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Mtb (Mycobacterium tuberculosis) is a pathogenic bacterium that causes tuberculosis infection (TB). Mtb -secreted proteins have recently been investigated as virulence factors, as well as therapeutic and vaccine possibilities. The early-secreted antigen target MTB48 is one of these proteins that has been explored as a cocktail antigen in the serodiagnosis of active tuberculosis. However, there exists no information about the function or control of MTB48's inflammatory activity in macrophages at the site of inflammation. As a result, the goal of this research was to figure out what processes are involved in MTB48's function. MTB48 stimulated inflammation in LPS induced macrophages at both the protein and mRNA levels, which was interesting. MTB48 aided LPS induced IB phosphorylation and NF-κB translocation. MTB48 also led to the phosphorylation of MAPK signaling protein. These findings imply that MTB48 can enhance inflammatory activity via NF-κB and MAPK signaling by upregulating COX-2, iNOS, NO and PGE 2. Many tuberculosis antigens have been tested for the development of rapid serological diagnosis. The results of this study suggest that MTB48 is a very high conservative antigen and is a major factor causing inflammatory reactions, suggesting that it can help control and diagnose tuberculosis. [Display omitted] • The function and control of the Mtb-secreted protein MTB48's inflammatory activity in macrophages at the site of inflammation. • MTB48 enhanced inflammatory activity via NF-κB and MAPK signaling by upregulating COX-2, iNOS, NO, and PGE2. • MTB48 could be a potential target for therapeutic and vaccine possibilities, as well as a diagnostic tool for active tuberculosis. • MTB48 is a highly conservative antigen and a major factor in causing inflammatory reactions. [ABSTRACT FROM AUTHOR]

Malaria is a parasitic infection responsible for high morbidity and mortality rates worldwide. During the disease, phagocytosis of infected red blood cells by the macrophages induces the production of reactive oxygen (ROS) and nitrogen species (RNS), culminating in parasite death. Curcumin (CUR) is a bioactive compound that has been demonstrated to reduce the production of pro-inflammatory cytokines and chemokines produced by macrophages but to reduce parasitemia in infected mice. Hence, the main purpose of this study is to investigate whether curcumin may interfere with macrophage function and polarization after Plasmodium berghei infection in vitro. In our findings, non-polarized macrophage (M0), classically activated (M1), and alternatively activated (M2) phenotypes showed significantly increased phagocytosis of infected red blood cells (iRBCs) when compared to phagocytosis of uninfected red blood cells (RBCs) 3 h after infection. After 24 h, M1 macrophages exposed to RBCs + CUR showed greater elimination capacity when compared to macrophages exposed to iRBCs + CUR, suggesting the interference of curcumin with the microbicidal activity. Additionally, curcumin increased the phagocytic activity of macrophages when used in non-inflammatory conditions (M0) and reduced the inducible nitric oxide synthase (iNOS) and arginase activities in all macrophage phenotypes infected (M0, M1, and M2), suggesting interference in arginine availability by curcumin and balance promotion in macrophage polarization in neutral phenotype (M0). These results support the view of curcumin treatment in malaria as an adjuvant, promoting a balance between pro- and anti-inflammatory responses for a better clinical outcome. [ABSTRACT FROM AUTHOR]

[Display omitted] The potentiation of pharmacological effects can be achieved through several strategies, such as the association of substances and delivery in nanostructured systems. In practice, potentiation can be measured by the law of mass action and joint evaluation of the combination index (CI) and dose–response curves. In this context, this study aimed to evaluate the anti-inflammatory effect of the association of β-caryophyllene and indomethacin in the free form and delivered in nanoemulsions using the in vitro model of LPS-stimulated murine macrophage. The results indicated potentiation of the anti-inflammatory effect of nanoemulsified substances compared to free substances, as well as synergistic action between the sesquiterpene and the selected NSAID. In comparison, the association of β-caryophyllene and indomethacin in the free form inhibited the production of nitric oxide by 50% at 48.60 µg/mL (CI = 0.21), while the nanoemulsified association of these substances resulted in an IC 50 of 1.45 µg/mL (CI = 0.14). In parallel, cytotoxicity assays on HaCaT and MRC-5 cell lines demonstrated the safety of IC 50 -equivalent concentrations of the anti-inflammatory action, and no irritating effects on the chorioallantoic membrane of embryonated eggs were observed (HET-CAM assay). The results suggest that β-caryophyllene may be an alternative to replace an inert oily core in nanoemulsion systems when anti-inflammatory effects are desirable. [ABSTRACT FROM AUTHOR]

Conventionally T-cell receptors (TCRs) have so far been considered as a T-lymphocyte privilege. However, recent findings also place TCR expression in non-lymphoid cells, namely neutrophils, eosinophils and macrophages. In order to examine the ectopic expression of TCR, this study focused on RAW 264.7 cells, which have been broadly used for their macrophage properties. Immunofluorescence staining detected 70% and 40% of the cells to express TCRαβ and TCRγδ respectively, which was also verified by RT-PCR experiments and confocal microscopy analysis. Interestingly, except from the predicted 292 and 288 bp gene products for the α- and γ-chain, additional products at 220 and 550 bp could be detected, respectively. RAW 264.7 cells also expressed the co-stimulatory CD4 and CD8 markers at a percentage of 61% and 14% respectively, which supported the expression of TCRs. However, only low numbers of cells expressed CD3ε and CD3ζ (9% and 7% respectively). Such observations contradicted the existing knowledge, and indicated that TCRs would be supported by other molecules for reaching the membrane and transducing their signal. Such candidate molecules could be the Fcγ receptors (FcγRs). Indeed, the FcγRII/III receptor was found to be expressed in 75% of the cells, which also expressed at a percentage of 25% major histocompatibility complex (MHC) class II molecules. Engagement of the FcγRII/III receptor by a recombinant IgG2aCH2 fragment, except from stimulating the macrophage-dependent properties of the cells, was shown to reduce expression of TCRαβ and γδ indicating that FcγRII/III was indeed used by TCRs for their transport to the cell membrane. In order to examine the ability of RAW 264.7 cells to simultaneously display antigen presenting- and T-cell properties, functional experiments as to antigen-specific antibody and IL-2 production were performed. In in vitro immunization assays in the presence of naïve B cells, RAW264.7 failed to promote antibody production. However, RAW 264.7 cells could compete with antigen-stimulated macrophages but not T cells when applied to a system of in vivo antigen-sensitized cells followed by an in vitro immunization protocol. Interestingly, simultaneous addition of antigen and the IgG2aCH2 fragment to RAW 264.7 cells could promote IL-2 production from the cells, indicating that FcγRII/III activation could also support TCR stimulation. Extrapolating these findings to cells of the myeloid origin, the above results dictate novel regulatory mechanisms towards the alteration of the immune response. [Display omitted] • RAW 264.7 macrophages express TCRαβ/TCRγδ and CD4/CD8 but insufficient CD3 levels. • Recombinant IgG2aCH2 decreases TCRαβ/TCRγδ indicating that FcγRII/III is involved in TCR membrane transport. • RAW 264.7 cells may compete macrophages but not T cells in an antigen specific antibody production. • Stimulation of RAW 264.7 cells with rIgG2aCH2 and antigen induces IL-2 production. • Novel regulatory pathways by TCR+ myeloid cells towards stimulation of immunity. [ABSTRACT FROM AUTHOR]

Host inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Introduction: Several experimental models have been designed to promote the development of new anti-inflammatory drugs. The in vitro model using RAW 264.7 cells has been widely used. However, there is still no consensus on which inflammatory mediators should initially be measured to screen for possible anti-inflammatory effects. To determine the rationality of measuring inflammatory mediators together with NO, such as the levels of tumor necrosis factor (TNF)-α, and interleukins (IL) 1β and 6, we carried out this systematic review (SR) and meta-analysis (MA). Methodology: We conducted this SR and MA in accordance with the Preferred Reporting of Systematic Reviews and Meta-Analysis and the Cochrane Handbook for Systematic Reviews of Intervention. This review was registered in the Open Science Framework (https://doi.org/10.17605/OSF.IO/8C3HT). Results: LPS-induced cells produced high NO levels compared to non-LPS induced, and this production was not related to cell density. TNF-α, IL-1β, and IL-6, also showed high levels after cells had been stimulated with LPS. Though with some restrictions, all studies were reliable, as the risk of bias was detected in the test compounds and systems. Conclusion: Measurement of NO levels may be sufficient to screen for possible anti-inflammatory action in the context of LPS-induced RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

Introduction: Sungkai (Peronema canescens, Jack.) contains polysaccharides, terpenoids, alkaloids, and polyphenols which have pharmacological activity as immunostimulants. Objective: This study aimed to see how the effect of Sungkai extract as an immunostimulant agent was carried out in vitro and in vivo. Materials and Methods: This study was conducted using two methods, namely in vivo and in vitro. In vivo research method was conducted to test the activity and phagocytic capacity of macrophage cells, the percentage of leukocytes, and the total number of leukocytes. This study used 30 male white mice as the test animals that were randomly divided into 5 treatment groups. Each group was consisting of 6 mice which were given different treatments. The negative control group was given with the 0.5% NaCMC suspension, the mice test substance group was given with the suspension of Sungkai ethanol extract with various doses of 800, 400, and 200 mg/kgBW, and lastly the comparison group was given with the Stimuno in a dose of 50 mg/kg orally for 7 days. On day 8, blood was taken from the mice's vein to count the number and percentage of its leukocytes, then followed by the intraperitoneal injection of a Staphylococcus aureus bacteria suspension. After 1 hour of administration of the bacterial suspension, the peritoneal fluid was taken to be observed for its activity and phagocytic capacity of macrophage cells. The in vitro research method was used to test the viability and immunostimulatory activity of RAW 264.7 cells with the Sungkai extraction at the concentration of 1.10, 100 g/m. This cell viability test using the microtetrazolium (MTT) method aims to see whether the Sungkai sample used is safe and not toxic to RAW 264.7 cells by observing at the cell viability value that should exceed >90%. The concentration of Sungkai extraction at 1.10, 100 g/mL was found to be safe and non-toxic to RAW 264.7 cells with a viability value of >90%. Thus, this concentration of Sungkai extraction can be performed for its immunostimulatory activity test on LPS induced of RAW 264.7 cells by observing their levels of IL-6 and TNF-a. (proinflammatory cytokines) were compared with the LPS alone as a control using the sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method. Results: The observations were analyzed by one-way ANOVA and Duncan's follow-up test (significance was taken at p<0.05). The results showed that variations in concentration increased significantly (p<0.05) on the activity and phagocytic capacity of macrophage cells, along with the total leukocyte cells. The percentage of leukocytes showed that the cells had a significant increase (p<0.05). It was found that the Sungkai extraction on 1.10, 100 g/mL could significantly increase the concentration of TNF- and IL-6 (p<0.05) which were tested by one-way ANOVA and followed by Duncan's post hoc test. Conclusion: Sungkai leaf extract (Peronemacanescsens Jack.) in a dose of800, 400, and 200 mg/kgBW has an immunostimulant effect both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Objective To explore the protective effect of exosomes derived from M2 macrophages (M2 Exos) on concanavalin (Con) A induced autoimmune hepatitis in mice. Methods RAW264.7 macrophages were treated with IL-4 (20 μg/L) for 24 h to obtain cell culture supernatant containing M2 Exos. M2 Exos were separated by ultracentrifugation and identified by transmission electron microscope, nanoparticle tracking analysis and Western blot assay. Immunofluorescence was used to detect whether M2 Exos were uptaken by RAW264.7 macrophages. Twelve C57BL/6J mice were randomly divided into PBS group, DiR-M0 Exos group, DiR-M2 Exos group and DiR dye control group. PBS solution, DiR-M0 Exos (100 μg), DiR-M2 Exos (100 μg) and DiR dye were given to corresponding groups through tail veins after the injection of Con A (15 mg/kg) for 1h. The distribution of Exos in liver, spleen, heart, lung, kidney and intestine of mice with autoimmune hepatitis was observed by vivo imaging system. Twenty C57BL/6J mice were randomly divided into control group (PBS solution), Con A group (15 mg/kg Con A), M0 Exos group (15 mg/kg Con A + 200 μg M0 Exos) and M2 Exos group (15 mg/kg Con A+200 μg M0 Exos), 5 mice in each group. Peripheral blood and liver tissues were collected 12 h after Con A injection. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automated biochemistry analyzer. The histological profiles of liver tissue were observed by hematoxylin-eosin (HE) staining. The mRNA expression levels of liver TNF-α and IL-6 were detected with quantitative real-time PCR (qPCR). The changes of liver macrophage subpopulations were detected with flow cytometric analysis. Results M2 Exos were successfully induced and isolated, which can be taken up by RAW264.7 macrophages in large quantities. M2 Exos were mainly absorbed by liver and spleen of mice through tail vein injection. Compared with Control group, the serum levels of ALT and AST were significantly increased in Con A group (both P＜0.05). The disordered liver structure and increased necrosis areas were found in Con A group. Meanwhile, the mRNA expression of TNF- α and IL-6 in liver increased (both P＜0.05). The infiltration of liver monocyte-derived macrophages (MoMFs) increased (P＜0.05). Compared with Con A group, the serum levels of ALT and AST were significantly decreased in M2 Exos group (both P＜0.05). M2 Exos group also showed lower liver TNF-α, IL-6 mRNA expression levels (both P＜0.05) and lower MoMFs infiltration. HE staining showed that the necrosis areas of liver cells were relieved in M2 Exos group. Conclusion M2 Exos plays a protective role in autoimmune hepatitis in model mice，and its mechanism may be related to the decreased production of liver inflammatory cytokines and decreased recruitment of MoMFs. [ABSTRACT FROM AUTHOR]

Mud lobsters are crustaceans from the genus Thalassina which are lesser known and seldom seen but are nevertheless an important organism to the mangrove ecosystem. In Malaysia and Thailand, mud lobsters are eaten by locals as treatment for asthma. It is traditionally believed that they are effective in reducing the number of asthma attacks and severity of asthma symptoms. However, the therapeutic potential of mud lobster extract remains unclear and has not been fully elucidated or reported in any scientific study. The objectives of this study are to investigate the anti-inflammatory potential of mud lobster, Thalassina anomala extracts (hexane, chloroform and methanol) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and to identify the potential bioactive compounds involved. An MTT assay was performed to determine the cytotoxicity of the T. anomala extracts on RAW 264.7 macrophages. Nitrite quantification assay and enzyme-linked immunosorbent assay (ELISA) were conducted to investigate the ability of the T. anomala extracts to suppress the secretion and expression of nitric oxide (NO), Prostaglandin E2 (PGE2) and proinflammatory cytokines (TNF-a, IL-6 and IL-1ß) in LPS-stimulated macrophages. GC-MS analysis was done to identify putative metabolites. The hexane extract of T. anomala showed anti-inflammatory activity by significantly inhibiting the LPS-induced production of NO, PGE2, interleukin- (IL-) 6, IL-1ß and tumour necrosis factor-alpha (TNF-a) in a concentration-dependent manner. Hexane extract treatment with 100 µg/mL has decreased the NO secretion into 37 µM. Meanwhile, hexane extract at concentration of 100 µg/mL able to significantly suppressed PGE2,TNF-a, IL-6 and IL-1ß production into 2015 pg/mL, 2406 pg/mL, 460 pg/mL and 9.6 pg/mL, respectively. GC-MS analysis of the hexane extract revealed the presence of 19 putative compounds. The identified compounds were reported to have anti-inflammatory, antioxidant and antibacterial activities. These results suggest that the hexane extract of T. anomala potentially has anti-inflammatory properties and concentration dependently suppressed NO, PGE2 and proinflammatory cytokines' production in LPS-stimulated macrophages. The findings provide a rational basis of the traditional use of mud lobster for inflammationassociated ailments. [ABSTRACT FROM AUTHOR]

Danshensu, isolated from Salvia miltiorrhiza (Danshen), is known to have anti-inflammatory properties. Therefore danshen is extensively used in many nutraceutical formulations. Reactive oxygen species are essential for the development of hypoxia-induced inflammation. Generation of reactive oxygen species by infiltrating macrophages is common in various diseases such as cardiovascular disease, neurodegenerative disease, tumor, and aging. To explore the mechanism underlying the attenuation of inflammation, we used RAW 264.7 cells as a model and hypoxia as an inducer of inflammation. The results showed the protective mechanism of danshensu on reactive oxygen species production, hypoxia-inducible factor 1-alpha expression, c-Jun N-terminal kinase phosphorylation, nuclear translocation of nuclear factor kappa B, and inducible nitric oxide synthase expression following hypoxia in RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate the inhibitory effect of 2,4 bis-[(4-ethoxyphenyl)azo] 5-(3-hydroxybenzylidene) thiazolidine-2,4-dione (TZD-OCH2CH3) on the cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. The effects of TZD-OCH2CH3 on COX-2 and iNOS mRNA expression in LPS-activated RAW 264.7 cells were detected by real time PCR. Also, to understand structure and substrate specificity, we have utilized molecular docking simulations (AutoDock Vina) and the active residues in the binding pocket were determined from COX-2 and iNOS. The treatment of RAW 264.7 cells with TZD-OCH2CH3 significantly inhibited LPS-induced COX-2 mRNA expression, corresponding to 46.1% and 61.06% at 30 and 60 µg/mL, respectively. The present study revealed that the TZD-OCH2CH3 had a little effect on iNOS mRNA expression. Meanwhile, the TZD-OCH2CH3 also could inhibit the production of NO compared to single LPS-stimulated cell. According to the results obtained, TZD-OCH2CH3 dramatically suppressed lipopolysaccharide (LPS) induced nitric oxide (NO) production after 24 h, in a concentration-dependent manner with an IC50 of 65 µg/mL. Our data suggest that TZD-OCH2CH3, as a functionally novel agent, inhibits the inflammatory pathway via suppression of COX-2 mRNA expression and also by the inhibition of the iNOS activity. Therefore, this compound could be suggested as a novel therapeutic strategy for inflammation-associated disorders. [ABSTRACT FROM AUTHOR]

The neonicotinoid imidacloprid was promoted in the market because of widespread resistance to other insecticides, plus its low mammalian impact and higher specific toxicity towards insects. This study aimed to evaluate the immunomodulatory effect of imidacloprid on macrophages. RAW 264.7 cells were incubated to 0–4000 mg/L of imidacloprid for 24 and 96 h. Imidacloprid presented a concentration-dependent cytotoxicity after 24 h and 96 h incubation for MTT reduction (3-(4,5-dimethyl-thiazol-2-yl)− 2,5-diphenyltetrazolium bromide) (EC 50 519.6 and 324.6 mg/L, respectively) and Neutral Red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) assays (EC 50 1139.0 and 324.2 mg/L, respectively). Moreover, imidacloprid decreased the cells' inflammatory response and promoted a mitochondrial depolarization. The complex II and succinate dehydrogenase (SDH) activities in RAW 264.7 cells incubated with imidacloprid increased more at 24 h. These results suggest that imidacloprid exerts an immunomodulatory effect and mitochondria can act as regulator of innate immune responses in the cytotoxicity mediated by the insecticide in RAW 264.7 cells. [Display omitted] • A commercial formulation of imidacloprid impaired the inflammatory response in RAW 264.7 cells; • The immunotoxic effects were associated with long term exposure to imidacloprid; • The mitochondria is a primary target of imidacloprid cytotoxicity. [ABSTRACT FROM AUTHOR]

Ginsenoside Rh2, a protopanaxadiol saponin from ginseng, has been reported to have strong anti-inflammatory activity. However, the concentration of ginsenoside Rh2 is very low (>0.001%) in the total ginseng extracted, which is not enough for production despite its high pharmacological effects. Thus, in this study, we evaluated the anti-inflammatory effect of ginsenoside Rh2-mix (GRh2-mix) on lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. From the high-performance liquid chromatography analysis, it was confirmed that the GRh2-mix was mainly composed of 20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3. The LPS-stimulated RAW 264.7 cells were treated with different concentrations of GRh2-mix (100, 200, 400, 500 μg/mL). The cell counting kit-8 assay showed that the GRh2-mix treatment increased cell proliferation in LPS-stimulated RAW 264.7 murine macrophage cells. The GRh2-mix inhibited nitric oxide production in a dose-dependent manner, suggesting an anti-inflammatory effect. Furthermore, reverse transcription polymerase chain reaction and Western blot results also indicated that the GRh2-mix suppressed inflammatory genes such as iNOS, TNF-α, COX-2, IL-1β, IL-6, and NF-κB. In summary, these results suggest that the GRh2-mix exhibits anti-inflammatory activity via the downregulation of the NF-κB pathway and has high efficiency with a simple production procedure. [ABSTRACT FROM AUTHOR]

Objective(s): In traditional Chinese medicine, gamboge can detoxify bodies, kill parasites, and act as a hemostatic agent. Recent studies have demonstrated that gambogic acid (GBA) suppressed inflammation in arthritis, and also presented antitumor effect. Thus, this study investigated the new biological properties of GBA on macrophages. Materials and Methods: RAW 264.7 cells were pretreated with GBA at different concentrations (10, 20, 40, 80, 160, 320 nM) for 24 hrs, and then cell viability was measured using Cell Counting Kit (CCK)-8 assays. Pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1α were determined using ELISA kits and qPCR. Then nitrite concentration was calculated according to a standard curve. At last, the effect of GBA on MAPK and NF-κB signaling pathways was assessed by western blot and luciferase reporter gene assay. Results: GBA (IC50: 260 nM) suppressed the TNF-α, IL-6 and IL-1β expression induced by lipopolysaccharide (LPS) in RAW 264.7 cells. The expression of TNF-α, IL-6 and IL-1&#; decreased to 30-50% and 70-75% in the high-dose (160 nM) and low-dose (40 and 80 nM) GBA groups, respectively. Furthermore, the nitric oxide (NO) production and the activation of NF-κB, ERK, and JNK pathways were significantly reduced in high-dose (160 nM) GBA only, while p38 pathway was inhibited at both low (40 and 80 nM) and high (160 nM) concentration of GBA. Conclusion: These data suggested that GBA inhibited LPS-induced production of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β mainly through the suppression of the p38 pathway. [ABSTRACT FROM AUTHOR]

The in vivo immunomodulatory activities of Tinospora crispa have been reported but its molecular mechanisms underlying its immunomodulatory properties remains obscure and the active constituents contributing to the activities have not been identified. The present study was aimed to investigate the immunomodulatory effects of T. crispa extract (TCE) and its chemical constituents on RAW 264.7 macrophages. Six known compounds including magnoflorine and syringin were isolated by various chromatographic techniques from TCE and their structures were determined spectroscopically. A validated HPLC method was used to quantify magnoflorine and syringin in the extract. The immunomodulatory effects of TCE and its isolated compounds on chemotaxis, phagocytosis, production of inflammatory mediators including reactive oxygen species (ROS), nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokines which include tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and monocyte chemoattractant protein-1 (MCP-1) on macrophages were assessed. TCE increased the chemotaxis and phagocytic activity of macrophages and significantly enhanced the production of ROS, NO and pro-inflammatory cytokines. All alkaloids isolated, specifically magnoflorine showed remarkable inducing effects on the chemotaxis, phagocytic activity, ROS and NO productions and the secretions of IL-1β, TNF-α, IL6, PGE2 and MCP-1. In contrast, syringin potently reduced the chemotaxis, phagocytic activity, ROS and NO productions and secretions of IL-1β, TNF-α, IL6, PGE2 and MCP-1. TCE showed strong immunostimulant effects on various components of the immune system and these activities were possibly contributed mainly by the alkaloids specifically magnoflorine. TCE has potential to be developed as an effective natural immunostimulant for improvement of immune-related disorders. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) causes acute respiratory tract infections in infants, the elderly and immunocompromised individuals. No licensed vaccine is available against RSV. We previously reported that intranasal immunization of rodents and lambs with a RSV vaccine candidate (ΔF/TriAdj) induces protective immunity with a good safety profile. ΔF/TriAdj promoted innate immune responses in respiratory mucosal tissues in vivo , by local chemokine and cytokine production, as well as infiltration and activation of immune cells including macrophages. The macrophage is an important cell type in context of both innate and adaptive immune responses against RSV. Therefore, we characterized the effects of ΔF/TriAdj on a murine macrophage cell line, RAW264.7, and bone marrow-derived macrophages (BMMs). A gene expression study of pattern recognition receptors (PRRs) revealed induction of endosomal and cytosolic receptors in RAW264.7 cells and BMMs by ΔF/TriAdj, but no up-regulation by ΔF in PBS. As a secondary response to the PRR gene expression, induction of several chemokines and pro-inflammatory cytokines, as well as up-regulation of MHC-II and co-stimulatory immune markers, was observed. To further investigate the mechanisms involved in ΔF/TriAdj-mediated secondary responses, we used relevant signal transduction pathway inhibitors. Based on inhibition studies at both transcript and protein levels, JNK, ERK1/2, CaMKII, PI3K and JAK pathways were clearly responsible for ΔF/TriAdj-mediated chemokine and pro-inflammatory cytokine responses, while the p38 and NF-κB pathways appeared to be not or minimally involved. ΔF/TriAdj induced IFN-β, which may participate in the JAK-STAT pathway to further amplify CXCL-10 production, which was strongly up-regulated. Blocking this pathway by a JAK inhibitor almost completely abrogated CXCL-10 production and caused a significant reduction in the cell surface expression of MHC-II and co-stimulatory immune markers. These data demonstrate that ΔF/TriAdj induces multiple signaling pathways in macrophages. [ABSTRACT FROM AUTHOR]

The objectives of this study were to determine the variety and amount of various functional components in Scutellaria barbata D. Don as well as study their anti-inflammatory activity on RAW 264.7 cells. Both ethanol and ethyl acetate extracts were shown to contain the functional components including phenolics, flavonoids, chlorophylls, and carotenoids, with the former mainly composed of phenolics and flavonoids, and the latter of carotenoids and chlorophylls. Both extracts could significantly inhibit (p < 0.05) the production of lipopolysaccharide-induced nitric oxide, prostaglandin E2, interlukin-6, and interlukin-1b, as well as the expressions of phosphor extracellular signal-regulated kinase and phosphor-c-Jun N-terminal kinase (p-JNK), but failed to retard tumor necrosis factor-α expression. Both ethanol and ethyl acetate extracts had a dose-dependent anti-inflammatory activity on RAW 264.7 cells. Furthermore, the anti-inflammatory efficiency can be varied for both ethanol and ethyl acetate extracts, which can be attributed to the presence of different varieties and amounts of functional components, as mentioned above. This finding suggested that S. Barbata extract may be used as an anti-inflammatory agent for possible future biomedical application. [ABSTRACT FROM AUTHOR]

Context:ZincL-carnosine (ZnC) is a chelate of Zn andL-carnosine and is used clinically in the treatment of peptic ulcer. Objective:In this study, we aim to investigate the involvement of heme oxygenase-1 (HO-1) in the anti-inflammatory effects of ZnC in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages. Materials and methods:We used immunoblotting analysis to evaluate the involvement of HO-1 in the anti-inflammatory effects of ZnC and the signaling pathway involved was measured using Dual luciferase reporter assay. Results:Results from immunoblotting analysis demonstrated that pretreatment of cells with ZnC enhanced the expression of HO-1 in RAW 264.7 cells. Pretreatment of cells with HO-1 inhibitor (tin protoporphyrin IX dichloride) significantly attenuated the inhibitory effects of ZnC on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and NF-κB activation in LPS-induced RAW 264.7 cells, suggesting that HO-1 play an important role in the suppression of inflammatory responses induced by ZnC. Furthermore, results from co-immunoprecipitation of Nrf2 and Keap1 and dual luciferase reporter assay showed that pretreatment of ZnC was able to activate the Nrf2 signaling pathway. Treatment of cells with p38 inhibitor (SB203580), c-Jun N-terminal kinase inhibitor (SP600125), and MEK 1/2 inhibitor (U0126) did not significantly suppress the induction of HO-1 by ZnC. Moreover, our present findings suggest that the effects of ZnC on NO production, HO-1 expression, and Nrf2 activation were attributed to its Zn subcomponent, but notl-carnosine. Conclusion:Pretreatment with ZnC was able to activate Nrf2/HO-1 signaling pathway, thus suppressing the expression of inflammatory mediators, such as NO and iNOS in LPS-induced RAW 264.7 cells. [ABSTRACT FROM PUBLISHER]

Ethnopharmocological relevance Cortex Eucommiae (CE), the bark of Eucommia ulmoides Oliv., has been traditionally used for its kidney-tonifying and bone- and tendon-enhancing properties in Korea, China, and Japan. CE has been historically prescribed for inflammatory conditions such as arthritis of the knee and ankle. Aim of the study Although CE has recently been shown to suppress inflammation in scientific studies, whether this effect involves modulation of the toll-like receptor 4 (TLR-4) pathway is currently unknown. Materials and methods The modulatory effect of CE on the TLR-4 pathway, both myeloid differentiation primary response gene 88 (Myd88)-dependent and independent, was investigated through real-time reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, and a reporter gene assay in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. Results CE dose-dependently inhibited nitric oxide production without significant cytotoxicity with an IC 50 of 356.23 μg/mL. In addition, CE down-regulated both LPS-induced mRNA and protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in a dose-dependent manner. CE suppressed LPS-induced activation of nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathways, which together comprise the Myd88-dependent TLR-4 pathway. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was also down-regulated by CE in a dose-dependent manner. CE additionally suppressed LPS-induced activation of interferon-β (IFN-β) and signal transducer and activator of transcription (STAT) pathway, which is associated with the Myd88-independent TLR-4 pathway. Conclusions CE down-regulated both Myd88-dependent and independent TLR-4 pathways, thus exerting anti-inflammatory effects. These results suggest that CE may be used as a potential therapeutic agent against chronic inflammatory diseases. [ABSTRACT FROM AUTHOR]

Most fungi that grow on damp building materials produce low molecular weight compounds, some of which are known to be toxic. In this study, we tested the hypothesis that exposure to some metabolites of fungi common on damp building materials would result in time-, dose-, and compound-specific responses in the production of various chemokines by RAW 264.7 cells. Cell cultures were exposed to a 10 − 7 M or 10 − 8 M metabolite dose for 2, 4, 8 or 24 h. Metabolite concentrations used were based on those that might be expected in alveolar macrophages due to inhalation exposure from living or working in a damp building. Compared to controls, exposure provoked significant time-, dose- and compound-specific responses manifest as differentially elevated secretion of three of nine cytokines tested in culture supernatant of treated cells. The greatest number of cytokines produced in response to the metabolites tested were in andrastin A-treated cells (GM-CSF, TGFβ1, Tnf-α) followed by koninginin A (TGFβ1 and Tnf-α) and phomenone (GM-CSF, TGFβ1). Chaetoglobosin A, chaetomugilin D and walleminone exposures each resulted in significant time-specific production of Tnf-α only. This investigation adds to a body of evidence supporting the role of low molecular weight compounds from damp building materials as pathogen associated molecular patterns (PAMPs). Along with fungal glucan and chitin, these compounds contribute to the non-allergy based respiratory outcomes for people living and working in damp buildings. [ABSTRACT FROM AUTHOR]

Hentriacontane, has various pharmacological effects including anti-inflammatory, antitumor and antimicrobial activities. Its anti-inflammatory potential has been demonstrated in peritoneal macrophages. However detailed studies on other models elucidating the mechanistic description of the mode of action has not been done. Hence, the aim of the present study is to evaluate the anti-inflammatory potential of hentriacontane both in - vivo (Balb/c mice) and in-vitro (RAW 264.7 cells). Cytokine inhibition of both pro-inflammatory (TNF-α, IL-6, MCP-1 and IL-1β) and anti-inflammatory (IL-10) cytokines was studied in RAW 264.7 cells and Balb/c mice. Suppressive potential of hentriacontane on NO, PGE 2 , LTB4 and on LPS induced translocation of NF-κB in RAW 264.7 cells was studied. Further investigations on the effect of hentriacontane on phagocytic index, carrageenan induced paw oedema in mice and on organ weight were done. It was found that hentriacontane significantly reduced all the parameters of inflammation in the experiments under study at all the concentrations, 10 μM, 5 μM and 1 μM ( in-vitro ) and 5 mg/kg, 2 mg/kg and 1 mg/kg ( in-vivo ). The highest concentration used in the two models presented the most significant results. The results indicate that hentriacontane is a potent suppressor of inflammatory cytokines and other mediators. Moreover it also has regulatory effect on NF-κB. Hence, hentriacontane is a potential candidate for investigations to develop anti-inflammatory drug. [ABSTRACT FROM AUTHOR]

Electroporation is the most widely employed method of gene transfer into macrophages which are hard to transfect. RAW 264.7 is a widely used cell line for studying macrophage responses. Electroporation of RAW 264.7 cells with commercial reagents although very efficient is expensive necessitating the development of cost effective alternatives. In this study, we have formulated an economical electroporation buffer for electroporation of RAW 264.7 cells compatible with commercial nucleofector apparatus. We observed that supplementation of membrane fusogenic agents such as Ficoll, PEG and membrane resealing agent, poloxamer P188, enhanced the transfection efficiency of macrophages to a level comparable to the commercially available solutions thereby providing us a cost effective solution for genetic manipulation of macrophages especially in large numbers. [ABSTRACT FROM AUTHOR]

Synthetic pyrethroids (SPs) are commonly used insecticides that have been detected in mammals, including humans, indicating a potential threat to human health. Bifenthrin (BF), as well as other pyrethroids, has been shown to possess neurotoxic, reproductive, hepatotoxic and nephrotoxic potential in mammals. However, studies regarding the immunotoxicity of BF and its mechanism are limited. In this study, we aim to exam the immunotoxicity of cis -BF on the murine macrophage cell line, RAW 264.7. MTT assay results demonstrated that cis -BF exposure induced apoptosis in RAW 264.7 cells in a concentration-dependent manner. We found that the expression of p53 and caspase-3 was up-regulated, while the expression of Bcl-2 was down-regulated during cis -BF-induced apoptosis. In addition, we also found that cis -BF exposure caused oxidative stress in RAW 264.7 cells in a dose-dependent manner. Interestingly, cis -BF exposure was found to inhibit the increase in transcription levels of IL-1β , IL-6 and TNF-α responding to LPS stimulation. We also found that the induced increase in IFN-β mRNA levels upon Sendai virus infection was blocked with cis -BF exposure. Finally, we found that cis -BF exposure increased ROS levels and dysregulated mRNA levels of oxidative stress-related genes in RAW 264.7 cells. The present study elucidates the immunotoxicity effect of cis -BF on macrophages and its possible underlying mechanism. The results from this study support the necessity to evaluate immune dysfunction in the risk assessment of cis -BF exposure. [ABSTRACT FROM AUTHOR]

We previously reported that palmitate induces receptor-interacting protein (RIP)1-dependent necrosis in RAW 264.7 macrophage cells. In response to death receptor stimuli, RIP1 is reported to activate RIP3, which causes the phosphorylation and translocation of mixed-lineage kinase domain-like (MLKL) protein to the plasma membrane, subsequent pore formation in the plasma membrane, and necrotic cell death. In the current study, we investigated the role of MLKL in palmitate-induced, RIP1/RIP3-dependent necrotic cell death in RAW 264.7 cells. The down-regulation of RIP1 or RIP3 by siRNA transfection protected the cells from palmitate-induced cell death. In addition, MLKL was phosphorylated at the serine residue and translocated to the plasma membrane in palmitate-treated cells. In these cells, MLKL was observed as aggregate dots on the plasma membrane. The findings also show that palmitate induced the formation of pores with varied shapes and sizes, and an increase in propidium iodide (PI) uptake and lactate dehydrogenase ( LDH) release. Furthermore, the down-regulation of MLKL by siRNA transfection significantly decreased palmitate-induced PI uptake and LDH release, resulting in protection against palmitate-induced necrotic cell death. The findings reported here indicate that palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation of RAW 264.7 cells in the plasma membrane, which could provide a new mechanism to explain the link between elevated levels of free fatty acids (FFAs), palmitate in particular, and macrophage death. [ABSTRACT FROM AUTHOR]

This research analyzed the effect of β-glucan that is expected to alleviate the production of the inflammatory mediator in macrophagocytes, which are processed by the lipopolysaccharide (LPS) of Escherichia. The incubated layer was used for a nitric oxide (NO) analysis. The DNA-binding activation of the small unit of nuclear factor-κB was measured using the enzyme-linked immunosorbent assay-based kit. In the RAW264.7 cells that were vitalized by Escherichia coli (E. coli) LPS, the β-glucan inhibited both the combatant and rendering phases of the inducible NO synthase (iNOS)-derived NO. β-Glucan increased the expression of the heme oxygenase-1 (HO-1) in the cells that were stimulated by E. coli LPS, and the HO-1 activation was inhibited by the tin protoporphyrin IX (SnPP). This shows that the NO production induced by LPS is related to the inhibition effect of β-glucan. The phosphorylation of c-Jun N-terminal kinases (JNK) and the p38 induced by the LPS were not influenced by the β-glucan, and the inhibitory κB-α (IκB-α) decomposition was not influenced either. Instead, β-glucan remarkably inhibited the phosphorylation of the signal transducer and activator of transcription-1 (STAT1) that was induced by the E. coli LPS. Overall, the β-glucan inhibited the production of NO in macrophagocytes that was vitalized by the E .coli LPS through the HO-1 induction and the STAT1 pathways inhibition in this research. As the host immune response control by β-glucan weakens the progress of the inflammatory disease, β-glucan can be used as an effective immunomodulator. [ABSTRACT FROM AUTHOR]

Protein interacting with C kinase 1 (PICK1) is a scaffolding protein mainly implicated in neurological diseases, however, the function of PICK1 in acute liver injury (ALI) remains unknown. Our study found a dramatical decrease in mRNA and protein levels of PICK1 in liver tissues and isolated Kupffer cells (KCs) from the liver in mice with ALI. Furthermore, pretreatment the mice with ALI with FSC-231, a pharmacological inhibitor of PICK1, could significantly augment inflammatory response. Furthermore, in vitro studies showed that both lipopolysaccharide (LPS) and interferon gamma (IFN-γ) significantly reduced the expression of PICK1, while IL-4 elevated its expression in RAW 264.7 cells. Additionally, over-expression of PICK1 inhibited the expression of M1 biomarkers by suppressing NF-κB activity, and enhanced the expression of M2 biomarkers by promoting STAT6 activity. In contrast, knockdown of PICK1 or FSC-231 pretreatment promoted M1 polarization and suppressed M2 polarization. Besides, caveolin-1 was identified as a potential target gene controlled by PICK1 in RAW 264.7 cells. Mechanistic investigation revealed a dual role of PICK1 in regulating macrophage polarization and implied PICK1 as a potential therapeutic target in ALI. [ABSTRACT FROM AUTHOR]

Owing to the serious adverse effects caused by pyrimethamine and sulfadiazine, the drugs commonly used to treat toxoplasmosis, there is a need for treatment alternatives for this disease. Nanotechnology has enabled significant advances toward this goal. This study was conducted to evaluate the activity of biogenic silver nanoparticles (AgNp-Bio) in RAW 264.7 murine macrophages infected with the RH strain of Toxoplasma gondii. The macrophages were infected with T. gondii tachyzoites and then treated with various concentrations of AgNp-Bio. The cells were evaluated by microscopy, and culture supernatants were collected for ELISA determination of their cytokine concentration. Treatment with 6 μM AgNp-Bio reduced the infection and parasite load in infected RAW 264.7 macrophages without being toxic to the cells. The treatment also induced the synthesis of reactive oxygen species and tumor necrosis factor-alpha (both pro-inflammatory mediators), which resulted in ultrastructural changes in the tachyzoites and their intramacrophagic destruction. Our findings suggest that AgNp-Bio affect T. gondii tachyzoites by activating microbicidal and pro-inflammatory mechanisms and may be a potential alternative treatment for toxoplasmosis. [ABSTRACT FROM AUTHOR]

Asthma is a heterogeneous airway inflammatory disease, which is associated with Th2 cytokine-driven inflammation and non-Th2, TNF-α mediated inflammation. Unlike Th2 mediated inflammation, TNF-α mediated asthma inflammation is generally insensitive to inhaled corticosteroids (ICS). ASHMITM, aqueous extract of three medicinal herbs— Ganoderma lucidum ( G. lucidum ), Sophora flavescens Ait ( S. flavescens ) and Glycyrrhiza uralensis Fischer ( G. uralensis ), showed a high safety profile and was clinically beneficial in asthma patients. It also suppresses both Th2 and TNF-α associated inflammation in murine asthma models. We previously determined that G. uralensis flavonoids are the key active compounds responsible for ASHMITM suppression of Th2 mediated inflammation. Until now, there are limited studies on anti-TNF-α compounds presented in ASHMITM. The objective of this study was to isolate and identify TNF-α inhibitory compounds in ASHMITM. Here we report that G. lucidum , but not the other two herbal extracts, S. flavescens or G. uralensis inhibited TNF-α production by murine macrophages; and that the methylene chloride (MC)–triterpenoid-enriched fraction, but not the polysaccharide-enriched fraction, contained the inhibitory compounds. Of the 15 triterpenoids isolated from the MC fraction, only ganoderic acid C1 (GAC1) significantly reduced TNF-α production by murine macrophages (RAW 264.7 cells) and peripheral blood mononuclear cells (PBMCs) from asthma patients. Inhibition was associated with down-regulation of NF-κB expression, and partial suppression of MAPK and AP-1 signaling pathways. Ganoderic acid C1 may have potential for treating TNF-α mediated inflammation in asthma and other inflammatory diseases. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Orostachys japonicus A. Berger (Crassulaceae) (OJ), well-known as Wa-song in Korea is a medicinal plant with immunoregulatory, anti-febrile, antidote, and anti-cancer activities. This study was aimed at evaluating the immunostimulatory effect of O. japonicus A. Berger and its possible mechanisms of action. Materials and methods To evaluate the effect of OJ aqueous extract on macrophage activity, we evaluated the modulation of macrophage activation state by observing structural (phagocytic activities) and the production of nitric oxide increase. The effect of OJ aqueous extract on RAW264.7 cell viability were assessed using Cell Counting Kit (CCK)-8 assay. HPLC analysis was performed to identify potential active compounds of this extract. Results The biological investigations indicated that OJ aqueous extract, among others, possessed the highest macrophage activation as indicated by NO production yield. The results showed that OJ aqueous extract exhibited antioxidant effects, which included scavenging activities against DPPH radicals. OJ aqueous extract increased the phagocytic activity of RAW 264.7 cells against IgG-opsonized red blood cells (RBC). The level of phosphorylated Syk kinase was increased in OJ aqueous extract-treated group as compared to control. Phosphorylation of PLC-γ was increased in the OJ aqueous extract-treated groups. Quercetin-3- O -rhamnose and kaempferol-3- O -rhamnose was detected in OJ aqueous extract by HPLC analysis. Conclusions OJ aqueous extract might play a pivotal ethnopharmacologic role as an immunostimulatory agent by promoting Fc gamma receptor (FcγR)-mediated phagocytosis of IgG-opsonized RBCs. On the basis of our results, OJ aqueous extract can enhance innate immunity and may serve as an adjuvant for tumor treatment. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the role of the toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), and stress activated protein kinases/Jun N-terminal kinase (SAPK/JNK) signalling pathways in the responses of RAW 264.7 macrophages to low-intensity microwaves (MW). Materials and methods: Three inhibitors of TLR4, SAPK/JNK, and NF-κB signalling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before MW treatment. Results: MW exposure resulted in stimulation of RAW 264.7 cell activity manifested by increases in cytokine production and the stimulation of cell signalling. The blocking of a key kinase of the NF-κB pathway by IKK Inhibitor XII resulted in decreased MW-induced TLR4 expression and increased SAPK/JNK and NF-κB phosphorylation in irradiated cells. In addition, IKK Inhibitor XII significantly decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1α (IL-1α), interleukin 6 (IL-6), and interleukin 10 (IL-10) production in both exposed and unexposed RAW 264.7 macrophages. Inhibitor SP6000125 did not prevent an MW effect on signal proteins with the exception of decreased SAPK/JNK phosphorylation in RAW 264.7 cells. Cytokine production was markedly decreased in MW-exposed cells cultured with SP6000125. The inhibitor of TLR4, CLI-095, did not affect signal proteins and cytokine production changes in MW-exposed cells. Conclusions: The results suggest that low-intensity MW promotes macrophage activity via mechanisms involving cellular signalling, particularly the NF-κB pathway. [ABSTRACT FROM AUTHOR]

Abstract: Three sulfated phenolic compounds, juglanin B (11R)-O-sulfate (1), myricetin 3´-O-sulfate (2), and ampelopsin 3´-O-sulfate (3), were isolated from the leaves of Myrica rubra. Compound 1 was a new sulfated lignan, 2 was a new sulfated flavone, and 3 was a known sulfated flavone. The structures of the new compounds (1 and 2) were determined by acid hydrolysis and spectroscopic methods, including IR, FAB-MS, 1D and 2D NMR. The inhibitory activities of compounds 1–3 and their hydrolysates (1a–3a) against LPS-induced cytokine (TNF-α, IL-1β, and IL-6) production in macrophage RAW 264.7 cells were evaluated. The 2 new compounds (1 and 2) and their aglycones (1a and 2a) significantly reduced LPS-induced expression of iNOS and COX-2 proteins. [Copyright &y& Elsevier]

Abstract: Numerous studies have shown various relationships between foods with a high nutritional value and a robust immune response, particularly studies that have focused on host protection and cytokine networks. This study aimed to clarify the role played by the procyanidin trimer C1 in innate and adaptive immunity. Procyanidin C1 did not exert cytotoxicity at concentrations ranging from 7.8 to 62.5μg/ml in macrophage cells; therefore, concentration of 62.5μg/ml was used as the maximum dose of procyanidin C1 throughout subsequent experiments. Procyanidin C1 enhanced inducible nitric oxide synthase-mediated nitric oxide production in a concentration-dependent manner. In addition, procyanidin C1 functionally induced macrophage activation by augmenting the expression of cell surface molecules (CD80, CD86, and MHC II) and proinflammatory cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) via activation of mitogen-activated protein kinase (MAPK), e.g., p38, ERK, and JNK and nuclear factor (NF)-κB signaling pathways. Interestingly, procyanidin C1 effectively polarized T helper type 1 (Th1) by secreting Th1-mediated cytokines (interferon-γ, IL-12p70, and IL-2) and inducing splenocyte proliferation, indicating that procyanidin C1 contributes to Th1 polarization of the immune response. Accordingly, these findings confirms that the procyanidin C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes, which suggests that procyanidin C1 regulates innate and adaptive immunity by macrophage activation and Th1 polarization. [Copyright &y& Elsevier]

Purpose: Laminaria japonica is a representative marine brown alga used as a culinary item in East Asia. L. japonica extract was shown to exert various biological activities; however, its anti-inflammatory activity has not been reported. The aim of this study is to investigate the molecular mechanisms underlying its anti-inflammatory action. Methods: Anti-inflammatory mechanisms of L. japonica n-hexane fraction (LHF) were assessed using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. An anti-inflammatory compound isolated from LHF by reverse-phase chromatography was identified using nuclear magnetic resonance (NMR) spectroscopy. Results: Our results indicate that LHF significantly inhibited LPS-stimulated nitric oxide (NO) and prostaglandin E (PGE) secretion in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) with no cytotoxicity. As results, levels of pro-inflammatory cytokines were significantly reduced by pretreatment of LHF in LPS-stimulated RAW 264.7 cells. Treatment of LHF strongly suppressed nuclear factor-κB (NF-κB) promoter-driven expression and nuclear translocation of NF-κB by preventing proteolytic degradation of inhibitor of κB (IκB)-α in LPS-stimulated RAW 264.7 cells. Moreover, LHF inhibited the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW 264.7 cells. One of the anti-inflammatory compounds was isolated from LHF and identified as fucoxanthin. Conclusions: These results indicate that the LHF-mediated inhibition of NO and PGE secretion in LPS-stimulated macrophages is regulated by NF-κB inactivation through inhibition of IκB-α, MAPKs, and Akt phosphorylation. LHF may be considered as a functional food candidate for the prevention or treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Abstract: Suppression of interleukin (IL)-6 production has beneficial effects against various inflammatory diseases. Through a rapid screening system, we found that nitazoxanide, or 2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide, which is a well-known antiparasitic agent, suppressed lipopolysaccharide (LPS)-induced production of IL-6 from RAW 264.7 cells and mouse peritoneal macrophages, with 50% inhibitory concentrations (IC50s) of 1.54mM and 0.17mM, respectively. Nitazoxanide also inhibited the LPS-induced expression of IL-6 mRNA in RAW 264.7 cells. To investigate the effects of nitazoxanide in vivo, we orally administered nitazoxanide at a dose of 100mg/kg to mice 2h before a 1-mL intraperitoneal injection of 4% thioglycollate (TG). Six hours after TG injection, plasma IL-6 levels were markedly lower (by 90%) than the levels in vehicle-treated mice. These data suggest that nitazoxanide could be a promising lead compound for agents against various diseases associated with overproduction of IL-6. [Copyright &y& Elsevier]

Abstract: The aim of this study is to investigate the inhibitory effects of aminoethyl-chitooligosaccharides (AE-COS) on oxidative stress in mouse macrophages (RAW 264.7 cells). The inhibitory effects of AE-COS on DNA and protein oxidation were studied in RAW 264.7 cells. Furthermore, free radical scavenging effect of AE-COS were determined in RAW264.7 cells by 2′,7′-dichlorofluorescein (DCF) intensity and intracellular glutathione (GSH) level. AE-COS also inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL-60). These results suggest that AE-COS acts as a potential free radical scavenger in RAW 264.7 cells. [Copyright &y& Elsevier]

Abstract: Four glucans (LNT-H, LNT-S, LNT-E, and LNT-B) were successively isolated from the fruiting bodies of Lentinus edodes by hot-water, sonication, enzyme, and NaOH. The chemical structures were characterized by GC, FTIR, and 13C NMR, which demonstrated that the four samples are (1→3)-β-d-glucans with (1→6)-glucopyranoside side groups having different molecular weight. The results of viscometry and atomic force microscopy (AFM) proved that LNT-H, LNT-S, LNT-E, and LNT-B exist as stiff triple helices in water and random coil in dimethyl sulfoxide (DMSO). The morphology of the original triple helix was almost dominated by linear shape, but the renatured LNT-B contains some branched structures in AFM image. Among the four glucans, LNT-H, LNT-E, and LNT-B are clearly contaminated by endotoxin, while LNT-S significantly exhibits nitric oxide (NO) inhibition effect in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner. Therefore, LNT-S is probably used as a promising molecule which can be helpful to the diseases associated with NO overproduction. This is the first primary report dealing with the suppression of LPS-induced NO from macrophage RAW 264.7 cells by Lentinan. [Copyright &y& Elsevier]

Abstract: We have recently reported that phlorofucofuroeckol A isolated from the edible brown algae Ecklonia stolonifera showed potential antioxidative and anti-inflammatory properties in macrophage stimulated by LPS treatments. In this study, we further investigated the pharmacological characteristic of phlorofucofuroeckol A in regulations of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 through regulatory and signaling pathways using LPS-treated RAW 264.7 cells. Treatment with 20μM of phlorofucofuroeckol A significantly decreased levels of iNOS and COX-2 mRNA induced by LPS stimulation. As results, levels of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were significantly reduced by treatments of phlorofucofuroeckol A in LPS-stimulated RAW 264.7 cells. Phlorofucofuroeckol A inhibited promoter activities of inflammatory-mediators (iNOS and COX-2) and transcriptional factors (nuclear factor-κB, NF-κB, and AP-1) in LPS-treated RAW 264.7 cells. Moreover, phlorofucofuroeckol A inhibited activation of Akt and p38 MAPK in LPS-treated RAW 264.7 cells. These results indicate that the phlorofucofuroeckol A regulates iNOS and COX-2 expressions through the NF-κB-dependent transcriptional control associated with inhibition of multiple signaling proteins, suggesting potential candidates of phloroglucinol derivatives for treatments of inflammatory diseases. [Copyright &y& Elsevier]

We previously reported that the increment of carbohydrate content in the Viscozyme® L (Novozyme Corp., Oklahoma City, OK, USA) extract of Lactobacillus brevis-fermented Ecklonia cava affected the inhibition of nitric oxide (NO) production and that it might be related to the polysaccharide compound. However, there is no report of anti-inflammatory effects of the polysaccharide or its biological mechanism. Here, we investigated the anti-inflammatory effects of the polysaccharide and its biological mechanism in lipopolysaccharide (LPS)-activated RAW 264.7 cells. The polysaccharide isolated from the Viscozyme extract of L. brevis-fermented E. cava (VLFEP) dose-dependently decreased LPS-stimulated NO production without cytotoxicity. Also, VLFEP significantly decreased the production of prostaglandin E2 (PGE2) at the 100 μg/mL concentration. In addition, VLFEP dose-dependently decreased the protein and mRNA expressions of inducible NO synthase, whereas it slightly decreased those of cyclooxygenase 2 and only at the 100 μg/mL concentration. Moreover, VLEFP dose-dependently decreased the productions and/or mRNA expressions of tumor necrosis factor-α and interleukin-6, compared with those of LPS only-stimulated cells. In further experiments, VLFEP considerably reduced the phosphorylation and degradation of inhibitory κB as well as the translocation of nuclear transcription factor-κB (NF-κB) p65 into the nucleus, and its DNA binding was markedly induced by LPS stimulation. This study suggests that VLFEP exerts anti-inflammatory effects by down-regulating the production and expression of pro-inflammatory cytokines and mediators via inhibiting the NF-κB pathway in LPS-stimulated RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

Abstract: We previously reported that extract of Rosa rugosa root and its active triterpenoids constituents exhibit anti-nociceptive and anti-inflammatory effects in animal models. However, little is known about the effects and the molecular mechanism of the 19α-hydroxyursane-type triterpenoids. Among the tested 19α-hydroxyursane-type triterpenoids (kaji-ichigoside F1, rosamultin, euscaphic acid, tormentic acid (TA)), TA was found to most potently inhibit the production of nitric oxide (NO) in RAW 264.7 cells. We investigated the anti-inflammatory effects and its underlying molecular mechanisms of TA in lipopolysaccaride (LPS)-stimulated RAW 264.7 cells. TA dose-dependently reduced the productions of NO, prostaglandin E2 (PGE2), and tumor necrosis factor-α (TNF-α) induced by LPS. In addition, TA significantly suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α at the mRNA and protein levels. Moreover, treatment with TA decreased LPS-induced DNA binding of nuclear factor-kappa B (NF-κB) and nuclear translocation of p65 and p50 subunits of NF-κB. Consistent with these findings, TA also suppressed the LPS-stimulated degradation and phosphorylation of inhibitor of kappa B-α (IκB-α). Taken together, these results suggest that the anti-inflammatory activity of TA is associated with the down-regulation of iNOS, COX-2, and TNF-α through the negative regulation of the NF-κB pathway in RAW 264.7 cells. [Copyright &y& Elsevier]

Background: Two major ways of macrophage (MΦ) activation can occur in radiation-induced pulmonary injury (RPI): classical and alternative MΦ activation, which play important roles in the pathogenesis of RPI. MΦ can produce chemokine MΦ inflammatory protein-1α (MIP-1α), while MIP-1α can recruit MΦ. The difference in the chemotactic ability of MIP-1α toward distinct activated MΦ is unclear. We speculated that there has been important interaction of MIP-1α with different activated MΦ, which might contribute to the pathogenesis of RPI.Methods: Classically and alternatively activated MΦ were produced by stimulating murine MΦ cell line RAW 264.7 cells with three different stimuli (LPS, IL-4 and IL-13); Then we used recombinant MIP-1α to attract two types of activated MΦ. In addition, we measured the ability of two types of activated MΦ to produce MIP-1α at the protein or mRNA level.Results: Chemotactic ability of recombinant MIP-1α toward IL-13-treated MΦ was the strongest, was moderate for IL-4-treated MΦ, and was weakest for LPS-stimulated MΦ (p<0.01). The ability of LPS-stimulated MΦ to secrete MIP-1α was significantly stronger than that of IL-4-treated or IL-13-treated MΦ (p<0.01). The ability of LPS-stimulated MΦ to express MIP-1α mRNA also was stronger than that of IL-4- or IL-13-stimulated MΦ (p<0.01).Conclusions: The chemotactic ability of MIP-1α toward alternatively activated MΦ (M2) was significantly greater than that for classically activated MΦ (M1). Meanwhile, both at the mRNA and protein level, the capacity of M1 to produce MIP-1α is better than that of M2. Thus, chemokine MIP-1α may play an important role in modulating the transition from radiation pneumonitis to pulmonary fibrosis in vivo, through the different chemotactic affinity for M1 and M2. [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acids (PUFAs) can affect various functions of the immune system including inflammatory responses. An oxidative burst of phagocytes accompanied by reactive oxygen species (ROS) and reactive nitrogen species (RNS) formation is one of the phagocyte functions that could be modulated by PUFAs. To investigate the effects of ω-3 (α-linolenic, docosahexaenoic, eicosapentaenoic) and ω-6 (arachidonic, linoleic) PUFAs on lipopolysaccharide (LPS)-stimulated ROS and RNS production by the murine macrophage cell line RAW 264.7. Murine peritoneal macrophages RAW 264.7 were stimulated with LPS (0.1 μg/ml) and treated with 0.1–100 μM ω-3 or ω-6 PUFAs for either 8 (ROS production) or 20 h (RNS production). The cytotoxicity of PUFAs was evaluated by an ATP (adenosine triphosphate) test after both 8 and 20 h of treatment with PUFAs. Changes in ROS production by LPS-treated macrophages subsequently activated with phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP) were determined by luminol-enhanced chemiluminescence, whilst the production of RNS was determined as the concentration of nitrites in cell supernatants (Griess reaction). Changes in inducible nitric oxide synthase (iNOS) expression were evaluated by Western blot analysis. The antioxidant properties of PUFAs were tested by TRAP (total peroxyl radical-trapping antioxidant parameter) assay. All PUFAs in 100 μM concentration except eicosapentaenoic acid decreased ROS production. The effect was most significant when docosahexaenoic acid was used. Arachidonic acid decreased PMA-activated ROS production even in 1 and 10 μM concentrations. On the other hand, 10 and 100 μM eicosapentaenoic acid potentiated ROS production. As concerns RNS production, all the fatty acids that were tested in a concentration of 100 μM decreased iNOS expression and nitrite accumulation. Fatty acids had no significant effect on the viability and proliferation of RAW 264.7 cells. The TRAP assay confirmed that none of the tested PUFAs exerted any significant antioxidant properties. High concentrations of PUFAs of both ω-3 and ω-6 groups can inhibit ROS and RNS formation by stimulated macrophages. The expression of iNOS can also be inhibited. This effect, together with the absence of antioxidant activity and cytotoxic properties, indicates that PUFAs can participate in the regulation of enzymes responsible for reactive species production. [ABSTRACT FROM AUTHOR]

Benzyl isothiocyanate (BITC) is detected in abundance in Brassica vegetables, and some previous studies have demonstrated that BITC may potentially function as a chemopreventive agent in humans. This study examined whether BITC inhibits lipopolysaccharide (LPS)-induced inflammatory responses in Raw 264.7 macrophages and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema formation. The treatment of macrophages with various concentrations of BITC resulted in a dose-dependent reduction in the LPS-induced secretion of interleukin (IL)-1β, TNF-α, and IL-6 and their corresponding mRNA levels, as well as in the production of nitric oxide and PGE2. Consistent with these findings, BITC inhibited the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins and their corresponding mRNAs. BITC inhibited LPS-induced phosphorylation and the degradation of the inhibitor of κBα, translocation of p65 into the nucleus, and the DNA binding activity and transcriptional activity of NFκB. Moreover, the LPS-stimulated phosphorylation of extracellular signal regulated kinase (ERK)1/2 and Akt was suppressed by BITC. BITC also inhibited ear edema formation and the protein expression of iNOS and COX-2 in mouse skin treated with TPA. We demonstrate that BITC is a potent anti-inflammatory agent, and the anti-inflammatory properties of BITC may result from the downregulation of NFκB signaling. [ABSTRACT FROM AUTHOR]

Abstract: The effect of toll-like receptor (TLR) 7 ligand pretreatment on the production of tumor necrosis factor (TNF)-α in response to TLR7 or TLR2 ligand was examined in order to establish a new TLR-mediated tolerance. RAW 264.7 macrophage-like cells were treated with imiquimod R837 as a TLR7 ligand for 18h, washed and incubated in fresh culture medium 6h. The second challenge with imiquimod R837 as a TLR7 ligand or Pam3CysSK4 as a TLR2 ligand resulted in reduced TNF-α production in TLR7 ligand-pretreated cells. There was impaired activation of NF-κB, p38 and stress-activated protein kinase (SAPK) in the tolerant cells. The expression of IRAK-M as a negative regulator of TLR signaling was markedly augmented in the tolerant cells while the interleukin-1 receptor-associated kinase (IRAK)-1 functioned normally. The involvement of IRAK-M in the TLR7-mediated tolerance is discussed. [Copyright &y& Elsevier]

This research was undertaken to find the in vitro inflammatory action of lansai A-D produced by Streptomyces sp. SUC1. We investigated the effects of lansai C not only on the formation of nitric oxide (NO), prostaglandin E2 (PGE2), tumour necrosis factor (TNF-α), interleukin (IL)-1α, IL-6 and IL-10, but also on inducible NO synthase and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells. The data obtained were consistent with the modulation of inducible nitric oxide synthase enzyme expression. A similar fashion was also observed when LPS-induced PGE2 release and COX-2 expression were tested. The significant inhibitory effects were shown in concentration-dependent manners. In addition, lansai C also mildly but significantly reduced the formation of TNF-α. These findings support the application of lansai C as anti-inflammatory agent. [ABSTRACT FROM AUTHOR]

Abstract: Murdannia bracteata has been used as a Taiwanese folk medicine for its anti-inflammatory properties. However, neither its active ingredients nor its anti-inflammatory actions are well defined. Nitric oxide (NO), overproduced by activated macrophages via inducible NO synthase (iNOS), is suggested to be a significant pathogenic factor in various inflammatory tissue injuries. In order to elucidate the anti-inflammatory actions of M. bracteata, the present study was designed to isolate its active constituents and examine its effects on iNOS in lipopolysaccharide (LPS)-activated macrophages. Two new hydroxybutenolides, bracteanolide A (1) and B (2), together with (+)-(R)-p-hydroxyphenyllactic acid (3) and isovitexin (4), were isolated and identified from M. bracteata by the NO production assay. All of the compounds inhibited NO production except 3. Their rank order of potency was 1 > 2 > 4. Among these, 1 significantly inhibited NO production, which is associated with its suppression on iNOS induction in a concentration-dependent manner, with an IC50 of 33.27±0.86μM. Nevertheless, isometric tension recordings in isolated endothelium-intact rat aorta revealed that 1–4 did not affect acetylcholine-induced endothelial NO-dependent relaxation, an index of endothelial NOS (eNOS) activity. The selective inhibition on iNOS provides a possible explanation for the anti-inflammatory use of M. bracteata. [Copyright &y& Elsevier]

This study presented a well-characterized biocompatibility profile of dental bonding agents in a mouse macrophage in vitro model. The cellular response to four different formulations of dental bonding resins and the cell viability was determined. Materials were prepared by photopolymerization and the unreacted monomers were extracted in a buffered medium. Murine macrophages were incubated in the extract medium for 24 h. Cellular viability was assessed by fluorescence microscopy and Wst-1 assay, while flow cytometry was used to quantify the apoptotic response. As an indicator of inflammatory responses, real-time polymerase chain reaction was utilized to quantify elevated cytokine production. These responses were monitored by quantifying the levels of tumor necrosis factor alpha (TNFα) and interleukin-1β (IL-1β) produced by the cells. Murine macrophage cells exposed to the unfilled resin systems containing glycidyl ether of bisphenol A (Bis-GMA) and 2-hydroxyethyl methacrylate (HEMA) had the most adverse response and cells exposed to the filled Bis-GMA and HEMA resin system were the most viable at photopolymerization times. Other resin systems displayed intermediate levels of viability when compared with the untreated control. The levels of IL-1β were significantly elevated in all samples. [ABSTRACT FROM AUTHOR]

Conventionally, a semi‐quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O 2 - ) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water‐soluble, yellow‐colored, nitroblue tetrazolium (Y‐NBT) by O 2 - . However, this assay is semi‐quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2 M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620 nm. The absorbance of dissolved NBT increased in proportion to cell number (r=0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O 2 - . The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB‐985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O 2 - produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O 2 - produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay. [ABSTRACT FROM AUTHOR]

Induction of COX-2 by catalase in smooth muscle cells, endothelial cells, and neuronal cells has been previously reported. However, the mechanism by which catalase up-regulates COX-2 remains poorly understood. In this study, we investigated the effect of catalase on induction of COX-2 in macrophages. The addition of catalase into Raw 264.7 macrophages induced COX-2 expression that was correlated with increased COX-2 transcription and mRNA stability. Catalase also induced activation of NF-κB, PI3K, ERKs, p38s, or JNKs. Catalase-induced COX-2 expression was abrogated by treatment of MG-132 (a NF-κB inhibitor) or LY294002 (a PI3K inhibitor), but not by treatment of PD98059 (an ERK inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor). Moreover, inhibition of PI3K by LY294002 caused partial decrease of catalase-induced COX-2 transcription and steady-state COX-2 transcript levels, but not COX-2 mRNA stability. Together, these results suggest that catalase induces the expression of COX-2 in Raw 264.7 macrophages, and the induction is related with activation of NF-κB transcription factor and PI3K signaling pathway. [Copyright &y& Elsevier]

Nitric oxide (NO) is involved in producing damage after exposure to radiation and also in the toxicity associated with bacterial endotoxin (lipopolysaccharide, LPS). We have evaluated different radioprotective thiols for their effects on LPS-stimulated NO production in mouse macrophage cells. RAW 264.7. Our results indicate that although thiols inhibited NO production in general, the degree of inhibition depended upon the thiol compound. Long-chain aminothiols like WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] exerted a strong inhibition; but its parent drug, amifostine, which protects mice against radiation lethality, was not as effective as WR-1065. Diethyl dithiocarbamate, which is less effective than amifostine as a radioprotector, strongly inhibited NO production from macrophages. These results indicate that the radioprotective potential of sulfhydryl compounds is not related to its ability to inhibit NO production by macrophages and suggest that some of the thiol radioprotectors may effectively ameliorate the fatal symptoms of hypotensive shock, associated with endotoxin (LPS)-induced NO production. [Copyright &y& Elsevier]

The synthesis of inflammation mediators produced from arachidonic acid is regulated primarily by the cellular concentration of free arachidonic acid. Since intracellular arachidonic acid is almost totally present as phospholipid esters, the concentration of intracellular arachidonic acid is primarily dependent on the balance between the release of arachidonic acid from membrane phospholipids and the uptake of arachidonic acid into membrane phospholipids. Cytosolic phospholipase A[sub 2] is a calciumdependent enzyme that catalyzes the stimulus-coupled hydrolysis of arachidonic acid from membrane phospholipids. Following exposure of macrophages to various foreign or endogenous stimulants, cytosolic phospholipase A[sub 2] is activated. Treatment with these compounds may also stimulate phospholipase D activity, and, in the presence of ethanol, phospholipase D catalyzes the synthesis of phosphatidylethanol. A cell-free system was used to evaluate the effect of phosphatidylethanol on cytosolic phospholipase A[sub 2] activity. Phosphatidylethanol (0.5 μM) added to 1-stearoyl-2-[[sup 3] H]-arachidonoyl-sn-glycero-3-phosphocholine vesicles stimulated cytosolic phospholipase A[sub 2] activity. However, high concentrations (20–100 μM) of phosphatidylethanol inhibited cytosolic phospholipase A[sub 2] activity. Phosphatidic acid, the normal phospholipase D product, also stimulated cytosolic phospholipase A[sub 2] activity at 0.5 μM, but had an inhibitory effect on cytosolic phospholipase A[sub 2] activity at concentrations of 50 and 100 μM. Ethanol (20–200 mM), the precursor of phosphatidylethanol, added directly to the assay did not alter cytosolic phospholipase A[sub 2] activity. These results suggest that phosphatidylethanol alters the physical properties of the substrate, and at lower concentrations of anionic phospholipids the substrate is more susceptible to hydrolysis. However, at high concentrations, phosphatidylethanol either reverses the alterations in physical properties of the substrate or phosphatidylethanol may be competing as the substrate. Both interactions may result in lower cytosolic phospholipase A[sub 2] activity.Copyright © 2000 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]