Sargachromenol Purified from Sargassum horneri Inhibits Inflammatory Responses via Activation of Nrf2/HO-1 Signaling in LPS-Stimulated Macrophages

In this study, we isolated sargachromenol (SC) from Sargassum horneri and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. SC did not show cytotoxicity at all concentrations and effectively increased the cell viability by reducing the nitric oxide (NO) and intracellular reactive oxygen species (ROS) production in LPS-stimulated RAW 264.7 macrophages. In addition, SC decreased the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and inflammatory mediators (iNOS and COX-2). Moreover, SC suppressed the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and mitogen-activated protein kinase (MAPK) signaling, whereas activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling in LPS-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effect of SC was abolished by the inhibition of HO-1 in LPS-stimulated RAW 264.7 macrophages. According to the results, this study suggests that the antioxidant capacity of SC leads to its anti-inflammatory effect and it potentially may be utilized in the nutraceutical and pharmaceutical sectors.     

Molecular Weight-Dependent Immunostimulative Activity of Low Molecular Weight Chitosan via Regulating NF-κB and AP-1 Signaling Pathways in RAW264.7 Macrophages

Chitosan and its derivatives such as low molecular weight chitosans (LMWCs) have been found to possess many important biological properties, such as antioxidant and antitumor effects. In our previous study, LMWCs were found to elicit a strong immunomodulatory response in macrophages dependent on molecular weight. Herein we further investigated the molecular weight-dependent immunostimulative activity of LMWCs and elucidated its mechanism of action on RAW264.7 macrophages. LMWCs (3 kDa and 50 kDa of molecular weight) could significantly enhance the mRNA expression levels of COX-2, IL-10 and MCP-1 in a molecular weight and concentration-dependent manner. The results suggested that LMWCs elicited a significant immunomodulatory response, which was dependent on the dose and the molecular weight. Regarding the possible molecular mechanism of action, LMWCs promoted the expression of the genes of key molecules in NF-κB and AP-1 pathways, including IKKβ, TRAF6 and JNK1, and induced the phosphorylation of protein IKBα in RAW264.7 macrophage. Moreover, LMWCs increased nuclear translocation of p65 and activation of activator protein-1 (AP-1, C-Jun and C-Fos) in a molecular weight-dependent manner. Taken together, our findings suggested that LMWCs exert immunostimulative activity via activation of NF-κB and AP-1 pathways in RAW264.7 macrophages in a molecular weight-dependent manner and that 3 kDa LMWC shows great potential as a novel agent for the treatment of immune suppression diseases and in future vaccines.     

Inhibitory Effect of N,N-Didesmethylgrossularine-1 on Inflammatory Cytokine Production in Lipopolysaccharide-Stimulated RAW 264.7 Cells

N,N-Didesmethylgrossularine-1 (DDMG-1), a compound with a rare α-carboline structure, was isolated from an Indonesian ascidian Polycarpa aurata as responsible for the observed inhibitory activity against TNF-α production in lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells. DDMG-1 inhibited the mRNA level of mTNF-α, IκB-α degradation, and binding of NF-κB to the target DNA site in LPS-stimulated RAW 264.7 cells. Moreover, DDMG-1 had an inhibitory effect on the production of IL-8, which is produced in CD14⁺-THP-1 cells stimulated by LPS. DDMG-1 is thus a promising drug candidate lead compound for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis.     

A Sulfated Polysaccharide from Saccharina japonica Suppresses LPS-Induced Inflammation Both in a Macrophage Cell Model via Blocking MAPK/NF-κB Signal Pathways In Vitro and a Zebrafish Model of Embryos and Larvae In Vivo

Inflammation is a complicated host-protective response to stimuli and toxic conditions, and is considered as a double-edged sword. A sulfated Saccharina japonica polysaccharide (LJPS) with a sulfate content of 9.07% showed significant inhibitory effects against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells and zebrafish. Its chemical and structural properties were investigated via HPLC, GC, FTIR, and NMR spectroscopy. In vitro experiments demonstrated that LJPS significantly inhibited the generation of nitric oxide (NO) and prostaglandin E₂ (PGE₂) via the downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and suppressed pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β production via the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways in LPS-induced RAW 264.7 cells. Moreover, LJPS showed strong protective effects against LPS-induced inflammatory responses in zebrafish, increasing the survival rate, reducing the heart rate and yolk sac edema size, and inhibiting cell death and the production of intracellular reactive oxygen species (ROS) and NO. Its convenience for large-scale production and significant anti-inflammatory activity indicated the potential application of LJPS in functional foods, cosmetics, and pharmaceutical industries.     

Anti-Inflammatory Effects of Sulfated Polysaccharide from Sargassum swartzii in Macrophages via Blocking TLR/NF-Κb Signal Transduction

This study involves enzymatic extraction of fucoidan from Sargassum swartzii and further purification via ion-exchange chromatography. The chemical and molecular characteristics of isolated fucoidan is evaluated concerning its anti-inflammatory potential in RAW 264.7 macrophages under LPS induced conditions. Structural properties of fucoidan were assessed via FTIR and NMR spectroscopy. NO production stimulated by LPS was significantly declined by fucoidan. This was witnessed to be achieved via fucoidan acting on mediators such as iNOS and COX-2 including pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), with dose dependent down-regulation. Further, the effect is exhibited by the suppression of TLR mediated MyD88, IKK complex, ultimately hindering NF-κB and MAPK activation, proposing its therapeutic applications in inflammation related disorders. The research findings provide an insight in relation to the sustainable utilization of fucoidan from marine brown algae S. swartzii as a potent anti-inflammatory agent in the nutritional, pharmaceutical, and cosmeceutical sectors.     

Penicillinolide A: A New Anti-Inflammatory Metabolite from the Marine Fungus Penicillium sp. SF-5292

In the course of studies on bioactive metabolites from marine fungi, a new 10-membered lactone, named penicillinolide A (1) was isolated from the organic extract of Penicillium sp. SF-5292 as a potential anti-inflammatory compound. The structure of penicillinolide A (1) was mainly determined by analysis of NMR and MS data and Mosher’s method. Penicillinolide A (1) inhibited the production of NO and PGE₂ due to inhibition of the expression of iNOS and COX-2. Penicillinolide A (1) also reduced TNF-α, IL-1β and IL-6 production, and these anti-inflammatory effects were shown to be correlated with the suppression of the phosphorylation and degradation of IκB-α, NF-κB nuclear translocation, and NF-κB DNA binding activity. In addition, using inhibitor tin protoporphyrin (SnPP), a competitive inhibitor of HO activity, it was verified that the inhibitory effects of compound 1 on the production of pro-inflammatory mediators and NF-κB DNA binding activity were partially associated with HO-1 expression through Nrf2 nuclear translocation.     

Anti-Fine Dust Effect of Fucoidan Extracted from Ecklonia maxima Laves in Macrophages via Inhibiting Inflammatory Signaling Pathways

Brown seaweeds contain fucoidan, which has numerous biological activities. Here, the anti-fine-dust activity of fucoidan extracted from Ecklonia maxima, an abundant brown seaweed from South Africa, was explored. Fourier transmittance infrared spectroscopy, high-performance anion-exchange chromatography with pulsed amperometric detection analysis of the monosaccharide content, and nuclear magnetic resonance were used for the structural characterization of the polysaccharides. The toll-like receptor (TLR)-mediated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were evaluated. The results revealed that E. maxima purified leaf fucoidan fraction 7 (EMLF7), which contained the highest sulfate content, showed the best anti-inflammatory activity by attenuating the TLR-mediated NF-κB/MAPK protein expressions in the particulate matter-stimulated cells. This was solidified by the successful reduction of Prostaglandin E₂, NO, and pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β. The current findings confirm the anti-inflammatory activity of EMLF7, as well as the potential use of E. maxima as a low-cost fucoidan source due to its abundance. This suggests its further application as a functional ingredient in consumer products.     

Immunoenhancing Effects of Cyclina sinensis Pentadecapeptide through Modulation of Signaling Pathways in Mice with Cyclophosphamide-Induced Immunosuppression

Our study aimed to investigate the immune-enhancing mechanism of the pentadecapeptide (RVAPEEHPVEGRYLV) from Cyclina sinensis (SCSP) in a cyclophosphamide (CTX)-induced murine model of immunosuppression. Our results showed that SCSP treatment significantly increased mouse body weight, immune organ indices, and the production of serum IL-6, IL-1β, and tumor necrosis factor (TNF)-α in CTX-treated mice. In addition, SCSP treatment enhanced the proliferation of splenic lymphocytes and peritoneal macrophages, as well as phagocytosis of the latter in a dose-dependent manner. Moreover, SCSP elevated the phosphorylation levels of p38, ERK, JNK, PI3K and Akt, and up-regulated IKKα, IKKβ, p50 NF-κB and p65 NF-κB protein levels, while down-regulating IκBα protein levels. Our results indicate that SCSP has immune-enhancing activities, and that it can activate the MAPK/NF-κB and PI3K/Akt pathways to enhance immunity in CTX-induced immunosuppressed mice.     

Morphological and Proteomic Analyses Reveal that Unsaturated Guluronate Oligosaccharide Modulates Multiple Functional Pathways in Murine Macrophage RAW264.7 Cells

Alginate is a natural polysaccharide extracted from various species of marine brown algae. Alginate-derived guluronate oligosaccharide (GOS) obtained by enzymatic depolymerization has various pharmacological functions. Previous studies have demonstrated that GOS can trigger the production of inducible nitric oxide synthase (iNOS)/nitric oxide (NO), reactive oxygen species (ROS) and tumor necrosis factor (TNF)-α by macrophages and that it is involved in the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. To expand upon the current knowledge regarding the molecular mechanisms associated with the GOS-induced immune response in macrophages, comparative proteomic analysis was employed together with two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and Western blot verification. Proteins showing significant differences in expression in GOS-treated cells were categorized into multiple functional pathways, including the NF-κB signaling pathway and pathways involved in inflammation, antioxidant activity, glycolysis, cytoskeletal processes and translational elongation. Moreover, GOS-stimulated changes in the morphologies and actin cytoskeleton organization of RAW264.7 cells were also investigated as possible adaptations to GOS. This study is the first to reveal GOS as a promising agent that can modulate the proper balance between the pro- and anti-inflammatory immune responses, and it provides new insights into pharmaceutical applications of polysaccharides.     

Chitosan Oligosaccharides Alleviate Colitis by Regulating Intestinal Microbiota and PPARγ/SIRT1-Mediated NF-κB Pathway

Chitosan oligosaccharides (COS) have been shown to have potential protective effects against colitis, but the mechanism underlying this effect has not been fully elucidated. In this study, COS were found to significantly attenuate dextran sodium sulfate-induced colitis in mice by decreasing disease activity index scores, downregulating pro-inflammatory cytokines, and upregulating Mucin-2 levels. COS also significantly inhibited the levels of nitric oxide (NO) and IL-6 in lipopolysaccharide-stimulated RAW 264.7 cells. Importantly, COS inhibited the activation of the NF-κB signaling pathway via activating PPARγ and SIRT1, thus reducing the production of NO and IL-6. The antagonist of PPARγ could abolish the anti-inflammatory effects of COS in LPS-treated cells. COS also activated SIRT1 to reduce the acetylation of p65 protein at lysine 310, which was reversed by silencing SIRT1 by siRNA. Moreover, COS treatment increased the diversity of intestinal microbiota and partly restored the Firmicutes/Bacteroidetes ratio. COS administration could optimize intestinal microbiota composition by increasing the abundance of norank_f_Muribaculaceae, Lactobacillus and Alistipes, while decreasing the abundance of Turicibacte. Furthermore, COS could also increase the levels of propionate and butyrate. Overall, COS can improve colitis by regulating intestinal microbiota and the PPARγ/SIRT1-mediated NF-κB pathway.     

PTP1B Inhibitory and Anti-Inflammatory Effects of Secondary Metabolites Isolated from the Marine-Derived Fungus Penicillium sp. JF-55

Protein tyrosine phosphatase 1B (PTP1B) plays a major role in the negative regulation of insulin signaling, and is thus considered as an attractive therapeutic target for the treatment of diabetes. Bioassay-guided investigation of the methylethylketone extract of marine-derived fungus Penicillium sp. JF-55 cultures afforded a new PTP1B inhibitory styrylpyrone-type metabolite named penstyrylpyrone (1), and two known metabolites, anhydrofulvic acid (2) and citromycetin (3). Compounds 1 and 2 inhibited PTP1B activity in a dose-dependent manner, and kinetic analyses of PTP1B inhibition suggested that these compounds inhibited PTP1B activity in a competitive manner. In an effort to gain more biological potential of the isolated compounds, the anti-inflammatory effects of compounds 1–3 were also evaluated. Among the tested compounds, only compound 1 inhibited the production of NO and PGE₂, due to the inhibition of the expression of iNOS and COX-2. Penstyrylpyrone (1) also reduced TNF-α and IL-1β production, and these anti-inflammatory effects were shown to be correlated with the suppression of the phosphorylation and degradation of IκB-α, NF-κB nuclear translocation, and NF-κB DNA binding activity. In addition, using inhibitor tin protoporphyrin (SnPP), an inhibitor of HO-1, it was verified that the inhibitory effects of penstyrylpyrone (1) on the pro-inflammatory mediators and NF-κB DNA binding activity were associated with the HO-1 expression. Therefore, these results suggest that penstyrylpyrone (1) suppresses PTP1B activity, as well as the production of pro-inflammatory mediators via NF-κB pathway, through expression of anti-inflammatory HO-1.     

Amentadione from the Alga Cystoseira usneoides as a Novel Osteoarthritis Protective Agent in an Ex Vivo Co-Culture OA Model

Osteoarthritis (OA) remains a prevalent chronic disease without effective prevention and treatment. Amentadione (YP), a meroditerpenoid purified from the alga Cystoseira usneoides, has demonstrated anti-inflammatory activity. Here, we investigated the YP anti-osteoarthritic potential, by using a novel OA preclinical drug development pipeline designed to evaluate the anti-inflammatory and anti-mineralizing activities of potential OA-protective compounds. The workflow was based on in vitro primary cell cultures followed by human cartilage explants assays and a new OA co-culture model, combining cartilage explants with synoviocytes under interleukin-1β (IL-1β) or hydroxyapatite (HAP) stimulation. A combination of gene expression analysis and measurement of inflammatory mediators showed that the proposed model mimicked early disease stages, while YP counteracted inflammatory responses by downregulation of COX-2 and IL-6, improved cartilage homeostasis by downregulation of MMP3 and the chondrocytes hypertrophic differentiation factors Col10 and Runx2. Importantly, YP downregulated NF-κB gene expression and decreased phosphorylated IkBα/total IkBα ratio in chondrocytes. These results indicate the co-culture as a relevant pre-clinical OA model, and strongly suggest YP as a cartilage protective factor by inhibiting inflammatory, mineralizing, catabolic and differentiation processes during OA development, through inhibition of NF-κB signaling pathways, with high therapeutic potential.     

New 3-Acyl Tetramic Acid Derivatives from the Deep-Sea-Derived Fungus Lecanicillium fusisporum

Seven rare C3-C6 reduced 3-acyl tetramic acid derivatives, lecanicilliumins A–G (1–7), along with the known analogue cladosporiumin D (8), were obtained from the extract of the deep-sea-derived fungus Lecanicillium fusisporum GXIMD00542 within the family Clavipitacae. Their structures were elucidated by extensive spectroscopic data analysis, quantum chemistry calculations and chemical reaction. Compounds 1, 2, 5–7 exhibited moderate anti-inflammatory activity against NF-κB production using lipopolysaccharide (LPS) induced RAW264.7 cells with EC₅₀ values range of 18.49–30.19 μM.     

Anti-Inflammatory Effects of Mytilus coruscus Polysaccharide on RAW264.7 Cells and DSS-Induced Colitis in Mice

Considerable literature has been published on polysaccharides, which play a critical role in regulating the pathogenesis of inflammation and immunity. In this essay, the anti-inflammatory effect of Mytilus coruscus polysaccharide (MP) on lipopolysaccharide-stimulated RAW264.7 cells and a dextran sulfate sodium (DSS)-induced ulcerative colitis model in mice was investigated. The results showed that MP effectively promoted the proliferation of RAW264.7 cells, ameliorated the excessive production of inflammatory cytokines (TNF-α, IL-6, and IL-10), and inhibited the activation of the NF-κB signaling pathway. For DSS-induced colitis in mice, MP can improve the clinical symptoms of colitis, inhibit the weight loss of mice, reduce the disease activity index, and have a positive effect on the shortening of the colon caused by DSS, meliorating intestinal barrier integrity and lowering inflammatory cytokines in serum. Moreover, MP makes a notable contribution to the richness and diversity of the intestinal microbial community, and also regulates the structural composition of the intestinal flora. Specifically, mice treated with MP showed a repaired Firmicutes/Bacteroidetes ratio and an increased abundance of some probiotics like Anaerotruncus, Lactobacillus, Desulfovibrio, Alistipe, Odoribacter, and Enterorhabdus in colon. These data suggest that the MP could be a promising dietary candidate for enhancing immunity and protecting against ulcerative colitis.     

Molecular Mechanism of Anti-Inflammatory Activities of a Novel Sulfated Galactofucan from Saccharina japonica

Seaweed of Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. In this study, fucoidan (LJNF3), purified from S. japonica, was found to be a novel sulfated galactofucan, with the monosaccharide of only fucose and galactose in a ratio of 79.22:20.78, and with an 11.36% content of sulfate groups. NMR spectroscopy showed that LJNF3 consists of (1→3)-α-l-fucopyranosyl-4-SO₃ residues and (1→6)-β-d-galactopyranose units. The molecular mechanism of the anti-inflammatory effect in RAW264.7 demonstrated that LJNF3 reduced the production of nitric oxide (NO), and down-regulated the expression of MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKβ) signaling pathways. In a zebrafish experiment assay, LJNF3 showed a significantly protective effect, by reducing the cell death rate, inhibiting NO to 59.43%, and decreasing about 40% of reactive oxygen species. This study indicated that LJNF3, which only consisted of fucose and galactose, had the potential to be developed in the biomedical, food and cosmetic industries.     

3-Bromo-4,5-dihydroxybenzaldehyde Isolated from Polysiphonia morrowii Suppresses TNF-α/IFN-γ-Stimulated Inflammation and Deterioration of Skin Barrier in HaCaT Keratinocytes

Polysiphonia morrowii is a well-known red alga that has promising pharmacological characteristics. The current study evaluates the protective effect of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) isolated from P. morrowii on tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated inflammation and skin barrier deterioration in HaCaT keratinocytes. The anti-inflammatory effect of BDB in TNF-α/IFN-γ-stimulated HaCaT keratinocytes is evaluated by investigating nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, inflammatory cytokines, and chemokines. Further, the interaction between BDB and the skin barrier functions in stimulated HaCaT keratinocytes is investigated. The findings of the study reveal that BDB dose-dependently increases cell viability while decreasing intracellular reactive oxygen species (ROS) production. BDB downregulates the expression of inflammatory cytokines, interleukin (IL)-6, -8, -13, IFN-γ, TNF-α, and chemokines, Eotaxin, macrophage-derived chemokine (MDC), regulated on activation, normal T cells expressed and secreted (RANTES), and thymus and activation-regulated chemokine (TARC) by modulating the MAPK and NF-κB signaling pathways in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. Furthermore, BDB increases the production of skin hydration proteins and tight junction proteins in stimulated HaCaT keratinocytes by preserving skin moisturization and tight junction stability. These findings imply that BDB exhibits a protective ability against inflammation and deterioration of skin barrier via suppressing the expression of inflammatory signaling in TNF-α/IFN-γ-stimulated HaCaT keratinocytes.