Macrophage-like Cell Line J774 Research Articles

In vitro characterization of anti-inflammatory activities of 3RS, 7R, 11R-phytanic acid.

The aim of the research described here was to investigate the in vitro immunomodulatory effects of 3RS, 7R, 11R-phytanic acid (3RS-PHY) from the perspective of efficacy against autoimmune diseases. 3RS-PHY is a milk component with strong agonist activity at the peroxisome proliferator activated receptor (PPAR). As PPAR is a therapeutic target for several human diseases, 3RS-PHY intake may have possible health benefits. Recently, we chemically synthesized a preparation of 3RS-PHY and demonstrated that 3RS-PHY inhibited T-cell production of interferon (IFN)-γ. However, the overall immunomodulatory effects were not evaluated. In this study, mouse splenocytes, purified T-cells and B-cells were stimulated by mitogens and incubated with 3RS-PHY, followed by evaluation of cytokine and antibody production. A macrophage-like cell line J774.1 was also incubated with 3RS-PHY to evaluate nitric oxide production. 3RS-PHY decreased mRNA levels not only of IFN-γ but also of interleukin (IL)-2, IL-10 and IL-17A in splenocytes and similar effects were confirmed at the protein level. In addition, 3RS-PHY had a direct action on T-cells with preferential inhibitory effects on Th1 and Th17 cytokines such as IFN-γ and IL-17A. Furthermore, 3RS-PHY suppressed antibody secretion by B-cells and nitric oxide production by J774.1 almost completely, indicating that 3RS-PHY is a bioactive fatty acid with anti-inflammatory properties. These findings encourage further investigations, including in vivo experiments, to evaluate whether 3RS-PHY actually shows the potential to prevent autoimmune diseases, and provide basic information to produce milk and dairy products with an increased 3RS-PHY concentration.

Hox-driven conditional immortalization of myeloid and lymphoid progenitors: Uses, advantages, and future potential.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

Two Bioactive Compounds, Uniformides A and B, Isolated from a Culture of Nocardia uniformis IFM0856T in the Presence of Animal Cells.

Two new peptides named uniformides A and B (1 and 2, respectively) were isolated from the cultured extracts of Nocardia uniformis IFM0856T in the presence of mouse macrophage-like cell line J774.1, in modified Czapek-Dox medium. These compounds were not produced in a culture containing only N. uniformis but in one that also included J774.1. Compounds 1 and 2 showed high cytotoxicity against J774.1 and suppressed the production of nitric oxide, IL-6, and IL-1β by inhibiting the NF-κB pathway.

Coculture of a Pathogenic Actinomycete and Animal Cells To Produce Nocarjamide, a Cyclic Nonapeptide with Wnt Signal-Activating Effect.

A coculture method with a pathogenic actinomycete of the genus Nocardia and an animal cell line was designed to reconstruct and emulate the initial infection state, and a new cyclic nonapeptide, named nocarjamide (1), was obtained by coculture of Nocardia tenerifensis IFM 10554T and the mouse macrophage-like cell line J774.1 in a modified Czapek-Dox medium. Nocarjamide (1) exhibited Wnt signal-activating effects.

Brazilian propolis ethanol extract and its component kaempferol induce myeloid-derived suppressor cells from macrophages of mice in vivo and in vitro

BackgroundBrazilian green propolis is produced by mixing secretions from Africanized honey bees with exudate, mainly from Baccharis dracunculifolia. Brazilian propolis is especially rich in flavonoids and cinammic acid derivatives, and it has been widely used in folk medicine owing to its anti-inflammatory, anti-viral, tumoricidal, and analgesic effects. Moreover, it is applied to prevent metabolic disorders, such as type 2 diabetes and arteriosclerosis. Previously, we demonstrated that propolis ethanol extract ameliorated type 2 diabetes in a mouse model through the resolution of adipose tissue inflammation. The aims of this study were to identify the immunosuppressive cells directly elicited by propolis extract and to evaluate the flavonoids that induce such cells.MethodsEthanol extract of Brazilian propolis (PEE; 100 mg/kg i.p., twice a week) was injected into lean or high fat-fed obese C57BL/6 mice or C57BL/6 ob/ob mice for one month. Subsequently, immune cells in visceral adipose tissue and the peritoneal cavity were monitored using FACS analysis. Isolated macrophages and the macrophage-like cell line J774.1 were treated with PEE and its constituent components, and the expression of immune suppressive myeloid markers were evaluated. Finally, we injected one of the identified compounds, kaempferol, into C57BL/6 mice and performed FACS analysis on the adipose tissue.ResultsIntraperitoneal treatment of PEE induces CD11b+, Gr-1+ myeloid-derived suppressor cells (MDSCs) in visceral adipose tissue and the peritoneal cavity of lean and obese mice. PEE directly stimulates cultured M1 macrophages to transdifferentiate into MDSCs. Among twelve compounds isolated from PEE, kaempferol has an exclusive effect on MDSCs induction in vitro. Accordingly, intraperitoneal injection of kaempferol causes accumulation of MDSCs in the visceral adipose tissue of mice.ConclusionBrazilian PEE and its compound kaempferol strongly induce MDSCs in visceral adipose tissue at a relatively early phase of inflammation. Given the strong anti-inflammatory action of MDSCs, the induction of MDSCs by PEE and kaempferol is expected to be useful for anti-diabetic and anti-inflammatory therapies.Graphical

Quantitative analysis of the anti-inflammatory activity of orengedokuto II: berberine is responsible for the inhibition of NO production.

Orengedokuto is a Kampo formula that has been used for removing "heat" and "poison" to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. We report here our analysis of the anti-inflammatory effect of the component crude drugs of orengedokuto and their constituents using the inhibition of nitric oxide (NO) production in the murine macrophage-like cell line J774.1. An initial comparison of NO production inhibitory activities of the extracts of the component crude drugs and their combinations revealed that the activity could be attributed to Phellodendron Bark and Coptis Rhizome. Berberine (1), the major constituent of these crude drugs, showed potent activity (IC50 4.73 ± 1.46μM). Quantitative analysis of 1 in the extracts of all combinations of component crude drugs revealed that the amount of 1 in each extract of the combination of Scutellaria Root with either Phellodendron Bark and/or Coptis Rhizome was lower than that in the corresponding mixtures of the extracts of the individual crude drugs and that 1 was present in the precipitates formed during the decoction process. To the contrary, the differences in the amounts of 1 were smaller in the extracts containing Gardenia Fruit. These results indicated that the constituents of Scutellaria Root precipitated with 1 and that the constituents of Gardenia Fruit dissolved the precipitates. To identify the constituents affecting the solubility of 1, we fractionated the hot-water extracts of Scutellaria Root based on solubility tests of 1 to give baicalin (2), wogonin (3) and oroxyloside (4), which formed precipitates with 1.

Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of animal cells.

A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from D-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a "co-culture" in which the genus Nocardia was cultured in the presence of an animal cell line.

Pharmacological evaluation for improvement of Kanazawa Sutra, medicinal thread for anal fistula.

Kanazawa Sutra (KanS) is a medicinal thread that is used for the treatment of anal fistula. It is used as a substitute for Kshara Sutra (KS) which is used in Ayurvedic medicine. KanS is composed of Ficus carica latex (FCL), Capsicum annuum tincture (CAT), Achyranthes fauriei Kshara (which is processed ash from the whole plant) and powdered Curcuma longa rhizome (CLR). In this study, we evaluated the ingredients of KanS by measuring nitric oxide (NO) production in murine macrophage-like cell line J774.1 as well as examining cytotoxicity to rat skeletal muscle myoblasts (L6) and L6 differentiation, with a view to improving its pharmacological effect. We focused on Mallotus japonicus bark (MJB), which is described in the Japanese Pharmacopeia and belongs to the Euphorbiaceae family. Its biological activities were evaluated in a similar manner to the evaluation of KanS ingredients. We found that MJB extracts showed similar biological activity to Euphorbia neriifolia latex (ENL), an ingredient of KS. We conclude that the NO inhibitory activity of KanS is mainly due to CLR, and its cytotoxicity to L6 and inhibitory activity on L6 differentiation are mainly due to CLR and FCL. As CAT has no characteristic activity, the biological activity and the anal fistula treatment ability of KanS would be improved by substituting MJB for CAT.

The Biological Properties of the Strain Francisella tularensis 15 NIIEG with Decreased Gene Expression sodB, Encoding Fe-Dependent Superoxide Dismutase

Relevance. Superoxide anion has bactericidal properties and is also an important inducer of proinflammatory cytokines in macrophages. We have created F. tularensis 15/sodBII strain with transiently decreased FeSOD synthesis level and more sensitive to oxidative stress. So we suggest that the modified vaccine strain have lower reactogenicity. Goal. Studying of effect of sodB gene expression modulation on biological properties of vaccine F. tularensis strain 15 NIIEG. Materials and methods. F. tularensis survival in macrophage-like cell line J774.1A and in spleen and liver of infected mice were analyzed through colony-forming unit enumeration. Strains reactogenicity was assessed by the dynamics of change in weight of infected mice. Efficacy of immune response generated by mutant strain of F. tularensis 15/sodBII was estimated with virulent F. tularensis strain Schu S4 infection in the BALB/c mice model. Results. Degree of protection was significantly more pronounced in the mice vaccinated with the strain F. tularensis with decreased sodB gene expression in comparison with parental F. tularensis strain NIIEG 15. Conclusions. The modified strain of F. tularensis 15/sodBII may be consider as a promising variant for development of a new tularemia vaccine with reduced reactogenicity.

Application of a new method, orthogonal projection to latent structure (OPLS) combined with principal component analysis (PCA), to screening of prostaglandin E2 production inhibitory flavonoids in Scutellaria Root.

A new method consisting of orthogonal projection to latent structure (OPLS) and modified principal component analysis (PCA) was applied to a screening of bioactive compounds from natural products. In this report, extracts of 52 Scutellaria Root (the root ofScutellaria baicalensis Georgi) samples were analyzed by high-performance liquid chromatography (HPLC), and their inhibitory activities towards prostaglandin E2 (PGE2) production in a murine macrophage-like cell line J774.1 were examined. Wogonin and oroxylin A were predicted to be strong inhibitors of PGE2 production by OPLS analysis of the data. However, 6-methoxywogonin, which has been reported to have inhibitory activity, was omitted. Modified PCA was then applied to these data as a filter to exclude compounds less relevant to the activity, and OPLS analysis was applied to the modified data. As a result, this method predicted wogonin, oroxylin A and 6-methoxywogonin to be strong inhibitors of PGE2 production without any prior knowledge. The predictions by the OPLS combined with PCA method of PGE2 production inhibitory activities of 52 samples showed good agreement with the actual data. This method is simple and effective and can be used in screening of bioactive natural compounds without any prior knowledge.

Cellular interaction of polygycerol dendrimers and those derivatives

Event Abstract Back to Event Cellular interaction of polygycerol dendrimers and those derivatives Tooru Ooya1 and Yuji Kawashima1 1 Kobe University, Department of Chemical Science and Engineering, Japan Introduction: Understanding of cellular events is crucial for the development of nanomedicine. Polyglycerol dendrimers (PGDs) and hyperbranched polyglycerol (HyPG) have been examined for various biomedical applications due to their positive biocompatibilities and unique physicochemical properties[1]. Although HyPG-based naogels were reported with showing a size-dependent cellular uptake[2], PGDs, which modulate the polarity of water[3]-[5], has not been subjected to cellular interaction. In this study, PGDs and glycerol dendrons (GDs)-modified liposomes were investigated to evaluate modulating cellular functions in terms of cellular uptake, death and activation. Materials and Methods: PGDs of generation 3 and 4 (Fig. 1) were prepared by our previous method. Cytotoxicity test was performed by MTT assay using HeLa cells. Fig. 1 Chemical Structure of PGDs of generations 3 and 4 Mono-stearyl PGD-G4 was mixed with Qdot655 organic quantum dots to obtain PGD-G4-coated Qdot655. Nitric oxide (NO) release from mouse macrophage-like cell line J774.1 stimulated by the addition of the PGD-G4-coated Qdot655 was evaluated by measuring emission intensity of the NO-reacted DAF-FM. GDylated liposomes were prepared and evaluated in terms of cellular uptake (Fig. 2). The obtained conjugates were introduced into bare liposomes, in which doxorubicin (DOX) was preliminarily encapsulated, by a post-insertion method. Cellular uptake amount of each liposomes was evaluated by flow cytometry. Fig. 2 Preparation of GD-modified liposome containing L-α-phosphatidylcholine, cholesterol, and DSPE-GD-G2. Results and Discussion: With increasing the generation of PGDs, T1 was significantly shorten at the same number of hydroxyl groups per one water molecule, whereas the T1 value of PGD-G4 was larger than that of PGD-G3. This result suggests that the amount of bound water located on PGD-G3 molecule was apparently larger than that on PGD-G4. PGD-G3 showed significant cytotoxicity comparing with PGD-G4, suggesting the amount of bound water state affected to the interaction with Hela cells. The results of NO release stimulated by the PGD-G4-coated Qdot655 suggest that the PGD-G4-coatedQdot655 might induce nitric oxide synthase activation. Cellular uptake behavior of DOX-loaded GDylated liposomes was significantly greater than that of PEGylated liposome. Particularly, GD-G2ylated liposomes were internalized into cells as well as PEGylated liposomes, but GD-G1ylated liposome was much greater than the other liposomes. Conclusion: Local density of hydoxy groups provided by PGDs induced possibly both cell death and cellular activation. GDylation of liposome is a valuable approach to modulating cellular uptake of the encapsulated biologically active compounds. This work was partially supported by Grant-in-Aid for Scientific Research on Innovative Areas (New Polymeric Materials Based on Element-Blocks) (No. 15H00748) from JSPS, Japan.

Mg-doped biphasic calcium phosphate by a solid state reaction route: Characterization and evaluation of cytotoxicity

Hydroxyapatite (HAP) and β-tricalcium phosphate (β-TCP) are widely used in tissue engineering because of their chemical similarity to the inorganic bone phase. In this work, we prepare biphasic calcium phosphate (BCP, a mixture of HAP and β-TCP) doped with different concentrations of magnesium to investigate the influence of magnesium on the BCP crystal structure. Magnesium is known to be an important element in the composition of bones and teeth. Recent research has shown that the doping of magnesium into BCP improves its bone metabolism and mechanical properties without affecting its biocompatibility. The samples were prepared by solid-state reaction from calcium carbonate, monobasic ammonium phosphate, and magnesium nitrate hexahydrate. Varying concentrations of magnesium were used and its modifications were examined by different characterization techniques. The phase composition and morphology of the ceramic powders were characterized by X-ray diffraction and scanning electron microscopy, respectively. The functional groups were analyzed using Fourier transform infrared spectroscopy and Raman spectroscopy. Cell viability experiments, using macrophage-like cell lines J774, showed that the synthesized Mg-doped BCP did not exhibit cytotoxicity regardless of the doses assayed or the different concentrations of magnesium used, suggesting it as a good material for potential biological applications.

Nitric oxide is an important regulator of heme oxygenase-1 expression in the lipopolysaccharide and interferon-γ-treated murine macrophage-like cell line J774.1/JA-4.

Heme oxygenase-1 (HO-1) catabolizes the degradation of heme into bilirubin, carbon monoxide, and iron ions. The HO-1 products provide antioxidant cytoprotection in addition to having potent antiinflammatory and immunomodulatory functions. HO-1 is induced by its substrate heme and environmental factors including oxidative and heat stresses. Although previous studies reported that lipopolysaccharide (LPS) induced the expression of both the HO-1 gene and its protein in macrophages, the major regulators of HO-1 expression remain unknown. To identify these regulators, we used two types of cell, the murine macrophage-like cell line J774.1/JA-4 and its LPS-resistant mutant, LPS1916. Based on a comparison of the results obtained with these cells, we found that nitric oxide (NO) was closely linked to the induction of HO-1. Real-time polymerase chain reaction (PCR) showed that the time course for inducible HO-1 mRNA by LPS or LPS+interferon (IFN)-γ was similar to that for inducible NO synthase (iNOS) mRNA. Furthermore, the expression of iNOS mRNA and protein increased earlier than that of HO-1 mRNA and protein. N-Nitro-L-arginine methyl ester, an NO synthase inhibitor, reduced both HO-1 expression and NO production in LPS+IFN-γ-treated JA-4 cells. Furthermore, NOC-12, an NO donor, significantly induced HO-1 expression not only in JA-4 but also in LPS1916 cells. Reactive oxygen species (ROS) scavengers, such as superoxide dismutase and catalase, did not affect HO-1 protein expression in LPS+IFN-γ-treated JA-4 cells. These results suggest that, among ROS, NO plays an important role in HO-1 induction in activated macrophages treated with LPS+IFN-γ.

Effects of strains of Lactococcus lactis on the production of nitric oxide and cytokines in murine macrophages.

Nitric oxide (NO) is a multifunctional mediator that is involved in a variety of pathologic and physiologic processes. Few studies have addressed the effect of lactic acid bacteria (LAB), especially Lactococcus lactis strains used in dairy products, on inducible nitric oxide synthase (iNOS) induction as a component of the host's gastrointestinal immune response. We investigated the ability of L. lactis strains to induce NO synthesis in the murine macrophage-like cell line J774.1 and in peritoneal macrophages from mice. The degree of NO induction was specific to the L. lactis strain used. Compared with the no-treatment control, heat treatment of L. lactis cells decreased NO and TNF-α levels but further stimulated interleukin (IL)-12 production. Adding L. lactis cells to peritoneal macrophages dose-dependently increased the production of NO and IL-10 but decreased that of IL-12p70. Adding L. lactis cells to interferon-γ-stimulated J774.1 cells enhanced cell death and the production of NO and IL-12p40, whereas addition of 1400W, a specific inhibitor of iNOS, decreased NO production and cell death. Conversely, adding 1400W to J774.1 cells further enhanced IL-12p40 production, suggesting that IL-12 production is perturbed by excess endogenous NO. IL-12 production is thought to be a marker of improved immunostimulation. Our results suggest that IL-12 production could be increased by limiting endogenous NO production.

A role for LHC1 in higher order structure and complement binding of the Cryptococcus neoformans capsule.

Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.

Comparative proteome profiling of host–pathogen interactions: insights into the adaptation mechanisms of Francisella tularensis in the host cell environment

The intracellular pathogens have the unique capacity to sense the host cell environment and to respond to it by alteration in gene expression and protein synthesis. Proteomic analysis of bacteria exposed directly to the host cell milieu might thus greatly contribute to the elucidation of processes leading to bacterial adaptation and proliferation inside the host cell. Here we have performed a global proteome analysis of a virulent Francisella tularensis subsp. holarctica strain during its intracellular cycle within the macrophage-like murine cell line J774.2 using the metabolic pulse-labeling of bacterial proteins with (35)S-methionine and (35)S-cysteine in various periods of infection. The two-dimensional gel analysis revealed macrophage-induced bacterial proteome changes in which 64 identified proteins were differentially expressed in comparison to controls grown in tissue culture medium. Nevertheless, activation of macrophages with interferon gamma before in vitro infection decreased the number of detected alterations in protein levels. Thus, these proteomic data indicate the F. tularensis ability to adapt to the intracellular hostile environment that is, however, diminished by prior interferon gamma treatment of host cells.

Activation of J774.1 murine macrophages by lactate dehydrogenase

We have already reported that lactate dehydrogenase (LDH) activates lymphocytes in vitro and in vivo. In this paper, we report the activating effects of LDH on the macrophage-like cell line J774.1. LDH was found to enhance production of IL-6 and TNF-α by J774.1 cells in a dose-dependent manner. Transcription levels of IL-6 and TNF-α in J774.1 cells were also enhanced by supplementation with LDH. From immunoblot analysis, it was revealed that LDH enhances the phosphorylation level of JNK in J774.1 cells. Moreover, the JNK inhibitor SP600125 decreased production of IL-6 and TNF-α induced by LDH. NF-κB translocation to the nucleus was also facilitated by LDH. These results was revealed that LDH enhances production of IL-6 and TNF-α by J774.1 cells via the increase of JNK phosphorylation and NF-κB translocation to the nucleus. Our data indicated that macrophages may be activated by LDH released from damaged tissues and cells in our body.

Potent effects of, and mechanisms for, modification of crosstalk between macrophages and adipocytes by lactobacilli

The murine macrophage-like cell line J774.1 was treated with heat-killed cells of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC 0356). Interleukin (IL)-6, IL-12, and tumor necrosis factor-α were profiled from the J774.1 cells using enzyme-linked immunosorbent assay methods. The conditioned medium from cultured J774.1 cells was transferred to the preadipocyte cell line 3T3-L1 (which is a mouse embryonic fibroblast-adipose-like cell line). Growth and differentiation of 3T3-L1 cells were monitored by analyzing lipid accumulation and expression of peroxisome proliferator-activated receptor (PPAR)-γ mRNA. The medium conditioned by 3T3-L1 cells was added to J774.1 cells and the cytokines in the supernatant analyzed. Compared with that of cells exposed to a PBS-conditioned medium, lipid accumulation in 3T3-L1 cells was significantly suppressed in a dose-dependent manner by each medium that had been conditioned with LGG and TMC0356. PPAR-γ mRNA expression in 3T3-L1 cells was also significantly downregulated (P < 0.01, P < 0.05, respectively). The conditioned medium of 3T3-L1 adipose phenotype significantly stimulated production of IL-6 and IL-12 in J774.1 cells treated with LGG and TMC0356. These results suggest that lactobacilli may suppress differentiation of preadipocytes through macrophage activation and alter the immune responses of macrophages to adipose cells.

A Quantitative Proteomic Profile of the Nrf2-Mediated Antioxidant Response of Macrophages to Oxidized LDL Determined by Multiplexed Selected Reaction Monitoring

The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.

Quantitative analysis of anti-inflammatory activity of orengedokuto: importance of combination of flavonoids in inhibition of PGE2 production in mouse macrophage-like cell line J774.1

Orengedokuto is a Kampo formula which has been used for removing "heat" and "poison" to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. In this report, we quantitatively analyzed the anti-inflammatory effect of the component crude drugs of orengedokuto and their constituents, using inhibition of prostaglandin E2 (PGE2) production in the murine macrophage-like cell line J774.1. First, we compared PGE2 production inhibitory activities of extracts of combinations of the component crude drugs, which showed that the activity could be ascribed to Scutellaria Root. Next, as baicalin (1), one of the major constituents of Scutellaria Root, did not show any activity, and baicalein (2), the aglycon of 1, showed only weak activity (IC50 92μM), a hot-water extract of Scutellaria Root was fractionated under the guidance of the activity to give wogonin (3) (IC50 28μM), 6-methoxywogonin (4) (IC50 7.2μM) and oroxylin A (5) (IC50 45μM) from the most active fraction. However, the activities of these compounds at concentrations equivalent to those in the extract were weaker than that of the extract, and none of these compounds alone could explain the activity of the extract. Therefore, we examined the activity of combinations of compounds 2-5. Comparison of all combinations of the four compounds in a ratio which is the same as in the extract revealed that wogonin (3) had an essential role in the activity, and a combination of baicalein (2) and wogonin (3), together with 6-methoxywogonin (4) or oroxylin A (5), was necessary to show activity equivalent to that of the extract.