Cytokines In Lipopolysaccharide Research Articles

Mesenchymal Stem Cells Suppress Inflammatory Cytokines in Lipopolysaccharide Exposed Preterm and Term Human Pregnant Myometrial Cells.

Objective The objective of this study was to determine the cytokine response in human pregnant preterm and term myometrial cells exposed to lipopolysaccharide (LPS) and cocultured with mesenchymal stem cells (MSCs). Study Design Myometrium was obtained at cesarean delivery in term and preterm patients. Human myometrial cells were exposed to 5 μg/mL LPS for 4 hours followed by 1 μg/mL LPS for 24 hours and were cocultured with MSCs for 24 hours. Culture supernatants were collected at 24 hours and expression of cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and IL-10, was quantified by enzyme-linked immunosorbent assay. Results There was significantly increased expression of the proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in preterm myometrial cells treated with LPS compared with untreated preterm myometrial cells. Coculture with MSCs significantly suppressed the proinflammatory cytokine levels in LPS-treated preterm versus treated term myometrial cells. Moreover, MSC cocultured preterm myometrial cells expressed increased levels of the anti-inflammatory cytokines TGF-β and IL-10 compared with treated term myometrial cells. Conclusion MSCs ameliorate LPS-mediated inflammation in preterm human myometrial cells compared with term myometrial cells. Immunomodulatory effects of MSCs mediated through anti-inflammatory cytokine regulation suggest a potential cell-based therapy for preterm birth.

NLRP3 INFLAMMASOME ACTIVATION IN MACROPHAGE (RAW 264.7) CELLS BY LIPOPOLISACCARIDE/NIGERICIN: REGULATORY EFFECT OF PSORALIDIN

Psoralidin, a prenylated coumestan, has been reported its anti-inflammatory effect, but the regulatory effects on inflammasome activation and pyroptosis-related cytokines are not clear. The aim of this study was to investigate the regulatory role of psoralidin on inflammasome activation and expression of pyroptosis-related cytokines in Lipopolysaccharide (LPS)/Nigericin-stimulated RAW 264.7 macrophages. Regulatory mechanism of psoralidin on inflammasome activation was evaluated using Western blotting for NLRP3, adaptor protein apoptosis-associated speckle-like protein (ASC)and Caspase-1. Pyroptosis-related cytokines were evaluated using RT-PCR for interleukin-1β (IL-1β)and interleukin-18 (IL18).Non-cytotoxic concentration of Psoralidin (5.0µM) significantly inhibited LPS/Nigericin induced inflamasome activation. In addition, pretreatment with psoralidin suppressed the LPS/Nigericin-induced production of IL-1β and IL18. Our results indicate that the regulatory effects of psoralidin on inflammasome activation and pyroptosis related cytokine production in RAW 264.7 macrophages are associated with suppression of NLRP3inflammasome activation and inhibition of the release of pyroptosis related cytokines.According to these results, Psoralidine may be considered as a potential therapeutic candidate for the prevention of inflammatory diseases or to support the treatment of inflammation.

Sulfur-containing spiroketals from Breynia disticha and evaluations of their anti-inflammatory effect.

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

Ramulus Cinnamomi essential oil exerts an anti-inflammatory effect on RAW264.7 cells through N-acylethanolamine acid amidase inhibition

Ethnopharmacological relevanceRamulus Cinnamomi, the dried twig of Cinnamomum cassia (L.) J.Presl., is a traditional Chinese medicine (TCM) with anti-inflammatory effects. The medicinal functions of Ramulus Cinnamomi essential oil (RCEO) have been confirmed, although the potential mechanisms by which RCEO exerts its anti-inflammatory effects have not been fully elucidated. Aim of the studyTo investigate whether N-acylethanolamine acid amidase (NAAA) mediates the anti-inflammatory effects of RCEO. Materials and methodsRCEO was extracted by steam distillation of Ramulus Cinnamomi, and NAAA activity was detected using HEK293 cells overexpressing NAAA. N-Palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), both of which are NAAA endogenous substrates, were detected by liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The anti-inflammatory effects of RCEO were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit. The nitric oxide (NO) in the cell supernatant was measured using the Griess method. The level of tumor necrosis factor-α (TNF-α) in the RAW264.7 cell supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The chemical composition of RCEO was assessed by gas chromatography-mass spectroscopy (GC-MS). The molecular docking study for (E)-cinnamaldehyde and NAAA was performed by using Discovery Studio 2019 software (DS2019). ResultsWe established a cell model for evaluating NAAA activity, and we found that RCEO inhibited the NAAA activity with an IC50 of 5.64 ± 0.62 μg/mL. RCEO significantly elevated PEA and OEA levels in NAAA-overexpressing HEK293 cells, suggesting that RCEO might prevent the degradation of cellular PEA and OEA by inhibiting the NAAA activity in NAAA-overexpressing HEK293 cells. In addition, RCEO also decreased NO and TNF-α cytokines in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, the GC-MS assay revealed that more than 93 components were identified in RCEO, of which (E)-cinnamaldehyde accounted for 64.88%. Further experiments showed that (E)-cinnamaldehyde and O-methoxycinnamaldehyde inhibited NAAA activity with an IC50 of 3.21 ± 0.03 and 9.62 ± 0.30 μg/mL, respectively, which may represent key components of RCEO that inhibit NAAA activity. Meanwhile, docking assays revealed that (E)-cinnamaldehyde occupies the catalytic cavity of NAAA and engages in a hydrogen bond interaction with the TRP181 and hydrophobic-related interactions with LEU152 of human NAAA. ConclusionsRCEO showed anti-inflammatory effects by inhibiting NAAA activity and elevating cellular PEA and OEA levels in NAAA-overexpressing HEK293 cells. (E)-cinnamaldehyde and O-methoxycinnamaldehyde, two components in RCEO, were identified as the main contributors of the anti-inflammatory effects of RCEO by modulating cellular PEA levels through NAAA inhibition.

Effects of fatty acid-ethanol amine (FA-EA) derivatives on lipid accumulation and inflammation.

This study aimed to investigate the effect of fatty acid-ethanol amine (FA-EA) derivatives (L1-L10) on the mitigation of intracellular lipid accumulation and downregulation of pro-inflammatory cytokines in vitro. First, the series of FA-EA derivatives were synthesized and characterized. Then, their cytotoxic, intracellular lipid accumulation and inhibition of pro-inflammatory cytokines were evaluated. The oil red O staining experiment showed that the tested compounds L4, L6, L8, L9, and L10 could reduce intracellular lipid accumulation induced by palmitic acid (PA). Moreover, ω-3/ω-6 PUFA-EA derivatives showed inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. ω-3/ω-6 PUFA-EA derivatives at a concentrations of 10μM could significantly decrease mRNA levels of IL-6, IL-1β, and TNF-α, inhibit NO production, and alleviate the protein expression of IL-1β in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These data suggest that ω-3 PUFA-EA derivatives can be beneficial for further pharmaceutical development to treat chronic low-grade inflammation diseases such as obesity.

Identification and Molecular Mechanism of Novel Immunomodulatory Peptides from Gelatin Hydrolysates: Molecular Docking, Dynamic Simulation, and Cell Experiments.

The purpose of this study was to identify donkey-hide gelatin-derived immunomodulatory peptides targeting Toll-like receptor 4-myeloid differentiation 2 (TLR4-MD2) and elucidate their binding modes using physicochemical property prediction, molecular docking, molecular dynamics simulations, and in vitro cell experiments. After hydrolyzing gelatin, 519 peptides were identified by liquid chromatography-tandem mass spectrometry. Peptides VQLSGEEK and GFSGLDGAKG bound to TLR4-MD2 with high binding affinity. In TLR4-MD2, Arg90, Ser118, Phe126, Tyr131, and Arg264 were key residues involved in the binding of these peptides. The RMSD and Rg values demonstrated that VQLSGEEK-TLR4-MD2 and GFSGLDGAKG-TLR4-MD2 complexes had stable and compact conformations. VQLSGEEK and GFSGLDGAKG were found to increase the cell viability and phagocytic activity of RAW264.7 macrophages; significantly promote the production of cytokines TNF-α, IL-1β, and IL-6 in cells; and inhibit the overproduction of nitric oxide (NO) and cytokines in lipopolysaccharide (LPS)-induced RAW264.7 cells. Our results provided preliminary evidence that VQLSGEEK and GFSGLDGAKG could function as two-way immunomodulatory peptides with immunostimulatory and anti-inflammatory activities.

Yomogin, Isolated from Artemisia iwayomogi, Inhibits Neuroinflammation Stimulated by Lipopolysaccharide via Regulating MAPK Pathway.

Neuroinflammation causes various neurological disorders, including depression and neurodegenerative diseases. Therefore, regulation of neuroinflammation is a promising therapeutic strategy for inflammation-related neurological disorders. This study aimed to investigate whether yomogin, isolated from Artemisia iwayomogi, has anti-neuroinflammatory effects. First, we evaluated the effects of yomogin by assessing pro-inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The results showed that yomogin inhibited the increase in neuroinflammatory factors, including nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6, and tumor necrosis factor-α, and suppressed phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase and p38, which participate in the mitogen-activated protein kinase (MAPK) pathway. To confirm these effects in vivo, we measured the activation of astrocyte and microglia in LPS-injected mouse brains. Results showed that yomogin treatment decreased astrocyte and microglia activations. Collectively, these results suggest that yomogin suppresses neuroinflammation by regulating the MAPK pathway and it could be a potential candidate for inflammation-mediated neurological diseases.

Design, synthesis, and bioactivity evaluation of novel 1-(4-(benzylsulfonyl)-2-nitrophenyl) derivatives as potential anti-inflammatory agents against LPS-induced acute lung injury

Acute lung injury (ALI) is a devastating disease with a high mortality rate of 30%–40%. There is an unmet clinical need owing to limited treatment strategies and little clinical benefit. The pathology of ALI indicates that reducing the inflammatory response could be a highly desirable strategy to treat ALI. In this study, we designed and synthesized 36 novel 1-(4-(benzylsulfonyl)-2-nitrophenyl) derivatives and evaluated their anti-inflammatory activities by measuring the release of cytokines in lipopolysaccharide (LPS)-challenged J774A.1 cells. Compounds 19, 20, and 39 potently reduced the release of IL-6 and TNF-α in J774A.1 cells. Additionally, 39 improved LPS-induced ALI in vivo and inhibited cytokine production in lung tissues. Furthermore, 39 reduced inflammatory infiltration and downregulated p-p65 levels in lung tissues. Thus, compound 39 could serve as a new lead structure for the development of anti-inflammatory drugs to treat ALI.

Spirulina platensis Suppressed iNOS and Proinflammatory Cytokines in Lipopolysaccharide-Induced BV2 Microglia.

The disease burden of neurodegenerative diseases is on the rise due to the aging population, and neuroinflammation is one of the underlying causes. Spirulina platensis is a well-known superfood with numerous reported bioactivities. However, the effect of S. platensis Universiti Malaya Algae Culture Collection 159 (UMACC 159) (a strain isolated from Israel) on proinflammatory mediators and cytokines remains unknown. In this study, we aimed to determine the anti-neuroinflammatory activity of S. platensis extracts and identify the potential bioactive compounds. S. platensis extracts (hexane, ethyl acetate, ethanol, and aqueous) were screened for phytochemical content and antioxidant activity. Ethanol extract was studied for its effect on proinflammatory mediators and cytokines in lipopolysaccharide (LPS)-induced BV2 microglia. The potential bioactive compounds were identified using liquid chromatography-mass spectrometric (LC-MS) analysis. Ethanol extract had the highest flavonoid content and antioxidant and nitric oxide (NO) inhibitory activity. Ethanol extract completely inhibited the production of NO via the downregulation of inducible NO synthase (iNOS) and significantly reduced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. Emmotin A, palmitic amide, and 1-monopalmitin, which might play an important role in cell signaling, have been identified. In conclusion, S. platensis ethanol extract inhibited neuroinflammation through the downregulation of NO, TNF-α and IL-6. This preliminary study provided insight into compound(s) isolation, which could contribute to the development of precision nutrition for disease management.

Anti-inflammatory compounds from the mangrove endophytic fungus Amorosia sp. SCSIO 41026.

Three new chlorinated compounds, including two propenylphenol derivatives, chlorophenol A and B (1 and 2), and one benzofuran derivative, chlorophenol C (3), together with 16 known compounds, were isolated from the mangrove endophytic fungus Amorosia sp. SCSIO 41026. 7-Chloro-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarine (4) and 2,4-dichloro-3-hydroxy-5-methoxy-toluene (5) were obtained as new natural products. Their structures were elucidated by physicochemical properties and extensive spectroscopic analysis. Compounds 1, 4, 7, 9, 13, 15, 16, and 19 possessed inhibitory effects against the excessive production of nitric oxide (NO) and pro-inflammatory cytokines in lipopolysaccharide (LPS)-challenged RAW264.7 macrophages without obvious cytotoxicity. Moreover, 5-chloro-6-hydroxymellein (13) further alleviated the pathological lung injury of LPS-administrated mice and protected RAW264.7 macrophages against LPS-induced inflammation through PI3K/AKT pathway in vivo. Our research laid the foundation for the application of compound 13 as a potential anti-inflammatory candidate.

Anti-Inflammatory Activity of Mulberry Leaf Flavonoids In Vitro and In Vivo.

Mulberry (Morus alba L.) is a flowering tree traditionally used in Chinese herbal medicine. Mulberry leaf flavonoids (MLFs) have been reported to exert important anti-inflammatory and antioxidant properties. The purpose of this study was to select the MLF with the best anti-inflammatory and antioxidative activities from MLFs eluted by different ethanol concentrations (30%, 50%, and 75%) and explore its pharmacological properties. Three types of MLFs inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 cells. All MLFs boosted the antioxidative capacity by decreasing the reactive oxygen species (ROS) production and the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and improving the metal ion chelating activity and reducing power. The results revealed that the MLFs eluted by 30% ethanol exhibited the best anti-inflammatory and antioxidative activities. A nontargeted metabolomic analysis was used to analyze 24 types of differential flavonoids between the MLFs. Quercetin, kaempferol, and their derivatives in 30%MLF were more abundant than the other two MLFs. Furthermore, we evaluated the pharmacological activities of 30%MLF in dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) mice. The 30%MLF could alleviate the clinical symptoms, reduce the secretion of inflammatory cytokines, and inhibit the activation of the inflammatory pathway in DSS-induced colitis mice. This study will provide valuable information for the development of MLFs eluted by 30% ethanol as a functional food.

Dioscin ameliorates inflammatory bowel disease by up-regulating miR-125a-5p to regulate macrophage polarization.

PurposeDioscin has been proven to have anti‐cancer, anti‐inflammatory, and anti‐infection roles. However, the role of Dioscin in inflammatory bowel disease (IBD) and its related mechanisms is unclear and needs further study.MethodsThe colitis model in mice was established. After Dioscin (20, 40, or 80 mg/kg) treatment, the colon length was measured by a ruler. Histopathology, inflammatory cytokines, gut permeability, tight junction proteins, macrophage infiltration, macrophage polarization, and miR‐125a‐5p level were detected by hematoxylin–eosin staining, enzyme‐linked immunosorbent assay, quantitative real‐time polymerase chain reaction (qRT‐PCR), FITC‐dextran, Western blot, and flow cytometry. In vitro experiments, after RAW264.7 cells induced by lipopolysaccharide (LPS)/interleukin‐4 (IL‐4), were treated with Dioscin and miR‐125a‐5p inhibitor, miR‐125a‐5p level, cell vitality, inflammatory cytokines, and M1/M2 marker genes were measured by qRT‐PCR and MTT assay.ResultsDioscin (20, 40, or 80 mg/kg) relieved DSS‐triggered colitis and restrained the serum and colon of pro‐inflammatory cytokines expression. Meanwhile, different concentrations' Dioscin weakened M1 macrophage polarization but facilitated tight junction protein expressions, M2 macrophage polarization, and miR‐125a‐5p level in colitic mice. Moreover, miR‐125a‐5p inhibitor reversed the modulation of Dioscin on miR‐125a‐5p expression, cell vitality, and inflammatory cytokines in lipopolysaccharide (LPS)‐induced RAW264.7 cells. We further discovered that Dioscin restrained M1 marker gene (CD16) expression while intensifying M2 marker genes (CD206 and Arginase‐1) expressions in vitro, which was reversed by miR‐125a‐5p inhibitor.ConclusionDioscin modulated macrophage polarization by increasing miR‐125a‐5p, thereby improving the intestinal epithelial barrier function and reducing IBD.

Effects of Poly‐Substituted Pyrroles on Pro‐Inflammatory Signaling in RAW264.7 Macrophages

Inflammation is the body’s natural means of protection against infection and the restoration to homeostasis after cellular and tissue damage from injury or metabolic stress. A multitude of complex signaling pathways are necessary for the activation of pro‐inflammatory signaling in macrophages, and the dysregulation of these pathways can contribute to disease states linked to chronic inflammation making them attractive targets for therapeutics. Our laboratory explores the potential of a class of poly‐substituted pyrrole compounds that are known microtubule depolymerizers to regulate the inflammatory response in macrophages. Previous work from our laboratory demonstrated that JG‐03‐14 (3,5‐dibromo‐4‐(3,4‐dimethoxyphenyl)‐1H‐pyrrole‐2‐carboxylic_acid ethyl ester) and its refined analog, NT‐07‐16, which possesses an additional methoxy group at the 2‐position of the phenyl ring decrease the production of pro‐inflammatory cytokines in lipopolysaccharide (LPS)‐activated RAW264.7 murine macrophages. Our results demonstrated that the reduction in pro‐inflammatory mediators is due, in part, to a decrease in the nuclear translocation of NFκB, a critical transcription factor for pro‐inflammatory signaling. These results are supported since microtubule dynamics is thought to be involved in NFκB nuclear translocation. Here we expand the scope of our previous work by exploring the effects of NT‐07‐16, and a potentially more potent stereoisomer mix of the NT analog, NT‐07‐45 on other pro‐inflammatory signaling pathways in the RAW264.7 macrophages. Recent studies have shown that exposure to NT‐07‐45 decreases the production of the antiviral and pro‐inflammatory cytokine interferon‐beta (IFN‐β) in macrophages. In addition, the NT analog alters the phosphorylation of the interferon regulatory factor (IRF3) which decreases its activation and could affect its ability to enter the nucleus and induce IFN‐β production. Our recent work has also found that exposure of RAW264.7 macrophages to the NT analogs alters the phosphorylation, and possibly the activation, of c‐Jun N‐terminal kinase (JNK), an important MAP‐Kinase (MAPK) involved in macrophage inflammatory signaling. These findings indicate that the decrease in the production of pro‐inflammatory mediators observed in RAW264.7 macrophages exposed to polysubstituted pyrroles, may be due to the inability of several transcription factors involved in the inflammatory response to efficiently translocate into the nucleus. While much is still to be learned about the anti‐inflammatory mechanisms of these pyrrole compounds it appears that the effects may go beyond their microtubule‐depolymerizing activity.

Anti-Inflammatory and Neuroprotective Mechanisms of GTS-21, an α7 Nicotinic Acetylcholine Receptor Agonist, in Neuroinflammation and Parkinson’s Disease Mouse Models

Neuroinflammation is crucial in the progression of neurodegenerative diseases. Thus, controlling neuroinflammation has been proposed as an important therapeutic strategy for neurodegenerative disease. In the present study, we examined the anti-inflammatory and neuroprotective effects of GTS-21, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, in neuroinflammation and Parkinson’s disease (PD) mouse models. GTS-21 inhibited the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary microglia. Further research revealed that GTS-21 has anti-inflammatory properties by inhibiting PI3K/Akt, NF-κB, and upregulating AMPK, Nrf2, CREB, and PPARγ signals. The effects of GTS-21 on these pro-/anti-inflammatory signaling molecules were reversed by treatment with an α7 nAChR antagonist, suggesting that the anti-inflammatory effects of GTS-21 are mediated through α7 nAChR activation. The anti-inflammatory and neuroprotective properties of GTS-21 were then confirmed in LPS-induced systemic inflammation and MPTP-induced PD model mice. In LPS-injected mouse brains, GTS-21 reduced microglial activation and production of proinflammatory markers. Furthermore, in the brains of MPTP-injected mice, GTS-21 restored locomotor activity and dopaminergic neuronal cell death while inhibiting microglial activation and pro-inflammatory gene expression. These findings suggest that GTS-21 has therapeutic potential in neuroinflammatory and neurodegenerative diseases such as PD.

Analgesic and Anti-Arthritic Activities of Polysaccharides in Chaenomeles speciosa.

Chaenomeles speciosa (Sweet) Nakai has been long used as a folk medicine for rheumatic diseases treatment. This study aimed to investigate the effects and underlying mechanism of polysaccharides in Chaenomeles speciosa (CSP) on the pro-inflammatory cytokines and MAPK pathway in complete Freund’s adjuvant (CFA)-induced arthritis and LPS-induced NR8383 cells. We used acetic acid (HAc)-induced writhing and CFA induced paw edema to determine the analgesic activity and anti-inflammatory activity, respectively. CFA rats were administered CSP (12.5, 25.0, and 50.0 mg/kg) daily for 3 weeks via oral gavage. The analgesic test was done using three different doses of the extract (50, 100, and 200 mg/kg). The anti-arthritic evaluation involved testing for paw swelling, swelling inhibition, and histological analysis in CFA rats. Finally, ELISA, western blot, qRT-PCR were done to determine the effect of CSP on the activation of MAPK pathway, production of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated NR838 macrophage cells. In pain models, oral uptake of CSP greatly reduced pain perception. Furthermore, in CFA rats, CSP substantially decreased paw swelling as well as synovial tissue proliferation and inflammatory cell infiltration. In addition, CSP was shown to inhibit pro-inflammatory cytokines (TNF-α, IL-1β, and COX-2) as well as JNK and ERK1/2 phosphorylation in LPS-stimulated NR8383 cells. Thus, pro-inflammatory cytokine secretion and MAPK signaling downregulation promoted the analgesic and anti-arthritic effects of CSP.

Diterpenoids Isolated from Podocarpus macrophyllus Inhibited the Inflammatory Mediators in LPS-Induced HT-29 and RAW 264.7 Cells.

Species of Podocarpus are used traditionally in their native areas for the treatment of fevers, asthma, coughs, cholera, chest pain, arthritis, rheumatism, and sexually transmitted diseases. To identify natural products having efficacy against inflammatory bowel disease (IBD), we identified a new, 16-hydroxy-4β-carboxy-O-β-D-glucopyranosyl-19-nor-totarol (4) together with three known diterpenoids from P. macrophyllus. Furthermore, all the extracts, fractions, and isolates 1–4 were investigated for their anti-inflammatory effects by assessing the expression on nitric oxide (NO) production and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 and HT-29 cells. Among them, nagilactone B (2) exhibited a potent anti-inflammatory effect against NO production on RAW 264.7 cells; therefore, nagilactone B was further assessed for anti-inflammatory activity. Western blot analysis revealed that nagilactone B significantly decreased the expression of LPS-stimulated protein, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and phosphorylated extracellular regulated kinase (pERK)1/2. In addition, nagilactone B downregulated tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels in LPS-induced macrophages and colonic epithelial cells. To our best knowledge, this is the first report on the inhibitory effect of nagilactone B (pure state) and rakanmakilactone G against NO production in LPS-stimulated RAW 264.7 cells. Thus, diterpenoids isolated from P. macrophyllus could be employed as potential therapeutic phytochemicals for IBD.

Optimization of 1,4-bis(arylsulfonamido)naphthalene-N,N'-diacetic acids as inhibitors of Keap1-Nrf2 protein-protein interaction to suppress neuroinflammation

The protein–protein interaction (PPI) between kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) is recognized as a promising target for the prevention and treatment of oxidative stress-related inflammatory diseases. Herein, a series of novel 1,4-bis(arylsulfonamido)naphthalene-N,N'-diacetic acid analogs (7p-t and 8c) were designed to further explore the structure–activity relationships of the series. Their activities were measured first with a fluorescence polarization (FP) assay and more potent compounds were further evaluated using a more sensitive time-resolved fluorescence energy transfer (TR-FRET) assay, demonstrating IC50 values between 7.2 and 31.3 nM. In cytotoxicity studies, the naphthalene derivatives did not show noticeable toxicity to human HepG2-C8 and mouse brain BV-2 microglia cells. Among them, compound 7q bearing oxygen-containing fused rings was shown to significantly stimulate the cellular Nrf2 signaling pathway, including activation of antioxidant response element (ARE)-controlled expression of Nrf2 target genes and proteins. More importantly, 7q suppressed up-regulation of several pro-inflammatory cytokines in lipopolysaccharide (LPS)-challenged BV-2 microglial cells, representing a potential therapeutic application for controlling neuroinflammatory disorders.

Alteration of inflammation cytokines in lipopolysaccharide – activated lymphocyte by crude extract of taurine from sea slug Paraonchidium via down-regulation of NF-kB pathway

Free amino acid taurine is found in sea slugs. It has been used as a preventive agent for cardiovascular disease and inflammatory disorders. Inflammation takes place in the presence of lipopolysaccharide (LPS), as a specific inducer, which provokes activation of molecular signals controlling the development of pro-inflammatory and anti-inflammatory cytokines. This current work investigated the effects of taurine extract from shell-less sea slugs on the inhibition of IFN-γ as pro-inflammatory cytokine and IL-10 as anti-inflammatory cytokine in LPS-induced mice lymphocytes. The crude taurine was extracted using water solvent maceration of the sea slug’s mucus. Culture of mice splenocytes in RPMI-1640 medium underwent for 5 days and they were then analysed by flow cytometry. As the result, crude taurine extracts substantially suppressed NF-kB expression in T cells, found in all concentrations. The best inhibitory effect was attributed to 500 μg/mL, which was significantly different over control groups (p<0.05). Additionally, the extract inhibited expression of CD4+ IFN-γ + pro-inflammatory cytokines at 500 μg/mL• being stronger than inhibition by control (p<0.05), while it improved expression of CD4+IL-10+ anti-inflammatory cytokines at 500 μg/mL compared with control (p<0.05). Our experimental results indicated potential use of taurine crude extract isolated from sea slug Paraonchidium as anti-inflammatory agent from marine source.

Characterization of Molecular Species and Anti-Inflammatory Activity of Purified Phospholipids from Antarctic Krill Oil.

The phospholipids (PLs) from Antarctic krill oil were purified (>97.2%) using adsorption column chromatography. Forty-nine PL molecular species were characterized by ultrahigh-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Most of molecular species contained eicosapentaenoic acid (EPA, 20:5), docosahexaenoic acid (DHA, 22:6), docosapentaenoic acid (DPA, 22:5), and arachidonic acid (AA, 20:4). Notably, a special species PC (20:5/22:6) (1298.17 nmol/g) and many ether PLs were detected. The Antarctic krill PL liposome (IC50 = 0.108 mg/mL) showed better anti-inflammatory activity than crude Antarctic krill oil (IC50 = 0.446 mg/mL). It could block NF-κB signaling pathway via suppression of IκB-α degradation and p65 activation and dose-dependently reduce the cellular content of inflammatory mediators including nitric oxide (NO), reactive oxygen species (ROS), and inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In addition, it can suppress carrageenan-induced mouse paw swelling. Results from the present study could provide a reference for better evaluation of nutritional and medicinal values of Antarctic krill oil.

N-acetyl-chitooligosaccharide attenuates inflammatory responses by suppression of NF-κB signaling, MAPK and NLRP3 inflammasome in macrophages

Abstract N-acetyl-chitooligosaccharide (NACOS), prepared from the shells of shrimp and crabs, has displayed various biological activities. As prebiotics and dietary supplements, these bioactive materials can be incorporated into functional foods and beverages. This study aimed to explore the anti-inflammatory effects and associated mechanism of NACOS in macrophages. We found that NACOS reduced the levels of inflammation-related cytokines in lipopolysaccharide stimulated RAW264.7 macrophages. NACOS attenuated NF-κB activation not only through the repressed IKK phosphorylation but also the blunted nuclear translocation of NF-κB subunit p65. Additionally, NACOS inhibited the level of p-JNK. Moreover, NACOS promoted NLRP3 degradation, accompanied by the blockade of caspase-1 activation and IL-1β maturation in human THP-1 cells. This study demonstrated for the first time that NACOS has a negative effect against NLRP3 protein and NLRP3 inflammasome activation. It was suggested that the pharmacological intervention of inflammatory responses by NACOS should be a promising approach against inflammation-related clinical complications.