Anti-Inflammatory Effect of Chamaecyparis obtusa (Siebold & Zucc.) Endl. Leaf Essential Oil.

Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) belongs to the Cupressaceae family and is native to East Asian regions. Essential oils extracted from the leaves, bark, branches, and roots of C. obtusa have both aesthetic and medicinal properties and are thus widely used. However, detailed analyses of the active ingredients of C. obtusa extract are lacking. In this study, the sabinene content in the hydro-distillation of C. obtusa leaf essential oil (COD) was analyzed using GC-MS, and the anti-inflammatory effect of COD was compared with that of pure sabinene. Cell viability was evaluated by MTT assay, and nitric oxide (NO) production was measured using Griess reagent. Relative mRNA and protein levels were analyzed using RT-qPCR and western blot, and secreted cytokines were analyzed using a cytokine array kit. The results showed that both COD and sabinene inhibited the expression of inducible nitric oxide synthase (iNOS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in lipopolysaccharide (LPS)-induced RAW 264.7 cells. COD and sabinene also reduced the production of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-27, IL-1 receptor antagonist (IL-1ra), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The anti-inflammatory mechanisms of COD and sabinene partially overlap, as COD was shown to inhibit MAPKs and the JAK/STAT axis, and sabinene inhibited MAPKs, thereby preventing LPS-induced macrophage activation.

Involvement of IL-17 A/IL-17 Receptor A with Neutrophil Recruitment and the Severity of Coronary Arteritis in Kawasaki Disease.

PURPOSE: To assess the role of the interleukin (IL)-17 A/IL-17 receptor A (IL-17RA) in Kawasaki disease (KD)-related coronary arteritis (CA). METHODS: In human study, the plasma levels of IL-17 A and coronary arteries were concurrently examined in acute KD patients. In vitro responses of human coronary endothelial cells to plasma stimulation were investigated with and without IL-17RA neutralization. A murine model of Lactobacillus casei cell-wall extract (LCWE)-induced CA using wild-type Balb/c and Il17ra-deficient mice were also inspected. RESULTS: The plasma levels of IL-17 A were significantly higher in KD patients before intravenous immunoglobulin therapy, especially in those with coronary artery lesion. The pre-IVIG IL-17 A levels positively correlated with maximal z scores of coronary diameters and plasma-induced endothelial mRNA levels of chemokine (C-X-C motif) ligand-1, IL-8, and IL-17RA. IL-17RA blockade significantly reduced such endothelial upregulations of aforementioned three genes and inducible nitric oxide synthase, and neutrophil transmigration. IL-17RA expression was enhanced on peripheral blood mononuclear cells in pre-IVIG KD patients, and in the aortic rings and spleens of the LCWE-stimulated mice. LCWE-induced CA composed of dual-positive Ly6G- and IL-17 A-stained infiltrates. Il17ra-deficient mice showed reduced CA severity with the fewer number of neutrophils and lower early inducible nitric oxide synthase and chemokine (C-X-C motif) ligand-1 mRNA expressions than Il17ra+/+ littermates, and absent IL-17RA upregulation at aortic roots. CONCLUSION: IL-17 A/IL-17RA axis may play a role in mediating aortic neutrophil chemoattraction, thus contributory to the severity of CA in both humans and mice. These findings may help to develop a new therapeutic strategy toward ameliorating KD-related CA.

Improved synthesis, molecular modeling and anti-inflammatory activity of new fluorinated dihydrofurano-naphthoquinone compounds.

A series of new fluorinated dihydrofurano-napthoquinone compounds were sucessfully synthesized in good yields using microwave-assisted multi-component reactions of 2-hydroxy-1,4-naphthoquinone, fluorinated aromatic aldehydes, and pyridinium bromide. The products were fully characterized using spectroscopic techniques and evaluated for their anti-inflammatory activity using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Among 12 new compounds, compounds 8b, 8d, and 8e showed high potent NO inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 values ranging from 1.54 to 3.92 µM. The levels of pro-inflammatory cytokines IL-1ß and IL-6 in LPS-stimulated RAW264.7 macrophages were remarkably decreased after the application of 8b, 8d, 8e and 8k. Molecular docking simulations revealed structure-activity relationships of 8b, 8d, and 8e toward NO synthase, cyclooxygenase (COX-2 over COX-1), and prostaglandin E synthase-1 (mPGES-1). Further physicochemical and pharmacokinetic computations also demonstrated the drug-like characteristics of synthesized compounds. These findings demonstrated the importance of fluorinated dihydrofurano-napthoquinone moieties in the development of potential anti-inflammatory agents.

iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.

Detection of nitric oxide-mediated metabolic effects using real-time extracellular flux analysis.

Dendritic cell (DC) activation is marked by key events including: (I) rapid induction and shifting of metabolism favoring glycolysis for generation of biosynthetic metabolic intermediates and (II) large scale changes in gene expression including the upregulation of the antimicrobial enzyme inducible nitric oxide synthase (iNOS) which produces the toxic gas nitric oxide (NO). Historically, acute metabolic reprogramming and NO-mediated effects on cellular metabolism have been studied at specific timepoints during the DC activation process, namely at times before and after NO production. However, no formal method of real time detection of NO-mediated effects on DC metabolism have been fully described. Here, using Real-Time Extracellular Flux Analysis, we experimentally establish the phenomenon of an NO-dependent mitochondrial respiration threshold, which shows how titration of an activating stimulus is inextricably linked to suppression of mitochondrial respiration in an NO-dependent manner. As part of this work, we explore the efficacy of two different iNOS inhibitors in blocking the iNOS reaction kinetically in real time and explore/discuss parameters and considerations for application using Real Time Extracellular Flux Analysis technology. In addition, we show, the temporal relationship between acute metabolic reprogramming and NO-mediated sustained metabolic reprogramming kinetically in single real-time assay. These findings provide a method for detection of NO-mediated metabolic effects in DCs and offer novel insight into the timing of the DC activation process with its associated key metabolic events, revealing a better understanding of the nuances of immune cell biology.

Screening and characterization of an anti-inflammatory pectic polysaccharide from Cucurbita moschata Duch.

Pectin polysaccharides have demonstrated diverse biological activities, however, the inflammatory potential of pectin polysaccharides extracted from Cucurbita moschata Duch remains unexplored. This study aims to extract, characterize and evaluate the effects of pumpkin pectin polysaccharide on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and dextran sulfate sodium (DSS)-induced colitis in mice, along with its underlying mechanism of action. Initially, we extracted three fractions of pectin polysaccharides from pumpkin and screened them for anti-inflammatory activity in LPS-induced macrophages, identifying CMDP-3a as the most potent anti-inflammatory fraction. Subsequently, CMDP-3a underwent comprehensive characterization through chromatography and spectroscopic analysis, revealing CMDP-3a as an RG-I-HG type pectin polysaccharide with →4)-α-D-GalpA-(1 â†’ and →4)-α-D-GalpA-(1 â†’ 2,4)-α-L-Rhap-(1 â†’ as the main chain. Further, in the LPS-induced RAW264.7 cells model, treatment with CMDP-3a significantly down-regulated the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines (IL-1ß, TNF-α, and IL-6) by inhibiting the MAPK and NF-κB signaling pathways. Finally, in a mouse colitis model, CMDP-3a administration obviously inhibited DSS-induced pathological alterations and reduced inflammatory cytokine expressions in the colonic tissues by down-regulating the TLR4/NF-κB and MAPK pathways. These findings provide a molecular basis for the potential application of CMDP-3a in reducing inflammatory responses.

Oxyimperatorin attenuates LPS-induced microglial activation in vitro and in vivo via suppressing NF-κB p65 signaling.

BACKGROUND: Microglia-mediated neuroinflammation is an important pathological feature in many neurological diseases; thus, suppressing microglial activation is considered a possible therapeutic strategy for reducing neuronal damage. Oxyimperatorin (OIMP) is a member of furanocoumarin, isolated from the medicinal herb Glehnia littoralis. However, it is unknown whether OIMP can suppress the neuroinflammation. PURPOSE: To investigate the neuroprotective activity of oxyimperatorin (OIMP) in LPS-induced neuroinflammation in vitro and in vivo models. METHODS: In vitro inflammation-related assays were performed with OIMP in LPS-induced BV-2 microglia. In addition, intraperitoneal injection of LPS-induced microglial activation in the mouse brain was used to validate the anti-neuroinflammatory activity of OIMP. RESULTS: OIMP was found to suppress LPS-induced neuroinflammation in vitro and in vivo. OIMP significantly attenuated LPS-induced the production of free radicals, inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokines in BV-2 microglia without causing cytotoxicity. In addition, OIMP could reduce the M1 pro-inflammatory transition in LPS-stimulated BV-2 microglia. The mechanistic study revealed that OIMP inhibited LPS-induced NF-κB p65 phosphorylation and nuclear translocation. However, OIMP did not affect LPS-induced IκB phosphorylation and degradation. In addition, OIMP also was able to reduce LPS-induced microglial activation in mice brain. CONCLUSION: Our findings suggest that OIMP suppresses microglia activation and attenuates the production of pro-inflammatory mediators and cytokines via inhibition of NF-κB p65 signaling.

Repurposed drug agomelatine is therapeutic against collagen-induced arthritis via iNOS targeting.

BACKGROUND: The most promising biologics tumor necrosis factor α (TNFα) inhibitors are effective in treating rheumatoid arthritis (RA) in only 50-70 % of the cases; thus, new drugs targeting TNFα-mediated inflammation are required. METHODS: Firstly, the drugs that could inhibit FLS proliferation and TNFα induced inflammatory cytokine production were screened. Secondly, treatment effects of the identified drugs were screened in collagen-induced arthritis (CIA) mouse model. Thirdly, the inhibitory effect of the identified drug, agomelatine (AOM), on TNFα induced inflammatory cytokine production and NF-κB activity were confirmed. Fourthly, bioinformatics was applied to predict the binding target of AOM and the binding was confirmed, and the already known inhibitor of target was used to test the treatment effect for CIA mouse model. Finally, the effect of AOM on signaling pathway was tested and on TNFα induced inflammatory cytokine production was observed after inhibiting the target. RESULTS: AOM effectively inhibited TNFα-induced NF-κB activation, NF-κB p65 translocation, and inflammatory cytokines production in vitro and was therapeutic against CIA. The mechanistic study indicated inducible nitric oxide synthase (iNOS) as the binding target of AOM. 1400 W, a known inhibitor of iNOS, could effectively treat CIA by decreasing iNOS activity and the levels of inflammatory cytokines. The inhibitory effect of AOM on TNFα-induced inflammation was further elucidated by 1400 W, or NF-κB p65 inhibitor JSH-23, indicating that AOM is therapeutic against CIA via iNOS/ERK/p65 signaling pathway after binding with iNOS. CONCLUSIONS: AOM is therapeutic against CIA via inhibition of the iNOS/ERK/p65 signaling pathway after binding with iNOS.

LPS priming-induced immune tolerance mitigates LPS-stimulated microglial activation and social avoidance behaviors in mice.

In this study, we investigated the regulatory mechanisms underlying the effects of LPS tolerance on the inflammatory homeostasis of immune cells. LPS priming-induced immune tolerance downregulated cyclooxygenase-2, and lowered the production of prostaglandin-E2 in microglial cells. In addition, LPS tolerance downregulated the expression of suppressor of cytokine signaling 3, and inducible nitric oxide synthase/nitric oxide; suppressed the LPS-mediated induction of tumor necrosis factor-α, interleukin (IL)-6, and IL-1; and reduced reactive oxygen species production in microglial cells. LPS stimulation increased the levels of the adaptive response-related proteins heme oxygenase-1 and superoxide dismutase 2, and the levels of heme oxygenase-1 (HO-1) enhanced after LPS priming. Systemic administration of low-dose LPS (0.5 mg/kg) to mice for 4 consecutive days attenuated high-dose LPS (5 mg/kg)-induced inflammatory response, microglial activation, and proinflammatory cytokine expression. Moreover, repeated exposure to low-dose LPS suppressed the recruitment of peripheral monocytes or macrophages to brain regions and downregulated the expression of proinflammatory cytokines. Notably, LPS-induced social avoidance behaviors in mice were mitigated by immune tolerance. In conclusion, immune tolerance may reduce proinflammatory cytokine expression and reactive oxygen species production. Our findings provide insights into the effects of endotoxin tolerance on innate immune cells and social behaviors.

Macrophage immunophenotypes in Jorge Lobo's disease and lepromatous leprosy- A comparative study.

Jorge Lobo's disease (JLD) and lepromatous leprosy (LL) share several clinical, histological and immunological features, especially a deficiency in the cellular immune response. Macrophages participate in innate and adaptive inflammatory immune responses, as well as in tissue regeneration and repair. Macrophage function deficiency results in maintenance of diseases. M1 macrophages produce pro-inflammatory mediators and M2 produce anti-inflammatory cytokines. To better understand JLD and LL pathogenesis, we studied the immunophenotype profile of macrophage subtypes in 52 JLD skin lesions, in comparison with 16 LL samples, using a panmacrophage (CD68) antibody and selective immunohistochemical markers for M1 (iNOS) and M2 (CD163, CD204) responses, HAM56 (resident/fixed macrophage) and MAC 387 (recently infiltrating macrophage) antibodies. We found no differences between the groups regarding the density of the CD163, CD204, MAC387+ immunostained cells, including iNOS, considered a M1 marker. But HAM56+ cell density was higher in LL samples. By comparing the M2 and M1 immunomarkers in each disease separately, some other differences were found. Our results reinforce a higher M2 response in JLD and LL patients, depicting predominant production of anti-inflammatory cytokines, but also some distinction in degree of macrophage activation. Significant amounts of iNOS + macrophages take part in the immune milieu of both LL and JLD samples, displaying impaired microbicidal activity, like alternatively activated M2 cells.

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.

Pyridazine and pyridazinone derivatives: Synthesis and in vitro investigation of their anti-inflammatory potential in LPS-induced RAW264.7 macrophages.

New pyridazine and pyridazinone derivatives 3a-g, 4a-f, 6a, and 6b were designed and synthesized. Cell viability of all compounds was established based on the viability of lipopolysaccharide-induced RAW264.7 macrophage cells determined via the MTT assay. In vitro inhibition assays on human COX-1 and COX-2 enzymes were conducted to probe the newly synthesized compounds' anti-inflammatory activity. The half maximal inhibitory concentration values for the most active compounds, 3d, 3e, and 4e towards COX-2 were 0.425, 0.519, and 0.356 µM, respectively, in comparison with celecoxib. The newly synthesized compounds' ability to inhibit the production of certain proinflammatory cytokines, such as inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-6, and prostaglandin-E2, was also estimated in lipopolysaccharide-induced macrophages (RAW264.7 cells). Compounds 3d and 3e were identified as the most potent cytokine production inhibitors. The results of molecular modeling studies suggested that these compounds were characterized by a reasonable binding affinity toward the active site of COX-2, when compared to a reference ligand. These results might be taken into consideration in further investigations into new anti-inflammatory agents.

Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages.

Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.

Isolation of New Diterpenoids from the Halophyte, Vitex rotundifolia, and their Antioxidant and Anti-inflammatory Activities.

In this study, three diterpenoids (1-3), including one known compound (1), were isolated from the fruits of Vitex rotundifolia and their structures were determined via spectroscopic analysis. In lipopolysaccharide-stimulated RAW264.7 cells, these compounds dose-dependently decreased the intracellular reactive oxygen species levels and nitric oxide production compared to those in the control cells. At 25 µM/mL, these compounds also diminished the protein expression of the pro-inflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase-2, and interleukin-6, with compound 3 exhibiting the most potent inhibitory effect.

Depth of invasion of oral squamous cell carcinoma in Nos2-knockout mice correlated to alterations in systemic inflammatory markers following 4NQO treatment.

BACKGROUND: Peripheral blood analysis is a non-invasive and low-cost technique of prognostic value for several diseases, including oral cancer. Considering the role of inducible nitric oxide synthase in tumor-associated inflammation, this study purposed to evaluate the influence of this enzyme on peripheral blood parameters and systemic inflammatory biomarkers during murine oral carcinogenesis. METHODS: A 50 µg/mL solution of 4-nitroquinoleine-N-oxide was provided to 15 C57BL/6J (Nos2+/+ ) and 16 B6.129P2-Nos2tm1Lau /J (Nos2-/- ) for 16 weeks. Animals were followed for 8 weeks after treatment. Blood samples and tongues were collected for hematological and histopathological analyses. Red blood cells, white blood cells, and platelet cell parameters were analyzed. The neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and the systemic immune-inflammation index were also calculated. The depth of invasion of all carcinomas was measured. RESULTS: Differences were found in several blood parameters. The depth of invasion in Nos2-/- was lower than in Nos2+/+ (p = 0.009), and strong correlations were found between depth of invasion and neutrophil count (ρ = -0.68, p = 0.017), lymphocyte count (ρ = 0.72, p = 0.011), neutrophil-to-lymphocyte ratio (ρ = -0.65, p = 0.025), platelet-to-lymphocyte ratio (ρ = -0.73, p = 0.013), and systemic immune-inflammation index (ρ = -0.67, p = 0.037) in Nos2-/- mice. CONCLUSION: Inducible nitric oxide synthase seems to have an important role in OSCC invasion and progression, which might be associated to alterations in immune-inflammatory cell dynamics evidenced by peripheral blood and systemic inflammatory biomarkers.

Design and synthesis of forsythin derivatives as anti-inflammatory agents for acute lung injury.

Acute lung injury (ALI) is a clinically high mortality disease, which has not yet been effectively treated. The development of anti-ALI drugs is imminent. ALI can be effectively treated by inhibiting the inflammatory cascade and reducing the inflammatory response in the lung. Forsythia suspense is a common Chinese herbal medicine with significant anti-inflammatory activity. Using forsythin as the parent, 27 Forsythin derivatives were designed and synthesized, and the anti-AIL activity of these compounds was evaluated. Among them, compound B5 has the best activity to inhibit the release of IL-6, and the inhibition rate reaches 91.79% at 25 µM, which was 7.5 times that of the parent forsythin. In addition, most of the compounds have no significant cytotoxicity in vitro. Further studies showed that compound B5 had a concentration-dependent inhibitory effect on NO, IL-6 and TNF-α. And the IC50 values of compound B5 for NO and IL-6 are 10.88 µM and 4.93 µM, respectively. We also found that B5 could significantly inhibit the expression of some immune-related cytotoxic factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, B5 inhibits NF-κB/MAPK signaling pathway. In vivo experiments showed that B5 could alleviate lung inflammation in LPS-induced ALI mice and inhibit IL-6, TNF-α, COX-2 and iNOS. In summary, B5 has anti-inflammatory effects and alleviates ALI by regulating inflammatory mediators and inhibiting MAPK and NF-κB signaling pathways.

Anti-Inflammatory and Immunomodulatory Properties of a Crude Polysaccharide Derived from Green Seaweed Halimeda tuna: Computational and Experimental Evidences.

In this study, we investigated for the first time the anti-inflammatory and immunomodulatory properties of crude polysaccharide (PSHT) extracted from green marine algae Halimeda tuna. PSHT exhibited anti-oxidant activity in vitro through scavenging 1, 1-diphenyl-2-picryl hydroxyl free radical, reducing Fe3+/ferricyanide complex, and inhibiting nitric oxide. PSHT maintained the erythrocyte membrane integrity and prevented hemolysis. Our results also showed that PSHT exerted a significant anti-edematic effect in vivo by decreasing advanced oxidation protein products and malondialdehyde levels and increasing the superoxide dismutase and glutathione peroxidase activities in rat's paw model and erythrocytes. Interestingly, PSHT increased the viability of murine RAW264.7 macrophages and exerted an anti-inflammatory effect on lipopolysaccharide-stimulated cells by decreasing pro-inflammatory molecule levels, including nitric oxide, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-α). Our findings indicate that PSHT could be used as a potential immunomodulatory, anti-inflammatory, anti-hemolytic, and anti-oxidant agent. These results could be explained by the computational findings showing that polysaccharide building blocks bound both cyclooxygenase-2 (COX-2) and TNF-α with acceptable affinities.

Pyruvate maintains and enhances the pro-inflammatory response of microglia caused by glucose deficiency in early stroke.

Pro-inflammatory microglia mainly rely on glycolysis to maintain cytokine production during ischemia, accompanied by an increase in inducible nitric oxide synthase (iNOS) and monocarboxylate transporter 1 (MCT1). The role of energy metabolism in the pro-inflammatory response of microglia is currently unclear. In this study, we tested the response of microglia in mice after cerebral ischemia and simulated an energy environment in vitro using low glucose culture medium. The research results indicate that the expression levels of iNOS and arginase 1 (ARG1) increase in the ischemic mouse brain, but the upregulation of MCT1 expression is mainly present in iNOS positive microglia. In microglia exposed to low glucose conditions, iNOS and MCT1 levels increased, while ARG1 levels decreased. Under the same conditions, knocking down MCT1 in microglia leads to a decrease in iNOS levels, while overexpression of MCT1 leads to the opposite result. The use of NF-κB inhibitors reduced the expression levels of iNOS and MCT1 in microglia. In summary, our data indicate that pyruvate maintains and enhances the NF-κB regulated pro-inflammatory response of microglia induced by low glucose.

Antioxidant and Anti-Inflammatory Activities of Lindera glauca Extracts.

INTRODUCTION: The current study investigated the antioxidant and anti-inflammatory effects of ethanol extracts from Lindera glauca twig (LGT) and leaf/stem (LGLS). METHODS: The antioxidant activities were measured by total content of polyphenol and flavonoid, DPPH radical scavenging, and ABTS+ radical scavenging activity. To evaluate the anti-inflammatory effect in the LPS-induced RAW 264.7 cells, protein and mRNA expression of major inflammatory factors were analyzed using Western blot analysis and RT-PCR. RESULTS: The total polyphenol content of LGT and LGLS was 88.45 ± 11.74 and 115.75 ± 7.87 GA mg/g, respectively. The total flavonoid content was 66 ± 2.89 and 74.33 ± 2.89 QE mg/g. Both LGT and LGLS showed high DPPH and ABTS+ radical scavenging activities. Neither LGT nor LGLS was cytotoxic to RAW 264.7 cells. The anti-inflammatory activities were measured by LPS-induced RAW 264.7 cells. LGT and LGLS showed inhibition of the LPS-induced production of nitric oxide (NO), inducible NO synthase, cyclooxygenase-2 at the protein and mRNA levels, as determined by Western blotting and RT-PCR, respectively. In addition, the release of tumor necrosis factor-α and interleukin-6 mRNA expression levels of these cytokines was reduced by LGT and LGLS. CONCLUSION: These results suggest that LGT and LGLS extracts have potential for use as a functional antioxidant and anti-inflammatory ingredient in cosmetic industry.

Synthesis and anti-inflammatory activity of novel firocoxib analogues with balanced COX inhibition.

The adverse effects caused by nonselective and selective cyclooxygenase-2 (COX-2) inhibitors remain a challenge for current anti-inflammatory medications. A balanced inhibition of COX-1/-2 represents a promising strategy for the development of novel COX-2 inhibitors. In this study, we present the design and synthesis of a novel series of firocoxib analogues incorporating an amide bond to facilitate essential hydrogen bonding with amino residues in COX-2. The synthesized analogs were evaluated for their inhibitory activity against both COX-1 and COX-2 enzymes. Among them, compound 9d demonstrated potent and balanced inhibition. Inhibition of COX enzymes by 9d in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages resulted in the suppression of the NF-κB signaling pathway to reduced expression of pro-inflammatory factors such as inducible nitric oxide synthase (iNOS), COX-2, nitric oxide (NO), and reactive oxygen species (ROS). The remarkable in vitro anti-inflammatory activity exhibited by 9d positions it as a promising candidate for further development as a novel lead compound for inflammation treatment.