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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]

Background: Excessive inflammation poses significant risks to human physical and mental health. Astilbe grandis, a traditional Miao medicine, is renowned for its anti-inflammatory properties. However, the specific anti-inflammatory effects and mechanisms of many compounds within this plant remain unclear. This study aims to investigate the anti-inflammatory effects and mechanisms of two characteristic oleanane triterpenoids, 3α-acetoxyolean-12-en-27-oic acid (1) and 3β-acetoxyolean-12-en-27-oic acid (2), isolated from Astilbe grandis, using lipopolysaccharide (LPS)-induced Macrophages. Methods: The anti-inflammatory effects and mechanisms of compounds 1 and 2 were investigated by establishing an LPS-induced inflammation model in RAW 264.7 cells and THP-1 cells. Nitric oxide (NO) levels were assessed using the Griess method. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1beta (IL-1β) were measured via enzymelinked immunosorbent assay (ELISA). The expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was determined using western blotting and quantitative real-time PCR (qRT-PCR). Additionally, the phosphorylation level of p65 in nuclear factor-kappa B (NF-κB) was assessed through western blotting. The nuclear translocation of NF-κB p65 was assessed through immunofluorescence staining. Finally, the binding affinity of the compounds to NF-κB p65 target was validated through molecular docking. Results: Compounds 1 and 2 significantly inhibited the expression of NO, TNF-α, IL-6, IL-1β, COX-2, and iNOS in LPS-induced Macrophages. Mechanistically, they attenuated the activation of the NF-κB signaling pathway by downregulating the phosphorylation level and nuclear translocation of p65. Conclusion: This study elucidates the anti-inflammatory activities and potential mechanism of the characteristic oleanane triterpenoids with C-14 carboxyl group, compounds 1 and 2, in LPS-induced Macrophages by inhibiting the NF-κB signaling pathway for the first time. These findings suggest that these two compounds hold promise as potential candidates for anti-inflammatory interventions in the future. [ABSTRACT FROM AUTHOR]

This study aimed to investigate a synergistic anti-inflammatory effect of a citrus flavonoid nobiletin and docosahexaenoic acid (DHA), one of n-3 long-chain polyunsaturated fatty acids, in combination. Simultaneous treatment with nobiletin and DHA synergistically inhibited nitric oxide production (combination index < 0.9) by mouse macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) without cytotoxicity. On the other hand, the inhibitory effect of nobiletin and DHA in combination on proinflammatory cytokine production was not synergistic. Neither nobiletin nor DHA affected the phagocytotic activity of RAW 264.7 cells stimulated with LPS. Immunoblot analysis revealed that the inhibition potency of DHA on the phosphorylation of ERK and p38 and nuclear translocation of NF-κB is markedly enhanced by simultaneously treating with nobiletin, which may lead to the synergistic anti-inflammatory effect. Overall, our findings show the potential of the synergistic anti-inflammatory effect of nobiletin and DHA in combination. [ABSTRACT FROM AUTHOR]

Abstract Background The global use of plastic materials has undergone rapid expansion, resulting in the substantial generation of degraded and synthetic microplastics and nanoplastics (MNPs), which have the potential to impose significant environmental burdens and cause harmful effects on living organisms. Despite this, the detrimental impacts of MNPs exposure towards host cells and tissues have not been thoroughly characterized. Results In the present study, we have elucidated a previously unidentified hepatotoxic effect of 20 nm synthetic polystyrene nanoparticles (PSNPs), rather than larger PS beads, by selectively inducing necroptosis in macrophages. Mechanistically, 20 nm PSNPs were rapidly internalized by macrophages and accumulated in the mitochondria, where they disrupted mitochondrial integrity, leading to heightened production of mitochondrial reactive oxygen species (mtROS). This elevated mtROS generation essentially triggered necroptosis in macrophages, resulting in enhanced crosstalk with hepatocytes, ultimately leading to hepatocyte damage. Additionally, it was demonstrated that PSNPs induced necroptosis and promoted acute liver injury in mice. This harmful effect was significantly mitigated by the administration of a necroptosis inhibitor or systemic depletion of macrophages prior to PSNPs injection. Conclusion Collectively, our study suggests a profound toxicity of environmental PSNP exposure by triggering macrophage necroptosis, which in turn induces hepatotoxicity via intercellular crosstalk between macrophages and hepatocytes in the hepatic microenvironment.

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]

Background: The global use of plastic materials has undergone rapid expansion, resulting in the substantial generation of degraded and synthetic microplastics and nanoplastics (MNPs), which have the potential to impose significant environmental burdens and cause harmful effects on living organisms. Despite this, the detrimental impacts of MNPs exposure towards host cells and tissues have not been thoroughly characterized. Results: In the present study, we have elucidated a previously unidentified hepatotoxic effect of 20 nm synthetic polystyrene nanoparticles (PSNPs), rather than larger PS beads, by selectively inducing necroptosis in macrophages. Mechanistically, 20 nm PSNPs were rapidly internalized by macrophages and accumulated in the mitochondria, where they disrupted mitochondrial integrity, leading to heightened production of mitochondrial reactive oxygen species (mtROS). This elevated mtROS generation essentially triggered necroptosis in macrophages, resulting in enhanced crosstalk with hepatocytes, ultimately leading to hepatocyte damage. Additionally, it was demonstrated that PSNPs induced necroptosis and promoted acute liver injury in mice. This harmful effect was significantly mitigated by the administration of a necroptosis inhibitor or systemic depletion of macrophages prior to PSNPs injection. Conclusion: Collectively, our study suggests a profound toxicity of environmental PSNP exposure by triggering macrophage necroptosis, which in turn induces hepatotoxicity via intercellular crosstalk between macrophages and hepatocytes in the hepatic microenvironment. [ABSTRACT FROM AUTHOR]

Ulcerative colitis is a chronic inflammatory disease caused by abnormal immune responses in the intestinal mucosa and gut microorganisms. Unlike other mugworts, Artemisia argyi H. (A. argyi H.) enhances antioxidant, anti-inflammatory, and anticancer effects, but the improvement effects against gut inflammation have not yet been reported. Therefore, this study aimed to confirm the alleviation of the inflammatory state in the gut by A. argyi H. fermented with Lactobacillus plantarum (FAA), using lipopolysaccharide (LPS)-induced RAW 264.7 cells and dextran sulfate sodium (DSS)-induced colitis models. In vitro, FAA (10, 50, 100, and 200 μg/mL) was pretreated into RAW 264.7 cells, followed with LPS (100 ng/mL), which induced the cell damage. Meanwhile, in vivo, FAA (100, 200 mg/kg/day) was orally administered into 6-week-old C57BL/6N mice for 3 weeks. During the last week of FAA administration, 2.5% DSS was used to induce colitis. The results showed that FAA reduced the production of nitric oxide (p < 0.0001), tumor necrosis factor (TNF)-α, interleukin (IL)-6 (p < 0.0001), and IL-1β (p < 0.0001) in the LPS-induced RAW 264.7 cells. Moreover, in the DSS-induced colitis model, FAA alleviated clinical symptoms (p < 0.001), inhibited the inflammatory state by reducing the production of TNF-α (p < 0.0001) and interferon-γ in intestinal immune cells (p < 0.0001), and strengthened the intestinal barrier by increasing the number of goblet cells (p < 0.0001). Furthermore, the anti-inflammatory effects were confirmed by the alleviation of histological damage (p < 0.001) and down-regulation of the expression of inflammatory proteins (TLR4, p < 0.0001; MyD88, p < 0.0001; Cox-2, p < 0.0001). These results suggest the potential of FAA as a dietary ingredient for preventing inflammation in the gut. [ABSTRACT FROM AUTHOR]

In this study, we green synthesized silver nanoparticles (Ag Nps) from Hybanthus enneaspermus leaves (HE-Ag NPs) and evaluated their antimicrobial and wound-healing properties. The synthesized HE-Ag NPs were characterized using various techniques, revealing face-centered polygonal structures, a well-dispersed appearance, and an average particle size of 42–51 nm. The antimicrobial effects of HE-Ag NPs and their embedded cotton fabrics were tested against several pathogens, showing effective inhibition of growth. The cytotoxicity and anti-inflammatory properties of HE-Ag NPs were assessed using MTT assays on L929 and RAW 264.7 cells and by measuring inflammatory cytokine levels in LPS-treated RAW 264.7 cells. HE-Ag NPs did not affect the viability of L929 and RAW 264.7 cells and significantly reduced inflammatory cytokine levels. In vivo studies using an excision wound model demonstrated that HE-Ag NPs-loaded ointment significantly increased hydroxyproline, total protein, and antioxidant levels and enhanced the wound contraction rate. These findings suggest that HE-Ag NPs have potent antimicrobial properties and promote wound healing, indicating their potential for use in topical ointments for wound care.

To explore structurally and pharmacologically interesting secondary metabolites from Psammosilene tunicoides, a crude extract of this plant was investigated, leading to the isolation of a new phytoecdysteroid, furoecdysterone (1) and four known phytosteroid compounds, ecdysterone (2), 22-deoxyecdysterone (3), β-sitosteryl D-glucoside (4), and β-sitosterol (5). Their structures were identified based on a detailed analysis of NMR spectra and MS data. Those compounds, except for 2, showed moderate NO inhibition activities on lipopolysaccharide (LPS)-induced release in RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

We demonstrated the effect of Ishige okamurae extract (IOE) on the receptor activator of nuclear factor-κB ligand (RANKL)-promoted osteoclastogenesis in RAW 264.7 cells and confirmed that IOE inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation. IOE inhibited protein expression of TRAP, metallopeptidase-9 (MMP-9), the calcitonin receptor (CTR), and cathepsin K (CTK). IOE treatment suppressed the expression of activated T cell cytoplasmic 1 and activator protein-1, thus controlling the expression of osteoclast-related factors. Moreover, IOE significantly reduced RANKL-phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). It also reduced the RANKL-induced phosphorylation of NF-κB and nuclear translocation of p65. IOE inhibited Dex-induced bone loss and osteoclast-related gene expression in zebrafish larvae. HPLC analysis shows that IOE consists of 3.13% and 3.42% DPHC and IPA, respectively. Our results show that IOE has inhibitory effects on osteoclastogenesis in vitro and in vivo and is a potential therapeutic for osteoporosis. [ABSTRACT FROM AUTHOR]

Cyanobacteria are among the oldest organisms colonizing Earth. Their great biodiversity and ability to biosynthesize secondary metabolites through a variety of routes makes them attractive resources for biotechnological applications and drug discovery. In this pioneer study, four filamentous cyanobacteria (Cephalothrix lacustris LEGE 15493, Leptolyngbya boryana LEGE 15486, Nodosilinea nodulosa LEGE 06104 and Leptothoe sp. LEGE 11479) were explored for their anti-inflammatory potential in cell and cell-free in vitro bioassays, involving different inflammatory mediators and enzymes. Extracts of different polarities were sequentially prepared and chemically characterized for their content of phycobiliproteins (PBPs) and carotenoids. HPLC-PDA analysis of the acetone extracts revealed β-carotene to be the dominant carotenoid (18.4–44.3 mg/g) and zeaxanthin as the dominant xanthophyll (52.7–192.9 mg/g), with Leptothoe sp. LEGE 11479 and Nodosilinea nodulosa LEGE 06104, respectively, being the richest strains. The PBP profile was in accordance with the color presented by the aqueous extracts, with Leptolyngbya boryana LEGE 15486 being the richest in phycocyanin (204.5 μg/mg) and Leptothoe sp. LEGE 11479 the richest in phycoerythrin (78.5 μg/mg). Aqueous extracts were more effective in superoxide anion radical scavenging, while acetone ones were more effective in scavenging nitric oxide radical (●NO) and in inhibiting lipoxygenase. Acetone extracts also reduced ●NO production in lipopolysaccharide-stimulated RAW 264.7 macrophages, with the mechanistic study suggesting a downregulation of the inducible nitric oxide synthase expression. Nodosilinea nodulosa LEGE 06104 and Leptothoe sp. LEGE 11479 acetone extracts presented the lowest IC50 values for the mentioned assays, pointing them out as promising resources for the development of new multi-target anti-inflammatory therapies. [ABSTRACT FROM AUTHOR]

Excessive osteoclast activity can promote periodontitis-associated bone destruction. The inhibitory mechanisms of Weissella cibaria strains CMU and CMS1 against periodontitis have not yet been fully elucidated. In this study, we aimed to investigate whether heat-killed (HK) W. cibaria CMU and CMS1 or their respective cell-free supernatants (CFSs) inhibit osteoclast differentiation and bone resorption in response to receptor activator of nuclear factor kappa-B ligand (RANKL)-treated RAW 264.7 cells. TRAP (tartrate-resistant acid phosphatase) staining and bone resorption assays revealed that both HK bacteria and CFSs significantly suppressed the number of TRAP-positive cells, TRAP activity, and bone pit formation compared to the RANKL-treated control (p < 0.05). HK bacteria dose-dependently inhibited osteoclastogenesis while selectively regulating certain genes in CFSs (p < 0.05). We found that disrupting the direct interaction between HK bacteria and RAW 264.7 cells abolished the inhibitory effect of HK bacteria on the expression of osteoclastogenesis-associated proteins (c-Fos, nuclear factor of activated T cells c1 (NFATc1), and cathepsin K). These results suggest that dead bacteria suppress osteoclast differentiation more effectively than the metabolites and may serve as beneficial agents in preventing periodontitis by inhibiting osteoclast differentiation via direct interaction with cells. [ABSTRACT FROM AUTHOR]

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

Introduction Aspirin is one of the most commonly used drugs worldwide. It is known to present antipyretic, anti-inflammatory and anti-thrombotic actions, making it extremely useful in a wide range of clinical contexts. Interestingly, homeopathically prepared Aspirin 15cH has been found to have a pro-thrombotic effect in rats, raising the hypothesis that Aspirin 15cH could also modulate the activity of inflammatory cells in different pathological processes. Objective Our objective was to assess what effect Aspirin 15cH has on RAW 264.7 macrophages in vitro. Methods The effects of Aspirin 15cH on biochemical and morphological activities of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were evaluated. These effects were compared with unchallenged macrophages (negative control), untreated LPS-stimulated macrophages, macrophages treated with succussed water (vehicle control), or aspirin 200 µg/mL (pharmacological inhibitor of LPS activity). Cell morphology (adhered cell area and cytoskeleton arrangements), cell viability, toll-like receptor-4 (TLR-4) expression, and the production of nitric oxide, cytokines and intracellular reactive oxygen species were assessed. ResultsAspirin 15cH reduced the number of cells expressing TLR-4 on the surface (p = 0.03) and induced a "columnar" morphology of macrophage pseudopods, indicating changes in cytoskeleton arrangement. When cells were treated with both Aspirin 15cH and LPS, cell morphology became heterogeneous, suggesting that sub-populations of cells had differing sensitivities to LPS or Aspirin 15cH. Exposure of the cells to LPS alone, succussed water or aspirin 200 µg/mL produced effects consistent with the literature. ConclusionAspirin 15cH, aspirin 200 µg/mL, LPS and succussed water appear to act as independent stimuli able to induce different patterns of macrophage response. Aspirin 15cH induced changes suggestive of M2 polarization of the macrophages (i.e., toward a wound healing or tissue repair, rather than inflammatory, phenotype). These preliminary findings need to be confirmed in further specific studies. [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]

Breynia spp. are a key source of sulfur-containing spiroketal glycosides with potential anti-inflammatory activity. In this study, three new sulfur-containing spiroketals – breynin J (1), epibreynin J (2), and probreynogenin (3) – along with four known compounds – probreynin I (4), phyllaemblic acid (5), breynin B (6), and epibreynin B (7) – were isolated from the roots of Breynia disticha. The structures of compounds 1–7 were elucidated by extensive 1D and 2D NMR spectroscopic analyses, including 1D total correlation spectroscopy (TOCSY), HSQC, HMBC, double quantum-filtered (DQF)-COSY, heteronuclear two-bond correlation (H2BC), and HSQC-TOCSY experiments, as well as high-resolution electrospray ionization HRESIMS analysis, and quantum chemical electronic CD calculations. Furthermore, the absolute configurations of sugar residues were determined by derivatization of the hydrolysates with ʟ-cysteine methyl ester and o-tolyl isothiocyanate followed by HPLC analysis. The anti-inflammatory effects of the isolated compounds were evaluated based on the mRNA levels of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. Compounds 1, 2, 6, and 7 inhibited the increase in interleukin (IL)-1β and IL-6 mRNA levels stimulated by LPS. Moreover, the most potent compound 7 was found to significantly inhibit the production of IL-1β and IL-6 proteins, as revealed by the analysis of culture supernatants.

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]

Cannabichromene (CBC), a non-psychoactive cannabinoid found in Cannabis sativa, has recently been shown to possess several medicinal properties. However, how CBC produces anti-inflammatory effects and the mechanisms of this remain poorly studied. Therefore, we extracted and purified the CBC from the Cannabis sativa cv. pink pepper (hemp cultivar). The efficacy of CBC in reducing inflammation in RAW 264.7 macrophages and a λ-carrageenan-induced mouse model was then evaluated. CBC had no cytotoxicity up to a concentration of 20 μM and inhibited nitric oxide production by approximately 50% at a concentration of 20 μM. In addition, CBC treatment significantly inhibited causes of inflammation such as inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) at both the mRNA and protein levels. Moreover, CBC suppressed LPS-stimulated inflammation in RAW 264.7 cells by downregulating the nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase pathways (MAPK). Furthermore, our in vivo experiments confirmed that the λ-carrageenan-induced increase in the levels of the cytokines iNOS, IL-1β, and IL-6 was abrogated following treatment with CBC. Therefore, CBC has potential anti-inflammatory effects and may be useful for preventing or treating inflammation. [ABSTRACT FROM AUTHOR]

Indian borage (Plectranthus amboinicus) is a herb that has been reported to have numerous pharmacological activities including anti-inflammatory and antioxidant activities. The murine macrophage RAW 264.7 cell line is commonly utilized for anti-inflammatory drug screening. However, the anti-inflammatory effect of Plectranthus ambonicus ethanol extract (PaE) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cell line has not been studied. This study is aimed at evaluating the antioxidant activity and anti-inflammatory effect of PaE in murine macrophage model. The anti-inflammatory activity was assessed via the inhibitory effect against NO production in RAW 264.7 cell line following LPS stimulation. The antioxidant activity of PaE was evaluated using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. The total flavonoid and phenolic contents were also evaluated using standard procedures. The antioxidant and anti-inflammatory activities of compounds in Plectranthus amboinicus were also predicted in silico. Results from the study has shown that PaE reduced nitric oxide production in LPS-stimulated RAW 264.7 cells. PaE also inhibited DPPH free radical and had a total flavonoid and phenolic contents of 2.04 ± 0.23 mgQE/g and 4.76 ± 0.03 mgGAE/g, respectively. From the in silico study, fifteen compounds from Plectranthus amboinicus were predicted to have strong antioxidant and anti-inflammatory activities. Thus, the findings from the present study has shown Indian borge as a plant with antiinflammatory and antioxidant potentials. However, further investigation of the mechanism of action and the identification of its bioactive components is needed. [ABSTRACT FROM AUTHOR]

We reported the discovery of six novel coumarins, toddasirins A–F (1 – 6), each endowed with modified isoprenyl or geranyl side chains, derived from the roots of Toddalia asiatica. Comprehensive structural elucidation was achieved through multispectroscopic analyses, single-crystal X-ray diffraction experiments, and advanced quantum mechanical electronic circular dichroism (ECD) calculations. Furthermore, the anti-inflammatory activity of these compounds was assessed. Notably, compounds 1 – 3 and 6 demonstrated notable inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells, with 50% inhibitory concentration (IC 50) values of 3.22, 4.78, 8.90, and 4.31 μmol·L−1, respectively. [ABSTRACT FROM AUTHOR]

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

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

Inflammatory diseases contribute to more than 50 % of global deaths. Research suggests that network pharmacology can reveal the biological mechanisms underlying inflammatory diseases and drug effects at the molecular level. The aim of the study was to clarify the biological mechanism of Cinnamomum zeylanicum essential oil (CZEO) and predict molecular targets of CZEO against inflammation by employing network pharmacology and in vitro assays. First, the genes related to inflammation were identified from the Genecards and Online Mendelian Inheritance in Man (OMIM) databases. The CZEO targets were obtained from the SwissTargetPrediction and Similarity Ensemble Approach (SEA) database. A total of 1057 CZEO and 526 anti-inflammation targets were obtained. The core hub target of CZEO anti-inflammatory was obtained using the protein–protein interaction network. KEGG pathway analysis suggested CZEO to exert anti-inflammatory effect mainly through Tumor necrosis factor, Toll-like receptor and IL-17 signalling pathway. Molecular docking of active ingredients-core targets interactions was modelled using Pyrx software. Docking and simulation studies revealed benzyl benzoate to exhibit good binding affinity towards IL8 protein. MTT assay revealed CZEO to have non-cytotoxic effect on RAW 264.7 cells. CZEO also inhibited the production of NO, PGE2, IL-6, IL-1β and TNF-α and promoted the activity of endogenous antioxidant enzymes in LPS-stimulated RAW 264.7 cells. Additionally, CZEO inhibited intracellular ROS generation, NF-kB nuclear translocation and modulated the expression of downstream genes involved in Toll-like receptor signalling pathway. The results deciphered the mechanism of CZEO in treating inflammation and provided a theoretical basis for its clinical application.

BackgroundPersistent inflammation following vascular injury drives neointimal hyperplasia (NIH). Specialized lipid mediators (SPM) mediate resolution which attenuates inflammation and downstream NIH. We investigated the effects of a synthetic analogue of resolvin D1 (RvD1) on vascular cells and in a model of rat carotid angioplasty.MethodsHuman venous VSMC and endothelial cells (EC) were employed in migration, cell shape, toxicity, proliferation and p65 nuclear translocation assays. Murine RAW 264.7 cells were utilized to test the effect of pro-resolving compounds on phagocytic activity. A model of rat carotid angioplasty was used to evaluate the effects of 17R/S-benzo-RvD1 (benzo-RvD1) and 17R-RvD1 applied to the adventitia via 25% Pluronic gel. Immunostaining was utilized to examine Ki67 expression and leukocyte recruitment. Morphometric analysis was performed on arteries harvested 14 days after injury.ResultsExposure to benzo-RvD1 attenuated PDGF- stimulated VSMC migration across a range of concentrations (0.1-100 nM), similar to that observed with 17R-RvD1. Pre-treatment with either Benzo-RvD1 or 17R-RvD1 (10, 100nM) attenuated PDGF-BB-induced VSMC cytoskeletal changes to nearly baseline dimensions. Benzo-RvD1 demonstrated modest anti-proliferative activity on VSMC and EC at various concentrations, without significant cytotoxicity. Benzo-RvD1 (10nM) inhibited p65 nuclear translocation in cytokine-stimulated EC by 21% (p

The intracellular bacterial pathogen Legionella pneumophila (L.p.) secretes ∼330 effector proteins into the host cell to sculpt an ER-derived replicative niche. We previously reported five L.p. effectors that inhibit IRE1, a key sensor of the homeostatic unfolded protein response (UPR) pathway. In this study, we discovered a subset of L.p. toxins that selectively activate the UPR sensor ATF6, resulting in its cleavage, nuclear translocation, and target gene transcription. In a deviation from the conventional model, this L.p-dependent activation of ATF6 does not require its transport to the Golgi or its cleavage by the S1P/S2P proteases. We believe that our findings highlight the unique regulatory control that L.p exerts upon the three UPR sensors and expand the repertoire of bacterial proteins that selectively perturb host homeostatic pathways.

Cancer therapy reduces tumor burden via tumor cell death ("debris"), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E2 receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.

A naturally lacto-fermented cucumber product was developed for use as anti-inflammatory functional foods. To explore the anti-inflammatory characteristics, water (CWE) and ethanol extracts (CEE) from this product were selected to assess their anti-inflammatory potential on RAW 264.7 macrophages in the absence or presence of lipopolysaccharide (LPS), using four different inflammatory models. Changes in pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokine secretions by treated macrophages were measured using ELISA. The results showed that both CWE and CEE had strong potential to inhibit LPS-stimulated inflammation in macrophages in a repair manner. CWE had a better effect than CEE. The total phenolic, flavonoid and saponin contents in CEE were significantly (P

This study aims to formulate naringenin (Nar)-loaded pH/temperature sensitive nanopolymers for therapeutic options for inflammatory diseases. For this aim, Nar loaded SNPs (NarSNPs) were synthesized by miniememulsion method and Scanning electron microscopy (SEM), Fourier-Transform Infrared Spectroscopy (FTIR) and, Dynamic Light Scattering (DLS) characterization experiments were conducted. The cytokines release (IL-6 and TNF-α) and cell viability were determined on lipopolysaccharide (LPS) induced RAW 264.7 macrophages. SNPs were determined as a potential naringenin carrier in terms of loading capacity, entrapment efficiency, pH/temperature dependent release, and preservation of bioactivity of Nar. According to our cell viability analyses, NarSNPs are safe at concentration 120 µg/mL. pH/temperature sensitive SNPs decreased the levels of the proinflammatory cytokines IL-6, TNF-α 3.95 and 4.35 times more efficiently than free Nar, respectively. These results indicate that SNPs increase bioactivity of Nar against adverse effects of inflammatory response.

Excessive osteoclast-mediated bone resorption is a critical cause of osteoporosis affecting many aging people worldwide. 5′-Methylthioadenosine (MTA) is a natural sulfur-containing nucleoside normally produced in prokaryotes, plants, yeast, and higher eukaryotes via polyamine metabolism. MTA affects various physiological responses particularly the inflammatory pathway in both normal and cancerous cells and modulates the activation of nuclear factor-κB involved in the osteoclastogenesis signalling process. While several studies have reported that natural products possess anti-osteoclastogenesis phenolics and flavonoids, the effect of nucleoside derivatives on osteoclastogenesis remains limited. Therefore, this study aimed to explore the molecular mechanisms by which MTA affects pre-osteoclastic RAW 264.7 cells as a potential alleviation compound for inflammation-mediated bone loss. Osteoclasts were established by incubating RAW264.7 macrophage cells with receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor, the vital cytokines for activation of osteoclast differentiation. Cell viability was measured using MTT assays at 24, 48, and 72 h. The suppressive effect of MTA on RANKL-induced osteoclast differentiation and function was assessed using tartrate-resistant acid phosphatase (TRAP) analysis, qRT-PCR, and pit formation, Western blot, and immunofluorescence assays. MTA showed dose-dependent anti-osteoclastogenic activity by inhibiting TRAP-positive cell and pit formation and reducing essential digestive enzymes, including TRAP, cathepsin K, and matrix metallopeptidase 9. MTA was observed to suppress the osteoclast transduction pathway through (RANKL)-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NFƘB); it attenuated NFƘB-P65 expression and down-regulated cFos proto-oncogene and nuclear factor of activated T cell c1 (NFATc1), the main regulators of osteoclasts. Moreover, the suppression of RANK (the initial receptor triggering several osteoclastogenic transduction pathways) was observed. Thus, this study highlights the potential of MTA as an effective therapeutic compound for restoring bone metabolic disease by inhibiting the RANK-NFATc1 signal pathway.

Cannabis sativa L. contains more than 80 cannabinoids, among which cannabidiol (CBD) is the main neuroactive component. We aimed to investigate the anti-inflammatory efficacy of CBD in vitro and in vivo isolated from "Pink pepper", a novel hemp cultivar, by repeating the method of selecting and cultivating individuals with the highest CBD content. We investigated the effects of CBD on inflammatory markers elevated by lipopolysaccharide (LPS) treatment in RAW 264.7 mouse macrophage cells through Western blot and RT-PCR. In addition, we confirmed these effects through the ELISA of inflamed paw tissue of a λ-carrageenan-induced mouse edema model that received an oral administration of CBD. CBD inhibited the LPS-induced phosphorylation of NF-κB and MAPK in RAW 264.7 and exhibited anti-inflammatory effects by participating in these pathways. In our in vivo study, we confirmed that CBD also inhibited the inflammatory mediators of proteins extracted from edematous mouse paw tissue. These results show that CBD isolated from "Pink pepper" exhibits potent anti-inflammatory effects. These anti-inflammatory effects of CBD have pharmacological and physiological significance, highlighting the industrial value of this novel cultivar. [ABSTRACT FROM AUTHOR]

Isaria cicadae, currently identified as Paecilomyces cicadae (Miq.) Samson (Cordycipitaceae, Hypocreales, Ascomycetes), is a parasitic fungus on cicadas and is considered a folk medicine in traditional medicine. In this research, the antioxidant activity of some extracts from the biomass and fruit bodies of Isaria cicadae F0004 isolated in Vietnam was investigated. The results showed that the Ethyl acetate (EA) fraction extract of the biomass of Isaria cicadae F0004 exhibited the best antioxidant activity in vitro, with IC50 values of 159.73 ± 7.33 µg/mL (DPPH assay) and 379.25 ± 7.33 µg/mL (ABTS assay). Meanwhile, the EA fraction extract of the fruit bodies of I. cicadae F0004 showed antioxidant activity with IC50 values of 90.8 ± 2.36 µg/mL (xanthine oxidase inhibition), 68.631 ± 0.632 µg/mL (protein membrane protection), 19.042 ± 1.072 µg/mL (lipid membrane protection). Furthermore, the EA fraction extract of the fruit bodies of I. cicadae F0004 exhibited a strong capability for DNA damage protection at 100 µg/mL concentration and OD4000 µg/mL = 0.422 ± 0.014 (reducing power). Therefore, the EA fraction extract of I. cicadae F0004 serves as an effective free radical scavenger and a potential source of natural antioxidants, and this study contributes more information on the biological activities of I. cicadae F0004 from local raw materials. [ABSTRACT FROM AUTHOR]

Mesosphaerum suaveolens (L.) Kuntze (Syn. Hyptis suaveolens (L.) Poit.) is a wild essential-oil-bearing plant having multiple uses in traditional medicine, perfumery, food, agriculture, and pharmaceutical industries. The present paper is the first report on the in vitro anti-inflammatory effects of the leaf essential oil of M. suaveolens (MSLEO) and unravels its molecular mechanism in LPS-stimulated RAW 264.7 macrophage cells. GC-MS analysis of the essential oil (EO) isolated from the leaves by hydro-distillation led to the identification of 48 constituents, accounting for 90.55% of the total oil, and β-caryophyllene (16.17%), phyllocladene (11.85%), abietatriene (11.46%), and spathulenol (7.89%) were found to be the major components. MSLEO treatment had no effect on the viability of RAW 264.7 cells up to a concentration of 100 μg/mL, and the EO was responsible for a reduction in proinflammatory cytokines like IL-6, IL-1β, and TNF-α, a decrease in intracellular ROS production, and the restoration of oxidative damage by elevating the levels of endogenous antioxidative enzymes like CAT, SOD, GPx, and GSH. RT-qPCR analysis indicated that MSLEO reduced the mRNA expression levels of iNOS and COX-2 as compared to the LPS-induced group. In addition, a confocal microscopy analysis showed that MSLEO inhibited the translocation of NF-κB from the cytosol to the nucleus. The results of this experiment demonstrate that MSLEO possesses significant anti-inflammatory potential by preventing the activation of NF-κB, which, in turn, inhibits the downstream expression of other inflammatory mediators associated with the activation of the NF-κB pathway in LPS-induced RAW 264.7 cells. Thus, the leaf essential oil of M. suaveolens may prove to be a promising therapeutic agent for the treatment of inflammation, and targeting the NF-κB signaling pathway may be considered as an attractive approach for anti-inflammatory therapies. [ABSTRACT FROM AUTHOR]

BACKGROUND: Jackfruit seed flour can be used as a cocoa aroma replacer with similar technological properties. The purpose of this study was to investigate the in vivo toxicity and in vitro antioxidant activity of fermented jackfruit seed flour (Fjs) and non‐alkaline cocoa powder (Nac). RESULTS: Fjs and Nac extracts (Fjs‐E and Nac‐E) were produced and submitted to in vitro gastrointestinal digestion producing digested fractions named Fjs‐D and Nac‐D, respectively. Nac‐E showed over two‐fold higher oxygen radical absorbance capacity (ORAC) than Fjs‐E. However, after simulated gastrointestinal digestion (in vitro), there were no significant differences between Nac‐D and Fjs‐D (P < 0.01). Similarly, the cellular antioxidant activity (CAA) of Nac‐D and Fjs‐D was not significantly different (P < 0.01). The anti‐inflammatory assay in transgenic RAW 264.7 murine macrophages showed that Fjs‐E did not affect cell viability up to 300 μg mL−1 (P > 0.05) and reduced by 15% the release of TNF‐α (P < 0.05). Fjs‐D did not affect cell viability up to 300 μg mL−1 (P > 0.05) and showed 58% reduction of NF‐κB activation (P < 0.05), with no effects on TNF‐α levels. Treatment with Nac‐E up to 300 μg mL−1 did not decrease cell viability (P > 0.05) and reduced the release of TNF‐α levels by 34% and 66% at 100 and 300 μg mL−1, respectively (P < 0.05). Nac‐D did not reduce the NF‐κB activation or TNF‐α levels at any tested concentration. CONCLUSION: Collectively, these findings indicate that Fjs is a safe and promising functional ingredient with biological activities even after gastrointestinal digestion. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Hibiscus syriacus L. extract (HSE) traditionally has been recognized as promising natural resources for the treatment of diseases related to hyper-inflammation. This study aimed to investigate the anti-inflammatory effect of HSE in LPS-stimulated Raw 264.7 cells. For this purpose, we determined the optimal extraction method and found that the ultrasonic extraction showed the most effective activity for reduction of free radical species by DPPH assay. Then, we performed PGE2 concentration analysis, COX-2 activity assay, proliferation assay, apoptosis detection, and western blotting analysis. HSE decreased PGE2 concentration and COX-2 activity and reduced the expression of COX-2 and IL6 in LPS stimulated Raw 264.7 cells. In addition, we found that HSE inhibited the proliferation and induced apoptosis with increase in the expression of pro-apoptotic proteins such as caspase-3, -7, -9, Bax, Bim, FOXO3, and p53 in LPS-stimulated Raw 264.7 cells. Taken together, we found that HSE effectively suppressed LPS-stimulated Raw 264.7 cells suggesting that HSE has the potential to treat inflammation via regulation of proinflammatory COX-2, IL6, and PGE2 and induction of apoptosis.

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are transient species that have broad actions in signaling and stress, but spatioanatomical understanding of their biology remains insufficient. Here, we report a tandem activity-based sensing and labeling strategy for H2O2 imaging that enables capture and permanent recording of localized H2O2 fluxes. Peroxy Green-1 Fluoromethyl (PG1-FM) is a diffusible small-molecule probe that senses H2O2 by a boronate oxidation reaction to trigger dual release and covalent labeling of a fluorescent product, thus preserving spatial information on local H2O2 changes. This unique reagent enables visualization of transcellular redox signaling in a microglia-neuron coculture cell model, where selective activation of microglia for ROS production increases H2O2 in nearby neurons. In addition to identifying ROS-mediated cell-to-cell communication, this work provides a starting point for the design of chemical probes that can achieve high spatial fidelity by combining activity-based sensing and labeling strategies.

Encapsulating genetic material into biocompatible polymeric microparticles is a means to improving gene transfection while simultaneously decreasing the tendency for inflammatory responses; and can be advantageous in terms of delivering material directly to the lungs via aerosolization for applications such as vaccinations. In this study, we investigated the advantages of using polymeric microparticles carrying the luciferase reporter gene in increasing transfection efficiency in the readily transfectable HEK293 cell line and the difficult to transfect RAW264.7 cell line. The results indicated that there was a limit to the ratio of nitrogen in polyethylenimine (PEI) to phosphate in DNA (N/P ratio) beyond which further increases in transgene expression no longer, or only marginally, occurred. Microparticles encapsulating PEI:DNA nanoplexes induced cellular toxicity in a dose-dependent manner. PEGylation increased transgene expression, likely related to enhanced degradation of particles. Furthermore, intra-tracheal instillation in rats allowed us to investigate the inflammatory response in the lung as a function of PEGylation, porosity, and size. Porosity did not influence cell counts in bronchoalveolar lavage fluid in the absence of PEG, but in particles containing PEG, non-porous particles recruited fewer inflammatory cells than their porous counterparts. Finally, both 1 μm and 10 μm porous PLA-PEG particles recruited more neutrophils than 4 μm particles. Thus, we have shown that PEGylation and lack of porosity are advantageous for faster release of genetic cargo from microparticles and a reduced inflammatory response, respectively.

Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis. These glycoproteins are widely involved in important biological processes, such as antigen presentation, cytokine production and glycosylation progression. Under the stimulation of the different pathogens, glycoproteins showed a dramatic change. We found that receptors in the Toll-like receptor pathway, such as Tlr2 and CD14, were increased under LPS and HSV stimulation. Glycosylation of those proteins was proven to influence their subcellular locations.

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.

Macrophages destroy pathogens and diseased cells through Fcγ receptor (FcγR)-driven phagocytosis of antibody-opsonized targets. Phagocytosis requires activation of multiple FcγRs, but the mechanism controlling the threshold for response is unclear. We developed a DNA origami-based engulfment system that allows precise nanoscale control of the number and spacing of ligands. When the number of ligands remains constant, reducing ligand spacing from 17.5 nm to 7 nm potently enhances engulfment, primarily by increasing efficiency of the engulfment-initiation process. Tighter ligand clustering increases receptor phosphorylation, as well as proximal downstream signals. Increasing the number of signaling domains recruited to a single ligand-receptor complex was not sufficient to recapitulate this effect, indicating that clustering of multiple receptors is required. Our results suggest that macrophages use information about local ligand densities to make critical engulfment decisions, which has implications for the mechanism of antibody-mediated phagocytosis and the design of immunotherapies.

Glycyl-tRNA synthetase 1 (GARS1), a cytosolic enzyme secreted from macrophages, promotes apoptosis in cancer cells. However, the mechanism underlying GARS1 secretion has not been elucidated. Here, we report that GARS1 is secreted through unique extracellular vesicles (EVs) with a hydrodynamic diameter of 20-58 nm (mean diameter: 36.9 nm) and a buoyant density of 1.13-1.17 g/ml. GARS1 was anchored to the surface of these EVs through palmitoylated C390 residue. Proteomic analysis identified 164 proteins that were uniquely enriched in the GARS1-containing EVs (GARS1-EVs). Among the identified factors, insulin-like growth factor II receptor, and vimentin also contributed to the anti-cancer activity of GARS1-EVs. This study identified the unique secretory vesicles containing GARS1 and various intracellular factors that are involved in the immunological defence response against tumorigenesis.

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multimodular scaffold, GIV (a.k.a. Girdin), titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress proinflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene-depletion studies confirmed that the presence of GIV ameliorates dextran sodium sulfate-induced colitis and sepsis-induced death. The antiinflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL) motif which binds the cytoplasmic TIR modules of TLR4 in a manner that precludes receptor dimerization; such dimerization is a prerequisite for proinflammatory signaling. Binding of GIV's TILL motif to TIR modules inhibits proinflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.

Ulcerative colitis is a chronic inflammatory disease caused by abnormal immune responses in the intestinal mucosa and gut microorganisms. Unlike other mugworts, Artemisia argyi H. (A. argyi H.) enhances antioxidant, anti-inflammatory, and anticancer effects, but the improvement effects against gut inflammation have not yet been reported. Therefore, this study aimed to confirm the alleviation of the inflammatory state in the gut by A. argyi H. fermented with Lactobacillus plantarum (FAA), using lipopolysaccharide (LPS)-induced RAW 264.7 cells and dextran sulfate sodium (DSS)-induced colitis models. In vitro, FAA (10, 50, 100, and 200 μg/mL) was pretreated into RAW 264.7 cells, followed with LPS (100 ng/mL), which induced the cell damage. Meanwhile, in vivo, FAA (100, 200 mg/kg/day) was orally administered into 6-week-old C57BL/6N mice for 3 weeks. During the last week of FAA administration, 2.5% DSS was used to induce colitis. The results showed that FAA reduced the production of nitric oxide (p < 0.0001), tumor necrosis factor (TNF)-α, interleukin (IL)-6 (p < 0.0001), and IL-1β (p < 0.0001) in the LPS-induced RAW 264.7 cells. Moreover, in the DSS-induced colitis model, FAA alleviated clinical symptoms (p < 0.001), inhibited the inflammatory state by reducing the production of TNF-α (p < 0.0001) and interferon-γ in intestinal immune cells (p < 0.0001), and strengthened the intestinal barrier by increasing the number of goblet cells (p < 0.0001). Furthermore, the anti-inflammatory effects were confirmed by the alleviation of histological damage (p < 0.001) and down-regulation of the expression of inflammatory proteins (TLR4, p < 0.0001; MyD88, p < 0.0001; Cox-2, p < 0.0001). These results suggest the potential of FAA as a dietary ingredient for preventing inflammation in the gut.

Excessive osteoclast activity can promote periodontitis-associated bone destruction. The inhibitory mechanisms of Weissella cibaria strains CMU and CMS1 against periodontitis have not yet been fully elucidated. In this study, we aimed to investigate whether heat-killed (HK) W. cibaria CMU and CMS1 or their respective cell-free supernatants (CFSs) inhibit osteoclast differentiation and bone resorption in response to receptor activator of nuclear factor kappa-B ligand (RANKL)-treated RAW 264.7 cells. TRAP (tartrate-resistant acid phosphatase) staining and bone resorption assays revealed that both HK bacteria and CFSs significantly suppressed the number of TRAP-positive cells, TRAP activity, and bone pit formation compared to the RANKL-treated control (p < 0.05). HK bacteria dose-dependently inhibited osteoclastogenesis while selectively regulating certain genes in CFSs (p < 0.05). We found that disrupting the direct interaction between HK bacteria and RAW 264.7 cells abolished the inhibitory effect of HK bacteria on the expression of osteoclastogenesis-associated proteins (c-Fos, nuclear factor of activated T cells c1 (NFATc1), and cathepsin K). These results suggest that dead bacteria suppress osteoclast differentiation more effectively than the metabolites and may serve as beneficial agents in preventing periodontitis by inhibiting osteoclast differentiation via direct interaction with cells.

Cyanobacteria are among the oldest organisms colonizing Earth. Their great biodiversity and ability to biosynthesize secondary metabolites through a variety of routes makes them attractive resources for biotechnological applications and drug discovery. In this pioneer study, four filamentous cyanobacteria (Cephalothrix lacustris LEGE 15493, Leptolyngbya boryana LEGE 15486, Nodosilinea nodulosa LEGE 06104 and Leptothoe sp. LEGE 11479) were explored for their anti-inflammatory potential in cell and cell-free in vitro bioassays, involving different inflammatory mediators and enzymes. Extracts of different polarities were sequentially prepared and chemically characterized for their content of phycobiliproteins (PBPs) and carotenoids. HPLC-PDA analysis of the acetone extracts revealed β-carotene to be the dominant carotenoid (18.4–44.3 mg/g) and zeaxanthin as the dominant xanthophyll (52.7–192.9 mg/g), with Leptothoe sp. LEGE 11479 and Nodosilinea nodulosa LEGE 06104, respectively, being the richest strains. The PBP profile was in accordance with the color presented by the aqueous extracts, with Leptolyngbya boryana LEGE 15486 being the richest in phycocyanin (204.5 μg/mg) and Leptothoe sp. LEGE 11479 the richest in phycoerythrin (78.5 μg/mg). Aqueous extracts were more effective in superoxide anion radical scavenging, while acetone ones were more effective in scavenging nitric oxide radical (●NO) and in inhibiting lipoxygenase. Acetone extracts also reduced ●NO production in lipopolysaccharide-stimulated RAW 264.7 macrophages, with the mechanistic study suggesting a downregulation of the inducible nitric oxide synthase expression. Nodosilinea nodulosa LEGE 06104 and Leptothoe sp. LEGE 11479 acetone extracts presented the lowest IC50 values for the mentioned assays, pointing them out as promising resources for the development of new multi-target anti-inflammatory therapies.

We demonstrated the effect of Ishige okamurae extract (IOE) on the receptor activator of nuclear factor-κB ligand (RANKL)-promoted osteoclastogenesis in RAW 264.7 cells and confirmed that IOE inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation. IOE inhibited protein expression of TRAP, metallopeptidase-9 (MMP-9), the calcitonin receptor (CTR), and cathepsin K (CTK). IOE treatment suppressed the expression of activated T cell cytoplasmic 1 and activator protein-1, thus controlling the expression of osteoclast-related factors. Moreover, IOE significantly reduced RANKL-phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). It also reduced the RANKL-induced phosphorylation of NF-κB and nuclear translocation of p65. IOE inhibited Dex-induced bone loss and osteoclast-related gene expression in zebrafish larvae. HPLC analysis shows that IOE consists of 3.13% and 3.42% DPHC and IPA, respectively. Our results show that IOE has inhibitory effects on osteoclastogenesis in vitro and in vivo and is a potential therapeutic for osteoporosis.

Exopolysaccharides (EPS) are biological macromolecules produced by Bifidobacterium to the outside of the cell wall during growth and metabolism. The EPS of Bifidobacterium have anti-inflammatory and antimicrobial activities and could regulate the immune system. In this study, the Bifidobacterium longum subsp. infantis E4 (B. infantis E4) has 21 specific genes for carbohydrate transport and metabolism compared with other 12 strains of B. infantis and could produce exopolysaccharides. The average molecular weights of EPS-1, EPS-2 and EPS-3 were 2.68 × 104, 3.79 × 104 and 4.72 × 104 Da, respectively. The EPS-1 mainly comprised of 7 kinds of monosaccharide, and EPS-2 and EPS-3 mainly comprised of 8 kinds of monosaccharide. The immunomodulatory and anti-inflammatory properties of EPS-1, EPS-2 and EPS-3 on RAW264.7 cells, spleen lymphocytes and NK cells of mouse were investigated in vitro of this study. EPS-1, EPS-2 and EPS-3 promoted the growth and increased phagocytic index of macrophage RAW264.7 at the concentration of 100 μg/mL. Subsequently, EPS-1, EPS-2 and EPS-3 groups were found to increase splenic lymphocyte proliferation and NK cell activity. Compared with the LPS group, EPS-1, EPS-2 and EPS-3 reduced the production of NO, IL-1β, IL-6, TNF-α and PGE-2 in RAW264.7 cells after in lipopolysaccharide (LPS) induced, with EPS-1 having the best effect. Meanwhile, EPS-1, EPS-2 and EPS-3 down-regulated the expression levels of LPS-induced NF-κβ signaling pathway-related genes p-p65, p-Iκβ, iNOS, COX-2 and reduced the nuclear translocation of p65 protein in RAW264.7 cells. In conclusion, our study suggested that EPS produced by B. infantis E4 has immunostimulatory and anti-inflammatory activities and may serve as a potential prebiotic to maintain future health.

This research investigated the anti-inflammatory effects of Purple Devil fruit’s chloroform extract (SAF-CFE) using LPS-stimulated RAW264.7 macrophage cells and in-vivo histamine- and carrageenan-induced paw edema models as well as membrane stabilization model. Writhing and licking tests for nociception and delayed-type hypersensitivity reaction (DTHR) for immunomodulation were accomplished. SwissADME, ProTox-II, and PASS tests assessed a ligand-receptor binding affinity, and network-pharmacological tests explored the modulatory linked-genes. The MCF-7 cells were strongly inhibited by SAF-CFE, which reduced LPS-induced PGE2, IL-6, TNF-α, and IL-1β expression. The upregulation of proapoptotic (p53 and Bax) and downregulation of antiapoptotic (Bcl-2) genes were observed by SAF-CFE. It significantly reduced inflammatory indexes in anti-inflammatory models. Tris (2,4-di-tert-butylphenyl) phosphate, a natural biometabolite from SAF-CFE, had the highest target receptor-binding and drug-likeness; while NOS2, PTGER1, TRPV1, HMGCR, and TBXAS1 hub genes were highly modulated by the SAF-CFE. The results demonstrate that SAF-CFE could be a functional food source for anti-inflammatory action.

During a search for natural inflammatory inhibitors, 1-O-acetylbritannilactone (ABL), a sesquiterpene lactone, was isolated from the flowers of Inula britannica. ABL significantly inhibited human neutrophil elastase (HNE) with a half-maximal inhibitory concentration (IC50) of 3.2 ± 0.3 µM, thus did so more effectively than the positive control material (epigallocatechin gallate) (IC50 7.2 ± 0.5 µM). An enzyme kinetic study was performed. ABL noncompetitively inhibited HNE with an inhibition constant Ki of 2.4 µM. ABL inhibited lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by RAW 264.7 cells in a dose-dependent manner, as well as the protein-level expression of inducible nitric oxide synthase and cyclooxygenase-2. The anti-inflammatory effect of ABL was confirmed using a transgenic Tg(mpx:EGFP) zebrafish larval model. The exposure of the larvae to ABL inhibited neutrophil recruitment to the site of injury after tail fin amputation. [ABSTRACT FROM AUTHOR]