Anti-inflammatory effects of benzenediamine derivate FC-98 on sepsis injury in mice via suppression of JNK, NF-κB and IRF3 signaling pathways.

FC-98, a synthesized benzenediamine derivate, was reported to regulate Toll-like receptor 9-induced activation of dendritic cells in our previous study. In this study, we evaluated the anti-inflammatory properties of FC-98 both in macrophages and in septic mouse models. By using enzyme-linked immunosorbent assay and real-time quantitative PCR, we found that FC-98 (6.25, 25 and 100µM) dose-dependently attenuated lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP-1) productions in RAW264.7 and primary mouse peritoneal macrophages. These inhibitory effects were not due to inducing cell cytotoxicity or altering LPS binding or TLR4 expression. Subsequently, western blot, immunofluorescence and luciferase reporter assays were used to investigate the underlying mechanisms of its anti-inflammatory activities. Results showed that FC-98 blocked activation of the c-Jun N-terminal kinase (JNK), nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) signaling pathways. In vivo, FC-98 (30 or 100mg/kg) was intraperitoneally administrated into LPS-induced or CLP-induced sepsis mice. It was observed to enhance the survival rate, inhibit pro-inflammatory mediator production, improve organ injuries and suppress bacterial propagation. In conclusion, FC-98 effectively inhibited macrophage inflammatory responses and ameliorated sepsis in mice through down-regulation of both MyD88 and TRIF-dependent pathways. These results suggest that FC-98 could be a promising therapeutic agent for inflammatory diseases.

A novel benzenediamine derivate rescued mice from experimental sepsis by attenuating proinflammatory mediators via IRAK4.

We designed and synthesized a novel benzenediamine derivate, FC-99, that was tested for its ability to protect mice from experimental sepsis. Moreover, we sought to determine whether FC-99 could control a bacterial infection and to clarify the mechanism by which FC-99 inhibited LPS-activated macrophages. The effects of FC-99 on inflammation were evaluated in two experimental sepsis models and in cultured macrophages. Microarrays and docking and molecular dynamics simulations were used to determine the target of FC-99. Surface plasmon resonance and molecular detection were performed to confirm the direct interaction of FC-99 with its target. FC-99 protected mice from experimental sepsis. The mice that received FC-99 exhibited a diminished inflammatory response, had a lower local bacterial burden, and experienced a significantly improved survival rate. Genome-wide transcriptional profiling of FC-99-treated macrophages identified IRAK4 as a drug-regulated gene involved in LPS/TLR4 signaling. A computer search and calculations indicated that IRAK4 directly interacted with FC-99. Surface plasmon resonance, IRAK4-regulated signaling pathway analysis, and gene expression profiling of proinflammatory mediators confirmed the direct interaction between FC-99 and IRAK4. FC-99 is a potential therapeutic molecule for sepsis that alleviated experimental sepsis by directly inhibiting IRAK4 activation, which represents a novel target for sepsis therapy.

Bis-N-norgliovictin, a small-molecule compound from marine fungus, inhibits LPS-induced inflammation in macrophages and improves survival in sepsis.

Sepsis is a highly lethal disorder characterized by systemic inflammation, and Toll-like receptor 4 (TLR4) in macrophages plays a crucial role in modulating innate immune response and outcome of sepsis. During the screening of natural products against inflammation, we identified bis-N-norgliovictin, a small-molecule compound isolated from marine-derived fungus, significantly inhibited lipopolysaccharide (LPS, ligand of TLR4)-induced tumor necrosis factor-α (TNF-α) production in RAW264.7 cells. In this study, we evaluated the effect of bis-N-norgliovictin on TLR4-mediated inflammation in mouse macrophages and LPS-induced sepsis model. In RAW264.7 and mouse peritoneal macrophages, bis-N-norgliovictin dose-dependently inhibited LPS-induced production of TNF-α, interleukin-6 (IL-6), interferon-ß (IFN-ß) and monocyte chemoattractant protein (MCP-1), but without suppressing cell viability. The anti-inflammatory effect was attributed to the down-regulation of TLR4-triggered myeloid differentiation primary response protein 88 (MyD88)-dependent and TIR-containing adapter inducing interferon-ß (TRIF)-dependent signaling pathways, including p38 and c-Jun N-terminal kinase (JNK) of mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) cascades. Importantly, bis-N-norgliovictin also protected mice against LPS-induced endotoxic shock. Intravenous injection of bis-N-norgliovictin 1h before LPS challenge dose-dependently inhibited LPS-induced increases in serum levels of TNF-α, IL-6, MCP-1 and IL-10, attenuated liver and lung injury and diminished M1 macrophage polarization in liver. Our results demonstrate that bis-N-norgliovictin exhibit potent anti-inflammatory effect both in vitro and in vivo. These findings suggest that bis-N-norgliovictin can be a useful therapeutic candidate for the treatment of sepsis and other inflammatory diseases.

Pre-treatment with IL-1ß enhances the efficacy of MSC transplantation in DSS-induced colitis.

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly due to their immunosuppressive properties. Although interleukin-1ß (IL-1ß) is one of the most important inflammatory mediators, growing evidence indicates that IL-1ß signaling elicits the immunosuppressive properties of MSCs. However, it remains unclear how IL-1ß signaling accomplishes this activity. Here, we focus on the therapeutic efficacy of IL-1ß-primed MSCs in the dextran sulfate sodium (DSS)-induced colitis model, in addition to the underlining mechanisms. We first found that IL-1ß-primed MSCs, without any observable phenotype change in vitro, significantly attenuated the development of DSS-induced murine colitis. Moreover, IL-1ß-primed MSCs modulated the balance of immune cells in the spleen and the mesenteric lymph nodes (MLNs) through elevating cyclooxygenase-2 (COX-2), IL-6 and IL-8 expression and influencing the polarization of peritoneal macrophages. Importantly, IL-1ß-primed MSCs possessed an enhanced ability to migrate to the inflammatory site of the gut via upregulation of chemokine receptor type 4 (CXCR4) expression. In summary, IL-1ß-primed MSCs have improved efficacy in treating DSS-induced colitis, which at least partly depends on their increased immunosuppressive capacities and enhanced migration ability.

Technetium-99 conjugated with methylene diphosphonate inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

1. In the present study, we investigated the effects of technetium-99 conjugated with methylene diphosphonate ((99)Tc-MDP), an agent used in radionuclide therapy, on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and explored the underlying mechanisms. 2. The murine macrophage cell line RAW264.7 and bone marrow-derived-macrophages from C57BL/6 mice (BMM) were used as models for osteoclastogenesis in vitro. The expression of some key factors in RANKL (50 ng/mL)-induced osteoclastogenesis in RAW264.7 cells was investigated by flow cytometry and real-time reverse transcription-polymerase chain reaction (RT-PCR). To detect multinucleated osteoclast formation, RAW264.7 cells were induced with RANKL for 4 days, whereas BMM were induced by 50 ng/mL RANKL and 20 ng/mL macrophage colony-stimulating factor for 7 days, before being stained with tartrate-resistant acid phosphatase. 3. Osteoclastogenesis was evaluated using the osteoclast markers CD51, matrix metalloproteinase (MMP)-9 and cathepsin K. At 0.01 µg/mL, (99)Tc-MDP significantly inhibited RANKL-induced osteoclastogenesis without any cytotoxicity. In addition, (99)Tc-MDP abolished the appearance of multinucleated osteoclasts. 4. Real-time RT-PCR analysis of transcription factor expression revealed that (99)Tc-MDP inhibited the expression of c-Fos and nuclear factor of activated T cells. In addition, (99)Tc-MDP inhibited the expression of the inflammatory factors interleukin (IL)-6, tumour necrosis factor-α and IL-1ß. Finally, (99)Tc-MDP inhibited the activation of mitogen-activated protein kinases in RAW264.7 cells following RANKL stimulation. 5. In conclusion, (99)Tc-MDP possesses anti-osteoclastogenic activity against RANKL-induced osteoclast formation.

Chaetoglobosin Fex from the marine-derived endophytic fungus inhibits induction of inflammatory mediators via Toll-like receptor 4 signaling in macrophages.

Chaetoglobosin Fex (Cha Fex), a cytochalasan-based alkaloid, was isolated from marine-derived endophytic fungus Chaetomium globosum QEN-14. The knowledge of its biological function is still limited. We investigated the effects and mechanism of Cha Fex on inflammatory mediators via Toll-like receptor 4 (TLR4) signaling in macrophages. Lipopolysaccharide (LPS), TLR4 ligand, was therefore designed to active TLR4 signaling pathway, and Cha Fex significantly inhibited the LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in peritoneal macrophages and murine macrophage cell line RAW264.7. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detection also found that Cha Fex down-regulated the mRNA expressions of these pro-inflammtory cytokines. Moreover, Cha Fex significantly attenuated the LPS-stimulated degradation of inhibitory kappa B-alpha and the subsequent translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) to the nucleus. Cha Fex also reduced the phosphorylations of extracellular-signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2). Furthermore, we confirmed that Cha Fex didn't affect LPS binding to the RAW264.7 cells and human monocytes, while Cha Fex was able to inhibit the increase of membrane-associated CD14 (mCD14) expression both on RAW cells and human monocytes induced by LPS to a certain degree. These results suggest that the anti-inflammatory property of Cha Fex may be attributed to NF-κB inhibition as well as the negative regulation of ERK1/2, p38, and JNK1/2 phosphorylations. On the other hand, these inhibitory effects may also be due to the blocking of mCD14 expression.

Effects of dietary chitosan and Bacillus subtilis on the growth performance, non-specific immunity and disease resistance of cobia, Rachycentron canadum.

The present study was performed to investigate the effects of various levels of dietary Bacillus subtilis and chitosan on the growth performance, non-specific immunity and protection against Vibrio harveyi infection in cobia, Rachycentron canadum. Fish were fed with the control diet and six different experimental diets containing three graded levels of B. subtilis at 2 × 10(10) CFU g(-1) (0.0, 1.0, 2.0 g kg(-1) diet) for each of two levels of chitosan (3.0 and 6.0 g kg(-1) diet). The results of 8 weeks feeding trial showed that the survival rate ranged from 81.3% to 84.0% with no significant difference (P > 0.05). The SGR (%) in the fish fed with dietary treatments was significantly higher than that of the control fish except diet 6 group with 2.0 g kg(-1)B. subtilis and 3.0 g kg(-1) chitosan. The serum lysozyme activities were significantly higher in 6.0 g kg(-1) chitosan groups and no significant differences were observed among B. subtilis levels. The serum ACP activities were significantly higher in 3.0 g kg(-1) chitosan groups at 0.0 and 1.0 g kg(-1)B. subtilis levels; at low chitosan level, the cobia fed diets with 1.0 g kg(-1)B. subtilis had significantly higher serum ACP activity, but at high chitosan level, the cobia fed diets with 2.0 g kg(-1)B. subtilis had significantly higher serum ACP activity. The phagocytosis and respiratory burst activity in the fish fed with dietary treatments was significantly higher than that of the control fish except diet 3 group with 6.0 g kg(-1) chitosan. Moreover, fish fed the diet containing 2.0 g kg(-1)B. subtilis and 6.0 g kg(-1) chitosan had significantly higher post-challenge survival on the 7th day following V. harveyi infection and post-challenge survival showed clearly the synergistic effect of chitosan and B. subtilis. Based on these results, the combination of 1.0 g kg(-1)B. subtilis and 6.0 g kg(-1) chitosan is optimal for the growth, innate immunity and disease resistance of cobia with an 8-week oral administration.

Black soot and the survival of Tibetan glaciers.

We find evidence that black soot aerosols deposited on Tibetan glaciers have been a significant contributing factor to observed rapid glacier retreat. Reduced black soot emissions, in addition to reduced greenhouse gases, may be required to avoid demise of Himalayan glaciers and retain the benefits of glaciers for seasonal fresh water supplies.