A novel antimicrobial peptide acting via formyl peptide receptor 2 shows therapeutic effects against rheumatoid arthritis.

In oriental medicine, centipede Scolopendra subspinipes mutilans has long been used as a remedy for rheumatoid arthritis (RA), a well-known chronic autoimmune disorder. However, the molecular identities of its bioactive components have not yet been extensively investigated. We sought to identify bioactive molecules that control RA with a centipede. A novel antimicrobial peptide (AMP) (scolopendrasin IX) was identified from Scolopendra subspinipes mutilans. Scolopendrasin IX markedly activated mouse neutrophils, by enhancing cytosolic calcium increase, chemotactic cellular migration, and generation of superoxide anion in neutrophils. As a target receptor for scolopendrasin IX, formyl peptide receptor (FPR)2 mediates neutrophil activation induced by the AMP. Furthermore, scolopendrasin IX administration strongly blocked the clinical phenotype of RA in an autoantibody-injected model. Mechanistically, the novel AMP inhibited inflammatory cytokine synthesis from the joints and neutrophil recruitment into the joint area. Collectively, we suggest that scolopendrasin IX is a novel potential therapeutic agent for the control of RA via FPR2.

Promotion of formyl peptide receptor 1-mediated neutrophil chemotactic migration by antimicrobial peptides isolated from the centipede Scolopendra subspinipes mutilans.

We investigated the effects of two antimicrobial peptides (AMPs) isolated from Scolopendra subspinipes mutilans on neutrophil activity. Stimulation of mouse neutrophils with the two AMPs elicited chemotactic migration of the cells in a pertussis toxin-sensitive manner. The two AMPs also stimulated activation of ERK and Akt, which contribute to chemotactic migration of neutrophils. We found that AMP-stimulated neutrophil chemotaxis was blocked by a formyl peptide receptor (FPR) 1 antagonist (cyclosporin H); moreover the two AMPs stimulated the chemotactic migration of FPR1-expressing RBL-2H3 cells but not of vector-expressing RBL-2H3 cells. We also found that the two AMPs stimulate neutrophil migration in vivo, and that this effect is blocked in FPR1-deficient mice. Taken together, our results suggest that the two AMPs stimulate neutrophils, leading to chemotactic migration through FPR1, and the two AMPs will be useful for the study of FPR1 signaling and neutrophil activation. [BMB Reports 2016; 49(9): 520-525].

A novel natural compound from garlic (Allium sativum L.) with therapeutic effects against experimental polymicrobial sepsis.

Sepsis is a serious, life-threatening, infectious disease. In this study, we demonstrate that sucrose methyl 3-formyl-4-methylpentanoate (SMFM), a novel natural compound isolated from garlic (Allium sativum L.), markedly enhances survival rates by inhibiting lung inflammation in a cecal ligation and puncture (CLP) experimental polymicrobial sepsis model. SMFM strongly reduced bacterial colony units from peritoneal fluid in CLP mice by stimulating the generation of reactive oxygen species. Lymphocyte apoptosis in spleens from CLP mice was also markedly decreased by SMFM administration. SMFM also significantly inhibited the production of proinflammatory cytokines, such as TNF-α, interleukin-1ß (IL-1ß) and IL-6, in CLP mice. Lipopolysaccharide-stimulated production of TNF-α and IL-6 were also strongly inhibited by SMFM in mouse bone marrow-derived macrophages. Taken together, our results indicate that SMFM has therapeutic effects against polymicrobial sepsis that are mediated by enhanced microbial killing and blockage of cytokine storm.

Ethyl linoleate from garlic attenuates lipopolysaccharide-induced pro-inflammatory cytokine production by inducing heme oxygenase-1 in RAW264.7 cells.

In the present study, an essential fatty acid, ethyl linoleate (ELA), was isolated from the cloves of Allium sativum, and its structure was elucidated by NMR and GC-MS analyses. In vitro systems were used to evaluate the anti-inflammatory activity of ELA. Our results indicate that ELA down-regulates inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and thereby reduces nitric oxide (NO) and prostaglandin E2 production in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Immunofluorescent microscopy and western blot analyses revealed that these effects were mediated by impaired translocation of nuclear factor (NF)-κB and inhibition of phosphorylation of mitogen activated protein kinases. Furthermore, ELA exerted its anti-inflammatory activity by inducing heme oxygenase-1 (HO-1) expression, as determined by HO-1 small interfering (Si) RNA system. Si RNA-mediated knock-down of HO-1 abrogated the inhibitory effects of ELA on the production of NO, TNF-α, IL-1ß, and IL-6 in LPS-induced macrophages. These findings indicate the potential therapeutic use of ELA as an anti-inflammatory agent.

Therapeutic effects of α-iso-cubebenol, a natural compound isolated from the Schisandra chinensis fruit, against sepsis.

α-Iso-cubebenol, a natural compound isolated from the Schisandra chinensis fruit, strongly enhances survival rate in cecal ligation and puncture (CLP) challenge-induced sepsis. Mechanistically, α-iso-cubebenol markedly reduces viable bacteria in the peritoneal fluid and peripheral blood, by increasing production of superoxide anion. α-Iso-cubebenol also significantly attenuates widespread immune cell apoptosis in a mouse CLP sepsis model, and inhibits the production of proinflammatory cytokines including interleukin-1ß (IL-1ß) and IL-6 in CLP mice and lipopolysaccharide-stimulated splenocytes. Taken together, the results indicate that α-iso-cubebenol can reverse the progression of septic shock by triggering multiple protective downstream signaling pathways to enhance microbial killing and maintain organ function and leukocyte survival.

α-Iso-cubebene, a natural compound isolated from Schisandra chinensis fruit, has therapeutic benefit against polymicrobial sepsis.

α-Iso-cubebene, a natural compound isolated from Schisandra chinensis fruit, strongly enhanced survival rate in cecal ligation and puncture (CLP) challenge-induced sepsis. The mechanism involved the marked reduction of viable bacteria in the peritoneal fluid, by virtue of increased phagocytic activity and production of hydrogen peroxide. α-Iso-cubebene also significantly attenuated lung inflammation and widespread immune cell apoptosis in a mouse CLP sepsis model, and inhibited the production of proinflammatory cytokines including tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in CLP mice and lipopolysaccharide-stimulated splenocytes. The results indicate that α-iso-cubebene can reverse the progression of toxic shock by triggering multiple protective downstream signaling pathways to enhance microbial killing and maintain organ function and leukocyte survival.

A WKYMVm-containing combination elicits potent anti-tumor activity in heterotopic cancer animal model.

The development of efficient anti-cancer therapy has been a topic of intense interest for several decades. Combined administration of certain molecules and immune cells has been shown to be an effective form of anti-cancer therapy. Here, we examined the effects of administering an immune stimulating peptide (WKYMVm), 5-fluoro-uracil (5-FU), and mature dendritic cells (mDCs) against heterotopic cancer animal model. Administration of the triple combination strongly reduced tumor volume in CT-26-inoculated heterotopic cancer animal model. The induced anti-tumor activity was well correlated with FAS expression, caspase-3 activation, and cancer cell apoptosis. The triple combination treatment caused recruitment of CD8 T lymphocytes and natural killer (NK) cells into the tumor. The production of two cytokines, IFN-γ and IL-12, were strongly stimulated by administration of the triple combination. Depletion of CD8 T lymphocytes or NK cells by administration of anti-CD8 or anti-asialoGM1 antibody inhibited the anti-tumor activity and cytokine production of the triple combination. The triple combination strongly inhibited metastasis of colon cancer cells in a heterotopic cancer animal model as well as in a metastatic cancer animal model, and enhanced the survival rate of the mice model. Adoptive transfer of CD8 T lymphocytes and NK cells further increased the survival rate. Taken together, we suggest that the use of triple combination therapy of WKYMVm, 5-FU, and mDCs may have implications in solid tumor and metastasis treatment.

Identification of a novel compound that stimulates intracellular calcium increase and CXCL8 production in human neutrophils from Schisandra chinensis.

Schisandra chinensis has been regarded as a useful material in the preventive or treatment of several human diseases. The study of the Schisandra chinensis' molecular identity has been prioritized because it has been found to regulate cellular responses. Here, we examined the effects from various extracts of Schisandra chinensis to monitor the relative intracellular calcium increase in human neutrophils. We identified an active molecule and structural configuration of a new active compound (alpha-iso-cubebene), based on the discovery of a cubebene backbone using NMR, MS, and CD spectral data. In terms of its functional aspect, we observed that alpha-iso-cubebene strongly stimulated CXCL8 production in human neutrophils. Also, alpha-iso-cubebene-induced CXCL8 production was almost completely inhibited by the calcium chelator, EGTA, thus highlighting the role of calcium signaling in the process. Taken together, our results demonstrate that alpha-iso-cubebene is a novel natural compound which stimulates intracellular calcium signaling and CXCL8 production. As a result, alpha-iso-cubebene should be useful for the development of an immune-modulating agent.

Phospholipase C, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astroglioma cells.

We show that epigallocatechin-3 gallate (EGCG), a major component of green tea, stimulates phospholipase D (PLD) activity in U87 human astroglioma cells. EGCG-induced PLD activation was abolished by the phospholipase C (PLC) inhibitor and a lipase inactive PLC-gamma1 mutant, which is dependent on intracellular or extracellular Ca(2+), with the possible involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). EGCG induced translocation of PLC-gamma1 from the cytosol to the membrane and PLC-gamma1 interaction with PLD1. EGCG regulates the activity of PLD by modulating the redox state of the cells, and antioxidants reverse this effect. Moreover, EGCG-induced PLD activation was reduced by PKC inhibitors or down-regulation of PKC. Taken together, these results show that, in human astroglioma cells, EGCG regulates PLD activity via a signaling pathway involving changes in the redox state that stimulates a PLC-gamma1 [Ins(1,4,5)P(3)-Ca(2+)]-CaM kinase II-PLD pathway and a PLC-gamma1 (diacylglycerol)-PKC-PLD pathway.