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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]

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]

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]

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]

[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]

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]

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]

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]

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]

In the present study, a novel water-soluble polysaccharide (DNP−1) was isolated and purified from the root of Duhaldea nervosa via column chromatography. Structural analyses indicated that DNP−1 had a linear backbone consisting of (2→1)-linked β-D- fructofuranosyl residues, ending with a (2→1) bonded α-D-glucopyranose. DNP−1 was a homogeneous polysaccharide with an average molecular weight of 3.7 kDa. Furthermore, the anti-inflammatory activity of DNP−1 was investigated in vitro. The concentration of pro-inflammatory cytokines, including NO, TNF-α, MCP-1, IL-2, and IL-6, in the DNP−1 treatment group was suppressed in LPS-induced RAW 264.7 cells. DNP−1 was able to improve inflammatory injury by inhibiting the secretion of pro-inflammatory cytokines. These investigations into this polysaccharide from the root of Duhaldea nervosa provide a scientific basis for the further development of this plant. The results indicate that this Duhaldea nervosa polysaccharide could be used as a potential natural source for the treatment of inflammatory injury. [ABSTRACT FROM AUTHOR]

Conventionally T-cell receptors (TCRs) have so far been considered as a T-lymphocyte privilege. However, recent findings also place TCR expression in non-lymphoid cells, namely neutrophils, eosinophils and macrophages. In order to examine the ectopic expression of TCR, this study focused on RAW 264.7 cells, which have been broadly used for their macrophage properties. Immunofluorescence staining detected 70% and 40% of the cells to express TCRαβ and TCRγδ respectively, which was also verified by RT-PCR experiments and confocal microscopy analysis. Interestingly, except from the predicted 292 and 288 bp gene products for the α- and γ-chain, additional products at 220 and 550 bp could be detected, respectively. RAW 264.7 cells also expressed the co-stimulatory CD4 and CD8 markers at a percentage of 61% and 14% respectively, which supported the expression of TCRs. However, only low numbers of cells expressed CD3ε and CD3ζ (9% and 7% respectively). Such observations contradicted the existing knowledge, and indicated that TCRs would be supported by other molecules for reaching the membrane and transducing their signal. Such candidate molecules could be the Fcγ receptors (FcγRs). Indeed, the FcγRII/III receptor was found to be expressed in 75% of the cells, which also expressed at a percentage of 25% major histocompatibility complex (MHC) class II molecules. Engagement of the FcγRII/III receptor by a recombinant IgG2aCH2 fragment, except from stimulating the macrophage-dependent properties of the cells, was shown to reduce expression of TCRαβ and γδ indicating that FcγRII/III was indeed used by TCRs for their transport to the cell membrane. In order to examine the ability of RAW 264.7 cells to simultaneously display antigen presenting- and T-cell properties, functional experiments as to antigen-specific antibody and IL-2 production were performed. In in vitro immunization assays in the presence of naïve B cells, RAW264.7 failed to promote antibody production. However, RAW 264.7 cells could compete with antigen-stimulated macrophages but not T cells when applied to a system of in vivo antigen-sensitized cells followed by an in vitro immunization protocol. Interestingly, simultaneous addition of antigen and the IgG2aCH2 fragment to RAW 264.7 cells could promote IL-2 production from the cells, indicating that FcγRII/III activation could also support TCR stimulation. Extrapolating these findings to cells of the myeloid origin, the above results dictate novel regulatory mechanisms towards the alteration of the immune response. [Display omitted] • RAW 264.7 macrophages express TCRαβ/TCRγδ and CD4/CD8 but insufficient CD3 levels. • Recombinant IgG2aCH2 decreases TCRαβ/TCRγδ indicating that FcγRII/III is involved in TCR membrane transport. • RAW 264.7 cells may compete macrophages but not T cells in an antigen specific antibody production. • Stimulation of RAW 264.7 cells with rIgG2aCH2 and antigen induces IL-2 production. • Novel regulatory pathways by TCR+ myeloid cells towards stimulation of immunity. [ABSTRACT FROM AUTHOR]

Inflammation causes a protective immune response, which can be observed by examining the inflammatory responses of macrophages. Macrophages release various immunostimulatory factors when destroying external pathogens. We induced lipopolysaccharides (LPS) in RAW 264.7 cells, a macrophage cell line, to determine whether Helixor-M can cause immuno-suppression. Helixor-M is known to have anticancer and immune effects. However, an indicator that regulates immunity has not been clearly confirmed. To this end, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to confirm Helixor-M was not cytotoxic. Western blotting and real-time polymerase chain reaction (RT-PCR) confirmed the anti-inflammatory effects. Additionally, immunofluorescence assay confirmed the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, a representative inflammatory pathway. Helixor-M was found to be non-cytotoxic, induce the NF-κB pathway, and reduce the levels of pro-inflammatory cytokine and mitogen-activated protein kinase (MAPK). We found Helixor-M affected the PI3K/AKT/JNK pathway. Therefore, we confirmed Helixor-M acts as an anti-inflammatory agent through NF-κB, TLR4 and PI3K inhibition and that it could be an effective immunosuppressive drug. [ABSTRACT FROM AUTHOR]

Red betel (Piper crocatum Ruiz and Pav) is a traditional Indonesian plant. The ethyl acetate fraction of red betel leaf has the potential as an anti-inflammatory. This research aimed to determine the anti-inflammatory effect of isolate compound from ethyl acetate fraction of Piper crocatum leaves on the production of cytokine pro-inflammatory TNF-a and ICAM-1 in lipopolysaccharide-induced RAW 264.7 cells. Cell viability was determined by the MTT method in the concentration range of 1.25; 2.5; 5; 10; 20, and 40 µg/ml. The TNF-a and ICAM-1 level was determined by the TNF-a and ICAM-1 assay kit as measured by an ELISA plate reader. Cell viability test of isolate compound from ethyl acetate fraction, the concentrations that gave viability percentages above 80% were 10; 5; 2.5 and 1.25 ug/ml. Isolate compound at concentrations of 10, 5, 2.5 and 1.25 ug/ml significantly inhibited TNF-a and ICAM-1 production with p<0.05 (p = 0.000). [ABSTRACT FROM AUTHOR]

Neocinnamomum caudatum (Lauraceae) plant is used in the traditional system of medicine and is considered a potential source of edible fruits, spices, flavoring agents and biodiesel. The leaves, bark and roots of the species are used by local communities for the treatment of inflammatory responses, such as allergies, sinusitis and urinary tract infections. However, there is no scientific evidence to support the molecular mechanism through which this plant exerts its anti-inflammatory effect. The aim of the current research was to characterize the chemical constituents of bark (NCB) and leaf (NCL) essential oil of N. caudatum and to elucidate its anti-inflammatory action in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Essential oils extracted by hydrodistillation were further subjected to gas chromatography mass spectrometry (GC-MS) analysis. The major constituents in bark essential oil identified as β-pinene (13.11%), α-cadinol (11.18%) and α-pinene (10.99%), whereas leaf essential oil was found to be rich in β-pinene (45.21%), myrcene (9.97%) and α-pinene (9.27%). Treatment with NCB and NCL at a concentration of 25 µg/mL exerted significant anti-inflammatory activity by significantly reducing LPS-triggered nitric oxide (NO) production to 45.86% and 61.64%, respectively, compared to the LPS-treated group. In the LPS-treated group, the production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β, decreased after treatment with essential oil, alleviating the mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. The essential oil also inhibited the production of intracellular ROS and attenuated the depletion of mitochondrial membrane potential in a concentration-dependent manner. Pretreatment with NCB also reduced nuclear factor kappa-B (NF-κB)/p65 translocation and elevated the levels of endogenous antioxidant enzymes in LPS-induced macrophages. The present findings, for the first time, demonstrate the anti-inflammatory potential of both bark and leaf essential oils of N. caudatum. The bark essential oil exhibited a significantly more important anti-inflammatory effect than the leaf essential oil and could be used as a potential therapeutic agent for the treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Host inflammatory responses are key to protection against injury; however, persistent inflammation is detrimental and contributes to morbidity and mortality. Herein, we demonstrated the anti-inflammatory role of Arteannuin-B (1) and its new spirocyclic-2-isoxazoline derivative JR-9 and their side effects in acute inflammatory condition in vivo using LPS-induced cytokines assay, carrageenan-induced paw edema, acetic acid-induced writhing and tail immersion. The results show that the spirocyclic-2-isoxazoline derivative is a potent anti-inflammatory agent with minimal cell toxicity as compared to Arteannuin-B. In addition, the efficacies of these compounds were also validated by flow cytometric, computational, and histopathological analysis. Our results show that the anti-inflammatory response of JR-9 significantly reduces the ability of mouse macrophages to produce NO, TNF-α, and IL-6 following LPS stimulation. Therefore, JR-9 is a prospective candidate for the development of anti-inflammatory drugs and its molecular mechanism is likely related to the regulation of NF-κB and MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Introduction: Osteoporosis is a major cause of bone fracture in post-menopausal women. We evaluated the effects of pristimerin treatment on ovariectomy- induced osteoporosis, and its possible molecular mechanism. Material and methods: Rats were ovariectomised and biochemical markers of bone formation were determined from serum samples. The microarchitectures of bone tissues were analyzed via micro-CT scans and Western blotting assays. The cytotoxic effects of pristimerin, the differentiation of osteoclasts, and bone reabsorption were evaluated in vitro using RAW 264.7 cells. Results: Treatment with pristimerin attenuated changes in markers of bone formation and reabsorption such as creatine kinase (CK), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), collagen type I fragments (CTX), bone Gla-protein (BGP), and osteocalcin (OC) in the serum of ovariectomised rats. It also appeared to restore the microarchitecture of bone tissue. The expression levels of TNF receptor-associated factor 6 (TRAF-6), nuclear factor k light chain enhancer of activated B cells (NF-κB p65), and receptor activator of nuclear factor-κB ligand (RANKL) protein were significantly lower, while those of Akt and PI3K were significantly higher, in the bone tissues of the pristimerin-treated group than in negative controls. Pristimerin had no cytotoxic effect on RAW 264.7 cells and reduced the differentiation of osteoclasts, bone reabsorption, and translocation of p65 in RANKL-stimulated RAW 264.7 cells in vitro. Conclusions: Pristimerin reduces the effects of osteoporosis by restoring the altered RANKL/TRAF-6/NF-κB pathway in ovariectomised rats. [ABSTRACT FROM AUTHOR]

Oxidative stress is strongly involved in the pathogenesis of neurodegenerative diseases, like Parkinson´s disease (PD). Particularly, an excess of reactive oxygen species (ROS) released by the cells promotes an oxidative stress condition, which is a main cause of tissue injury leading to nervous system dysfunction. In this work, the antioxidant, neuroprotective and anti-inflammatory activities of different fractions from the brown seaweed Bifurcaria bifurcata are presented and related with their chemical profile. The antioxidant capacity was evaluated by the Folin-Ciocalteu method, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Neuroprotective capacity was evaluated to prevent neurological cell death mediated by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y cells, and their anti-inflammatory effects on RAW 264.7 macrophages. The ethyl acetate fractions (100 µg mL−1) exhibited significant antioxidant and neuroprotective activities in the in vitro models assayed. Furthermore, two of the most polar fractions obtained with methanol and water also evidenced a significant neuroprotective potential. Bifurcaria bifurcata fractions treatment decreased ROS production, mitochondrial dysfunction, and Caspase-3 activity. Regarding the anti-inflammatory potential, five fractions (100 µg mL−1) inhibited nitric oxide (NO) production and reduced the interleukin – 6 (IL-6) and tumor necrosis factor (TNF-α) levels. Mannitol, identified as the major component of the most bioactive fraction, protected SH-SY5Y cells against the 6-OHDA neurotoxicity mediating ROS generation mitigation, mitochondrial dysfunction, and DNA damage, together with the Caspase-3 activity inhibition. Results suggest that B. bifurcata is a relevant source of neuroprotective agents, with particular interest for preventive therapeutics. [ABSTRACT FROM AUTHOR]

Bioactive peptides are naturally found in various foods and were shown to have various distinct physiological as well as medicinal benefits. In this study shrimp peptide hydrolysate (SPH) was prepared to investigate its immunomodulatory effect against cyclophosphamide (CTX) induced immunosuppressed mice. The SPH effect was also analyzed on murine macrophage (RAW264.7 cells). The findings show that SPH stimulates macrophages to form multiple pseudopodia, has no cytotoxic effect, and increases phagocytic activity in RAW264.7 cells. Furthermore, the immunosuppressed in‐vivo model illustrates the improvement in various aspects, that is body weight, escalation in immune organ index, and ameliorates histopathological transformation of thymus along with the spleen. SPH enhances cell‐mediated immunity by facilitating splenocyte proliferation and inhibit excessive apoptosis. Moreover, the significant outcome had been observed with the upregulation of cytokines interferon‐gamma (IFN‐ϒ), interleukin‐2 (IL‐2) level and simultaneously downregulate certain genes include interleukin‐4 (IL‐4) and interleukin‐10 (IL‐10). Additionally, SPH expedites cellular immunity by enhancing the regulation of immunoglobulin A (IgA) and immunoglobulin M (IgM). However, these findings support the hypothesis that SPH is an effective immunomodulatory agent capable of preventing immune system hypofunction. It is necessary to investigate the detailed mechanism to rule out any unforeseen effects of SPH in future research. Practical applications: Chemotherapy medications, despite their dominating detrimental effects of damaging immunological organs such as the spleen and thymus, extend the treatment process as well as the destruction of the self‐immune system. This study found that SPH is an effective immunomodulatory agent capable of avoiding immune organ hypofunction and improving cell mediate immunity by enhancing macrophage activation, phagocytosis, spleenocyte proliferation, suppressing apoptosis, and elevating cytokines and antibodies. As a result, SPH can be utilized as a nutritional and functional dietary supplement to boost immunological modulation in combination with chemotherapy medications in order to lessen their adverse effects. [ABSTRACT FROM AUTHOR]

Antrodia camphorata is an endemic mushroom in Taiwan. This study was designed to screen anti-inflammatory compounds from the methanolic extract of the mycelium of A. camphorata on nitric oxide (NO) production in RAW 264.7 cells induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA) known to be present in viral infection. A combination of bioactivity-guided isolation with an NMR-based identification led to the isolation of 4-acetylantroquinonol B (1), along with seven compounds. The structure of new compounds (4 and 5) was elucidated by spectroscopic experiments, including MS, IR, and NMR analysis. The anti-inflammatory activity of all isolated compounds was assessed at non-cytotoxic concentrations. 4-Acetylantroquinonol B (1) was the most potent compound against poly I:C-induced NO production in RAW 264.7 cells with an IC50 value of 0.57 ± 0.06 μM. [ABSTRACT FROM AUTHOR]

Introduction: Several experimental models have been designed to promote the development of new anti-inflammatory drugs. The in vitro model using RAW 264.7 cells has been widely used. However, there is still no consensus on which inflammatory mediators should initially be measured to screen for possible anti-inflammatory effects. To determine the rationality of measuring inflammatory mediators together with NO, such as the levels of tumor necrosis factor (TNF)-α, and interleukins (IL) 1β and 6, we carried out this systematic review (SR) and meta-analysis (MA). Methodology: We conducted this SR and MA in accordance with the Preferred Reporting of Systematic Reviews and Meta-Analysis and the Cochrane Handbook for Systematic Reviews of Intervention. This review was registered in the Open Science Framework (https://doi.org/10.17605/OSF.IO/8C3HT). Results: LPS-induced cells produced high NO levels compared to non-LPS induced, and this production was not related to cell density. TNF-α, IL-1β, and IL-6, also showed high levels after cells had been stimulated with LPS. Though with some restrictions, all studies were reliable, as the risk of bias was detected in the test compounds and systems. Conclusion: Measurement of NO levels may be sufficient to screen for possible anti-inflammatory action in the context of LPS-induced RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

Punica granatum L. (Punicaceae) is a popular fruit all over the world. Owning to its enriched polyphenols, P. granatum has been widely used in treating inflammation-related diseases, such as cardiovascular diseases and cancer. Twenty polyphenols, containing nine unreported ones, named punicagranins A–I (1–9), along with eleven known isolates (10–20), were obtained from the peels. Their detailed structures were elucidated based on UV, IR, NMR, MS, optical rotation, ECD analyses and chemical evidence. The potential anti-inflammatory activities of all polyphenols were examined on a lipopolysaccharide (LPS)-induced inflammatory macrophages model, which indicated that enhancing nitric oxide (NO) production in response to inflammation stimulated in RAW 264.7 cells was controlled by compounds 1, 3, 5–8, 10, 11, 14 and 16–20 in a concentration-dependent manner. The investigation of structure–activity relationships for tannins 6–8 and 12–20 suggested that HHDP, flavogallonyl and/or gallagyl were key groups for NO production inhibitory activity. Western blotting indicated that compounds 6–8 could down-regulate the phosphorylation levels of proteins p38 MAPK, IKKα/β, IκBα and NF-κB p65 as well as inhibit the levels of inflammation-related cytokines and mediators, such as IL-6, TNF-α, iNOS and COX-2, at the concentration of 30 μM. In conclusion, polyphenols are proposed to be the potential anti-inflammatory active ingredients in P. granatum peels, and their molecular mechanism is likely related to the regulation of the p38 MAPK and NF-κB signaling pathways. [ABSTRACT FROM AUTHOR]

It has been reported that 4,5-dihydropyrazole and thiazole derivatives have many biological functions, especially in the aspect of anti-inflammation. According to the strategy of pharmacophore combination, we introduced thiazolinone and dihydropyrazole moiety into steroid skeleton to design and synthesize a novel series of D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives, and assessed their in vitro anti-inflammatory profiles against Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. The anti-inflammatory activities assay demonstrated that compound 12e was considered as the most effective anti-inflammatory drug, which suppressed the expression of pro-inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), it also dose-dependently inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 macrophage cells. Furthermore, the results of the Western blot analysis showed a correlation between the inhibition of the Nuclear factor-kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs) signaling pathways and the suppressive effects of compound 12e on pro-inflammatory cytokines. Molecular docking studies of compound 12e into the COX-2 protein receptor (PDB ID: 5IKQ) active site was performed to rationalize their COX-2 inhibitory potency. The results were found to be in line with the biological findings as they exerted more favorable interactions compared to that of dexamethasone (DXM), explaining their remarkable COX-2 inhibitory activity. The findings revealed that these candidates could be identified as potent anti-inflammatory agents, compound 12e could be a promising drug for the treatment of inflammatory diseases. [Display omitted] • This study provided a novel, efficient and simple synthesis of steroidal 4,5-dihydropyrazole thiazolinone derivatives. • In vitro activity assay screened compound 12e with excellent anti-inflammatory biological activity and low cytotoxicity. • 12e inhibited LPS-induced expression of the pro-inflammatory cytokines iNOS, COX-2, TNF-α and IL-6 on RAW 264.7 cells. • 12e exerted anti-inflammatory effects through down-regulation of NF-κB/MAPKs pathway. [ABSTRACT FROM AUTHOR]

Inflammation, an important biological protective response to tissue damage or microbial invasion, is considered to be an alarming signal for the progress of varied biological complications. Based on the previous reports in the literature that proved the noticeable efficacy of pyrazole and thiazole scaffold as well as nitrogen heterocyclic based compounds against acute and chronic inflammatory disease, a new set of novel D-ring substituted steroidal 4,5-dihydropyrazole thiazole derivatives were synthesized and evaluated their anti-inflammatory activities in vitro. Preliminary structure-activity relationship (SAR) analysis was conducted by their inhibitory activities against nitric oxide (NO) release in lipopolysaccharide (LPS)-induced RAW 264.7 cells, and the optimal compound 12b [3β-hydroxy-pregn-5-en-17β-yl-5′- (o- chlorophenyl)− 1′-(4′′- phenyl -[1′′, 3′′]- thiazol-2′′- yl) − 4′,5′-dihydro − 1′H-pyrazol − 3′- yl] exhibited more potent anti-inflammatory activity than the positive control treatment methylprednisolone (MPS), with an IC 50 value of 2.59 μM on NO production and low cytotoxicity against RAW 264.7 cells. In further mechanism study, our results showed that compound 12b significantly suppressed the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and inhibited the expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) through blocking NF-κB p65 nuclear translocation and phosphorylation of IκBα. Compound 12b also attenuated LPS-induced activation of c-Jun amino-terminal kinase (JNK) and p38 phosphorylation in RAW 264.7 cells. Molecular docking study revealed the strong binding affinity of compound 12b to the active site of the COX-2 proteins, which confirmed that compound 12b acted as an anti-inflammatory mediator. These results indicate that steroidal derivatives bearing 4,5-dihydropyrazole thiazole structure might be considered for further research and scaffold optimization in designing anti-inflammatory drugs and compound 12b might be a promising therapeutic anti-inflammatory drug candidate. [Display omitted] • This study provided a novel series of D-ring substituted steroidal 4,5-dihydropyrazole thiazole derivatives. • The structures of all targeted synthesized compounds had been characterized. • The anti-inflammatory biological activity of targeted synthesized compounds was assayed. • In vitro activity assay screened compound 12b with excellent anti-inflammatory biological activity in LPS-induced RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Tetrahydropiperic acid (THPA) can effectively attenuate the inflammatory response, both in-vitro and in-vivo suggesting, THPA as a potential novel anti-inflammatory drug candidate in the treatment of inflammatory disorders. • THPA significantly decreased the production of NO and pro-inflammatory cytokines (IL-6, IL-1β & TNF-α) in LPS stimulated RAW 264.7 macrophages and in-vivo experimental model. • THPA effectively decreased carrageenan-induced paw edema and showed protective effect in LPS challenged mice. • The anti-inflammatory potential of THPA is attributed to downregulation of NF-κB signaling 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-1β) 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Efficient siRNAs can help control the emergence of drug-resistant viruses. • Conserved regions of HIV-1 vpr gene were identified. • Using a comprehensive strategy, anti -HIV siRNAs were designed and ranked. • Molecular docking simulation appears to be crucial in identifying the most efficient siRNAs. [ABSTRACT FROM AUTHOR]

Objectives Hydrolysable tannin fraction (HTF) derived from Terminalia chebula fruit pericarps was assessed for its anti-inflammatory potential in LPS-induced RAW 264.7 cells. Its molecular mechanism was also established and compared with individual tannins – chebulagic acid (CH) and corilagin (CO). Methods The effect of HTF on LPS-stimulated RAW 264.7 cells was studied by estimating the release of NO, ROS, cytokines and changes in nuclear morphology by DAPI staining. Furthermore, the effect of HTF, CO and CH was compared with the expression of p65, p38 and pERK proteins by immunoblotting and the mRNA transcript level of COX-2, iNOS and TNF-α by quantitative PCR. The in-silico interactions of various hydrolysable tannins present in HTF with molecular targets of inflammation were studied using Maestro software. Key findings HTF at the dose levels of 25, 50 and 100 µg/ml was able to decrease the release of NO, ROS and cytokines from LPS-induced RAW 264.7 cells without disturbing the cell nuclear morphology. Investigation of molecular mechanism revealed that inhibition of NF-κB and MAPK signalling pathways was responsible for its anti-inflammatory action. The effect of HTF was higher than the individual tannins CH and CO. Conclusion HTF can be developed as an effective anti-inflammatory agent. [ABSTRACT FROM AUTHOR]

Exogenous ganglioside GM1 has been reported to exert an immunomodulatory effect. We investigated the anti-inflammatory effect of GM1 ganglioside on endotoxin-induced uveitis (EIU) in rats and RAW 264.7 macrophages. Methods: EIU was induced in Lewis rats by administering a subcutaneous injection of lipopolysaccharide (LPS). GM1 was injected intraperitoneally for three consecutive days prior to the LPS injection. Twenty-four hours after the LPS injection, the integrity of the blood-aqueous barrier was evaluated by determining the protein concentration and number of infiltrating cells in the aqueous humor (AqH). Immunohistochemical and Western blot analyses of the iris-ciliary body (ICB) were performed to evaluate the effect of GM1 on the LPS-induced expression of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1). The effect of GM1 on proinflammatory mediators and signaling cascades was examined in LPS-stimulated RAW 264.7 cells using Western blotting and immunofluorescence staining to further clarify the underlying anti-inflammatory mechanism. Results: GM1 significantly reduced the protein concentration and number of infiltrating cells in the AqH of rats with EIU. GM1 also decreased the LPS-induced expression of the ICAM-1 and COX-2 proteins in the ICB. In RAW 264.7 cells, GM1 inhibited the proinflammatory mediators induced by LPS, including inducible nitric oxide synthase (iNOS), COX-2, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and this inhibitory effect was potentially mediated by suppressing transforming growth factor-β-activated kinase 1 (TAK1) and reactive oxygen species (ROS)-mediated activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Conclusions: Based on this study, GM1 may be a potential anti-inflammatory agent for ocular inflammatory diseases. [ABSTRACT FROM AUTHOR]

[Display omitted] • Non-starch polysaccharide (ECPs-I) was extracted from finger millet. • Deproteinated non-starch polysaccharide (ECPs-II) prepared by proteinase K. • The molecular weight of polysaccharides were found to range from 43 to 120 kDa. • The extracted polysaccharides did not show any toxic effects on RAW 264.7 cells. • ECPs-II has significantly enhance the RAW 264.7 cells activation. The structural elucidation and investigation of the immunostimulatory effects of crude polysaccharides (ECPs), non-starch polysaccharides (ECPs-I), and deproteinated non-starch polysaccharides (ECPs-II) derived from finger millet (Eleusine coracana) were performed. The extracted ECPs, ECPs-I, and ECPs-II primarily comprised different levels of carbohydrate (82.9, 41.9 and 62.0%), protein (5.3, 38.4 and 22.0%) and uronic acid (0.5, 2.4 and 0.7%). Monosaccharide composition analysis showed that glucose (88.2 and 40.6) was the main sugar unit present in ECPs and ECPs-I, followed by galactose, arabinose, xylose, rhamnose, and mannose; however, ECPs-II contained galactose (42.2%) as a major unit. The average molecular weights of ECPs, ECPs-I, and ECPs-II were found to be 120, 78 and 43 kDa, respectively. The polysaccharides as well as non-starch polysaccharides did not show any toxic effects on RAW 264.7 cells. ECPs-II treatment significantly enhanced the activation of RAW 264.7 cells by inducing nitric oxide (NO) production, upregulating inducible nitric oxide synthase (iNOS) and various pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha (TNF-α). Furthermore, the RAW 264.7 cells were activated after ECPs-II treatment via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase signaling (MAPK) pathways induced by a cluster of differentiation 40 (CD40) expression. The structure of ECPs-II mainly consists of (1→3,4)-linked galactopyranosyl and (1→3,5) arabinopyranosyl. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) is mainly characterized by the activation of inflammatory cells and chronic inflammatory responses after cell injury. Pyroptosis is a form of programmed cell death (PCD) accompanied by the release of inflammatory factors. Many studies have shown that pyroptosis plays an important role in AS. Increasing evidence also indicates that long non-coding RNA H19 (lncRNA H19) involved in AS. However, whether the role of lncRNA H19 in AS is related to pyroptosis and the underlying mechanisms are largely unknown. In this study, we found that oxidized low-density lipoprotein (ox-LDL) induced pyroptosis and decreased the expression of lncRNA H19 in Raw 264.7 cells. Besides, silencing endogenous lncRNA H19 increased inflammatory responses and pyroptosis while exogenous overexpression of lncRNA H19 reversed this effect. Notably, we identified that the inhibitor of caspase-1 (XV-765) completely abrogated the silencing endogenous lncRNA H19 mediated pyroptosis. In addition, we found that lncRNA H19 inhibited ox-LDL-induced activation of nuclear factor-kappa B (NF-κB), mitochondrial dysfunction, and reduced the production of reactive oxygen species (ROS). Moreover, VX-765 impaired the silencing endogenous lncRNA H19 mediated pyroptosis. Overall, these findings indicated that lncRNA H19 may play an important role in pyroptosis and may serve as a potential therapeutic target for AS. [ABSTRACT FROM AUTHOR]

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

The usefulness of sirolimus (SIR) in the treatment of diseases that involve retinal degeneration like age-related macular degeneration (AMD) has been well documented. However, the problem still remains probably owing to the peculiar environment of the eye and/or unfavourable physiochemical profile of SIR. In the present work, we aimed to fabricate sirolimus loaded PLGA nanoparticles (SIR-PLGA-NP) and chitosan decorated PLGA nanoparticles (SIR-CH-PLGA-NP) to be administered via non-invasive subconjunctival route. Both the nanoparticles were characterized in terms of size, zeta potential, DSC, FTIR and XRD analysis. Quality by Design (QbD) approach was employed during the preparation of nanoparticles and the presence of chitosan coating was confirmed through thermogravimetric analysis and contact angle studies. Cationic polymer modification showed sustained in-vitro SIR release and enhanced ex-vivo scleral permeation and penetration. Further, SIR-CH-PLGA-NP revealed enhanced cellular uptake and thus, reduced lipopolysaccharide (LPS)-induced free-radicals generation by RAW 264.7 cells. The prepared nanoparticles were devoid of residual solvent and were found to be safe in HET-CAM analysis, RBCs damage analysis and histopathology studies. Moreover, high anti-angiogenic potential was observed in SIR-CH-PLGA-NP compared with SIR-PLGA-NP in chorioallantoic membrane (CAM) test. Overall, the current work opens up an avenue for further investigation of CH-PLGA-NP as SIR nanocarrier in the treatment of AMD. [Display omitted] • Quality by Design (QbD) approach was employed in preparation of nanoparticles. • Sirolimus loaded PLGA nanoparticles were successfully functionalized with chitosan. • Chitosan functionalization enhanced cellular uptake and scleral penetration. • SIR-CH-PLGA nanoparticles were safe with higher anti-angiogenic potential. [ABSTRACT FROM AUTHOR]

Introduction: Sungkai (Peronema canescens, Jack.) contains polysaccharides, terpenoids, alkaloids, and polyphenols which have pharmacological activity as immunostimulants. Objective: This study aimed to see how the effect of Sungkai extract as an immunostimulant agent was carried out in vitro and in vivo. Materials and Methods: This study was conducted using two methods, namely in vivo and in vitro. In vivo research method was conducted to test the activity and phagocytic capacity of macrophage cells, the percentage of leukocytes, and the total number of leukocytes. This study used 30 male white mice as the test animals that were randomly divided into 5 treatment groups. Each group was consisting of 6 mice which were given different treatments. The negative control group was given with the 0.5% NaCMC suspension, the mice test substance group was given with the suspension of Sungkai ethanol extract with various doses of 800, 400, and 200 mg/kgBW, and lastly the comparison group was given with the Stimuno in a dose of 50 mg/kg orally for 7 days. On day 8, blood was taken from the mice's vein to count the number and percentage of its leukocytes, then followed by the intraperitoneal injection of a Staphylococcus aureus bacteria suspension. After 1 hour of administration of the bacterial suspension, the peritoneal fluid was taken to be observed for its activity and phagocytic capacity of macrophage cells. The in vitro research method was used to test the viability and immunostimulatory activity of RAW 264.7 cells with the Sungkai extraction at the concentration of 1.10, 100 g/m. This cell viability test using the microtetrazolium (MTT) method aims to see whether the Sungkai sample used is safe and not toxic to RAW 264.7 cells by observing at the cell viability value that should exceed >90%. The concentration of Sungkai extraction at 1.10, 100 g/mL was found to be safe and non-toxic to RAW 264.7 cells with a viability value of >90%. Thus, this concentration of Sungkai extraction can be performed for its immunostimulatory activity test on LPS induced of RAW 264.7 cells by observing their levels of IL-6 and TNF-a. (proinflammatory cytokines) were compared with the LPS alone as a control using the sandwich ELISA (Enzyme-Linked Immunosorbent Assay) method. Results: The observations were analyzed by one-way ANOVA and Duncan's follow-up test (significance was taken at p<0.05). The results showed that variations in concentration increased significantly (p<0.05) on the activity and phagocytic capacity of macrophage cells, along with the total leukocyte cells. The percentage of leukocytes showed that the cells had a significant increase (p<0.05). It was found that the Sungkai extraction on 1.10, 100 g/mL could significantly increase the concentration of TNF- and IL-6 (p<0.05) which were tested by one-way ANOVA and followed by Duncan's post hoc test. Conclusion: Sungkai leaf extract (Peronemacanescsens Jack.) in a dose of800, 400, and 200 mg/kgBW has an immunostimulant effect both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Objective To explore the protective effect of exosomes derived from M2 macrophages (M2 Exos) on concanavalin (Con) A induced autoimmune hepatitis in mice. Methods RAW264.7 macrophages were treated with IL-4 (20 μg/L) for 24 h to obtain cell culture supernatant containing M2 Exos. M2 Exos were separated by ultracentrifugation and identified by transmission electron microscope, nanoparticle tracking analysis and Western blot assay. Immunofluorescence was used to detect whether M2 Exos were uptaken by RAW264.7 macrophages. Twelve C57BL/6J mice were randomly divided into PBS group, DiR-M0 Exos group, DiR-M2 Exos group and DiR dye control group. PBS solution, DiR-M0 Exos (100 μg), DiR-M2 Exos (100 μg) and DiR dye were given to corresponding groups through tail veins after the injection of Con A (15 mg/kg) for 1h. The distribution of Exos in liver, spleen, heart, lung, kidney and intestine of mice with autoimmune hepatitis was observed by vivo imaging system. Twenty C57BL/6J mice were randomly divided into control group (PBS solution), Con A group (15 mg/kg Con A), M0 Exos group (15 mg/kg Con A + 200 μg M0 Exos) and M2 Exos group (15 mg/kg Con A+200 μg M0 Exos), 5 mice in each group. Peripheral blood and liver tissues were collected 12 h after Con A injection. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automated biochemistry analyzer. The histological profiles of liver tissue were observed by hematoxylin-eosin (HE) staining. The mRNA expression levels of liver TNF-α and IL-6 were detected with quantitative real-time PCR (qPCR). The changes of liver macrophage subpopulations were detected with flow cytometric analysis. Results M2 Exos were successfully induced and isolated, which can be taken up by RAW264.7 macrophages in large quantities. M2 Exos were mainly absorbed by liver and spleen of mice through tail vein injection. Compared with Control group, the serum levels of ALT and AST were significantly increased in Con A group (both P＜0.05). The disordered liver structure and increased necrosis areas were found in Con A group. Meanwhile, the mRNA expression of TNF- α and IL-6 in liver increased (both P＜0.05). The infiltration of liver monocyte-derived macrophages (MoMFs) increased (P＜0.05). Compared with Con A group, the serum levels of ALT and AST were significantly decreased in M2 Exos group (both P＜0.05). M2 Exos group also showed lower liver TNF-α, IL-6 mRNA expression levels (both P＜0.05) and lower MoMFs infiltration. HE staining showed that the necrosis areas of liver cells were relieved in M2 Exos group. Conclusion M2 Exos plays a protective role in autoimmune hepatitis in model mice，and its mechanism may be related to the decreased production of liver inflammatory cytokines and decreased recruitment of MoMFs. [ABSTRACT FROM AUTHOR]

This work presents an investigation on the composition and structure of polysaccharides from the roots of Tetrastigma hemsleyanum (THP) and its associated antioxidant activity. It further explores the protective effect of THP on RAW264.7 cells against cytotoxicity induced by H2O2. Ion chromatography (IC) revealed that THP contained glucose, arabinose, mannose, glucuronic acid, galactose and galacturonic acid, in different molar ratios. Furthermore, gel permeation chromatography-refractive index-multiangle laser light scattering (GPC-RI-MALS) was employed to deduce the relative molecular mass (Mw) of the polysaccharide, which was 177.1 ± 1.8 kDa. Fourier transform infrared spectroscopy (FT-IR) and Congo red binding assay highlighted that the THP had a steady α-triple helix conformation. Similarly, assays of antioxidant activity disclosed that THP had reasonable concentration-dependent hydroxyl radical and superoxide radical scavenging activities, peroxidation inhibition ability and ferrous ion chelating potency, in addition to a significant 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity. Moreover, THP could protect RAW264.7 cells against H2O2-induced cytotoxicity by decreasing intracellular ROS levels, reducing catalase (CAT) and superoxide dismutase (SOD) activities, increasing lactate dehydrogenase (LDH) activity and increment in malondialdehyde (MDA) level. Data retrieved from the in vitro models explicitly established the antioxidant capability of polysaccharides from T. hemsleyanum root extracts. [ABSTRACT FROM AUTHOR]

Mud lobsters are crustaceans from the genus Thalassina which are lesser known and seldom seen but are nevertheless an important organism to the mangrove ecosystem. In Malaysia and Thailand, mud lobsters are eaten by locals as treatment for asthma. It is traditionally believed that they are effective in reducing the number of asthma attacks and severity of asthma symptoms. However, the therapeutic potential of mud lobster extract remains unclear and has not been fully elucidated or reported in any scientific study. The objectives of this study are to investigate the anti-inflammatory potential of mud lobster, Thalassina anomala extracts (hexane, chloroform and methanol) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and to identify the potential bioactive compounds involved. An MTT assay was performed to determine the cytotoxicity of the T. anomala extracts on RAW 264.7 macrophages. Nitrite quantification assay and enzyme-linked immunosorbent assay (ELISA) were conducted to investigate the ability of the T. anomala extracts to suppress the secretion and expression of nitric oxide (NO), Prostaglandin E2 (PGE2) and proinflammatory cytokines (TNF-a, IL-6 and IL-1ß) in LPS-stimulated macrophages. GC-MS analysis was done to identify putative metabolites. The hexane extract of T. anomala showed anti-inflammatory activity by significantly inhibiting the LPS-induced production of NO, PGE2, interleukin- (IL-) 6, IL-1ß and tumour necrosis factor-alpha (TNF-a) in a concentration-dependent manner. Hexane extract treatment with 100 µg/mL has decreased the NO secretion into 37 µM. Meanwhile, hexane extract at concentration of 100 µg/mL able to significantly suppressed PGE2,TNF-a, IL-6 and IL-1ß production into 2015 pg/mL, 2406 pg/mL, 460 pg/mL and 9.6 pg/mL, respectively. GC-MS analysis of the hexane extract revealed the presence of 19 putative compounds. The identified compounds were reported to have anti-inflammatory, antioxidant and antibacterial activities. These results suggest that the hexane extract of T. anomala potentially has anti-inflammatory properties and concentration dependently suppressed NO, PGE2 and proinflammatory cytokines' production in LPS-stimulated macrophages. The findings provide a rational basis of the traditional use of mud lobster for inflammationassociated ailments. [ABSTRACT FROM AUTHOR]

The immunomodulating effect of Phenolic acid bound arabinoxylans (PCA‐AXs) extracted from Little (PCA‐AX‐L) and Kodo (PCA‐AX‐K) millet seeds in RAW 264.7 cells were investigated. The PCA‐AXs were extracted from millets and their chemical characterization were carried out by GC‐MS, HPLC, and FT‐IR. The immunomodulatory effect of PCA‐AXs in RAW 264.7 cells were investigated by estimating ROS, NO, and cytokines TNF‐α, IL‐1β, IL‐6, and evaluation of molecular mechanism by q‐PCR & western blotting techniques. The xylose: arabinose ratio of PCA‐AX‐L and PCA‐AX‐K were 1.48:1.0 and 2.26:1.0, respectively. The phenolic acids content was higher in PCA‐AX‐K than PCA‐AX‐L determined by HPLC. FT‐IR analysis confirms the presence of α‐glucosidic linkage with the degree of substitution of xylan backbone by arabinose residues. The evaluation of immunomodulating effect of PCA‐AXs revealed that the PCA‐AX‐L–treated cells showed higher release of NO, ROS and cytokines than PCA‐AX‐K–treated cells. The mRNA expressions of TNF‐α, iNOS and COX‐2 were upregulated by PCA‐AX‐L and downregulated by PCA‐AX‐K in dose‐dependent manner. Furthermore, in western blotting, the ERK and NF‐κB were found to be activated by PCA‐AX‐L and inhibited by PCA‐AX‐K. Our findings suggest that the high branched arabinoxylans of PCA‐AX‐L could modulate the immune response in RAW 264.7 cells through activation of ERK and NF‐κB signaling pathways and acts as an immunostimulant. The higher phenolic content in PCA‐AX‐K could modulate the immune response by downregulation of ERK and NF‐κB signaling pathways and thus, it could act as an immunomodulator. Practical applications: Millets are the richest source of arabinoxylans in which they are known to be bound with phenolic acids (PCA‐AX). Arabinoxylans derived from rice and wheat is known immunomodulators. This study was focused to evaluate the immunomodulatory property of PCA‐AX derived from two different millets little and kodo. The study results clearly indicated the immune stimulatory action of PCA‐AX‐L and immunomodulatory action of PCA‐AX‐K. The explored mechanism indicated that the PCA‐AXs modulate NF‐κB & ERK pathways for their immunomodulatory action. [ABSTRACT FROM AUTHOR]

Oldenlandia umbellata L., belonging to the Rubiaceae family, is an annual plant possessing anti-inflammatory and antipyretic, anti-nociceptive, anti-bacterial, anti-helminthic, antioxidant and hepatoprotective activities and used in traditional medicine to treat inflammation and respiratory diseases. The present study aims to evaluate the anti-osteoporotic effect of Methanolic extract of O.umbellata in MG-63 cells and RANKL-stimulated RAW 264.7 cells. The methanolic extract from the aerial parts of O.umbellata was subjected to metabolite profiling. The anti-osteoporotic effect of MOU was assessed in MG-63 cells and RANKL-stimulated RAW 264.7 cells. In MG-63 cells, the proliferative effect of MOU was evaluated using MTT assay, ALP assay, Alizarin red staining, ELISA and western blot. Similarly, the anti-osteoclastogenic effect of MOU was assessed in RANKL-stimulated RAW 264.7 cells via MTT, TRAP staining and western blot. LC-MS metabolite profiling showed the presence of 59 phytoconstituents including scandoside, scandoside methyl ester, deacetylasperuloside, asperulosidic acid, and cedrelopsin in MOU. In MG-63 cells, MOU has increased the proliferation of osteoblast cells and ALP activity, thereby increasing bone mineralization. ELISA results showed increased levels of osteogenic markers such as osteocalcin and osteopontin in the culture media. Western blot analysis showed inhibition of GSK3β protein expression and increased the expression levels of β-catenin, Runx-2, col 1 and osterix, promoting osteoblast differentiation. In RANKL-stimulated RAW 264.7 cells, MOU did not elicit any significant cytotoxicity; instead, it suppressed the osteoclastogenesis reducing the osteoclast number. MOU has reduced TRAP activity in a dose-dependent manner. MOU inhibited the TRAF6, NFATc1, c-Jun, C-fos and cathepsin K expression, thereby inhibiting osteoclast formation. In conclusion, MOU promoted osteoblast differentiation via inhibiting GSK3β and activating Wnt/β catenin signalling and its transcription factors, including β catenin, Runx2 and Osterix. Similarly, MOU inhibited osteoclast formation by inhibiting the expression of TRAF6, NFATc1, c-Jun, C-fos and cathepsin K in RANK-RANKL signalling. Finally, it can be emphasised that O.umbellata is a potential source of therapeutic leads for the treatment of osteoporosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Purpose: To investigate the potential anti-inflammatory effects of the seeds of Opuntina humifusa and its active constituents. Methods: The extract of O. humifusa seeds was tested for the inhibition of nitric oxide (NO) production in liposaccharide (LPS)-stimulated RAW 264.7 cells using Griess reagent. The active constituents were isolated using bioassay-guided isolation methods. The effects of the active constituent on NO, proinflammatory cytokines, nuclear factor-kappa B (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) were evaluated by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Results: The seed extract of O. humifusa significantly attenuated LPS-induced NO production in RAW 264.7 cells (p < 0.05). Bioassay-guided fractionation resulted in the isolation of isoamericanin A as an active constituent. Isoamericanin A reduced LPS-induced production of NO, iNOS, and proinflammatory cytokines (TNF-α and IL-6) in a concentration-dependent manner (p < 0.05). Furthermore, the effect was accompanied by decreased translocation of NF-κB from the cytosol to the nucleus and the decreased phosphorylation of IκB in the cytosol induced by LPS (p < 0.05). Conclusion: The seed extract of O. humifusa and its active constituent, isoamericanin A, have antiinflammatory effects in LPS-stimulated RAW 264.7 cells, suggesting that they have potentials as antiinflammatory agents. [ABSTRACT FROM AUTHOR]

Fine dust-associated complications related to human health are one of the major issues in the East-Asia region. In the present study, we evaluated anti-inflammatory effect of dieckol in fine dust-stimulated RAW 264.7 macrophage cells and its biological mechanism. The result showed that the exposure of macrophages to fine dust induced the productions of nitric oxide (NO), prostaglandin E2 (PGE2), and the mRNA expression levels of inflammatory mediators such as inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 whereas it was significantly attenuated by the treatment of dieckol. In addition, fine dust increased the pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α that were down-regulated by dieckol. Interestingly, dieckol protected macrophages against cell damages via reducing the fine dust-caused intracellular reactive oxygen species (ROS) generation via induction of superoxide dismutase production as well as the HO-1/Nrf2 signaling activation. In conclusion, dieckol protects RAW 264.7 cells against fine dust-induced inflammation and oxidative stress via inducing anti-inflammatory and antioxidant mechanisms. [ABSTRACT FROM AUTHOR]

We previously demonstrated that 2-hydroxypropyltrimethyl ammonium chloride chitosan (HACC) promoted the production of nitric oxide (NO) and proinflammatory cytokines by activating the mitogen-activated protein kinases (MAPK) and Janus kinase (JAK)/STAT pathways in RAW 264.7 cells, indicating good immunomodulatory activity of HACC. In this study, to further investigate the immunomodulatory mechanisms of HACC, we determined the roles of phosphatidylinositol 3-kinase (PI3K)/Akt, activating protein (AP-1) and nuclear factor kappa B (NF-κB) in HACC-induced activation of RAW 264.7 cells by the western blotting. The results suggest that HACC promoted the phosphorylation of p85 and Akt. Furthermore, c-Jun and p65 were also increased after the treatment of RAW 264.7 cells with HACC, indicating the translocation of NF-κB and AP-1 from cytoplasm to nucleus. In addition, as scanning electron microscopy (SEM) analysis shows, the cell morphology changed after HACC treatment. These findings indicate that HACC activated MAPK, JAK/STAT, and PI3K/Akt signaling pathways dependent on AP-1 and NF-κB activation in RAW 264.7 cells, ultimately leading to the increase of NO and cytokines. [ABSTRACT FROM AUTHOR]

Lilium lancifolium is native to Northeast Asia and its bulbs have been used for medicinal treatment. Moreover, Japan has been using L. lancifolium bulbs more actively as food ingredients than Korea. Therefore, this study was to investigate the characteristics of Korean L. lancifolium bulbs, with respect to food component and functionality. As a result of proximate composition analysis, L. lancifolium bulbs have an abundant carbohydrate content. HPLC analysis indicated p‐coumaric acid and ferulic acid contents of Korean L. lancifolium extract were 1.14 ± 0.01, 1.46 ± 0.00 mg/g, but only p‐coumaric acid was less detected in Japanese extract. Also, Korean L. lancifolium bulbs extract exhibited significant antioxidant effects, as evaluated with antioxidant activity and compound, than Japanese extract. Furthermore, Korean L. lancifolium bulbs extract significantly inhibited pro‐inflammatory protein expressions through MyD88 dependent pathway. Therefore, these results suggested Korean L. lancifolium bulbs have the potential to being functional food ingredients. Practical applications: Lilium lancifolium is a perennial plant belonging to the Liliaceae family. The storage organ of L. lancifolium is surrounded by several fleshy nodes at the base of the stem, called the bulb, which has been used as food or medicine to treat pneumonia and bronchitis. L. lancifolium is widely found in countries of Northeast Asia, such as Korea, Japan, and China, and its bulbs have been studied for presence of bioactive compounds that have important functional activities. The bioactive compounds in the L. lancifolium bulbs may vary from region to region. In this study, the difference observed in the contents of different bioactive compounds and the efficacy of anti‐inflammatory effects of L. lancifolium bulbs from different regions were consistent in this regard. As a comparative study of food materials by region, these L. lancifolium bulbs have the potential to be used as a food material for preventing inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: Zanthoxylum rhetsa is a plant used in traditional medicine and is known to possess health benefits such as antibacterial, antidiabetic, and anti-diarrheal activities. Objectives: The objective of this study was to explore and demonstrate the anti-inflammatory activity of various solvent fractions of Z. rhetsa bark. Materials and Methods: The effect of crude methanolic extract and its fractions (hexane, chloroform, ethyl acetate, and butanol) on the levels of pro-inflammatory cytokines (interleukin-1β, tumor necrosis factor-alpha, and interleukin-6) and inflammatory factors was tested via targeting nuclear factor-kappa B (NF-κB) signaling pathways in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Results: Treatment with all the solvent fractions at various concentrations (50, 100, and 200 μg/ml) suppressed pro-inflammatory cytokine levels in a dose-dependent manner. Among the fractions, the chloroform fractions exhibited a significant inhibition of LPS-induced inflammation. Moreover, these fractions effectively suppressed the expression of various NF-κB signaling targets, including NF-κB, nitric oxide synthase, and cyclooxygenase-2 as well as inhibited the degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (inhibitor of kappa B alpha [IκBα]). This anti-inflammatory effect was mediated by the prevention of IκBα degradation, as this protein prevents the translocation of NF-κB from the cytoplasm to the nucleus, initiating the transcription of pro-inflammatory genes. Conclusion: Thus, the Z. rhetsa chloroform fraction may be an effective natural anti-inflammatory agent against inflammation-associated diseases. [ABSTRACT FROM AUTHOR]

Danshensu, isolated from Salvia miltiorrhiza (Danshen), is known to have anti-inflammatory properties. Therefore danshen is extensively used in many nutraceutical formulations. Reactive oxygen species are essential for the development of hypoxia-induced inflammation. Generation of reactive oxygen species by infiltrating macrophages is common in various diseases such as cardiovascular disease, neurodegenerative disease, tumor, and aging. To explore the mechanism underlying the attenuation of inflammation, we used RAW 264.7 cells as a model and hypoxia as an inducer of inflammation. The results showed the protective mechanism of danshensu on reactive oxygen species production, hypoxia-inducible factor 1-alpha expression, c-Jun N-terminal kinase phosphorylation, nuclear translocation of nuclear factor kappa B, and inducible nitric oxide synthase expression following hypoxia in RAW 264.7 cells. [ABSTRACT FROM AUTHOR]

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

Ganoderma lucidum immunomodulatory protein (FIP‐glu) is an active ingredient with potential immunoregulatory functions. The study was conducted to explore the immunomodulatory activities of recombinant FIP‐glu (rFIP‐glu) and its possible mechanism in macrophage RAW264.7 cells. In vitro assays of biological activity indicated that rFIP‐glu significantly activated RAW264.7 cells and possessed proinflammatory and anti‐inflammatory abilities. RNA sequencing analysis and Western blot analysis showed that macrophage activation involved PI3K/Akt and MAPK pathways. Furthermore, real‐time quantitative polymerase chain reaction indicated that the PI3K inhibitor LY294002 blocked the messenger RNA (mRNA) levels of MCP‐1 (CCL‐2), the MEK1/2 inhibitor U0126 reduced the mRNA levels of TNF‐α and MCP‐1 (CCL‐2), and the JNK1/2/3 inhibitor SP600125 prevented the upregulation of inducible nitric oxide synthase mRNA in rFIP‐glu‐induced cells. rFIP‐glu did not mediate these inflammatory effects through a general pathway but rather through a different pathway for a different inflammatory mediator. These data imply that rFIP‐glu possessed immunomodulatory activity in macrophages, which was mediated through PI3K/Akt and MAPK pathways. [ABSTRACT FROM AUTHOR]

Histone acetyltransferase (HAT) activity is well established to regulate inflammatory responses. In contrast, the mechanisms by which natural nutritional extracts influence epigenetic mechanisms to regulate inflammation have not yet been thoroughly investigated. Thus, in the present study, we observed that the anti-HAT activity exerted by an ethanol extract of Ligularia fischeri (ELF) inhibited inflammation. Specifically, we used a cell-free system to show that ELF attenuates HAT activity. We also demonstrated that ELF decreases lipopolysaccharide (LPS)-induced HAT mRNA and protein expression levels in Raw 264.7 cells, and thereby attenuates inflammation-induced patterns of hyperacetylation at nonhistone and histone-H4 proteins. Interestingly, we found that ELF blocked p65 translocation in LPS-stimulated Raw 264.7 cells by attenuating acetylation at lysine residue 310 of p65. Finally, we investigated whether ELF reduces the inflammatory cytokines, IL-6, IL-1β, and TNFα, using its HAT inhibitor activity. Taken together, these results suggest that ELF negatively regulates inflammatory responses by inhibiting HATs and HAT activity. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases involve chronic intestinal inflammation which is mostly caused by Crohn's disease and ulcerative colitis (UC) conditions. Patients having UC are prone to colorectal cancer and dysplastic polyps, and also have sporadic adenomas. Syringic acid (SA) possesses many health benefits including antioxidant, anti‐bacterial, and anti‐cancer. This study was aimed to identify the effects of SA on UC, using a murine experimental model. Clinical symptoms and weight loss were significantly reduced in mice induced with dextran sulfate sodium (DSS) and treated with SA, compared to untreated mice. The effects of SA exhibited in DSS‐induced mice were associated with significant decrease in the expressions levels of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), pro‐inflammatory cytokines (tumor necrosis factor [TNF‐α], interleukin [IL‐1β and IL‐6]), remarkable amelioration of colonic architectural disruption, and a significant reduction in the activity of colonic myeloperoxidase. To further confirm the anti‐inflammatory property of SA, RAW 264.7 cells were stimulated with lipopolysaccharides. SA dose‐dependently inhibited the cytokines TNF‐α, IL‐1β, and IL‐6. It also decreased the expressions of p65‐NF‐κB and IκB, thus reducing the activation and nuclear accumulation of p‐STAT‐3Y705. This prohibited the degradation of inhibitory protein, IκB, as well as inhibited the nuclear translocation of p65‐NF‐κB in colonic tissue. It was concluded that SA has a potential to limit inflammation via inhibiting the p65‐NF‐κB and STAT3 signaling; hence it can be used for therapeutic purposes. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate the inhibitory effect of 2,4 bis-[(4-ethoxyphenyl)azo] 5-(3-hydroxybenzylidene) thiazolidine-2,4-dione (TZD-OCH2CH3) on the cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. The effects of TZD-OCH2CH3 on COX-2 and iNOS mRNA expression in LPS-activated RAW 264.7 cells were detected by real time PCR. Also, to understand structure and substrate specificity, we have utilized molecular docking simulations (AutoDock Vina) and the active residues in the binding pocket were determined from COX-2 and iNOS. The treatment of RAW 264.7 cells with TZD-OCH2CH3 significantly inhibited LPS-induced COX-2 mRNA expression, corresponding to 46.1% and 61.06% at 30 and 60 µg/mL, respectively. The present study revealed that the TZD-OCH2CH3 had a little effect on iNOS mRNA expression. Meanwhile, the TZD-OCH2CH3 also could inhibit the production of NO compared to single LPS-stimulated cell. According to the results obtained, TZD-OCH2CH3 dramatically suppressed lipopolysaccharide (LPS) induced nitric oxide (NO) production after 24 h, in a concentration-dependent manner with an IC50 of 65 µg/mL. Our data suggest that TZD-OCH2CH3, as a functionally novel agent, inhibits the inflammatory pathway via suppression of COX-2 mRNA expression and also by the inhibition of the iNOS activity. Therefore, this compound could be suggested as a novel therapeutic strategy for inflammation-associated disorders. [ABSTRACT FROM AUTHOR]

The variation in the extracellular metabolites of RAW 264.7 cells obtained from different passage numbers (passage 9, 12 and 14) was examined. The impact of different harvesting protocols (trypsinization and scraping) on recovery of intracellular metabolites was then assessed. The similarity and variation in the cell metabolome was investigated using 1H NMR metabolic profiling modeled using multivariate data analysis. The characterization and quantification of metabolites was performed to determine the passage-related and harvesting-dependent effects on impacted metabolic networks. The trypsinized RAW cells from lower passages gave higher intensities of most identified metabolites, including asparagine, serine and tryptophan. Principal component analysis revealed variation between cells from different passages and harvesting methods, as indicated by the formation of clusters in score plot. Analysis of S-plots revealed metabolites that acted as biomarkers in discriminating cells from different passages including acetate, serine, lactate and choline. Meanwhile lactate, glutamine and pyruvate served as biomarkers for differentiating trypsinized and scraped cells. In passage-dependent effects, glycolysis and TCA cycle were influential, whereas glycerophospholipid metabolism was affected by the harvesting method. Overall, it is proposed that typsinized RAW cells from lower passage numbers are more appropriate when conducting experiments related to NMR metabolomics. • The metabolome of RAW 264.7 cells were evaluated. • The different passage number influenced the extracellular metabolites. • The harvesting techniques affecting the intracellular metabolites. • RAW cells from lower passage showed higher intensities of several metabolites. • Trypsin treatment demonstrated higher concentration of metabolites in RAW cells. [ABSTRACT FROM AUTHOR]