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\nPlain Language SummaryIn this study, we investigated the inhibitory effects of radiofrequency exposure on RANKL-induced osteoclast differentiation in RAW264.7 cells, along with the underlying mechanisms. RAW264.7 cells were subjected to radiofrequency exposure at three distinct power densities: 50 µW/cm2, 150 µW/cm2, and 450 µW/cm2. The results showed that, among the three dosage levels, exposure to 150 µW/cm2 of radiofrequency radiation significantly reduced the proliferation capacity of RAW264.7 cells. RF exposure at three power densities resulted in significant increases in the level of osteoclast apoptosis and notable decreases in osteoclast differentiation. Notably, the most pronounced effects on apoptosis, differentiation in RAW 264.7 cells were observed at the 150 µW/cm2 power density. These effects were accompanied by concurrent decreases in mRNA and protein levels of osteoclast-specific genes, including RANK, NFATc1, and TRACP. Furthermore, radiofrequency exposure at power density of 150 µW/cm2 induced a significant decrease in cytoplasmic NF-κB protein levels while increasing its nuclear fraction, thereby counteracting the effects of RANKL-induced NF-κB activation. These data suggest that radiofrequency exerts inhibitory properties on RANKL-induced NF-κB transcriptional activity, subsequently indirectly suppressing the expression of downstream NF-κB target genes, such as NFATc1 and TRACP. In conclusion, our study demonstrates that radiofrequency radiation effectively inhibits osteoclast differentiation by modulating the NF-κB signaling pathway. These findings have important implications for potential therapeutic interventions in osteoporosis.Osteoporosis is a common bone disease where bones become weak and brittle, often leading to fractures. It frequently occurs in older adults, especially postmenopausal women, due to low estrogen levels and inadequate calcium intake. This causes increased activity of bone cells called osteoclasts which break down bone tissue, resulting in severe bone loss. Currently, the primary treatment is long-term use of medications like bisphosphonates. However, these drugs can have side effects. The main adverse reactions include fever, vomiting, rash, diarrhea, dizziness, abdominal pain, musculoskeletal pain, headache, allergic-like reactions, indigestion, edema, and ocular symptoms.This study explored using radiofrequency (RF) radiation as a safe, non-invasive alternative therapy for osteoporosis. RF radiation is a type of energy used in communications like cell phones and WiFi. We tested whether exposure to 900MHz RF radiation could inhibit the formation and activity of osteoclasts to prevent excessive bone breakdown.We treated osteoclast precursor cells with RANKL, a protein that stimulates osteoclast formation. Cells were then exposed to RF radiation at various intensities. The results showed that medium-level RF radiation (150 μW/cm2) significantly suppressed RANKL-induced osteoclast differentiation and bone resorption capacity. This effect was like the osteoclast inhibition seen with estrogen treatment.Further analysis revealed that RF radiation blocks the activation of NF-κB, a key signaling molecule that promotes osteoclast formation when RANKL is present. This in turn reduced production of downstream signals like NFATc1 and TRACP which are essential for osteoclast differentiation.In summary, this study demonstrates that medium-intensity RF radiation could potentially prevent excessive osteoclastic bone resorption in osteoporosis patients by interfering with NF-κB signaling cascade. The research highlights RF radiation’s promise as a novel, non-invasive osteoporosis therapy.Osteoporosis is a common bone disease where bones become weak and brittle, often leading to fractures. It frequently occurs in older adults, especially postmenopausal women, due to low estrogen levels and inadequate calcium intake. This causes increased activity of bone cells called osteoclasts which break down bone tissue, resulting in severe bone loss. Currently, the primary treatment is long-term use of medications like bisphosphonates. However, these drugs can have side effects. The main adverse reactions include fever, vomiting, rash, diarrhea, dizziness, abdominal pain, musculoskeletal pain, headache, allergic-like reactions, indigestion, edema, and ocular symptoms.This study explored using radiofrequency (RF) radiation as a safe, non-invasive alternative therapy for osteoporosis. RF radiation is a type of energy used in communications like cell phones and WiFi. We tested whether exposure to 900MHz RF radiation could inhibit the formation and activity of osteoclasts to prevent excessive bone breakdown.We treated osteoclast precursor cells with RANKL, a protein that stimulates osteoclast formation. Cells were then exposed to RF radiation at various intensities. The results showed that medium-level RF radiation (150 μW/cm2) significantly suppressed RANKL-induced osteoclast differentiation and bone resorption capacity. This effect was like the osteoclast inhibition seen with estrogen treatment.Further analysis revealed that RF radiation blocks the activation of NF-κB, a key signaling molecule that promotes osteoclast formation when RANKL is present. This in turn reduced production of downstream signals like NFATc1 and TRACP which are essential for osteoclast differentiation.In summary, this study demonstrates that medium-intensity RF radiation could potentially prevent excessive osteoclastic bone resorption in osteoporosis patients by interfering with NF-κB signaling cascade. The research highlights RF radiation’s promise as a novel, non-invasive osteoporosis therapy. [ABSTRACT FROM AUTHOR]

Three new steroids (1–3) and 13 reported analogs (4–16) were extracted from Datura metel L. pericarps. Structure analysis of these extracted compounds was performed by 1 D-NMR and 2 D-NMR spectroscopy, and their spectra were compared with those of similar compounds previously described in the literature. The extracted steroids (1–3) and known compounds (4–16) were evaluated for anti-inflammatory activity against LPS-activated RAW 264.7 cells. Compounds 5, 7, 9, 12 and 15 showed potential anti-inflammatory activity with IC50 less than 35 μM, while compounds 3 and 11 showed weak anti-inflammatory activity with IC50 less than 100 μM. [ABSTRACT FROM AUTHOR]

While a range of pharmacological agents are currently used to alleviate inflammation, the clinical administration of many of these anti-inflammatory drugs is associated with high rates of adverse side effects that make them poorly suited to long-term use. Therefore, there is a critical need for the development of novel anti-inflammatory agents. Natural compounds and derivatives like ethyl ferulate have risen to prominence as a foundation for many drug discovery efforts owing to their structural diversity and wide-ranging biological activities. In the present study, 24 ethyl ferulate derivatives were synthesized. Their anti-inflammatory activity was evaluated in vitro using RAW264.7 cells and CCK-8, ELISA, and Western blotting assays. These analyses revealed that most of the synthesized compounds exhibited moderate to high anti-inflammatory activities. In particular, c10 and c23 exerted more pronounced activity than ethyl ferulate or dexamethasone with respect to the suppression of tumour necrosis factor-α production by RAW264.7 cells through the targeting of the NF-κB and MAPK signalling pathways, suggesting that these compounds warrant further investigation. [ABSTRACT FROM AUTHOR]

Based on the inhibition of mitochondrial permeability transition pore (mPTP) opening, puerarin (PUE) has a good potential to reduce myocardial ischemia/reperfusion injury (MI/RI). However, the lack of targeting of free PUE makes it difficult to reach the mitochondria. In this paper, we constructed matrix metalloproteinase-targeting peptide (MMP-TP) and triphenylphosphonium (TPP) cation co-modified liposomes loaded with PUE (PUE@T/M-L) for mitochondria-targeted drug delivery. PUE@T/M-L had a favorable particle size of 144.9 ± 0.8 nm, an encapsulation efficiency of 78.9 ± 0.6%, and a sustainedrelease behavior. The results of cytofluorimetric experiments showed that MMP-TP and TPP doublemodified liposomes (T/M-L) enhanced intracellular uptake, escaped lysosomal capture, and promoted drug targeting into mitochondria. In addition, PUE@T/M-L enhanced the viability of hypoxia-reoxygenation (H/R) injured H9c2 cells by inhibiting mPTP opening and reactive oxygen species (ROS) production, reducing Bax expression and increasing Bcl-2 expression. It was inferred that PUE@T/M-L delivered PUE into the mitochondria of H/R injured H9c2 cells, resulting in a significant increase in cellular potency. Based on the ability of MMP-TP to bind the elevated expression of matrix metalloproteinases (MMPs), T/M-L had excellent tropism for Lipopolysaccharide (LPS) -stimulated macrophages and can significantly reduce TNF-a and ROS levels, thus allowing both drug accumulation in ischemic cardiomyocytes and reducing inflammatory stimulation during MI/RI. Fluorescence imaging results of the targeting effect using a DiR probe also indicated that DiR@T/M-L could accumulate and retain in the ischemic myocardium. Taken together, these results demonstrated the promising application of PUE@T/M-L for mitochondria-targeted drug delivery to achieve maximum therapeutic efficacy of PUE. [ABSTRACT FROM AUTHOR]

Using various chromatographic methods, four new phenolics, coroffesters A-D (1 − 4) were isolated from the fruit of Cornus officinalis (Cornaceae). Their structures (1 − 4) were elucidated unambiguously by spectroscopic methods such as one- and two-dimensional nuclear magnetic resonance (1 D- and 2 D-NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The anti-inflammatory activity of the isolated compounds was also evaluated. All compounds (1 − 4) showed moderate inhibitory activity against NO production in a dose-dependent manner in RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

Bilberry (Vaccinium myrtillus L.) is a medicinal fruit known for its high content of anthocyanins, flavonoids, vitamins, ascorbic acid, carotenoids, tocopherols and phenolic acid. Bilberry has long been used in oriental medicine for the prevention and treatment of various diseases, such as visual acuity, hyperglycemia, dyslipidemia, diabetes, cancer, obesity, inflammatory and cardiovascular diseases. In this study, we investigated using the antioxidant and anti-inflammatory of 70% ethanol extracts of bilberry in vitro. Antioxidant activities were measured with total phenolic, flavonoid and ascorbic acid contents. The bilberry extract (BE) showed significant free radical scavenging activities and inhibited oxidation of linoleic acid in a dose-dependent manner. BE also suppressed nitric oxide (NO) generation and reversed pro-inflammatory cytokines such as iNOS, COX-2, TNF-α and IL-6 in LPS-induced RAW 264.7 cells. From these results, we suggest that BE is a natural powerful antioxidant, and the anti-inflammatory agent being a rich source of anthocyanins. [ABSTRACT FROM AUTHOR]

Aims: Owhadi is a popular commercial pistachio cultivar in Iran which could be an attractive source for natural bioactive compounds with health-promoting activity. Methods: The hulls subjected to fractionation and ethyl acetate fraction was a phenolic-enriched fraction (PEF). The PEF was encapsulated in nanoliposomes (PEF-NLs) as a newly developed delivery system. The phytochemical analysis of PEF-NLs confirmed the presence of phenolic and flavonoid compounds. Results: The PEF-NLs indicated the strong antioxidant activity through up-regulation of the antioxidant-related genes in the murine hepatocyte. The PEF-NLs indicated the notable anti-inflammatory activity by scavenging the nitric oxide (NO) and reducing the NO production in the murine macrophage cells. The PEF-NLs have also exhibited the anti-melanogenic activity through direct tyrosinase enzyme inhibition and by modulating melanin biosynthesis genes in B16F10 melanoma cells. Conclusion: The PEF-NLs possessed the promising potential to be used for controlling skin pigmentation disorders and as a skin-whitening agent in the cosmetic industry. [ABSTRACT FROM AUTHOR]

Context: Chatuphalatika (CTPT), is a Thai herbal formulation mixture of Phyllanthus emblica Linn. (Euphorbiaceae), Terminalia belerica Linn. (Combretaceae), T. chebula and the fruit of T. arjuna (Roxb.) Wight & Arn. CTPT is considered to exert anti-inflammatory and antihyperuricemic effects, but there have been no reports to demonstrate these pharmacological effects in a quantitative manner. Objectives: To investigate the antioxidative, anti-inflammatory and antihyperuricemic effects of CTPT. Materials and methods: Antioxidant activities of CTPT extracts were measured in vitro by DPPH, ABTS and FRAP assays, and anti-inflammatory effect by measuring inflammatory mediator production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages. The mechanism of the hypouricemic effect was investigated using oxonate-induced hyperuricemic ddY mice treated with oral administrations of CTPT at 250, 500 and 1000 mg/kg. Results: Antioxidant activities of CTPT measured by ABTS and FRAP assays were 1.35 g TEAC/g extract and 10.3 mmol/100 g extract, respectively. IC50 for the inhibition of DPPH radical was 13.8 µg/mL. CTPT (10 µg/mL) significantly downregulated the mRNA expression of TNF-α and iNOS in RAW 264.7 cells. Lineweaver-Burk analysis of the enzyme kinetics showed that CTPT inhibited xanthine oxidase (XOD) activity in a noncompetitive manner with the Ki of 576.9 µg/mL. Oral administration of CTPT (1000 mg/kg) significantly suppressed uric acid production by inhibiting hepatic XOD activity, and decreased plasma uric acid levels in hyperuricemic mice by approximately 40% (p < 0.05). Conclusions: This study demonstrated for the first time the antioxidative, anti-inflammatory and antihyperuricemic effects of CTPT in vivo and in vitro, suggesting a possibility of using CTPT for the treatment of hyperuricemia in gout. [ABSTRACT FROM AUTHOR]

Context:Immune dysregulation has been implicated in the pathogenesis of many diseases. Macrophages play a crucial role contributing to the onset, progression, and resolution of inflammation. Macrophage inflammatory mediators are of considerable interest as potential targets to treat inflammatory diseases. Objective:The present study was conducted to elucidate the anti-inflammatory mechanism of 2′,4-dihydroxy-3′,4′,6′-trimethoxychalcone (1), the major chalcone isolated fromChromolaena odorata(L.) R.M.King & H.Rob, against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. Materials and methods:Cell viability, nitric oxide (NO), and proinflammatory cytokines of LPS-activated RAW 264.7 cells were measured by MTT, Griess, and ELISA assays, respectively. Cell lysates were subjected to Western blotting for investigation of protein expression. Results and discussion:Treatment with the major chalcone1significantly attenuated the production of NO and proinflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6 in a dose-dependent manner. The chalcone suppressed nuclear factor-κB (NF-κB) stimulation by preventing activation of inhibitor κB kinase (IKK) α/β, degradation of inhibitor κB (IκB) α, and translocation of p65 NF-κB into the nucleus. Additionally, the chalcone markedly repressed the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but no further inhibition was detected for c-Jun N-terminal activated kinases or extracellular regulated kinases. Thus, suppression of NF-κB and p38 MAPK activation may be the core mechanism underlying the anti-inflammatory activity of 2′,4-dihydroxy-3′,4′,6′-trimethoxychalcone (1). Conclusion:These findings provide evidence that 2′,4-dihydroxy-3′,4′,6′-trimethoxychalcone (1) possesses anti-inflammatory activity via targeting proinflammatory macrophages. This anti-inflammatory chalcone is a promising compound for reducing inflammation. [ABSTRACT FROM PUBLISHER]

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]

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]

The aim of this study was to examine the effects of ultraviolet A (UV-A) irradiation-induced damage on cultured macrophage RAW 264.7 cells and determine which components produced these manifestations. RAW 264.7 cells were irradiated with 365 nm UV-A using a light-emitting diode (LED). Cell viability and damage were determined using a calcein-AM and propidium iodide dual-staining assay and lactate dehydrogenase leakage, respectively. Intracellular reactive oxygen species (ROS) were measured by H2DCF-DA. The components of ROS in each medium were measured using an electron paramagnetic resonance (EPR) spectrometer in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC). While UV-A irradiation for 2 min significantly suppressed cell growth, LDH leakage did not occur. Addition ofN-acetyl cysteine restored inhibition of cell proliferation, and reduced intracellular ROS levels. The EPR signal in the presence of TPC increased with time but was decreased by sodium azide. In addition, a typical EPR spectrum was obtained in the presence of DMPO, indicating the presence of a hydroxyradical. The spectrum was diminished by L-histidine. Data suggest that ROS generated in cells or culture medium by UV-A irradiation is predominantly singlet oxygen, and this singlet oxygen suppressed cell proliferation. [ABSTRACT FROM AUTHOR]

The present study was designed to examine and compare the effects of three suppressors on the cytokine response in tandem with examining: the synthesis of inducible forms of heat shock proteins; HSP72 and HSP90 α; activities of NF- κB and SAPK/JNK signaling pathways; and TLR4 expression. Pre-treatment with inhibitors offers promise as protective means to lower the activity of these cascades, thereby circumventing the formation of excessive amounts of pro-inflammatory molecules. Three inhibitors of TLR4, SAPK/JNK, and NF- κB signaling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before the Escherichia coli lipopolysaccharide (LPS) application. Treatments of RAW 264.7 cells with each of the inhibitors resulted in a reduced response to LPS as was visualized by a decrease of TNF- α, IL-1, and IFN- γ production. In addition, inhibitors of the NF- κB and SAPK/JNK signaling reduced IL-6 production in LPS-treated cells, whereas the IKK inhibitor XII also decreased IL-10 production. Further, the NO production in LPS-stimulated macrophages was significantly reduced following application of CLI-095 or IKK inhibitor XII. The results also showed that the inhibitors suppressed TLR4 production and decreased phosphorylation of NF- κB and SAPK/JNK proteins, thereby preventing the activation NF- κB and SAPK/JNK signaling pathways in LPS-activated cells. In addition, the production of inducible heat shock proteins, HSP72 and HSP90- α, was reduced in LPS-stimulated RAW 264.7 cells pre-treated with inhibitors. These results suggest that inhibitors CLI-095, SP600125, and IKK inhibitor XII demonstrate potential effectiveness in the reduction of the inflammatory response by mechanisms involving both the cellular defense system and cellular signaling. In conclusion, suppressor of NF- κB cascade, IKK inhibitor XII, seems to be the most effective anti-toxic agent among studied inhibitors. [ABSTRACT FROM AUTHOR]

Edible bird's nest (EBN) is a popular delicacy among the Chinese diaspora associated with various health claims. This study investigated the anti-inflammatory effects of white-EBN acid hydrolysate on tumour necrosis factor-alpha (TNF-α) and nitric oxide (NO) generation, while examining its cytotoxic effect on a lipopolysaccharide-stimulated RAW 264.7 macrophage cell line. Processed commercial EBN and unprocessed raw EBN collected from different geographical zones and at different harvesting seasons were studied. The cytotoxic effect was determined by cell viability assessed by MTS assay, while NO production was determined by the Griess reaction and TNF-α concentration was measured using an ELISA kit. The results showed that both commercial and raw EBN inhibited TNF-α and NO generation, the highest inhibition 58% and 63%, respectively without significant cytotoxic effect was brought about by raw EBN from the South zone. The results suggest that EBN may possess anti-inflammatory properties that should be further studied. [ABSTRACT FROM AUTHOR]

In inflammation, proinflammatory cytokines induce the formation of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS), and compounds that inhibit NO production have anti-inflammatory effects. In the present study, we investigated the effects of lansai C and D on NO production in lipopolysaccharide-induced RAW 264.7 cells, and evaluated the mechanisms of action of the compounds. Lansai C and D inhibited iNOS protein and mRNA expression and also NO production in a dose-dependent manner. These compounds inhibited the activation of nuclear factor-κB, which is a significant transcription factor for iNOS and also inhibited the activation of the signal transducer and activator of transcription-1, another important transcription factor for iNOS. The present study characterises the effects and mechanisms of lansais on iNOS expression and NO production in activated macrophages. The results explain the pharmacological efficacy of lansais as anti-inflammatory compounds. [ABSTRACT FROM AUTHOR]

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]

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]

Interleukin-1 β (IL-I β), a cytokine involved in homeostatic processes such as the immune system and inflammatory reactions, is a potent inducer of nitric oxide. The nonapeptide of human IL-I β (VQGEESNDK, position 163-171, specific radioprotective domain-SRD) has been shown to retain radioprotective, immunostimulatory, and adjuvant activities of the native molecule without any inflammatory and pyrogenic properties. Unlike the parent IL-I, SRD did not induce nitric oxide (NO) in control or irradiated RAW 264.7 cells nor did it affect inducible nitric oxide synthase (iNOS) as shown by ELISA based mRNA assay (Quantikine). A lipophillic derivative of the SRD (a palmitoyl residue at the amino terminus of the SRD) was synthesized (palmitoyl specific radioprotective domain, P-SRD) to find out if this structural derivatization would restore the NO-inducing ability of IL-I. Surprisingly, P-SRD not only did not induce NO, but significantly inhibited lipopolysaccharide (LPS) stimulated nitric oxide (NO) production. Quantikine studies indicated that P-SRD also inhibited iNOS in LPS stimulated macrophage cells, suggesting that decrease in NO production in the presence of P-SRD was the result of iNOS mRNA inhibition. These results indicate that N-palmitoylation of SRD may effectively ameliorate potentially fatal symptoms of LPS-induced endotoxemic hypotensive shock associated with IL-I without inflammatory and pyrogenic toxic side effects. [ABSTRACT FROM AUTHOR]