Isolated Mouse Peritoneal Macrophages Research Articles

Immunogenic properties of nickel-doped maghemite nanoparticles and the implication for cancer immunotherapy

Nanoparticles are commonly used in diagnostics and therapy. They are also increasingly being implemented in cancer immunotherapy because of their ability to deliver drugs and modulate the immune system. However, the effect of nanoparticles on immune cells involved in the anti-tumor immune response is not well understood. The study reported here showed that nickel-doped maghemite nanoparticles (FN NP) are differentially cytotoxic to cultured mouse and human cancer cell lines, causing their death without negatively impacting the subsequent anticancer immune response. It also found that FN NP induced cell death in the mouse colorectal cancer cell line CT26 and human prostate cancer cell line PC-3, but not in the human prostate cancer cell line LNCaP. The induced cancer cell death did not affect the phenotype and responsivity of the isolated mouse peritoneal macrophages, or ex vivo-generated mouse bone marrow-derived, or human monocyte-derived dendritic cells. Additionally, the induced cancer cell death did not prevent the ex vivo-generated mouse or human dendritic cells from stimulating lymphocytes and enriching cell cultures with cancer cell-reactive T-cells. In conclusion, this study shows that FN NP could be a valuable platform for targeting cancer cells without causing immunosuppressive effects on the subsequent anticancer immune response.

Selenium Ameliorates Acetaminophen-Induced Oxidative Stress via MAPK and Nrf2 Pathways in Mice.

Overdose of acetaminophen (paracetamol), a widely used non-prescriptive analgesic and antipyretic medication, is one of the main causes of drug-induced acute liver failure around the world. Oxidative stress contributes to this hepatotoxicity. Antioxidants are known to protect the liver from oxidative stress. Selenium, a potent antioxidant, is a commonly used micronutrient. Here, we evaluated the protective effect of selenium on acetaminophen-induced hepatotoxicity. Treating Wistar albino mice with sodium selenite (1mg/kg) before or after inducing hepatotoxicity with acetaminophen (150mg/kg) significantly reduced the levels of liver injury biomarkers such as serum glutamate oxaloacetate transaminase and serum glutamate pyruvate transaminase. In addition, selenium-treated mice showed decreased levels of oxidative stress markers such as protein carbonyls and myeloperoxidase. Acetaminophen treatment stimulated all three mitogen-activated protein kinases (MAPKs) and Keap1 and decreased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 in liver and in isolated mouse peritoneal macrophages, which was reversed by selenium treatment. Our findings suggest that the reactive oxygen species-mediated Nrf2 and MAPK pathways are critical players in acetaminophen-induced hepatotoxicity. These key findings offer an alternative therapeutic target for addressing acetaminophen-induced hepatotoxicity.

Oridonin attenuates the progression of atherosclerosis by inhibiting NLRP3 and activating Nrf2 in apolipoprotein E-deficient mice

Oridonin, a well-known traditional Chinese herbal medicinal product isolated from Isodon rubescens (Hemsl.) H.Hara, has many potential properties, including anti-inflammatory and antioxidant activities. However, there is no evidence whether oridonin have a protective effect on atherosclerosis. This study focused on the effects of oridonin on oxidative stress and inflammation generated from atherosclerosis. The therapeutic effect on atherosclerosis was evaluated by intraperitoneal injection of oridonin in a high-fat fed ApoE−/− mouse model. We isolated mouse peritoneal macrophages and detected the effect of oridonin on oxidized low-density lipoprotein-induced lipid deposition. Oil red O staining, Masson's staining, dihydroethidium fluorescence staining, immunohistochemical staining, western blotting analysis, immunofluorescence, enzyme-linked immunosorbent assay and quantitative real-time PCR were used to evaluate the effect on atherosclerosis and explore the mechanisms. Oridonin treatment significantly alleviated the progression of atherosclerosis, reduced macrophage infiltration and stabilized plaques. Oridonin could significantly inhibit inflammation associated with NLRP3 activation. Oridonin significantly reduced oxidative stress by blocking Nrf2 ubiquitination and degradation. We also found that oridonin could prevent the formation of foam cells by increasing lipid efflux protein and reducing lipid uptake protein in macrophages. Oridonin has a protective effect on atherosclerosis in ApoE−/− mice, which may be related to the inhibition of NLRP3 and the stabilization of Nrf2. Therefore, oridonin may be a potential therapeutic agent for atherosclerosis.

The Potential Effects of Scrophularia Striata Boiss on COVID-19

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) rapidly spread worldwide. The exact mechanisms involved in the pathogenesis of Coronavirus Disease 2019 (COVID-19) are not fully understood. In addition to classical treatment, herbal medicine may effectively manage COVID-19 symptoms and supportive therapy. Scrophularia striata Boiss is a perennial herbaceous plant with immunomodulatory and anti-inflammatory properties. The extract of S. striata Boiss has revealed immunomodulatory effects on reducing T helper 2 (Th2) cytokines, including IL-4 and IL-5, that may be used to manage allergic diseases and asthma. Scrophularia striata Boiss reduces IL-4, and IL-5 may alleviate allergic diseases and asthma. The ethyl acetate extract of S. striata Boiss inhibits the production of IL-1β, TNF-α, and PGE2. Quercetin inhibits proinflammatory mediators while increasing anti-inflammatory mediators. Ethanolic extract of S. striata can significantly reduce NO production in the isolated mouse peritoneal macrophages. Moreover, S. striata Boiss has 3 essential flavonoids: quercetin, isorhamnetin-3-O-rutinoside, and nepitrin. Of them, quercetin inhibits TNF-α, IL-1β, IL-6, PGE2, COX-2, and NO, whereas increases anti-inflammatory cytokines. Therefore, S. striata Boiss is recommended for clinical trial studies in patients with COVID-19 and other viral diseases.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-β-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1β release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1β release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

P38 MAP-kinase inhibitor protects against platelet-activating factor-induced death in mice

Platelet-activating factor (PAF) is a potent inflammatory agonist. In Swiss albino mice, intraperitoneal injection of PAF causes sudden death with oxidative stress and disseminated intravascular coagulation (DIC), characterized by prolonged prothrombin time, thrombocytopenia, reduced fibrinogen content, and increased levels of fibrinogen degradation products. However, the underlying mechanism(s) is unknown. The PAF-R antagonist WEB-2086 protected mice against PAF-induced death by reducing DIC and oxidative stress. Accordingly, general antioxidants such as ascorbic acid, α-tocopherol, gallic acid, and N-acetylcysteine partially protected mice from PAF-induced death. N-acetylcysteine, a clinically used antioxidant, prevented death in 67% of mice, ameliorated DIC characteristics and histological alterations in the liver, and reduced oxidative stress. WEB-2086 suppressed H2O2-mediated oxidative stress in isolated mouse peritoneal macrophages, suggesting that PAF signaling may be a downstream effector of reactive oxygen species generation. PAF stimulated all three (ERK, JNK, and p38) of the MAP-kinases, which were also inhibited by N-acetylcysteine. Furthermore, a JNK inhibitor (SP600125) and ERK inhibitor (SCH772984) partially protected mice against PAF-induced death, whereas a p38 MAP-kinase inhibitor (SB203580) provided complete protection against DIC and death. In human platelets, which have the canonical PAF-R and functional MAP-kinases, JNK and p38 inhibitors abolished PAF-induced platelet aggregation, but the ERK inhibitor was ineffective. Our studies identify p38 MAP-kinase as a critical, but unrecognized component in PAF-induced mortality in mice. These findings suggest an alternative therapeutic strategy to address PAF-mediated pathogenicity, which plays a role in a broad range of inflammatory diseases.

Flavonoid Enriched Fraction of Campylandra aurantiaca Attenuates Carbon Tetrachloride Induced Oxidative DNA Damage in Mouse Peritoneal Macrophages in Animal Model.

Recent studies have sought to draw attention of biological activity of Campylandra aurantiaca. The aim of the present study was to evaluate the effect of flavonoid enriched fraction of Campylandra aurantiaca (FEFCA) on in vitro and in vivo antioxidant and DNA protective effect in mouse peritoneal macrophages cells. FEFCA was characterized by HPLC analysis. The in vitro antioxidant activities of FEFCA was measured by different in vitro assays like 1, 1-diphenyl-2-picrylhydrazil radical (DPPH), superoxide anions, nitric oxide and hydroxyl radicals scavenging methods. Isolated mouse peritoneal macrophages were oxidized by carbon tetra chloride (CCl4) in animal model; subsequently the protective effect of FEFCA was determined in terms of estimation of antioxidant enzyme and the damage to DNA of the cells. FEFCA exhibited both in vitro antioxidant activities in a concentration dependent manner. FEFCA significantly (*p<0.05) attenuated the oxidative DNA damage of mouse peritoneal macrophage cells induced by CCl4 in an in vivo assay. Therefore FEFCA showed good free radical scavenging activity as well as reduced oxidative DNA damage in mouse peritoneal macrophages in animal model.

Modulation of macrophage functionality induced in vitro by chlorpyrifos and carbendazim pesticides

The immune response is the first defense against pathogens; however, it is very sensitive and can be impacted on by agrochemicals such as carbamate and organophosphate pesticides widely present in the environment. To understand how pesticides can affect immune cell function in vitro, this study investigated the effects of chlorpyrifos (CPF) and carbendazim (CBZ), the most commonly used pesticides worldwide, on murine immune cell (i.e. macrophage) functions, including lysosomal enzyme activity and pro-inflammatory cytokines (IL-1β and TNFα) and nitric oxide (NO) production by isolated mouse peritoneal macrophages. This study showed for the first time that CPF and CBZ dose-relatedly reduced macrophage lysosomal enzyme activity and LPS-induced production of IL-1β, TNFα and NO. In general, the effects caused by CPF appeared more pronounced than those by CBZ. Collectively, these results demonstrated that CPF and CBZ exhibited marked immunomodulatory effects and could act as potent immunosuppressive factors in vitro. This inhibition of macrophage pro-inflammatory function may be an integral part of the underlying mode of action related to pesticide-induced immunosuppression.

Impact of Glutathione Peroxidase-1 Deficiency on Macrophage Foam Cell Formation and Proliferation: Implications for Atherogenesis

Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE−/− mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE−/− mice after 12 weeks on a Western type diet revealed that both macrophages and – even though to a less extent – smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1−/−ApoE−/− mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.

Stercurensin inhibits nuclear factor‐κB‐dependent inflammatory signals through attenuation of TAK1–TAB1 complex formation

We identified a chalcone, 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (stercurensin), as an active compound isolated from the leaves of Syzygium samarangense. In the present study, the anti-inflammatory effects and underlying mechanisms of stercurensin were examined using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and mice. To determine the effects of stercurensin in vitro, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were analyzed by RT-PCR and immunoblotting. Nuclear factor-κB (NF-κB) activation and its upstream signaling cascades were also investigated using a dual-luciferase reporter assay, electrophoretic mobility shift assay, immunoblotting, immunofluorescence, and immunoprecipitation. To verify the effects of stercurensin in vivo, the mRNA expression levels of iNOS and COX-2 were evaluated in isolated mouse peritoneal macrophages by quantitative real-time PCR, and the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were assessed in serum samples from mice using a Luminex system. Pretreatment with stercurensin reduced LPS-induced iNOS and COX-2 expression, thereby inhibiting nitric oxide (NO) and prostaglandin E(2) production, respectively. In addition, an inhibitory effect of stercurensin on NF-κB activation was shown by the recovery of LPS-induced inhibitor of κB (I-κB) degradation after blocking the transforming growth factor-β-activated kinase 1 (TAK1)/I-κB kinase signaling pathway. In mouse models, stercurensin negatively regulated NF-κB-dependent pro-inflammatory mediators and cytokines. These results demonstrate that stercurensin modulates NF-κB-dependent inflammatory pathways through the attenuation of TAK1-TAB1 complex formation. Our findings demonstrating the anti-inflammatory effects of stercurensin in vitro and in vivo will aid in understanding the pharmacology and mode of action of stercurensin.

Suppression of nitric oxide production in activated murine peritoneal macrophages in vitro and ex vivo by Scrophularia striata ethanolic extract

Ethnopharmacological relevanceScrophularia striata (Scrophulariaceae), a traditional Iranian medicine, has been used for the treatment of allergy, rheumatics and chronic inflammatory disorders. Aim of the studyIn the present study, we investigated the in vitro and ex vivo suppressive effects of Scrophularia striata ethanolic extract on nitric oxide production in mouse peritoneal macrophages. Materials and methodsPeritoneal macrophages were harvested by lavaging with ice cold phosphate buffer saline. Macrophages obtained from mice not treated were cultured with 10μg/mL lipopolysaccaride (LPS), 20U/mL interferon-γ (IFN-γ), and various concentrations of Scrophularia striata extract for the in vitro experiments and those obtained from mice treated with different doses of the extract for 7 days were cultured with 10μg/mL LPS, 20U/mL IFN-γ for the in vivo experiments. Nitrit levels were measured by using the diazotization method based on the Griess reaction, which is an indirect assay for NO production. ResultsIn vitro exposure of mouse peritoneal macrophages with various concentrations of Scrophularia striata extract (10, 50 and 100μg/mL) significantly suppressed NO production in a dose-dependent manner. In vivo administration of Scrophularia striata extract (50 and 100mg/kg) to Balb/c mice inhibited LPS and IFN-γ induced production of NO in the isolated mouse peritoneal macrophages ex vivo in a dose-dependent manner. Exposure to Scrophularia striata extract had no effect on cell viability. ConclusionThe results of the study demonstrated that the Scrophularia striata extract inhibit NO production in activated murine macrophages and we suggest that Scrophularia striata may be used in treating the inflammatory diseases.

Regulation of Podosome Formation in Macrophages by a Splice Variant of the Sodium Channel SCN8A

Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.

Protective Effect of Polysaccharide Fractions from Radix A. Sinensis against tert-Butylhydroperoxide Induced Oxidative Injury in Murine Peritoneal Macrophages

Three Angelica sinensis polysaccharide fractions (APFs), named APF1, APF2 and APF3, were isolated and purified from Radix A. sinensis and their antioxidant activities were evaluated in isolated mouse peritoneal macrophages by pretreatment with APFs before exposure to 0.2 mM tertbutylhydroperoxide (t-BHP). The results showed that pretreatment of the macrophages with APFs as low as 10 microg/ml could significantly enhance t-BHP-decreased cell survival, intracellular glutathione (GSH) content and superoxide dismutase (SOD) activity, and also inhibited t-BHP-increased lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation (p < 0.05), and APF3 was the most active fraction, followed by APF2 and APF1 in decreasing order. Furthermore, we found for the first time that the bound-protein in APF3 was associated closely with the protective effects and the polysaccharide inhibited the excess NO release from t-BHP-activated macrophages to protect host cells.

Acanthopanax senticosus inhibits nitric oxide production in murine macrophages In Vitro and In Vivo

Excess nitric oxide (NO) production has been implicated in inflammatory diseases. The present study investigated the inhibitory effect of the stem bark extract of Acanthopanax senticosus (A. senticosus) on NO production in murine macrophages in vitro and in vivo. In vitro exposure of RAW264.7 cells to 1, 10, 50, 100, 250, 500 and 1000 microg/mL of A. senticosus extract significantly suppressed NO production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in a dose-dependent manner. In vitro exposure of mouse resident peritoneal macrophages to 1, 10, 100 and 1000 microg/mL of A. senticosus extract significantly suppressed NO production induced by LPS and IFN-gamma in a dose-dependent manner. In vivo administration of A. senticosus extract (50, 100 and 200 mg/kg) to KM mice dose-dependently inhibited LPS and IFN-gamma induced production of NO in isolated mouse peritoneal macrophages ex vivo. Exposure to A. senticosus extract had no effect on cell viability and systemic toxicity. The results demonstrated that the stem bark extract of A. senticosus extract inhibits NO production in murine macrophages in vitro and in vivo.

Granuloma formation in vitro requires B-1 cells and is modulated by Paracoccidioides brasiliensis gp43 antigen

The mechanisms that determine granuloma formation and the significance of this type of inflammatory response in the pathogenesis of fungal diseases such as paracoccidioidomycosis are far from fully understood. We developed a granuloma model in vitro using beads to evaluate the role of isolated mouse peritoneal macrophages and B-1 cells. We also investigated granuloma formation in the presence of gp43, the main antigenic component of Paracoccidioides brasiliensis, which is secreted exocellularly. To determine whether B-1 cells, macrophages, or both, participate in granuloma formation, peritoneal cells from Xid mice, which lack B-1 cells, were used. Granuloma-like structures were not formed with Xid peritoneal cells or with cells from wild type mice that had their peritoneal and pleural cavities irradiated before the cultures were established. Granulomas were observed either when total adherent peritoneal cells or when isolated B-1 cells were added to macrophage cultures. The data strongly suggest that an interaction of B-1 cells and macrophages plays an important role in granuloma-like formation in this experimental model and that the presence of gp43 strongly stimulates this response.

Macrophage activation by polysaccharide fraction isolated from Salicornia herbacea

We demonstrate that polysaccharides isolated from Salicornia herbacea (Salicornia polysaccharides, SPS) significantly induces nitric oxide (NO) production and inducible NO synthase (iNOS) transcription through the activation of nuclear factor-κB/Rel (NF-κB/Rel). SPS dose-dependently induced the production of NO in isolated mouse peritoneal macrophages and RAW 264.7, a mouse macrophage-like cell line. Moreover, iNOS gene expression was strongly induced by SPS in RAW 264.7 cells. To further investigate the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of SPS on the activation of transcription factors including NF-κB/Rel and Oct, whose binding sites were located in the promoter of iNOS gene. Treatment of RAW 264.7 cells with SPS produced strong induction of NF-κB/Rel-dependent reporter gene expression, whereas Oct-dependent gene expression was not affected by SPS. Nuclear translocation and DNA binding activity of NF-κB/Rel was significantly induced by SPS. The treatment with NF-κB SN50, an inhibitor of NF-κB/Rel nuclear translocation, effectively inhibited the activation of NF-κB/Rel binding complexes and NO production. In conclusion, we demonstrate that SPS stimulates macrophages to express iNOS gene through the activation of NF-κB/Rel.

Macrophage activation by polysaccharide isolated from Astragalus membranaceus

We show that APS, a polysaccharide isolated from the roots of Astragalus membranaceus, significantly induces nitric oxide (NO) production and inducible NO synthase (iNOS) transcription through the activation of nuclear factor-κB/Rel (NF-κB/Rel). In vivo administration of APS induced NO production by peritoneal macrophages of B6C3F1 mice. APS also dose-dependently induced the production of NO in isolated mouse peritoneal macrophages and RAW 264.7, a mouse macrophage-like cell line. Moreover, iNOS protein and mRNA transcription were strongly induced by APS in RAW 264.7 cells. To further investigate the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of APS on the activation of transcription factors including NF-κB/Rel and Oct, whose binding sites were located in the promoter of iNOS gene. Treatment of RAW 264.7 cells with APS produced strong induction of NF-κB/Rel-dependent reporter gene expression, whereas Oct-dependent gene expression was not affected by APS. Nuclear translocation and DNA binding activity of NF-κB/Rel was significantly induced by APS. The treatment with NF-κB SN50, an inhibitor of NF-κB/Rel nuclear translocation, effectively inhibited the activation of NF-κB/Rel binding complexes and NO production. In conclusion, we demonstrate that APS stimulates macrophages to express iNOS gene through the activation of NF-κB/Rel.

Hyperlipidemia in Concert With Hyperglycemia Stimulates the Proliferation of Macrophages in Atherosclerotic Lesions

Hyperglycemia and hyperlipidemia are important risk factors for diabetes-accelerated atherosclerosis. Macrophage proliferation has been implicated in the progression of atherosclerosis. We therefore investigated the effects of hyperglycemia and hyperlipidemia on macrophage proliferation in murine atherosclerotic lesions and isolated primary macrophages. Hyperglycemic LDL receptor-deficient mice that were fed a cholesterol-free diet for 12 weeks did not have elevated cholesterol levels compared with nondiabetic mice, and there was no evidence of increased macrophage proliferation in atherosclerotic lesions. Moreover, elevated glucose levels did not increase proliferation of isolated mouse peritoneal macrophages. In contrast, hyperglycemic LDL receptor-deficient mice that were fed a cholesterol-rich diet showed increased cholesterol levels concomitant with macrophage proliferation in atherosclerotic lesions. Glucose promoted lipid and protein oxidation of LDL in vitro. Glucose-oxidized LDL resulted in phosphorylation of extracellular signal-regulated kinase and protein kinase B/Akt and stimulated proliferation of isolated macrophages. The mitogenic effect of glucose-oxidized LDL was mediated by CD36 and by extracellular signal-regulated kinase activation induced by protein kinase C-dependent and phosphatidylinositol 3-kinase-dependent pathways. Thus, hyperglycemia is not sufficient to stimulate macrophage proliferation in lesions of atherosclerosis or in isolated macrophages. A combination of hyperglycemia and hyperlipidemia, however, stimulates macrophage proliferation by a pathway that may involve the glucose-dependent oxidation of LDL.

Glabridin, an Isoflavan from Licorice Root, Inhibits Inducible Nitric-Oxide Synthase Expression and Improves Survival of Mice in Experimental Model of Septic Shock

(R)-4-(3,4-Dihydro-8,8-dimethyl)-2H,8H-benzo[1,2-b:3,4-b']dipyran-3yl)-1,3-benzenediol (glabridin), a flavonoid present in licorice extract, is known to have antimicrobial, anti-inflammatory, and cardiovascular protective activities. In the present study, we report the inhibitory effect of glabridin on nitric oxide (NO) production and inducible nitric oxide (iNOS) gene expression in murine macrophages. Glabridin attenuated lipopolysaccharide (LPS)-induced NO production in isolated mouse peritoneal macrophages and RAW 264.7 cells, a mouse macrophage-like cell line. Moreover, iNOS mRNA expression was also blocked by glabridin treatment in LPS-stimulated RAW 264.7 cells. Further study demonstrated that the LPS-induced nuclear factor (NF)-kappaB/Rel DNA binding activity and NF-kappaB/Rel-dependent reporter gene activity were significantly inhibited by glabridin in RAW 264.7 cells and that this effect was mediated through the inhibition of inhibitory factor-kappaB degradation and p65 nuclear translocation. Moreover, reactive oxygen species generation was also suppressed by glabridin treatment in RAW 264.7 cells. In contrast, the activity of mitogen-activated protein kinases was unaffected by glabridin treatment. In animal model, in vivo administration of glabridin increased the rate of survival of LPS-treated mice and inhibited LPS-induced increase in plasma concentrations of nitrite/nitrate and tumor necrosis factor-alpha. Collectively, these data suggest that glabridin inhibits NO production and iNOS gene expression by blocking NF-kappaB/Rel activation and that this effect was mediated, at least in part, by inhibiting reactive oxygen species generation. Furthermore, in vivo anti-inflammatory effect of glabridin suggests a possible therapeutic application of this agent in inflammatory diseases.

Extract of the seed coat of Tamarindus indica inhibits nitric oxide production by murine macrophages in vitro and in vivo

The seed coat extract of Tamarindus indica, a polyphenolic flavonoid, has been shown to have antioxidant properties. The present studies investigated the inhibitory effect of the seed coat extract of T. indica on nitric oxide production in vitro using a murine macrophage-like cell line, RAW 264.7, and in vitro and in vivo using freshly isolated B6C3F1 mouse peritoneal macrophages. In vitro exposure of RAW 264.7 cells or peritoneal macrophages to 0.2–200 μg/mL of T. indica extract significantly attenuated (as much as 68%) nitric oxide production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) in a concentration-dependent manner. In vivo administration of T. indica extrac t (100–500 mg/kg) to B6C3F1 mice dose-dependently suppressed TPA, LPS and/or IFN-γ induced production of nitric oxide in isolated mouse peritoneal macrophages in the absence of any effect on body weight. Exposure to T. indica extract had no effect on cell viability as assessed by the MTT assay. In B6C3F1 mice, preliminary safety studies demonstrated a decrease in body weight at only the highest dose tested (1000 mg/kg) without alterations in hematology, serum chemistry or selected organ weights or effects on NK cell activity. A significant decrease in body weight was observed in BALB/c mice exposed to concentrations of extract of 250 mg/kg or higher. Oral exposure of BALB/c mice to T. indica extract did not modulate the development of T cell-mediated sensitization to DNFB or HCA as measured by the local lymph node assay, or dermal irritation to nonanoic acid or DNFB. These studies suggest that in mice, T. indica extract at concentrations up to 500 mg/kg may modulate nitric oxide production in the absence of overt acute toxicity.