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.     

Chitosan Nanoparticles Attenuate Hydrogen Peroxide-Induced Stress Injury in Mouse Macrophage RAW264.7 Cells

This study was carried out to investigate the protective effects of chitosan nanoparticles (CNP) against hydrogen peroxide (H₂O₂)-induced oxidative damage in murine macrophages RAW264.7 cells. After 24 h pre-incubation with CNP (25–200 μg/mL) and chitosan (CS) (50–200 μg/mL, as controls), the viability loss in RAW264.7 cells induced by H₂O₂ (500 μM) for 12 h was markedly restored in a concentration-dependent manner as measured by MTT assay (P < 0.05) and decreased in cellular LDH release (P < 0.05). Moreover, CNP also exerted preventive effects on suppressing the production of lipid peroxidation such as malondialdehyde (MDA) (P < 0.05), restoring activities of endogenous antioxidant including superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) (P < 0.05), along with increasing total antioxidant capacity (T-AOC) (P < 0.05). In addition, pre-incubation of CNP with RAW264.7 cells for 24 h resulted in the increase of the gene expression level of endogenous antioxidant enzymes, such as MnSOD and GSH-Px (P < 0.05). At the same concentration, CNP significantly decreased LDH release and MDA (P < 0.05) as well as increased MnSOD, GSH-Px, and T-AOC activities (P < 0.05) as compared to CS. Taken together, our findings suggest that CNP can more effectively protect RAW264.7 cells against oxidative stress by H₂O₂ as compared to CS, which might be used as a potential natural compound-based antioxidant in the functional food and pharmaceutical industries.     

Immunostimulative Activity of Low Molecular Weight Chitosans in RAW264.7 Macrophages

Chitosan and its derivatives such as low molecular weight chitosans (LMWCs) have been reported to exert many biological activities, such as antioxidant and antitumor effects. However, complex and molecular weight dependent effects of chitosan remain controversial and the mechanisms that mediate these complex effects are still poorly defined. This study was carried out to investigate the immunostimulative effect of different molecular weight chitosan in RAW264.7 macrophages. Our data suggested that two LMWCs (molecular weight of 3 kDa and 50 kDa) both possessed immunostimulative activity, which was dependent on dose and, at the higher doses, also on the molecular weight. LMWCs could significantly enhance the the pinocytic activity, and induce the production of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interferon-γ (IFN-γ), nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in a molecular weight and concentration-dependent manner. LMWCs were further showed to promote the expression of the genes including iNOS, TNF-α. Taken together, our findings suggested that LMWCs elicited significantly immunomodulatory response through up-regulating mRNA expression of proinflammatory cytokines and activated RAW264.7 macrophage in a molecular weight- and concentration-dependent manner.     

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.     

Total Synthesis and Anti-Inflammatory Bioactivity of (−)-Majusculoic Acid and Its Derivatives

The first total synthesis of marine natural product, (−)-majusculoic acid (1) and its seven analogs (9–15), was accomplished in three to ten steps with a yield of 3% to 28%. The strategy featured the application of the conformational controlled establishment of the trans-cyclopropane and stereochemical controlled bromo-olefination or olefination by Horner–Wadsworth–Emmons (HWE) reaction. The potential anti-inflammatory activity of the eight compounds (1 and 9–15) was evaluated by determining the nitric oxide (NO) production in the lipopolysaccharide (LPS)-induced mouse macrophages RAW264.7. (−)-Majusculoic acid (1), methyl majusculoate (9), and (1R,2R)-2-((3E,5Z)-6-bromonona-3,5-dien-1-yl)cyclopropane-1-carboxylic acid (12) showed significant effect with inhibition rates of 33.68%, 35.75%, and 43.01%, respectively. Moreover, they did not show cytotoxicity against RAW264.7 cells, indicating that they might be potential anti-inflammatory agents.     

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.     

Capgermacrenes A and B,Bioactive Secondary Metabolites from a Bornean Soft Coral,Capnella sp.

Two new bicyclogermacrenes, capgermacrenes A (1) and B (2), were isolated with two known compounds, palustrol (3) and litseagermacrane (4), from a population of Bornean soft coral Capnella sp. The structures of these metabolites were elucidated based on spectroscopic data. Compound 1 was found to inhibit the accumulation of the LPS-induced pro-inflammatory IL-1β and NO production by down-regulating the expression of iNOS protein in RAW 264.7 macrophages.     

Astaxanthin Exerts Immunomodulatory Effect by Regulating SDH-HIF-1α Axis and Reprogramming Mitochondrial Metabolism in LPS-Stimulated RAW264.7 Cells

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in cell membranes and mitochondria, which consist of the bilayer molecules. Targeting mitochondria to ameliorate inflammatory diseases by regulating mitochondrial metabolism has become possible and topical. Although AX has been shown to have anti-inflammatory effects in various cells, the mechanisms are quite different. In particular, the role of AX on mitochondrial metabolism in macrophages is still unknown. In this study, we investigated the effect of AX on mitochondria-mediated inflammation and its mechanisms in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. AX attenuated the mitochondrial O₂⁻ production and maintained the mitochondrial membrane potential, implying that AX preserved mitochondrial homeostasis to avoid LPS stimulation-induced mitochondrial dysfunction. Additionally, AX prevented the decrease in mitochondrial complexes I, II, and III, which were caused by LPS stimulation. Especially, AX inhibited the reduction in mitochondrial succinate dehydrogenase (SDH; complex II) activity and upregulated the protein and mRNA level of SDH complex, subunit B. Furthermore, AX blocked the IL-1β expression by regulating the SDH-HIF-1α axis and suppressed the energy shift from an OXPHOS phenotype to a glycolysis phenotype. These findings revealed important effects of AX on mitochondrial enzymes as well as on mitochondrial energy metabolism in the immune response. In addition, these raised the possibility that AX plays an important role in other diseases caused by SDH mutation and metabolic disorders.     

In Vitro and In Vivo Anti-Inflammatory Effects of Sulfated Polysaccharides Isolated from the Edible Brown Seaweed,Sargassum fulvellum

In the present study, the in vitro and in vivo anti-inflammatory effects of the sulfated polysaccharides isolated from Sargassum fulvellum (SFPS) were evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish. The results indicated that SFPS improved the viability of LPS-stimulated RAW 264.7 macrophages from 80.02 to 86.80, 90.09, and 94.62% at the concentration of 25, 50, and 100 µg/mL, respectively. Also, SFPS remarkably and concentration-dependently decreased the production levels of inflammatory molecules including nitric oxide (NO), tumor necrosis factor-alpha, prostaglandin E₂, interleukin-1 beta, and interleukin-6 in LPS-treated RAW 264.7 macrophages. In addition, SFPS significantly inhibited the expression levels of cyclooxygenase-2 and inducible nitric oxide synthase in LPS-treated RAW 264.7 macrophages. Furthermore, the in vivo test results indicated that SFPS improved the survival rate of LPS-treated zebrafish from 53.33 to 56.67, 60.00, and 70.00% at the concentration of 25, 50, and 100 µg/mL, respectively. In addition, SFPS effectively reduced cell death, reactive oxygen species, and NO levels in LPS-stimulated zebrafish. Taken together, these results suggested that SFPS possesses strong in vitro and in vivo anti-inflammatory activities, and could be used as an ingredient to develop anti-inflammatory agents in the functional food and pharmaceutical industries.     

New Ophiobolins from the Deep-Sea Derived Fungus Aspergillus sp. WHU0154 and Their Anti-Inflammatory Effects

Deep-sea fungi have become a new arsenal for the discovery of leading compounds. Here five new ophiobolins 1–5, together with six known analogues 6–11, obtained from a deep-sea derived fungus WHU0154. Their structures were determined by analyses of IR, HR-ESI-MS, and NMR spectra, along with experimental and calculated electronic circular dichroism (ECD) analysis. Pharmacological studies showed that compounds 4 and 6 exhibited obvious inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine macrophage RAW264.7 cells. Mechanical study revealed that compound 6 could inhibit the inducible nitric oxide synthase (iNOS) level in LPS-stimulated RAW264.7 cells. In addition, compounds 6, 9, and 10 could significantly inhibit the expression of cyclooxygenase 2 (COX 2) in LPS-induced RAW264.7 cells. Preliminary structure-activity relationship (SAR) analyses revealed that the aldehyde group at C-21 and the α, β-unsaturated ketone functionality at A ring in ophiobolins were vital for their anti-inflammatory effects. Together, the results demonstrated that ophiobolins, especially for compound 6, exhibited strong anti-inflammatory effects and shed light on the discovery of ophiobolins as new anti-inflammatory agents.     

Inhibition of Lipopolysaccharide-Induced Inflammatory Signaling by Soft Coral-Derived Prostaglandin A2 in RAW264.7 Cells

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria and causes inflammatory diseases. We searched MeOH extracts of collected marine organisms for inhibitors of LPS-induced nitric oxide (NO) production in RAW264.7 cells and identified prostaglandin A₂ (PGA₂) as an active compound from the MeOH extract of the soft coral Lobophytum sp. PGA₂ inhibited the production of NO and reduced the expression of inducible NO synthase (iNOS) in LPS-stimulated RAW264.7 cells. Although short preincubation with PGA₂ did not inhibit LPS-induced degradation and resynthesis of IκBα, the suppressive effect of PGA₂ was observed only after a prolonged incubation period prior to LPS treatment. In addition, PGA₂-inhibited NO production was negated by the addition of the EP4 antagonist L161982. Thus, PGA₂ was identified as an inhibitor of LPS-induced inflammatory signaling in RAW264.7 cells.     

Continuous Production of Biogenic Magnetite Nanoparticles by the Marine Bacterium Magnetovibrio blakemorei Strain MV-1T with a Nitrous Oxide Injection Strategy

Magnetotactic bacteria (MTB) produce magnetosomes, which are membrane-embedded magnetic nanoparticles. Despite their technological applicability, the production of magnetite magnetosomes depends on the cultivation of MTB, which results in low yields. Thus, strategies for the large-scale cultivation of MTB need to be improved. Here, we describe a new approach for bioreactor cultivation of Magnetovibrio blakemorei strain MV-1^T. Firstly, a fed-batch with a supplementation of iron source and N₂O injection in 24-h pulses was established. After 120 h of cultivation, the production of magnetite reached 24.5 mg∙L⁻¹. The maximum productivity (16.8 mg∙L⁻¹∙day⁻¹) was reached between 48 and 72 h. However, the productivity and mean number of magnetosomes per cell decreased after 72 h. Therefore, continuous culture in the chemostat was established. In the continuous process, magnetite production and productivity were 27.1 mg∙L⁻¹ and 22.7 mg∙L⁻¹∙day⁻¹, respectively, at 120 h. This new approach prevented a decrease in magnetite production in comparison to the fed-batch strategy.     

Ferroptosis Related Immunomodulatory Effect of a Novel Extracellular Polysaccharides from Marine Fungus Aureobasidium melanogenum

Marine fungi represent an important and sustainable resource, from which the search for novel biological substances for application in the pharmacy or food industry offers great potential. In our research, novel polysaccharide (AUM-1) was obtained from marine Aureobasidium melanogenum SCAU-266 were obtained and the molecular weight of AUM-1 was determined to be 8000 Da with 97.30% of glucose, 1.9% of mannose, and 0.08% galactose, owing to a potential backbone of α-D-Glcp-(1→2)-α-D-Manp-(1→4)-α-D-Glcp-(1→6)-(SO₃⁻)-4-α-D-Glcp-(1→6)-1-β-D-Glcp-1→2)-α-D-Glcp-(1→6)-β-D-Glcp-1→6)-α-D-Glcp-1→4)-α-D-Glcp-6→1)-[α-D-Glcp-4]₂₆→1)-α-D-Glcp and two side chains that consisted of α-D-Glcp-1 and α-D-Glcp-(1→6)-α-D-Glcp residues. The immunomodulatory effect of AUM-1 was identified. Then, the potential molecular mechanism by which AUM-1 may be connected to ferroptosis was indicated by metabonomics, and the expression of COX2, SLC7A11, GPX4, ACSL4, FTH1, and ROS were further verified. Thus, we first speculated that AUM-1 has a potential effect on the ferroptosis-related immunomodulatory property in RAW 264.7 cells by adjusting the expression of GPX4, regulated glutathione (oxidative), directly causing lipid peroxidation owing to the higher ROS level through the glutamate metabolism and TCA cycle. Thus, the ferroptosis related immunomodulatory effect of AUM-1 was obtained.     

Thallusin Quantification in Marine Bacteria and Algae Cultures

Thallusin, a highly biologically active, phytohormone-like and bacterial compound-inducing morphogenesis of the green tide-forming macroalga Ulva (Chlorophyta), was determined in bacteria and algae cultures. A sensitive and selective method was developed for quantification based on ultra-high-performance liquid chromatography coupled with electrospray ionization and a high-resolution mass spectrometer. Upon C₁₈ solid phase extraction of the water samples, thallusin was derivatized with iodomethane to inhibit the formation of Fe–thallusin complexes interfering with the chromatographic separation. The concentration of thallusin was quantified during the relevant phases of the bacterial growth of Maribacter spp., ranging from 0.16 ± 0.01 amol cell⁻¹ (at the peak of the exponential growth phase) to 0.86 ± 0.13 amol cell⁻¹ (late stationary phase), indicating its accumulation in the growth medium. Finally, we directly determined the concentration of thallusin in algal culture to validate our approach for monitoring applications. Detection and quantification limits of 2.5 and 7.4 pmol L⁻¹, respectively, were reached, which allow for quantifying ecologically relevant thallusin concentrations. Our approach will enable the surveying of thallusin in culture and in nature and will thus contribute to the chemical monitoring of aquaculture.