Anti-inflammatory actions of aspirin-triggered resolvin D1 (AT-RvD1) in bronchial epithelial cells stimulated by cigarette smoke extract.

Smoking causes several diseases such as chronic obstructive pulmonary disease (COPD). Aspirin-triggered-resolvin D1 (AT-RvD1) is a lipid mediator produced during the resolution of inflammation and demonstrates anti-inflammatory and pro-resolution effects in several inflammatory experimental models including in the airways. Here we evaluated the role of AT-RvD1 (100 nM) in bronchial epithelial cells (BEAS-2B) stimulated by cigarette smoke extract (CSE; 1%; 1 cigarette) for 24 h. CSE induced the productions of IL-1ß, TNF-α, IL-10, IL-4 and IFN-γ as well as the activations of NF-κB and STAT3 and the expression of ALX/FPR2 receptor. AT-RvD1 reduced the IL-1ß and TNF-α production and increased the production of IFN-γ. These effects were reversed BOC2, an antagonist of ALX/FPR2 receptor for AT-RvD1. The production of IL-4 and IL-10 were not altered by AT-RvD1. In addition, AT-RvD1 reduced the phosphorylation of NF-κB and STAT3 when compared to CSE-stimulated BEAS-2B cells. No alteration of ALX/FPR2 expression was observed by AT-RvD1 when compared to CSE group. In the human monocytic leukemia cell line, the relative number of copies of IL-1ß and IL-4 was significantly higher in CSE + AT-RvD1 group compared CSE group, however, the expression of M1 cytokine was more pronounced than M2 profile. AT-RvD1 could be an important target for the reduction of inflammation in the airways associated with smoking.

Biodegradable arginine based steroid-surfactants: Cationic green agents for hydrophobic ion-pairing.

The aim of this study was to evaluate the safety and efficacy for hydrophobic ion-pairing of surfactants based on arginine (Arg). The prepared Arg-cholesteryl ester (ACE) and Arg-diosgenyl ester (ADE) were characterized regarding solubility, pKa, critical micellar concentration (CMC), biodegradability as well as membrane- and aquatic toxicity using DOTAP as reference. The ability for hydrophobic ion-pairing was evaluated and the lipophilicity of formed complexes was determined. NMR, FT-IR and MS confirmed successful synthesis of Arg-surfactants. The slightly soluble single-charged Arg-surfactants (pH < pKa3 (ACE = 10.42 ± 0.52; ADE = 10.38 ± 0.27)) showed CMCs of 27.17 µM for ACE and 35.67 µM for ADE. CMCs of the sparingly soluble double-charged species (pH < pKa2 (ACE = 5.30 ± 0.20; ADE = 5.55 ± 0.06)) were determined at concentrations of ≥ 250 µM for ACE and ≥ 850 µM for ADE. The enzymatic- and environmental biodegradability was proven by an entire cleavage of Arg-surfactants within 24 h, whereas DOTAP remained stable. Arg-surfactants exhibited lower membrane- (> 2-fold) and aquatic toxicity (> 15-fold) than DOTAP. The complexes formed with Arg-surfactants and insulin showed higher lipophilicity than the DOTAP-complex. According to these results, Arg-surfactants might be a promising safe tool for the delivery of peptide drugs.

Discovery of thiosemicarbazone derivatives as effective New Delhi metallo-ß-lactamase-1 (NDM-1) inhibitors against NDM-1 producing clinical isolates.

New Delhi metallo-ß-lactamase-1 (NDM-1) is capable of hydrolyzing nearly all ß-lactam antibiotics, posing an emerging threat to public health. There are currently less effective treatment options for treating NDM-1 positive "superbug", and no promising NDM-1 inhibitors were used in clinical practice. In this study, structure-activity relationship based on thiosemicarbazone derivatives was systematically characterized and their potential activities combined with meropenem (MEM) were evaluated. Compounds 19bg and 19bh exhibited excellent activity against 10 NDM-positive isolate clinical isolates in reversing MEM resistance. Further studies demonstrated compounds 19bg and 19bh were uncompetitive NDM-1 inhibitors with Ki = 0.63 and 0.44 µmol/L, respectively. Molecular docking speculated that compounds 19bg and 19bh were most likely to bind in the allosteric pocket which would affect the catalytic effect of NDM-1 on the substrate meropenem. Toxicity evaluation experiment showed that no hemolysis activities even at concentrations of 1000 mg/mL against red blood cells. In vivo experimental results showed combination of MEM and compound 19bh was markedly effective in treating infections caused by NDM-1 positive strain and prolonging the survival time of sepsis mice. Our finding showed that compound 19bh might be a promising lead in developing new inhibitor to treat NDM-1 producing superbug.

Discovery of thiosemicarbazone derivatives as effective New Delhi metallo-β-lactamase-1 (NDM-1) inhibitors against NDM-1 producing clinical isolates

@#New Delhi metallo-β-lactamase-1 (NDM-1) is capable of hydrolyzing nearly all β-lactam antibiotics, posing an emerging threat to public health. There are currently less effective treatment options for treating NDM-1 positive “superbug”, and no promising NDM-1 inhibitors were used in clinical practice. In this study, structure–activity relationship based on thiosemicarbazone derivatives was systematically characterized and their potential activities combined with meropenem (MEM) were evaluated. Compounds 19bg and 19bh exhibited excellent activity against 10 NDM-positive isolate clinical isolates in reversing MEM resistance. Further studies demonstrated compounds 19bg and 19bh were uncompetitive NDM-1 inhibitors with Ki = 0.63 and 0.44 μmol/L, respectively. Molecular docking speculated that compounds 19bg and 19bh were most likely to bind in the allosteric pocket which would affect the catalytic effect of NDM-1 on the substrate meropenem. Toxicity evaluation experiment showed that no hemolysis activities even at concentrations of 1000 mg/mL against red blood cells. In vivo experimental results showed combination of MEM and compound 19bh was markedly effective in treating infections caused by NDM-1 positive strain and prolonging the survival time of sepsis mice. Our finding showed that compound 19bh might be a promising lead in developing new inhibitor to treat NDM-1 producing superbug.

D-Peptide analogues of Boc-Phe-Leu-Phe-Leu-Phe-COOH induce neovascularization via endothelial N-formyl peptide receptor 3.

N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune cells in response to pathogen-associated molecular patterns. Three FPRs have been identified in humans (FPR1-FPR3), characterized by different ligand properties, biological function and cellular distribution. Recent findings from our laboratory have shown that the peptide BOC-FLFLF (L-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor by binding to various angiogenic growth factors, including vascular endothelial growth factor-A165 (VEGF). Here we show that the all-D-enantiomer of L-BOC2 (D-BOC2) is devoid of any VEGF antagonist activity. At variance, D-BOC2, as well as the D-FLFLF and succinimidyl (Succ)-D-FLFLF (D-Succ-F3) D-peptide variants, is endowed with a pro-angiogenic potential. In particular, the D-peptide D-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebrafish embryos and adult mice. This activity is related to the capacity of D-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the effects exerted by D-Succ-F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in HUVECs. Finally, D-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion, our data demonstrate that the angiogenic activity of D-Succ-F3 is due to the engagement and activation of FPR3 expressed by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.

N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) inhibits the angiogenic activity of heparin-binding growth factors.

The peptides N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) and BOC-Met-Leu-Phe (BOC1) are widely used antagonists of formyl peptide receptors (FPRs), BOC2 acting as an FPR1/FPR2 antagonist whereas BOC1 inhibits FPR1 only. Extensive investigations have been performed by using these FPR antagonists as a tool to assess the role of FPRs in physiological and pathological conditions. Based on previous observations from our laboratory, we assessed the possibility that BOC2 may exert also a direct inhibitory effect on the angiogenic activity of vascular endothelial growth factor-A (VEGF-A). Our data demonstrate that BOC2, but not BOC1, inhibits the angiogenic activity of heparin-binding VEGF-A165 with no effect on the activity of the non-heparin-binding VEGF-A121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A165, thus competing for heparin interaction and preventing the binding of VEGF-A165 to tyrosine kinase receptor VEGFR2, its phosphorylation and downstream signaling. In addition, BOC2 inhibits the interaction of a variety of heparin-binding angiogenic growth factors with heparin, including fibroblast growth factor 2 (FGF2) whose angiogenic activity is blocked by the compound. Accordingly, BOC2 suppresses the angiogenic potential of human tumor cell lines that co-express VEGF-A and FGF2. Thus, BOC2 appears to act as a novel multi-heparin-binding growth factor antagonist. These findings caution about the interpretation of FPR-focusing experimental data obtained with this compound and set the basis for the design of novel BOC2-derived, FPR independent multi-target angiogenesis inhibitors.

BML-111 attenuats acute lung injury induced by intestine ischemia-reperfusion via inhibiting p38 MAPK/NF-κB signaling pathway / 实用医学杂志

Objective This study aims to investigate the effect of Lipoxin A4 receptor on acute lung injury (ALI) induced by intestine ischemia-reperfusion (IIR). Methods Thirty-two 8-week old SD rats were randomly divided into four groups: sham, intestine ischemia-reperfusion (IIR), IIR + BML111 (BML-111), Boc-2 + IIR +BML111 (Boc-2). BML-111 (1 mg/kg) was given intraperitoneally at the onset of reperfusion in the BML-111 and the Boc-2 group. Boc-2 (50 μg/kg) was given intraperitoneally after anesthesia in the Boc-2 group. Rats were subjected to superior mesenteric artery occlusion consisting of 45-min ischemia and 6-h reperfusion, and the sham laparotomy was served as controls. The lung pathology was assayed by the H&E staining. Lung water content was detected using dry/wet ratio. Concentrations of TNF-α, IL-1β, and IL-6 in lung tissue were determined by ELISA. The protein expression of p38 MAPK and NF-κB of lung was assayed by western blot. Results IIR induced serious ALI, with poor lung pathology and increased lung water content, elevation of TNF-α, IL-1β, and IL-6 levels in lung, accompanied with activation of p38 MAPK/NF-κB pathway. However, BML-111 could inhibit the activation of p38 MAPK/NF-κB pathway, leading to the reductions of TNF-α, IL-1β, and IL-6 in lung and attenuation of IIR-induced ALI. Conclusion BML-111 treatment could attenuate inflammation in lung after IIR injury via inactivating the p38 MAPK/NF-κB signaling pathway.

Involvement of the spinal NALP1 inflammasome in neuropathic pain and aspirin-triggered-15-epi-lipoxin A4 induced analgesia.

Neuroinflammation plays an important role in nerve-injury-induced neuropathic pain, but the explicit molecular mechanisms of neuroinflammation in neuropathic pain remain unclear. As one of the most critical inflammatory cytokines, interleukin-1ß (IL-1ß) has been regarded as broadly involved in the pathology of neuropathic pain. The inflammasome caspase-1 platform is one primary mechanism responsible for the maturation of IL-1ß. Lipoxins, a type of endogenous anti-inflammatory lipid, have proved to be effective in relieving neuropathic pain behaviors. The present study was designed to examine whether the inflammasome caspase-1 IL-1ß platform is involved in chronic constriction injury (CCI)-induced neuropathic pain and in lipoxin-induced analgesia. After rats were subjected to the CCI surgery, mature IL-1ß was significantly increased in the ipsilateral spinal cord, and the inflammasome platform consisting of NALP1 (NAcht leucine-rich-repeat protein 1), caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) was also activated in spinal astrocytes and neurons, especially at the superficial laminae of the spinal dorsal horn; The aspirin-triggered-15-epi-lipoxin A4 (ATL), which shares the potent actions of the endogenous lipoxins, was administered to the CCI rats. Repeated intrathecal injection with ATL markedly attenuated the CCI-induced thermal hyperalgesia and significantly inhibited NALP1 inflammasome activation, caspase-1 cleavage, and IL-1ß maturation. These results suggested that spinal NALP1 inflammasome was involved in the CCI-induced neuropathic pain and that the analgesic effect of ATL was associated with suppressing NALP1 inflammasome activation.

Folate-modified poly(2-ethyl-2-oxazoline) as hydrophilic corona in polymeric micelles for enhanced intracellular doxorubicin delivery.

The transmembrane transport of drug loaded micelles to intracellular compartment is quite crucial for efficient drug delivery. In the current study, we investigated the cellular internalization and anticancer activity of doxorubicin loaded micelles with folate modified stealthy PEOz corona. Folate-decorated micelles incorporating doxorubicin were characterized for particle size, degree of folate decoration, drug loading content and encapsulation efficiency, morphology, and surface charge. The targeting capability and cell viability were assessed using HeLa, KB, A549 and MCF-7/ADR cell lines. In vitro study clearly illustrated the folate receptor (FR) mediated targeting of FA modified micelles to FR-positive human HeLa, KB and MCF-7/ADR cells, while specific delivery to FR-negative A549 cells was not apparently increased at the same experimental conditions. Cytotoxicity assay showed 60% and 58% decrease in IC50 values for HeLa and KB cells, while only a slight decrease for A549 cells, following treatment with folate modified formulations. The enhanced intracellular delivery of FA modified micelles in MCF-7/ADR cells was also observed. In vivo antitumor tests revealed DOX entrapped FA-PEOz-PCL micelles effectively inhibited the tumor growth and reduced the toxicity to mice compared with free DOX. The current study showed that the targeted nano-vector improved cytotoxicity of DOX and suggested that this novel PEOz endowed stealthy micelle system held great promise in tumor targeted therapy.

Dual role of lipoxin A4 in pneumosepsis pathogenesis.

Lipoxin A4 (LXA4) is an endogenous lipid mediator with potent anti-inflammatory actions but its role in infectious processes is not well understood. We investigated the involvement of LXA4 and its receptor FPR2/ALX in the septic inflammatory dysregulation. Pneumosepsis was induced in mice by inoculation of Klebsiella pneumoniae. LXA4 levels and FPR2/ALX expression in the infectious focus as well as the effects of treatment with receptor agonists (LXA4 and BML-111) and antagonists (BOC-2 and WRW(4)) in early (1h) and late (24h) sepsis were studied. Sepsis induced an early increase in LXA4, FPR2/ALX lung expression, local and systemic infection and inflammation, and mortality. Treatment with BOC-2 in early sepsis increased leukocyte migration to the focus, and reduced bacterial load and dissemination. Inhibition of 5- and 15-lipoxygenase in early sepsis also increased leukocyte migration. Early treatment with WRW(4) and BOC-2 improved survival. Treatment with authentic LXA4 or BML-111 in early sepsis decreased cell migration and worsened the infection. In late sepsis, treatment with BOC-2 had no effect, but LXA4 improved the survival rate by reducing the excessive inflammatory response, this effect being abolished by pretreatment with BOC-2. Thus, the anti-inflammatory and pro-resolution mediator LXA4 and its receptor FPR2/ALX levels were increased in the early phase of sepsis, contributing to the septic inflammatory dysregulation. In addition, LXA4 has a dual role in sepsis and that its beneficial or harmful effects are critically dependent on the time. Therefore, a proper interference with LXA4 system may be a new therapeutic avenue to treat sepsis.

The development of a new class of inhibitors for betaine-homocysteine S-methyltransferase.

Betaine-homocysteine S-methyltransferase (BHMT) is an important zinc-dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. In the liver, BHMT performs to half of the homocysteine remethylation. In this study, we systematically investigated the tolerance of the enzyme for modifications at the "homocysteine" part of the previously reported potent inhibitor (R,S)-5-(3-amino-3-carboxy-propylsulfanyl)-pentanoic acid (1). In the new compounds, which are S-alkylated homocysteine derivatives, we replaced the carboxylic group in the "homocysteine" part of inhibitor 1 with different isosteric moieties (tetrazole and oxadiazolone); we suppressed the carboxylic negative charge by amidations; we enhanced acidity by replacing the carboxylate with phosphonic or phosphinic acids; and we introduced pyrrolidine steric constraints. Some of these compounds display high affinity toward human BHMT and may be useful for further pharmacological studies of this enzyme. Although none of the new compounds were more potent inhibitors than the reference inhibitor 1, this study helped to completely define the structural requirements of the active site of BHMT and revealed the remarkable selectivity of the enzyme for homocysteine.