Lipidomics of phospholipase A2 reveals exquisite specificity in macrophages.

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze phospholipids at their sn-2 fatty acyl position. Our laboratory has demonstrated that PLA2 enzymes regulate membrane remodeling and cell signaling by their specificity toward their phospholipid substrates at the molecular level. Recent in vitro studies show that each type of PLA2, including Group IVA cytosolic PLA2 (cPLA2), Group V secreted PLA2 (sPLA2), Group VIA calcium independent PLA2 (iPLA2) and Group VIIA lipoprotein-associated PLA2, also known as platelet-activating factor acetyl hydrolase, can discriminate exquisitely between fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse PUFA precursors of inflammatory metabolites. We now determined PLA2 specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a role in which specific phospholipids are hydrolyzed by each enzyme type. We used PLA2 stereospecific inhibitors in tandem with a novel UPLC-MS/MS-based lipidomics platform to quantify more than a thousand unique phospholipid molecular species demonstrating cPLA2, sPLA2, and iPLA2 activity and specificity toward the phospholipids in living cells. The observed specificity follows the in vitro capability of the enzymes and can reflect the enrichment of certain phospholipid species in specific membrane locations where particular PLA2's associate. For assaying, we target 20:4-PI for cPLA2, 22:6-PG for sPLA2, and 18:2-PC for iPLA2. These new results provide great insight into the physiological role of PLA2 enzymes in cell membrane remodeling and could shed light on how PLA2 enzymes underpin inflammation and other lipid-related diseases.

A novel tRNA-derived fragment tRF-3023b suppresses inflammation in RAW264.7 cells by targeting Cul4a through NF-κB signaling.

The role of transfer RNA (tRNA)-derived fragment (tRF) in various diseases has been established. However, the effect of tRF-3023b on inflammation remains unclear. Inflammation was imitated in RAW264.7 cells by adding Lipopolysaccharide (LPS). Cells were first divided into control, LPS, and LPS + Bulleyaconitine A (BLA) groups. The contents of TNF-α, IL-6, and MCP-1 were quantified using ELISA. The levels of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), and the phosphorylation of nuclear factor-kappa B (NF-κB)-P65 (p-P65) were detected by Western blotting. RNA sequencing was utilized to find differentially expressed tRFs (DE-tRFs) among three groups. The levels of various tRFs were checked by quantitative real-time PCR (qRT-PCR). Cell cycle and apoptosis were checked by flow cytometry. Dluciferase reporter assay was applied to predict and confirm the interaction between tRF-3023b and Cullin 4A (Cul4a), subsequently RNA pull-down followed by mass spectrometry analysis were conducted. BLA treatment decreased the contents of TNF-α, IL-6, MCP-1, and the expression levels of COX2, iNOS, p-P65. We found 6 DE-tRFs in LPS + BLA group compared to LPS group, tRF-3023b was high expression in control and BLA groups, and the lowest in LPS group. Cul4a was a direct target of tRF-3023b. tRF-3023b mimic affected the cell cycle distribution, promoted cells apoptosis, and suppressed the TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. The suppression of Cul4a affected the cell cycle distribution, resulted in an increase of cell apoptosis while a decrease of TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. Furthermore, Cul4a overexpression reversed the effect of tRF-3023b mimic. Cul4a knockdown reversed the effect of tRF-3023b inhibitor. Our study positions tRF-3023b as a compelling candidate, through its interaction with Cul4a, the underlying mechanism on inflammation maybe related to NF-κB pathway. The study provides a basis for exploring new therapeutic strategies for inflammation.

Exploring the potential of structural modeling and molecular docking for efficient siRNA screening: A promising approach to Combat viral mutants, with a focus on HIV-1.

RNA interference (RNAi) holds immense potential for sequence-specific downregulation of disease-related genes. Small interfering RNA (siRNA) therapy has made remarkable strides, with FDA approval for treating specific human diseases, showcasing its promising future in disease treatment. Designing highly efficient siRNAs is a critical step in this process. Previous studies have introduced various algorithms and parameters for siRNA design and scoring. However, these attempts have often fallen short of meeting all essential criteria or required modifications, resulting in variable and unclear effectiveness of screened siRNAs, particularly against viral mutants with non-conserved short sequences. In this study, we present a fully optimized siRNA screening system considering all necessary parameters. Notably, we highlight the critical role of molecular docking simulations between siRNA and two functional domains of the Argonaute protein (PAZ and PIWI) in identifying the most efficient siRNAs, since the appropriate interaction between the guide siRNA strand and the RISC complex is crucial. Through our stringent method, we designed approximately 50 potential siRNAs targeting the HIV-1 vpr gene. Evaluation through XTT, qRT-PCR, and flow cytometry analysis on RAW 264.7 macrophage stable cells revealed negligible cytotoxicity and exceptional gene-silencing efficiency at both the transcriptional and translational levels for the top-ranked screened siRNAs. Given the growing interest in siRNA-based therapeutics, we anticipate that the insights from this study will contribute to improving treatment strategies against mutant viruses, particularly HIV-1.

Synthesis, characterization, and anti-inflammatory activity of tetrahydropiperine, piperic acid, and tetrahydropiperic acid via down regulation of NF-κB pathway.

The present study describes the synthesis, characterization, and evaluation of tetrahydropiperine (THP), piperic acid (PA), and tetrahydropiperic acid (THPA) as anti-inflammatory agents. THPA demonstrated potent anti-inflammatory activity among all the compounds. The anti-inflammatory potential was investigated in both in-vitro and in-vivo experimental models. Our findings demonstrated that THPA effectively suppressed the production of pro-inflammatory mediators, including nitric oxide and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in both in vitro and in vivo. Additionally, THPA attenuated the expression of i-NOS and COX-2 in RAW 264.7 macrophages. The oral administration of THPA significantly reduced carrageenan induced paw edema thickness and alleviated liver, lung, and kidney injury induced by LPS. THPA also reduced the infiltration of inflammatory cells, prevented the occurrence of significant lesions, and mitigated tissue damage. Moreover, THPA significantly improved the survival rate of mice challenged with LPS. Our western blot studies also found that LPS induced NF-κB activation was downregulated by treatment with THPA in an in vivo system. These results collectively illustrated the potential of THPA as a therapeutic agent for treating inflammatory diseases.

Design, synthesis and anti-rheumatoid arthritis activity of target TLR4 inhibitors.

A series of 1-(2-oxocyclohexyl)butane-1, 3-dione derivatives were designed and synthesized as TLR4 inhibitors by modifying the core structure of the lead compound ((6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalen-2-yl) carbamate)). In vitro, compound 3p significantly inhibited the proliferation of rat synovial cells, inhibited the proliferation of LPS-induced RAW264.7 cells, and inhibited TLR4 activity, with IC50 values of 1.21 ± 0.09 µM, 0.73 ± 0.05 µM and 0.43 ± 0.03 µM, respectively, which was superior to the positive control methotrexate. In vivo anti-rheumatoid arthritis evaluation, compound 3p can significantly inhibit the inflammatory factors TNF-α, IL-1ß and IL-6, so as to achieve the therapeutic purpose. In the preliminary mechanism study, compound 3p has obvious regulatory effects on the abnormal increase of TLR4, JAK2 and STAT3 protein and gene expression related to inflammatory signaling pathway in RAW264.7 cells. In summary, this study aims to develop more effective candidates for rheumatoid arthritis.

Anti-inflammatory effects of Olive (olea europaea L.) fruit extract in LPS-stimulated RAW264.7 cells via MAPK and NF-κB signal pathways.

BACKGROUND: Olive is an evergreen tree of Oleaceae Olea with numerous bioactive components. While the anti-inflammatory properties of olive oil and the derivatives are well-documented, there remains a dearth of in-depth researches on the immunosuppressive effects of olive fruit water extract. This study aimed to elucidate the dose-effect relationship and underlying molecular mechanisms of olive fruit extract in mediating anti-inflammatory responses. METHODS AND RESULTS: The impacts of olive fruit extract on the release of nitric oxide (NO), tumor necrosis factor (TNF-α), interleukins-6 (IL-6) and reactive oxygen species (ROS) were assessed in RAW264.7 cells induced by lipopolysaccharide (LPS). For deeper understanding, the expression of genes encoding inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α and IL-6 was quantitatively tested. Additionally, the expression patterns of MAPK and NF-κB pathways were further observed to analyze the action mechanisms. Results suggested that olive fruit extract (200, 500, 1000 µg/mL) markedly exhibited a dose-dependent reduction in the generation of NO, TNF-α, IL-6 and ROS, as well as the expression of correlative genes studied. The activation of ERK, JNK, p38, IκB-α and p65 were all suppressed when p65 nuclear translocation was further restricted by olive fruit extract in NF-κB and MAPK signal pathways. CONCLUSIONS: Olive fruit extract targeted imposing restrictions on the signal transduction of key proteins in NF-κB and MAPK pathways, and thereby lowered the level of inflammatory mediators, which put an enormous hindrance to inflammatory development. Accordingly, it is reasonable to consider olive fruit as a potent ingredient in immunomodulatory products.

7,8-dihydroxyflavone displayed antioxidant effect through activating HO-1 expression and inhibiting caspase-3/PARP activation in RAW264.7 cells.

Flavonoids, which contain a benzo-γ-pyrone (C6-C3-C6) skeleton, have been reported to exhibit effective antioxidant ability. This study aimed to compare the antioxidant activities of 7,8-dihydroxyflavone (7,8-DHF) and 7-hydroxyflavone (7-HF) in H2 O2 , lipopolysaccharide (LPS), or tert-butyl hydroperoxide (t-BHP)-induced RAW264.7 cells, respectively. The antioxidant capacities of 7,8-DHF and 7-HF were firstly evaluated by 2,2-azinobis-3-ethyl-benzothiazoline-6-sulphonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Then, reactive oxygen species (ROS), super oxide dismutase (SOD), and malondialdehyde (MDA) productions in H2 O2 , LPS, or t-BHP-induced RAW264.7 cells were tested and compared, respectively. Finally, the antioxidant mechanisms of 7-HF and 7,8-DHF were initially investigated by western blot. Our results showed that 7,8-DHF possessed stronger free-radical scavenging capacity than 7-HF. Both 7,8-DHF and 7-HF suppressed MDA production and ROS accumulation, improved the activity of SOD in H2 O2 , LPS, or t-BHP-induced RAW264.7 cells, respectively. And 7,8-DHF exerted a better antioxidant effect than 7-HF, especially in t-BHP-induced oxidative stress. Mechanically, 7,8-DHF prevented the activation of poly ADP-ribosepolymerase and caspase-3, meanwhile markedly upregulated the expression of HO-1 protein in t-BHP-induced oxidative stress. These results suggested that 7,8-DHF might serve as a potential pharmaceutical drug against oxidative stress injury.

Cynaropicrin attenuates inflammatory cytokines in LPS-induced RAW264.7 cells and ovalbumin-induced asthmatic mice.

This study examines the anti-inflammatory activity of cynaropicrin against lipopolysaccharide (LPS) in vitro and ovalbumin (OVA)-challenged asthma in mice. Cynaropicrin's antimicrobial effects were tested on Escherichia coli (E. coli) and Streptococcus pyogenes (S. pyogenes) using the disc diffusion technique. Cytotoxicity was assessed with an (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. The anti-inflammatory property was evaluated in LPS-induced RAW264.7 cells, while OVA-challenged asthmatic mice were treated with 10 mg/kg of cynaropicrin. Key inflammatory and antioxidant markers were quantified, and lung histology was examined to confirm therapeutic roles. The antimicrobial studies proved that cynaropicrin effectively inhibited the growth of E. coli and S. pyogenes. Cynaropicrin displayed no cytotoxicity on RAW264.7 cells. Furthermore, it significantly inhibited inflammatory cytokine synthesis upon LPS induction. Cynaropicrin treatment decreased the inflammatory cell counts and also suppressed specific allergic markers in OVA-challenged mice. It also decreased nitric oxide and myeloperoxidase levels and reduced pulmonary edema. Cynaropicrin increased antioxidant levels and decreased proinflammatory cytokines in the asthmatic mice. Lung histological examination confirms the ameliorative potency of cynaropicrin against OVA-induced asthmatic pulmonary inflammation in mice. Our findings suggest cynaropicrin possesses significant ameliorative potency against allergen-induced pulmonary inflammation.

Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

Acute exposure to polystyrene nanoparticles promotes liver injury by inducing mitochondrial ROS-dependent necroptosis and augmenting macrophage-hepatocyte crosstalk.

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.

Molecular modification of chlorogenic acid via radiolysis with inhibitory effects on NO production.

The molecular modification of chlorogenic acid (1) through γ-irradiation resulted in the formation of five new products: chlorogenosins A (2), B (3), C (4), D (5), and E (6) along with known compounds rosmarinosin B (7), protocatechuic acid (8), and protocatechuic aldehyde (9). The structures of the new compounds were elucidated using spectroscopic methods, including one-dimensional and two-dimensional nuclear magnetic resonance, high-resolution electrospray ionization mass spectroscopy, and circular dichroism spectroscopy. The potential anti-inflammatory activities of all the isolated compounds were determined by evaluating their inhibitory effects on the nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages. Notably, compounds 2 and 3, which contained two hydroxymethyl functionalities instead of the trans-olefinic moiety present in the original chlorogenic acid, exhibited stronger inhibitory effects on NO production than that of the original compound. These findings suggest that the predominant chemical changes induced in chlorogenic acid by γ-irradiation may enhance its anti-inflammatory properties.

In Vitro Insights into the Role of 7,8-Epoxy-11-Sinulariolide Acetate Isolated from Soft Coral Sinularia siaesensis in the Potential Attenuation of Inflammation and Osteoclastogenesis.

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the process of bone remodeling. Excessive osteoclast differentiation plays a pivotal role in the pathogenesis of bone diseases such as rheumatoid arthritis and osteoporosis. In the present study, we examined whether 7,8-epoxy-11-sinulariolide acetate (Esa), a marine natural product present in soft coral Sinularia siaesensis, attenuates inflammation and osteoclastogenesis in vitro. The results indicated that Esa significantly inhibited lipopolysaccharide (LPS)-induced inflammation model of RAW264.7 cells and suppressed receptor activator for nuclear factor-κB ligand (RANKL)-triggered osteoclastogenesis. Esa significantly down-regulated the protein expression of iNOS, COX-2, and TNF-α by inhibiting the NF-κB/MAPK/PI3K pathways and reducing the release of reactive oxygen species (ROS) in RAW264.7 macrophages. Besides, Esa treatment significantly inhibited osteoclast differentiation and suppressed the expression of osteoclast-specific markers such as NFATC1, MMP-9, and CTSK proteins. These findings suggest that Esa may be a potential agent for the maintenance of bone homeostasis associated with inflammation.

Anti-Inflammatory Activity of Cyanobacteria Pigment Extracts: Physiological Free Radical Scavenging and Modulation of iNOS and LOX Activity.

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 ß-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.

Effect of Ishige okamurae Extract on Osteoclastogenesis In Vitro and In Vivo.

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.

Reposition: Focalizing ß-Alanine Metabolism and the Anti-Inflammatory Effects of Its Metabolite Based on Multi-Omics Datasets.

The incorporation of multi-omics data methodologies facilitates the concurrent examination of proteins, metabolites, and genes associated with inflammation, thereby leveraging multi-dimensional biological data to achieve a comprehensive understanding of the complexities involved in the progression of inflammation. Inspired by ensemble learning principles, we implemented ID normalization preprocessing, categorical sampling homogenization, and pathway enrichment across each sample matrix derived from multi-omics datasets available in the literature, directing our focus on inflammation-related targets within lipopolysaccharide (LPS)-stimulated RAW264.7 cells towards ß-alanine metabolism. Additionally, through the use of LPS-treated RAW264.7 cells, we tentatively validated the anti-inflammatory properties of the metabolite Ureidopropionic acid, originating from ß-alanine metabolism, by evaluating cell viability, nitric oxide production levels, and mRNA expression of inflammatory biomarkers. In conclusion, our research represents the first instance of an integrated analysis of multi-omics datasets pertaining to LPS-stimulated RAW264.7 cells as documented in the literature, underscoring the pivotal role of ß-alanine metabolism in cellular inflammation and successfully identifying Ureidopropionic acid as a novel anti-inflammatory compound. Moreover, the findings from database predictions and molecular docking studies indicated that the inflammatory-related pathways and proteins may serve as potential mechanistic targets for Ureidopropionic acid.

Anti-inflammatory activity of ß-glucans from different sources before and after fermentation by fecal bacteria in vitro.

BACKGROUND: ß-Glucans are widely sourced and have various physiological effects, including anti-inflammatory effects. However, the strength of the anti-inflammatory activity of ß-glucans from different sources remains unknown due to the lack of rapid and effective biomarkers. This study therefore aimed to screen out the ß-glucans with strong anti-inflammatory activity from five different sources and to further screen out possible biomarkers in metabolites after fermenting the ß-glucans with gut microorganisms. RESULTS: The results showed that all five ß-glucans inhibited the production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators and suppressed the mRNA expression level of TLR4/MyD88. Their anti-inflammatory mechanisms involved the inhibition of intracellular reactive oxygen species (ROS) production and suppression of mRNA expression of the NF-κB pathway and JNK pathway. Among them, barley ß-glucan exhibited the strongest anti-inflammatory effect, followed by Ganoderma lucidum ß-glucan. Enhanced anti-inflammatory activity of ß-glucan was found after fermentation and may be related to the increased abundance of metabolites such as vanillin, dihydroxyphenylacetic acid, caffeic acid, acetic acid, butyric acid, and lactic acid. They were strongly positively correlated to the abundance of beneficial bacteria such as Blautia, suggesting that the production of those metabolites may be responsible for the flourishing of the beneficial bacteria. CONCLUSION: In conclusion, barley was a preferred raw material for the preparation of ß-glucans with strong anti-inflammatory activity. Vanillin, dihydroxyphenylacetic acid, caffeic acid, acetic acid, butyric acid, and lactic acid were the possible biomarkers that could be utilized to evaluate the anti-inflammatory effect of ß-glucans. © 2023 Society of Chemical Industry.

Radiofrequency field inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells via modulating the NF-κB signaling pathway.

In 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.

Up-regulation of inflammatory reactions by MPT32, a secreted protein of Mycobacterium tuberculosis in RAW264.7 macrophages.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is still one of the global health burdens. The occurrence of various cases and multidrug resistance confirm that TB has not been completely conquered. For these reasons, the present research has been conducted to explore TB vaccine and drug candidate possibility using Mtb-secreted proteins. Among these proteins, MPT32 is known to have antigenicity and immunogenicity. There has not been a report on the host immune responses and regulation in macrophage cells. The present study was conducted with MPT32 in RAW 264.7 murine macrophage cells that control immune responses by sensing pathogen invasion and environmental change. We have found that MPT32 could activate lipopolysaccharide (LPS)-induced gene expression of metalloproteinase-9 (MMP-9) and inflammation in RAW 264.7 cells. After treating cells with MPT32, the increase in pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß (IL-1ß) and IL-6, was observed. In addition, activated macrophages expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to generate various inflammatory mediator molecules, such as nitric oxide (NO). The increase in iNOS and COX-2 levels, which are up-regulators of MMP-9 expression, was also confirmed. The biochemical events are involved in the downstream of activated MAPK signaling and translocation of NF-κ B transcription factor. The present results prove the immunomodulatory effect of MPT32 in the RAW 264.7 murine macrophage cells. it claims the possibility of a TB vaccination and drug candidate using MPT32, contributing to the prevention of TB.

TLR2 mediates autophagy through ERK signaling pathway in Chlamydia psittaci CPSIT_p7 protein-stimulated RAW264.7 cells.

Chlamydia psittaci is a zoonotic pathogen found in birds and humans. Macrophages, major components of the innate immune system, can resist chlamydial infections and trigger adaptive immune responses. However, the molecular mechanisms underlying the action of macrophages against C. psittaci infection are not well understood. This study investigated the roles and mechanisms of plasmid-encoded protein CPSIT_p7 of C. psittaci in regulating autophagy in RAW264.7 cells. The results demonstrated that stimulation of RAW264.7 with C. psittaci plasmid protein CPSIT_p7 induced the expressions of the autophagy signaling primary regulators LC3 and Beclin1, which could also significantly induce the phosphorylation levels of ERK, JNK, p38, and Akt. Next, siRNA knockdown of TLR2 resulted in significant downregulation of CPSIT_p7-triggered autophagy in RAW264.7 cells. Moreover, the extracellular regulated protein kinase (ERK) inhibitor PD98059 markedly reduced autophagy in CPSIT_p7-stimulated macrophages. In summary, these results indicated that TLR2 plays an essential role in the induction of autophagy through the ERK signaling pathway in CPSIT_p7-stimulated RAW264.7 cells.

New chromones from the roots of Saposhnikovia divaricata (Turcz.) Schischk with anti-inflammatory activity.

Fifteen new chromones, sadivamones A-E (1-5), cimifugin monoacetate (6), sadivamones F-N (7-15), together with fifteen known chromones (16-30), were isolated from the ethyl acetate portions of 70% ethanol extract of Saposhnikovia divaricata (Turcz.) Schischk roots. The structures of the isolates were determined using 1D/2D NMR data and electron circular dichroism (ECD) calculations. Meanwhile, LPS induced RAW264.7 inflammatory cell model was used to determine the potential anti-inflammatory activity of all the isolated compounds in vitro. The results showed that compounds 2, 8, 12-13, 18, 20-22, 24, and 27 significantly inhibited the production of lipopolysaccharide (LPS)-induced NO in macrophages. To determine the signaling pathways involved in the suppression of NO production by compounds 8, 12 and 13, we investigated ERK and c-Jun N-terminal protein kinase (JNK) expression by western blot analysis. Further mechanistic studies demonstrated that compounds 12 and 13 inhibited the phosphorylation of ERK and the activation of ERK and JNK signaling in RAW264.7 cells via MAPK signaling pathways. Taken together, compounds 12 and 13 may be valuable candidates for the treatment of inflammatory diseases.