vivo experiments we have used the LPS-induced endotoxin shock model in BALB/c mice, an established model for systemic inflammation in which macrophages are the major source of the proinflammatory cytokines responsible for the development of the shock. Administration of antalarmin, a synthetic CRH receptor 1 (CRHR1) antagonist, prior to LPS prolonged survival in a statistically significant manner. The effect was more evident at the early stages of endotoxin shock. CRHR1 blockade suppressed LPS-induced elevation of the macrophage-derived cytokines TNF-α, IL-1β, and IL-6, confirming the role of CRH signals in cytokine expression. In conclusion, our data suggest that CRH signals play an early and crucial role in augmenting LPS-induced proinflammatory cytokine production by macrophages. Our data suggest that the diffuse neuroendocrine system via CRH directly affects the immune system at the level of macrophage activation and cytokine production.
Derivatives of oleanolic acid, ursolic acid and glycyrrhetinic acid substituted with electron withdrawing groups at the 2-position in the A-ring which also contains a 1-en-3-one structure are potent inhibitors of cancer cell growth. In this study, we have compared the effects of several 2-substituted analogs of triterpenoid acid methyl esters derived from ursolic and glycyrrhetinic acid on proliferation of KU7 and 253JB-V bladder and Panc-1 and Panc-28 pancreatic cancer cells. The results show that the 2-cyano and 2-trifluoromethyl derivatives were the most active compounds. The glycyrrhetinic acid derivatives with the rearranged C-ring containing the 9(11)-en-12-one structure were generally more active than the corresponding 12-en-11-one isomers. However, differences in growth inhibitory IC50 values were highly variable and dependent on the 2- substitutent (CN vs. CF3) and cancer cell context.
glycyrrhetinate analogs; growth inhibition; bladder cancer; pancreatic cancer
Lipid A is the active center of bacterial lipopolysaccharide (LPS), which exhibits diverse biological activities via the production of various mediators. We investigated the production of nitric oxide (NO), one of the mediators, by a murine macrophage cell line, RAW264.7, upon stimulation with a series of monosaccharide lipid A analogues to elucidate the relationship of structure and activity in NO production. The production of other representative mediators, such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was also investigated to compare the structural requirements for the production of these cytokines with those for the production of NO. Structure-activity relationships in NO production correlated well with those in the production of TNF-α and IL-6. Among the lipid A analogues possessing different numbers of acyl groups on a 4-O-phosphono-d-glucosamine backbone, compounds like GLA-60 that possess three tetradecanoyl (C14) groups exhibited stronger activities in the production of the mediators than compounds possessing four or two C14 groups. Time course study of the production of these mediators showed that production of NO started and peaked later than those of TNF-α and IL-6. Neither neutralization of TNF-α activity by antibody nor suppression of TNF-α production by pentoxifylline showed a significant suppressive effect on production of NO and IL-6 upon stimulation with LPS or lipid A analogues. Neutralization of IL-6 activity by antibody showed no significant suppressive effect on production of NO and TNF-α. A monosaccharide lipid A analogue (GLA-58) which exhibited no detectable agonistic activity showed a suppressive effect on the production of all three mediators upon stimulation with LPS or lipid A analogues. These results indicate that signals for NO production by LPS agonists in murine macrophages are transduced in good correlation with those for production of TNF-α and IL-6, although they are not transduced via production of those cytokines.
The anti-inflammatory activity of Antrodia camphorata (AC) grown on germinated brown rice (CBR) extract was evaluated in vitro and in vivo. CBR suppressed the release of nitric oxide (NO) and prostaglandin (PG) E2 from lipopolysaccharide-(LPS-)stimulated RAW264.7 cells. CBR inhibited the level of inducible nitric oxide synthase (iNOS) and cyclooxygenase-(COX-)2 proteins, and it activated p38-MAPK, extracellular signal-related kinases (ERK), and NF-κB in LPS-stimulated RAW264.7 macrophages. LPS-induced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expression was reduced in CBR-treated RAW264.7 cells. In concert with in vitro data, CBR suppressed the levels of dextran-sulfate-sodium-(DSS-)induced iNOS and COX-2 proteins in the colon tissue. CBR treatment inhibited activated p38-MAPK, ERK, and NF-κB proteins in the colon tissue of DSS-induced mice. TNF-α and IL-6 mRNA expression was reduced in DSS+CBR-treated mice. The disease activity index and histological scores were significantly lower in CBR-treated mice (500 mg/kg/day) than in DSS-treated mice (P < 0.05 versus DSS). This is the first report of anti-inflammatory activity of CBR in DSS-induced acute colitis. These results suggest that CBR is a promising, potential agent for preventing acute colitis through the inhibition of NF-κB signaling and its upstream signaling molecules, including MAPKs.
Bacterial lipopolysaccharide and some cytokines can activate macrophages to secrete nitric oxide. Macrophage-derived nitric oxide is a key cytotoxic factor for microbicidal and tumoricidal processes. We report here that a monoclonal antibody specific for beta interferon inhibited lipopolysaccharide-induced nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages and macrophage-like cell line RAW 264.7. In addition, exogenous added beta interferon enabled lipopolysaccharide-hyporesponsive thioglycolate-elicited C3H/HeJ peritoneal macrophages to produce nitric oxide in response to lipopolysaccharide. These data support the concept that beta interferon provides an essential signal(s) for lipopolysaccharide-triggered nitric oxide production by mouse macrophages. Heat-killed Staphylococcus aureus, a gram-positive bacterium which was unable to initiate nitric oxide production in thioglycolate-elicited C3HeB/FeJ peritoneal macrophages in vitro, promoted nitric oxide formation in the presence of beta interferon, suggesting that beta interferon may be a general cofactor necessary for bacterium-derived stimulus-induced nitric oxide production in these macrophages. However, neither beta interferon nor tumor necrosis factor alpha, alone or in combination, triggered nitric oxide production in thioglycolate-elicited mouse peritoneal macrophages, demonstrating that these macrophage-derived cytokines, while necessary, were not sufficient by themselves for the induction of nitric oxide production in these cells. On the other hand, gamma interferon and tumor necrosis factor alpha acted together to induce nitric oxide production in vitro in the absence of lipopolysaccharide in thioglycolate-elicited mouse peritoneal macrophages, indicating that these two types of interferons provided different signals during the activation of these macrophages.
Glycyrrhizic acid (GA), the main component of radix glycyrrhizae, has a variety of pharmacological activities. In the present study, suspensions of GA nanoparticles with the average particle size about 200nm were prepared by a supercritical antisolvent (SAS) process. Comparative studies were undertaken using lipopolysaccardide(LPS)-stimulated mouse macrophages RAW 264.7 as in vitro inflammatory model. Several important inflammation mediators such as NO, PGE2, TNF-α and IL-6 were examined. These markers were highly stimulated by LPS and were inhibited both by nano-GA and unprocessed GA in a dose-dependent manner, especially PGE2 and TNF-α. However nano-GA and unprocessed GA inhibited NO only at a high concentration. In general, we found that GA nanoparticle suspensions exhibited much better anti-inflammatory activities compared to unprocessed GA.
glycyrrhizic acid; nanoparticle; mouse macrophages RAW 264.7; inflammatory cytokines
Glycyrrhizin (GA) and primary metabolite 18β-glycyrrhetinic acid (GRA) are pharmacologically active components of the medicinal licorice root, and both have been shown to have antiviral and immunomodulatory properties. Although these properties are well established, the mechanisms of action are not completely understood. In this study, GA and GRA were tested for the ability to inhibit rotavirus replication in cell culture, toward a long term goal of discovering natural compounds that may complement existing vaccines.
Epithelial cells were treated with GA or GRA various times pre- or post-infection and virus yields were measured by immunofluorescent focus assay. Levels of viral proteins VP2, VP6, and NSP2 in GRA treated cells were measured by immunoblot to determine if there was an effect of GRA treatment on the accumulation of viral protein.
GRA treatment reduced rotavirus yields by 99% when added to infected cultures post-- virus adsorption, whereas virus yields in GA treated cultures were similar to mock treated controls. Time of addition experiments indicated that GRA-mediated replication inhibition likely occurs at a step or steps subsequent to virus entry. The amounts of VP2, VP6 and NSP2 were substantially reduced when GRA was added to cultures up to two hours post-entry.
GRA, but not GA, has significant antiviral activity against rotavirus replication in vitro, and studies to determine whether GRA attenuates rotavirus replication in vivo are underway.
Rotavirus; Licorice; 18beta-glycyrrhetinic acid; Antiviral
It has been suggested that Echinacea has anti-inflammatory activity in vivo. Nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta are important mediators in the inflammatory response. The effect of alcohol extracts of E. angustifolia (EA), E. pallida (EPA) and E. purpurea (EP) on the production of these inflammatory mediators in both LPS-stimulated RAW 264.7 macrophages in vitro and murine peritoneal exudate cells (PECs) in vivo were investigated. As macrophages produce these inflammatory mediators in response to pathogenic infection, parallel cultures of macrophages were studied for phagocytosis and intracellular killing of Salmonella enterica. EPA and EP in vitro inhibited NO production and TNF-α release in a dose-dependent manner. RAW 264.7 cells treated with EA or EP showed decreased killing over 24 h, although EA enhanced bacterial phagocytosis. Upon bacterial infection, RAW 264.7 cells produce high levels of NO; however, an Echinacea-mediated decrease in NO production was observed. Echinacea alcohol extracts administered orally at 130 mg/kg per day for seven days had a weak effect on NO production and phagocytosis by LPS-stimulated PECs. The results indicated that all Echinacea species significantly decreased inflammatory mediators in vitro, however, only EA and EP reduced bacterial killing. Oral administration of Echinacea alcohol extracts did not adversely affect the development and anti-bacterial function of inflammatory PECs in vivo, however, NO production was decreased during bacterial infection of PECs.
Echinacea; macrophage; nitric oxide; phagocytosis; tumor necrosis factor-alpha