LPS-stimulated Nitric Oxide Production Research Articles

Isolation, structural characterization and biological activities of polysaccharides from Chondrus crispus

Biomedical applications often exploit the biodegradability and biocompatibility of natural polysaccharides. In this study, polysaccharide extracts from Chondrus crispus (CC) were characterized using HPLC-SEC, FTIR and 1H NMR spectroscopy. The proliferative and migrative capacities of various extracts on RAW264.7 cells, along with their impact on nitric oxide (NO) production and phagocytosis, were assessed. Polysaccharide bioactivity was also explored using HDF and HaCaT cell lines. This study additionally investigated the anti-inflammatory effects of CC fractions on Caco-2 cells, examining their impact on cytokines (TNF-α, TGF-β1) and chemokine (MCP-1) expression. CC-derived polysaccharides effectively modulated inflammatory activity and improved scratch or exclusion wound healing. Hot (95 °C) extracted polysaccharides fraction CC-2B (λ and, κ-carrageenan) stimulated RAW264.7 cell proliferation, while cold (25 °C) extracted polysaccharides fraction CC-1A (0.5 μg/μL) inhibited cell proliferation at 24 h (69.7 ± 4.8%, p<0.001), while exhibiting increased phagocytosis activity. Furthermore, RAW264.7 cells treated with hot-extracted fraction CC-2B showed a significant reduction in LPS-stimulated NO production (p<0.0001) over 24 h. HaCaT and HDF cell proliferation were stimulated when treated with fraction CC-2B (p<0.0001). Moreover, Caco-2 cells treated with all fractions displayed enhanced TGF-β1 expression and reduced TNF-α expression at 24 h, indicating the potential of these fractions for future medical applications. Polysaccharide cold-extracted fraction (CC-1A) exhibited highly effective anticoagulant properties. All polysaccharide fractions (0.13 μg/μL) appeared to be non-cytotoxic for Caco-2 cells during 24 h. CC-derived polysaccharides have potential applications in the medical (wound healing, gastrointestinal distress), food, cosmetic, and pharmaceutical industries.

Penidihydrocitrinins A-C: New Polyketides from the Deep-Sea-Derived Penicillium citrinum W17 and Their Anti-Inflammatory and Anti-Osteoporotic Bioactivities.

Three new polyketides (penidihydrocitrinins A-C, 1-3) and fourteen known compounds (4-17) were isolated from the deep-sea-derived Penicillium citrinum W17. Their structures were elucidated by comprehensive analyses of 1D and 2D NMR, HRESIMS, and ECD calculations. Compounds 1-17 were evaluated for their anti-inflammatory and anti-osteoporotic bioactivities. All isolates exhibited significant inhibitory effects on LPS-stimulated nitric oxide production in murine brain microglial BV-2 cells in a dose-response manner. Notably, compound 14 displayed the strongest effect with the IC50 value of 4.7 µM. Additionally, compounds 6, 7, and 8 significantly enhanced osteoblast mineralization, which was comparable to that of the positive control, purmorphamine. Furthermore, these three compounds also suppressed osteoclastogenesis in a dose-dependent manner under the concentrations of 2.5 μM, 5.0 μM, and 10 μM.

The Effects of 2'-Hydroxy-3,6'-Dimethoxychalcone on Melanogenesis and Inflammation.

In this study, we demonstrated that 2'-hydroxy-3,6'-dimethoxychalcone (3,6'-DMC) alleviated α-MSH-induced melanogenesis and lipopolysaccharides (LPS)-induced inflammation in mouse B16F10 and RAW 264.7 cells. In vitro analysis results showed that the melanin content and intracellular tyrosinase activity were significantly decreased by 3,6'-DMC, without cytotoxicity, via decreases in tyrosinase and the tyrosinase-related protein 1 (TRP-1) and TRP-2 melanogenic proteins, as well as the downregulation of microphthalmia-associated transcription factor (MITF) expression through the upregulation of the phosphorylation of extracellular-signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3β (GSK-3β)/catenin, and downregulation of the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and protein kinase A (PKA). Furthermore, we investigated the effect of 3,6'-DMC on macrophage RAW264.7 cells with LPS stimulation. 3,6'-DMC significantly inhibited LPS-stimulated nitric oxide production. 3,6'-DMC also suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 on the protein level. In addition, 3,6'-DMC decreased the production of the tumor necrosis factor-α and interleukin-6. Successively, our mechanistic studies revealed that 3,6'-DMC also suppressed the LPS-induced phosphorylation of the inhibitor of IκBα, p38MAPK, ERK, and JNK. The Western blot assay results showed that 3,6'-DMC suppresses LPS-induced p65 translocation from cytosol to the nucleus. Finally, the topical applicability of 3,6'-DMC was tested through primary skin irritation, and it was found that 3,6'-DMC, at 5 and 10 μM concentrations, did not cause any adverse effects. Therefore, 3,6'-DMC may provide a potential candidate for preventing and treating melanogenic and inflammatory skin diseases.

The inhibitory effects of Bakuchicin on ovalbumin-induced allergic airway inflammation via regulating M2 macrophage polarization

Abstract Asthma is an airway inflammatory disease caused by the activation of many immune cells, including macrophages. Bakuchicin (BKC), a furanocoumarin, has anti-inflammatory properties. This study aimed to evaluate the effects of BKC on allergic airway inflammation and demonstrate the mechanisms of macrophage activation and differentiation. We assessed the effects of BKC by evaluating inflammatory markers in an ovalbumin (OVA)-induced airway inflammation mouse model and macrophage cells. BKC treatment alleviated histological changes, mast cell infiltration, and collagen deposition, and reduced the levels of myeloperoxidase, eosinophil peroxidase, β-hexosaminidase, and transforming growth factor-β in the bronchoalveolar fluid (BALF) and the level of immunoglobulins in serum. BKC ameliorated inflammatory symptoms, including the level of inducible nitric oxide synthase (iNOS) in the lung and the expression and secretion of pro-inflammatory cytokines in serum. BKC treatment reduced not only the level of Th2 cytokines but M2 macrophage marker expression and population in the BALF. In vitro, BKC inhibited LPS-stimulated production of nitric oxide and expression of pro-inflammatory cytokines through inhibition of iNOS, COX-2, Akt, MAPKs, IκBα, and NF-κB in J774 A.1 mouse macrophages. Moreover, BKC suppressed IL-4-induced M2 macrophage polarization by inhibiting arginase-1, YM-1, and Fizz-1 and regulating sirtuin 2 levels in bone marrow derived macrophages. Collectively, BKC may help in the prevention and treatment of airway inflammation by regulating macrophage activation and polarization.

Lignans and Phenylethanoid Glycosides from the Aerial Parts of Pogostemon cablin.

Two new lignans (1-2) and a new octaketide (12), together with twenty-nine known compounds (3-11, 13-32) were isolated and identified from the aerial part of Pogostemon cablin. Their chemical structures were revealed mainly through NMR and MS data. The absolute configuration of 1 and 2 was deduced by comparing its experimental CD with the calculated ECD spectra. The inhibitory activities of the isolated compounds on LPS-stimulated nitric oxide (NO) production in RAW 264.7 cells were investigated. At a concentration of 25 μM, compounds 1 and 11 showed approximately equal NO inhibitory effects to that of aminoguanidine.

Assessing the Anti-inflammatory Effects of Bacopa-Derived Bioactive Compounds Using Network Pharmacology and In Vitro Studies.

Bacopa monnieri is reported as a potent Indian medicinal plant that possesses numerous pharmacological activities due to the presence of various bioactive compounds. These pharmacological activities were used in the ancient medicine system to cure inflammatory conditions. Bacopa has the ability to reduce acute pain and inflammation by inhibiting the enzyme cyclo-oxygenase-2 (COX-2) and reducing COX-2-arbitrated prostanoid mediators. Moreover, the anti-inflammatory property may also be associated with the neuroprotective activity of Bacopa. Considering this importance, the current pilot study focused on the anti-inflammatory potential of various phytocompounds of bacopa and their interaction with inflammation responsible genes such as COX2, iNOS, LOX, STAT3, CCR1, and MMP9 through pharmacology analysis of its systems. Docking results revealed that, quercetin (QR) showed significant binding energies with inflammatory genes. Hence, we selected QR as a potential phytocompound for further in vitro experiments. This existing study aimed to evaluate the efficacy of QR as a potent anti-inflammatory compound against lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The in vitro analysis concludes that QR effectively reduces the production of nitric oxide (NO) in LPS-induced RAW264.7 cells and downregulates the expression of COX-2 and iNOS genes due to the inhibitory potential of QR on LPS-stimulated NO production.

Mycobacterium tuberculosis glycolipoprotein LprG inhibits inflammation through NF-κB signaling of ERK1/2 and JNK in LPS-induced murine macrophage cells.

Mycobacterium tuberoculosis (Mtb) is a contagious pathogen that causes human tuberculosis (TB). TB is a major global health threat that causes 9.6 million illnesses and 1.5 million deaths per year. Recent studies have suggested Mtb-secreted proteins as new candidates for therapeutic drugs and vaccines. LprG is a Mtb-secreted surface glycolipoprotein encoded by lprG (Rv1411c), which forms an operon with Rv1410c, where Rv1410c encodes P55, an efflux pump membrane protein. Various in vitro and in vivo studies have reported on the target-binding activity, cell envelope biosynthesis, and mycobacterial virulence of LprG. However, the anti-inflammatory effect of LprG in macrophages has not yet been investigated. In this study, we demonstrated that LprG can suppress lipopolysaccharide (LPS)-induced inflammation in a macrophage model. LprG inhibited LPS-stimulated nitric oxide (NO) production. LprG also suppressed expression of inducible cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) at the transcriptional and protein levels. In addition, LprG decreased mRNA expression of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, LprG attenuated nuclear factor kappa-B (NF-κB) translocation and IκB phosphorylation. Moreover, LprG specifically inhibited phosphorylated kinases such as c-Jun N-terminal kinase (p-JNK) and extracellular signal-regulated kinase 1/2 (p-ERK1/2), but not p-p38. Taken together, these results suggest that LprG inhibits LPS-stimulated inflammation via downregulation of NO, COX-2, iNOS, and pro-inflammatory cytokines through the NF-κB, AP-1, and MAPK signaling pathways. The present study will aid in the development of anti-inflammatory medications using Mtb. The organism, which has long been regarded as a human pathogenic or human health-threating agent, can be utilized as a future medical resource.

A novel mechanism of Korean Red Ginseng-mediated anti-inflammatory action via targeting caspase-11 non-canonical inflammasome in macrophages

BackgroundKorean Red Ginseng (KRG) was reported to play an anti-inflammatory role, however, previous studies largely focused on the effects of KRG on priming step, the inflammation-preparing step, and the anti-inflammatory effect of KRG on triggering, the inflammation-activating step has been poorly understood. This study demonstrated anti-inflammatory role of KRG in caspase-11 non-canonical inflammasome activation in macrophages during triggering of inflammatory responses. MethodsCaspase-11 non-canonical inflammasome-activated J774A.1 macrophages were established by priming with Pam3CSK4 and triggering with lipopolysaccharide (LPS). Cell viability and pyroptosis were examined by MTT and lactate dehydrogenase (LDH) assays. Nitric oxide (NO)-inhibitory effect of KRG was assessed using a NO production assay. Expression and proteolytic cleavage of proteins were examined by Western blotting analysis. In vivo anti-inflammatory action of KRG was evaluated with the LPS-injected sepsis model in mice. ResultsKRG reduced LPS-stimulated NO production in J774A.1 cells and suppressed pyroptosis and IL-1β secretion in caspase-11 non-canonical inflammasome-activated J774A.1 cells. Mechanistic studies demonstrated that KRG suppressed the direct interaction between LPS and caspase-11 and inhibited proteolytic processing of both caspase-11 and gasdermin D in caspase-11 non-canonical inflammasome-activated J774A.1 cells. Furthermore, KRG significantly ameliorated LPS-mediated lethal septic shock in mice. ConclusionThe results demonstrate a novel mechanism of KRG-mediated anti-inflammatory action that operates through targeting the caspase-11 non-canonical inflammasome at triggering step of macrophage-mediated inflammatory response.

Macluraxanthone B inhibits LPS-induced inflammatory responses in RAW264.7 and BV2 cells by regulating the NF-κB and MAPK signaling pathways

Objective The prenylated xanthones compounds, macluraxanthone B (MCXB) was isolated from the MeOH extracts of Cudrania tricuspidata. In this study, we investigated the effect of MCXB on inflammatory response. Materials and Methods Anti-inflammatory effects of MCXB were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 and BV2 cells. We observed their anti-inflammatory effects by ELISA, western blot analysis, and immunofluorescence. Results MCXB significantly inhibited the LPS-stimulated production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-6 (IL-6), and tumor necrosis factor (TNF)-α in RAW264.7 and BV2 cells. MCXB also reduced the LPS-induced expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 proteins. Incubating cells with MCXB prevented subsequent activation of the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting the nuclear localization and DNA-binding activity of the p65 subunit induced by LPS. MCXB inhibited the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinases (MAPKs) in RAW264.7 and BV2 cells. MCXB induced the expression of heme oxygenase (HO)-1 protein, and the inhibitory effect of MCXB on nitric oxide production was partially reversed by a selective HO-1 inhibitor. Discussion and conclusions Our results suggested that the anti-inflammatory effect of MCXB is partly regulated by HO-1 induction. In conclusion, MCXB could be a useful candidate for the development of therapeutic and preventive agents to treat inflammatory diseases.

Immunomodulatory Activity of Phyllanthus maderaspatensis in LPS-Stimulated Mouse Macrophage RAW 264.7 Cells

Phyllanthus species (Family Euphorbiaceae) has been used in traditional medicine of several countries as a cure for numerous diseases, including jaundice and hepatitis. This study is an attempt to evaluate the immunomodulatory activity of various fractions, column eluents of ethyl acetate fraction, and their polyphenols. Phyllanthus maderaspatensis were standardized using high-performance liquid chromatography to identify and quantify polyphenols, and purification of polyphenols was carried out using vacuum liquid chromatography. Subsequently, we tested various fractions, column eluents of ethyl acetate fraction, and polyphenols in vitro to assess their impact on nitric oxide (NO) production in LPS-stimulated mouse macrophage RAW 264.7 cells. The ethyl acetate fraction (100 μg mL−1) had a more significant stimulatory effect on LPS-stimulated NO production by the RAW 264.7 cells. We found that the ethyl acetate fraction contains a high amount of catechin, quercetin, ellagic acid kaempferol, and rutin, which are responsible for immunomodulation. The ethyl acetate fraction at concentrations of 25 and 50 μg mL−1 had a significant inhibitory effect and 100 μg mL−1 had a more significant stimulatory effect when compared with the LPS control. The percentage of inhibition by LPS control ranged from zero percentage, kaempferol ranged from 45.4% at 50 μg mL−1 to 41.88% at 100 μg mL−1, catechin ranged from 50% at 50 μg mL−1 to 35.28% at 100 μg mL−1, rutin ranged from 36.2% at 50 μg mL−1 to 47.44% at 100 μg mL−1, gallic acid ranged from 28.4% at 50 μg mL−1 to 50.9% at 100 μg mL−1, ellagic acid ranged from 45.12% at 50 μg mL−1 to 38.64% at 100 μg mL−1, and purified quercetin ranged from 26.2% at 50 μg mL−1to 45.48% at 100 μg mL−1. As NO plays an important role in the immune function, polyphenols’ treatment could modulate several aspects of host defense mechanisms owing to the stimulation of the inducible nitric oxide synthase.

Bioassay-guided study of the anti-inflammatory effect of Anoectochilus burmannicus ethanolic extract in RAW 264.7 cells

Ethnopharmacological relevanceAnoectochilus species is a small terrestrial orchid found in tropical and subtropical rain forest. These orchids are traditionally used extensively in China, Taiwan, and Vietnam due to their medicinal properties and therapeutic benefits. They are employed for treatment in different systems, such as stomach disorders, chest pain, arthritis, tumor, piles, boils, menstrual disorders, and inflammation. Aqueous extract of Anoectochilus burmannicus (AB) has been previously reported to exhibit anti-inflammatory activities, however there is a lack of evidence regarding its bioactive compounds and the mechanism of its actions. Aim of the studyThe objectives of this study were to identify the anti-inflammatory compound(s) in an ethanolic extract of AB and to determine its anti-inflammatory mechanisms in LPS-stimulated macrophages and also its safety. Materials and methodsThe ethanolic extract of AB (ABE) was prepared and subsequently subjected to polarity-dependent extraction using n-hexane and ethyl acetate, which would result in isolation of the n-hexane (ABH), ethyl acetate (ABEA), and residue or aqueous (ABA) fractions. The AB fractions were investigated to determine total phenolic and flavonoid content, antioxidant capacity, toxicity, and safety in RAW 264.7 macrophages, human PBMCs, and RBCs. After extraction anti-inflammation screening of each extract was performed by nitric oxide (NO) production assay. The active fractions were further examined for their effect on proinflammatory mediators. In addition, kinsenoside content in the active fractions was identified using LC-MS/MS. Cellular toxicity and genotoxicity of AB were also tested using the wing spot test in Drosophila melanogaster. ResultsThe data showed that ABEA had the highest phenolic content and level of antioxidant activities. ABE, ABEA, and ABA, but not ABH, significantly inhibited the LPS-stimulated NO production in the macrophages. Both ABEA and ABA reduced LPS-mediated expression of TNF-α, IL-6, iNOS, and COX-2 at both mRNA and protein levels. Besides, only ABEA notably diminished the LPS-stimulated p65 phosphorylation required for nuclear translocation and transcriptional activation of the nuclear factor-κB (NF-κB). Interestingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed ABA contained a high level of kinsenoside, a likely anti-inflammatory compound, while ABE and ABEA might require other compounds in combination with kinsenoside for the inhibition of inflammation. It was shown that all active fractions were neither cytotoxic nor genotoxic. ConclusionOur study demonstrated that the hydrophilic fractions of AB exhibit anti-inflammatory activity in LPS-stimulated macrophages. The mechanism used by the AB involves the scavenging of free radicals and the reduction of proinflammatory mediators, including IL-1β, IL-6, TNF-α, NO, iNOS and COX-2. The anti-inflammatory action of AB involves the suppression of the NF-κB signaling pathway by some unknown component(s) present in ABEA. This study found that kinsenoside is a major active compound in ABA which could be used as a biomarker for the quality control of the plant extraction. This study provides convincing significant information in vitro regarding the anti-inflammatory mechanism and preliminary evidence of the safety of Anoectochilus burmanicus. Therefore, the knowledge acquired from this study would provide supportive evidence for the development and standardization of the use of the extract of this plant as alternative medicine or functional food to prevent or treat non-communicable chronic diseases related to chronic inflammation.

Wheatgrass inhibits the lipopolysaccharide-stimulated inflammatory effect in RAW 264.7 macrophages

Inflammation is a multifaceted set of cellular communications generated against foreign infection, toxic influence or autoimmune injury. The present study investigates the anti-inflammatory effect of wheatgrass extract against the harmful impact of lipopolysaccharide (LPS) in macrophage cells, i.e., RAW 264.7 cells. Our results indicate that 5- and 7- days old wheatgrass extracts inhibit the LPS-stimulated production of nitric oxide. Moreover, wheatgrass extract significantly downregulates the mRNA expression of LPS-stimulated various pro-inflammatory markers, tumor necrosis factor-α, interleukin-6, interleukin-1β, AP-1 and also iNOS-2 and COX-2. Our flow cytometry analyses confirmed that wheatgrass extract prevents the generation of reactive oxygen species in LPS-stimulated RAW 264.7 cells, thus arresting oxidative stress in cells. The immunoblot analyses also confirmed a significant reduction in the expression of inflammatory proteins, namely, iNOS-2 and COX-2, in wheatgrass extract-treated cells, compared to LPS-stimulated condition. The NF-κB transactivation assay further confirmed the inhibitory effect of wheatgrass extracts on the LPS-stimulated expression of NF-κB. Molecular docking based studies showed the plausible binding of two significant wheatgrass constituents, i.e., apigenin and myo-inositol with COX-2 protein, with binding energies of −10.59 kcal/mol and −7.88 kcal/mol, respectively. Based on the above results, wheatgrass may be considered as a potential therapeutic candidate for preventing inflammation.

N-acetylcysteine reduced the immunotoxicity effects induced in vitro by azoxystrobin and iprodione fungicides in mice.

Azoxystrobin (AZO) and Iprodione (IPR) fungicides are extensively used worldwide, and therefore, contaminate all environmental compartments. The toxicity and the mechanisms by which they affected immune cells are complex and remain unknown. This study investigated the impact of AZO and IPR on the in vitro function of mice peritoneal macrophages including lysosomal enzyme activity and tumor necrosis factor (TNF)α and nitric oxide (NO) production in response to lipopolysaccharide (LPS) stimulation, the proliferation of mice splenocytes stimulated by concanavalin (Con)A and LPS, and the production of the Th1cytokine interferon-gamma (IFNγ) and the Th2 cytokine interleukin (IL)-4 and IL-10 by ConA-activated splenocytes. This is the first report indicating that AZO and IPR fungicides dose-dependently inhibited mice macrophage lysosomal enzyme activity and LPS-stimulated production of TNFα and NO. Mitogen-induced proliferation of mice splenocytes was also suppressed by AZO and IPR in a dose-dependent manner. More pronounced impact was observed on ConA-induced response. The production of IFNγ by ConA-stimulated splenocytes was dose-dependently inhibited; however, the production of IL-4 and IL-10 increased in the same conditions. These results suggested that AZO and IPR polarized Th1/Th2 cytokine balance towards Th2 response. Overall, marked immunosuppressive effects were observed for AZO. The immunomodulatory effects caused by AZO and IPR were partially reversed by the pharmacological antioxidant N-acetylcysteine (NAC), suggesting that both fungicides exerted their actions through, at least in part, oxidative stress-dependent mechanism. Collectively, our data showed that AZO and IPR fungicides exerted potent immunomodulatory effects in vitro with eventually strong consequences on immune response and immunologically based diseases.

Anti-inflammatory principles from Balanophora laxiflora

Eighteen known compounds, including lignans, phenylpropanoids, triterpenoids, and phytosterols, were isolated from Balanophora laxiflora (Balanophoraceae). The inhibitory activities of these compounds on the production of nitric oxide (NO), a pro-inflammatory mediator, were evaluated. Compounds isolariciresinol (1), ethyl caffeate (13) and ferulic aldehyde (16) showed potent to mild inhibitory activities on LPS-stimulated NO production in RAW 264.7 macrophages with IC 50 values of 0.81, 7.29, and 98.67 μM, respectively. The results showed that compound 1 had a potent effect on TNF-α production with an IC 50 value of 0.87 μM, but practically no effect on IL-6 synthesis. In addition, an inhibitory effect of compound 1 on nuclear factor-κB (NF-κB) activation was shown by the repression of LPS-stimulated β-lactamase reporter activity. Taken together, these results demonstrated that compound 1 modulates the production of inflammatory mediators through the attenuation of NF-κB transcription signaling.

Acetate Suppresses Lipopolysaccharide-stimulated Nitric Oxide Production in Primary Rat Microglia but not in BV-2 Microglia Cells.

To show that acetate attenuates neuroinflammatory responses in activated microglia. Dietary acetate supplementation alleviates neuroglial activation in a rat model of neuroinflammation induced by intraventricular administration of lipopolysaccharide (LPS). However, the precise mechanism(s) underlying the anti-inflammatory effect of acetate, is not fully understood. To determine whether acetate has inhibitory effects on LPS-induced neuroinflammatory responses in microglia. We examined LPS-stimulated nitric oxide (NO) production in primary rat microglia and BV-2 cells. Protein expression of inducible NO synthase (iNOS) was determined by western blot analysis. The intracellular generation of reactive oxygen species (ROS) and glutathione (GSH) were also evaluated. In primary microglia, acetate decreased LPS-stimulated NO production in a dose-dependent manner, reaching significance at greater than 10 mM, and cell viability was not affected. Acetate suppressed LPS-induced expression of iNOS protein concomitantly with the decrease in NO. The LPS-induced increase in intracellular ROS production was attenuated by acetate. In addition, acetate prevented LPS-induced reduction of GSH. Notably, such suppressive effects of acetate on NO and ROS production were not observed in BV-2 cells. These findings suggest that acetate may alleviate neuroinflammatory responses by attenuating NO and ROS production in primary microglia but not in BV-2 cells. Other: All animals received humane care, and the animal protocols used in this study were approved by the Ethics Committees for Animal Experimentation.

Ruxolitinib protects lipopolysaccharide (LPS)-induced sepsis through inhibition of nitric oxide production in mice.

BackgroundRuxolitinib is an inhibitor of Janus kinases (JAK) 1/2. It was authorised recently by the U.S. Food and Drug Administration (FDA) as a new Myelofibrosis treatment. In this study, we identified ruxolitinib as a new inhibitor of nitric oxide (NO) production in response to lipopolysaccharide (LPS)stimulation of RAW 264.7 cells.MethodsIn vitro direct effects of ruxolitinib were determined through NO production on RAW 264.7 cells. Also the expression level of iNOS, TNF-α and IL-6 were detected by Western Blotting and qRT-PCR. In vivo therapeutic effects of ruxolitinib on sepsis were evaluated by an endotoxemia model with C57 mice. The survival was calculated and histopathological damage of organs was observed by HE. Cytokines in serum were detected by Mouse Cytokine Array Panel.ResultsRuxolitinib was found to significantly reduce NO production, inducible nitric oxide synthase (iNOS), TNF-α, and IL-6 expression, suggesting that ruxolitinib blocks LPS signaling that leads to pro-inflammatory factor expression. Furthermore, the inhibitory effects of ruxolitinib contributed to the survival of septic mice by 70% and pro-inflammatory cytokines in serum declined apparently. The results taken together indicate that ruxolitinib can significantly suppress LPS-stimulated NO production and improve the survival of septic mice, perhaps by interfering with the NF-κB pathway.ConclusionsThese findings suggest ruxolitinib might be a possible therapeutic candidate for sepsis therapy.

Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes

Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-β1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13–induced expressions of chIL-13Rα2 and TGF-β1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-β1 secretion. Importantly, the newly developed anti–chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors.

Immunomodulatory properties of photopolymerizable fucoidan and carrageenans

Innovative approaches to the control of immune response to tissue engineering scaffolds is of high priority. IL-10, an anti-inflammatory cytokine, has traditionally been conjugated to synthetic polymers for local immunomodulation. Marine-sulfated polysaccharides have been reported to possess anti-inflammatory properties. In the present work, it was hypothesized that photopolymerizable fucoidan and carrageenan play similar roles as the IL-10. The polysaccharides were functionalized with methacrylate groups. Their immunomodulatory properties were evaluated and compared relative to IL-10. The polysaccharides were characterized by NMR spectroscopy, revealing 12–13 % functionalization. The data revealed that fucoidan had the same activity as the IL-10 in decreasing LPS- and IFN-γ-stimulated CD86 expression. In addition, fucoidan had a protective role against LPS- and IFN-γ-induced cell growth inhibition. All polysaccharides demonstrated ∼90 % superoxide radical scavenging and they considerably decreased LPS-stimulated nitric oxide production. These results suggest that photopolymerizable fucoidan can be an alternative to IL-10 in the design of immunomodulatory biomaterials.

Vitexnegheteroin M, a new phenolic glycoside from Vitex negundo var. heterophylla

The investigation of chemical constituents from the leaves and seeds of Vitex negundo var. heterophylla resulted in the isolation of one new phenolic glycoside, vitexnegheteroin M (1), together with eleven known compounds (2-12). Their structures were elucidated through extensive spectroscopic analyses including 1D, 2D NMR, HR-ESI-MS, and by comparison with the literature. Among them, compound 3 was obtained from the Vitex genus for the first time, and compounds 4-8, and 10-12 were isolated from this plant for the first time. Their anti-inflammatory activities were investigated by measuring nitric oxide (NO) production in lipopolysaccharide (LPS)-induced murine microglial BV-2 cells. As the obtained results, compound 9 showed the strongest inhibitory activity against LPS-stimulated NO production in BV-2 cells with IC50 value ranging from 8.65 ± 0.67 μM.

Anti-inflammatory activities of Maillard reaction products from whey protein isolate fermented by Lactobacillus gasseri 4M13 in lipopolysaccharide-stimulated RAW264.7 cells

Maillard reaction products formed from whey protein isolate (WPI) and sugar have been shown to have an anti-inflammatory effect in vitro. Here, we incubated WPI and galactose (GWA) in an aqueous solution at 65°C for 24 h to produce a glycated conjugate, which was then fermented using Lactobacillus gasseri 4M13 to obtain the fermented product (F-GWA). We demonstrated that F-GWA had an anti-inflammatory effect on lipopolysaccharide (LPS)-stimulated RAW264.7 cells. It reduced both LPS-stimulated nitric oxide production and LPS-stimulated increases in the gene expression levels of tumor necrosis factor-α and cyclooxygenase-2 in a dose-dependent manner. Furthermore, F-GWA inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, members of the mitogen-activated protein kinase family. The glycation process was evaluated by measuring fluorescence intensity and the furosine concentration during the Maillard reaction to form GWA. The protein modifications of WPI were analyzed using MALDI-TOF tandem mass spectrometry. We found that the combination of the Maillard reaction and L. gasseri 4M13 fermentation increased the prebiotic properties of GWA as well as organic acid production, compared with the nonreacted WPI and galactose.