Palmitoylethanolamide Modulation of Microglia Activation: Characterization of Mechanisms of Action and Implication for Its Neuroprotective Effects

Palmitoylethanolamide (PEA) is an endogenous lipid produced on demand by neurons and glial cells that displays neuroprotective properties. It is well known that inflammation and neuronal damage are strictly related processes and that microglia play a pivotal role in their regulation. The aim of the present work was to assess whether PEA could exert its neuroprotective and anti-inflammatory effects through the modulation of microglia reactive phenotypes. In N9 microglial cells, the pre-incubation with PEA blunted the increase of M1 pro-inflammatory markers induced by lipopolysaccharide (LPS), concomitantly increasing those M2 anti-inflammatory markers. Images of microglial cells were processed to obtain a set of morphological parameters that highlighted the ability of PEA to inhibit the LPS-induced M1 polarization and suggested that PEA might induce the anti-inflammatory M2a phenotype. Functionally, PEA prevented Ca²⁺ transients in both N9 cells and primary microglia and antagonized the neuronal hyperexcitability induced by LPS, as revealed by multi-electrode array (MEA) measurements on primary cortical cultures of neurons, microglia, and astrocyte. Finally, the investigation of the molecular pathway indicated that PEA effects are not mediated by toll-like receptor 4 (TLR4); on the contrary, a partial involvement of cannabinoid type 2 receptor (CB2R) was shown by using a selective receptor inverse agonist.     

Syntheses and properties of the PET‐co‐PEA copolyester

An aliphatic‐aromatic random‐block copolyester of poly(ethylene terephthalate) (PET), and poly(enthylene adipate) (PEA), PET‐co‐PEA, was synthesized via melt polycondensation. The chemical structure of the products were characterized by two kinds of spectroscopic techniques (Fourier transform infrared and ¹H‐NMR). The thermal properties of the copolyester were characterized by thermogravimetry analysis, differential scanning calorimetry, wide‐angle X‐ray diffraction, and polarized optical microscopy. It was found that the crystallization ability, melting point, glass transition temperature of the random‐block coplyester decreased apparently. Meanwhile, the tensile strength and hydrolysis performance were measured as well. The result showed that the random‐block copolyesters PET‐co‐PEA displayed excellent properties in elasticity and strength. In addition, the potential degradability was found in hydrolysis measurement. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44967.     

Palmitoylethanolamide Inhibits Glutamate Release in Rat Cerebrocortical Nerve Terminals

The effect of palmitoylethanolamide (PEA), an endogenous fatty acid amide displaying neuroprotective actions, on glutamate release from rat cerebrocortical nerve terminals (synaptosomes) was investigated. PEA inhibited the Ca²⁺-dependent release of glutamate, which was triggered by exposing synaptosomes to the potassium channel blocker 4-aminopyridine. This release inhibition was concentration dependent, associated with a reduction in cytosolic Ca²⁺ concentration, and not due to a change in synaptosomal membrane potential. The glutamate release-inhibiting effect of PEA was prevented by the Cav2.1 (P/Q-type) channel blocker ω-agatoxin IVA or the protein kinase A inhibitor H89, not affected by the intracellular Ca²⁺ release inhibitors dantrolene and {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157, and partially antagonized by the cannabinoid CB1 receptor antagonist AM281. Based on these results, we suggest that PEA exerts its presynaptic inhibition, likely through a reduction in the Ca²⁺ influx mediated by Cav2.1 (P/Q-type) channels, thereby inhibiting the release of glutamate from rat cortical nerve terminals. This release inhibition might be linked to the activation of presynaptic cannabinoid CB1 receptors and the suppression of the protein kinase A pathway.     

O‐(Triazolyl)methyl Carbamates as a Novel and Potent Class of Fatty Acid Amide Hydrolase (FAAH) Inhibitors

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O‐aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O‐(1,2,3‐triazol‐4‐yl)methyl carbamate derivatives were designed and synthesized exploiting a copper‐ catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure–activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC₅₀ values in the single‐digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug‐like properties.     

Synthesis and Antimicrobial Activity of Short Analogues of the Marine Antimicrobial Peptide Turgencin A: Effects of SAR Optimizations,Cys-Cys Cyclization and Lipopeptide Modifications

We have synthesised short analogues of the marine antimicrobial peptide Turgencin A from the colonial Arctic ascidian Synoicum turgens. In this study, we focused on a central, cationic 12-residue Cys-Cys loop region within the sequence. Modified (tryptophan- and arginine-enriched) linear peptides were compared with Cys-Cys cyclic derivatives, and both linear and Cys-cyclic peptides were N-terminally acylated with octanoic acid (C₈), decanoic acid (C₁₀) or dodecanoic acid (C₁₂). The highest antimicrobial potency was achieved by introducing dodecanoic acid to a cyclic Turgencin A analogue with low intrinsic hydrophobicity, and by introducing octanoic acid to a cyclic analogue displaying a higher intrinsic hydrophobicity. Among all tested synthetic Turgencin A lipopeptide analogues, the most promising candidates regarding both antimicrobial and haemolytic activity were C₁₂-cTurg-1 and C₈-cTurg-2. These optimized cyclic lipopeptides displayed minimum inhibitory concentrations of 4 µg/mL against Staphylococcus aureus, Escherichia coli and the fungus Rhodothorula sp. Mode of action studies on bacteria showed a rapid membrane disruption and bactericidal effect of the cyclic lipopeptides. Haemolytic activity against human erythrocytes was low, indicating favorable selective targeting of bacterial cells.     

Development of Fragment‐Based n‐FABS NMR Screening Applied to the Membrane Enzyme FAAH

Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n‐fluorine atoms for biochemical screening (n‐FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n‐FABS to the discovery of novel fragment hits, targeting the membrane‐bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP‐FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n‐FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM –μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment‐based functional assay to a membrane protein.     

Effect of decrosslinking and annealing on interpenetrating polymer networks prepared from poly(ethyl acrylate)/polystyrene combinations

Interpenetrating polymer networks, IPN polymers, were prepared from poly(ethyl acrylate), PEA, and polystyrene, PS. Part or all of the crosslinking monomer was acrylic acid anhydride, AAA, which was subsequently hydrolysed and the samples annealed. Modulus-temperature, modulus-time (during annealing) determinations, and transmission electron microscopy were performed on the freshly made samples, after hydrolysis, and after annealing. The studies show that the phase continuity of the PS component is increased relative to the PEA component on annealing, and the separation of the phases becomes more pronounced. A new morphology was observed with the totally decrosslinked, chemically induced blend, with the PEA showing both a continuous and a discontinuous aspect.     

Rheological and mechanical properties of poly (ethylene acrylic acid) and low density polyethylene blends

Rheological properties of poly (ethylene‐acrylic acid) (PEA) and low density poly ethylene (LDPE) blends having varied amounts of LDPE from 0 to 100% have been evaluated at different temperatures (115, 120, and 130°C) and shear rates (61.33–613.30 s^?1) using a Monsanto processability tester. A reduction in the melt viscosity of the PEA/LDPE blends was noticed with increasing the shear rate. The observed positive deviation in the experimental melt viscosities of the blends is an indication of the synergy present in the blends during melt processing. The activation energy (E_a) of flow calculated using Arrhenius relation for PEA, LDPE, and their respective blends lies in the range 29.98–40.56 kJ mol^?1. The experimental activation energy of flow of the blends was higher than that obtained from the additivity rule. Highest activation energy was noticed for the blends containing 60–80% by weight of LDPE in PEA/LDPE blends, which is an indication for the miscibility of the blends at these ratios. The physicomechanical properties such as density, tensile behavior, tear strength, and hardness (Shore A) of PEA, LDPE, and their blends have been evaluated as a function of varying amounts of LDPE. The obtained physicomechanical properties of the PEA/LDPE blends lie in between that of pure polymers. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Tensile yield properties of starch-filled poly(ester amide) materials

Composite materials were prepared with granular corn starch (CS) or potato starch (PS) and poly(ester amide) resin (PEA), with starch volume fractions (?) up to 0.40. Tensile yield properties were evaluated at strain rates of 0.0017-0.05 s⁻¹. Yield stress of the CS-PEA materials increased with strain rate and starch content. The strain rate effect became more pronounced as the starch content increased. A crossover effect was observed with PS-PEA materials: at low strain rates, the yield stress decreased with increasing ?, and increased with ? at higher strain rates. This crossover suggests that the time scale of debonding in the PS-PEA materials is comparable to the time scale of the tension test. The addition of either CS or PS to PEA induced a distinct maximum in the stress-strain curve at yield compared to the neat PEA. Debonding of starch granules from the PEA matrix occurred at lower stresses in the PS-PEA materials than the CS-PEA. In PS-PEA, debonding occurred in bands similar in appearance to shear bands throughout the tensile specimen. After yielding, the cross-section area decreased as the debonded zones coalesced. In the CS-PEA materials, debonding zones were more diffuse, and a distinct neck formed at yield. Yield stress data for the CS-PEA materials could be shifted with respect to strain rate to construct a master curve, indicating that yield properties at these strain rates were determined by the matrix response rather than debonding as observed in other starch-filled materials.     

Barley β-Glucans-Containing Food Enhances Probiotic Performances of Beneficial Bacteria

Currently, the majority of prebiotics in the market are derived from non-digestible oligosaccharides. Very few studies have focused on non-digestible long chain complex polysaccharides in relation to their potential as novel prebiotics. Cereals β-glucans have been investigated for immune-modulating properties and beneficial effects on obesity, cardiovascular diseases, diabetes, and cholesterol levels. Moreover, β-glucans have been reported to be highly fermentable by the intestinal microbiota in the caecum and colon, and can enhance both growth rate and lactic acid production of microbes isolated from the human intestine. In this work, we report the effects of food matrices containing barley β-glucans on growth and probiotic features of four Lactobacillus strains. Such matrices were able to improve the growth rate of the tested bacteria both in unstressed conditions and, importantly, after exposure to in vitro simulation of the digestive tract. Moreover, the effect of β-glucans-containing food on bacterial adhesion to enterocyte-like cells was analyzed and a positive influence on probiotic-enterocyte interaction was observed.     

Preparation of nano-TiO2 from TiCl4 by dialysis hydrolysis

The hydrolysis of TiCl₄ was achieved by using a dialysis membrane to make H⁺ and Cl⁻ penetrate out slowly, which produced hydrogel. By this method, no reactant is required and no by-product occurs. The hydrogel was dried by different methods and calcined to obtain nano-TiO₂ powder. The samples were characterized by transmission electron microscope (TEM), X-ray diffraction (XRD), BET surface area and pore size distribution. For comparison, TiCl₄ was also hydrolyzed to prepare nano-TiO₂ by other methods such as heating and adding ammonia. The experiments indicate that hydrolysis and drying have important effects on the properties of product. The hydrogel obtained by dialysis hydrolysis is anatase with a higher phase transformation temperature in calcination and the gel washed with ethanol has better porosity and bigger specific area than the products prepared by other hydrolysis methods and the same washing. In this paper, the drying methods of washing with ethanol and azeotropic distillation with n-butanol were improved by an ensuing rewashing with cyclohexane containing a little of surfactant Span-80 and chemical dehydration with acetic oxide, which further improve the porosity and surface area of nano-TiO₂ powder.     

Studies on blends of melt‐processable liquid crystalline polymers and thermoplastics. I. Blend of polyesteramide with polyethylene

The processability characteristics, physicomechanical properties, and thermal decomposition characteristics of blends of low‐density polyethylene (LDPE) and polyesteramide (PEA), a thermotropic liquid crystalline polymer, were studied using various analytical techniques. Studies on a Brabender Plasticorder at temperatures ranging from 170 to 230°C showed good melt processability for the blends. The melt rheology of the blends containing 0–15% of PEA at 170°C was studied using a capillary rheometer (Goettfert) fitted with a circular die (L/D = 30/1) at apparent shear rates ranging from 12 to 2300 s⁻¹. The samples containing PEA showed a comparatively lower die swell at high shear rates. X‐ray diffraction measurements showed a reduction in crystallinity of LDPE in the presence of 2–4% of PEA. Scanning electron microscopic evaluation of the morphology of the fractured surface of the blend showed some degree of orientation, but not to the level typical of LCPs. However, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) indicated significant improvement in the resistance to thermooxidative decomposition of LDPE modified with PEA. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 1811–1817, 2000     

The Critical Role of Membrane Cholesterol in Salmonella-Induced Autophagy in Intestinal Epithelial Cells

It was previously observed that plasma membrane cholesterol plays a critical role in the Salmonella-induced phosphatidylinositol 3-kinase-dependent (PI3K)-dependent anti-inflammatory response in intestinal epithelial cells (IECs). The PI3K/Akt pathway is associated with autophagy which has emerged as a critical mechanism of host defense against several intracellular bacterial pathogens. Plasma membrane contributes directly to the formation of early Atg16L1-positive autophagosome precursors. Therefore, this study aimed to investigate the role of plasma membrane cholesterol on the Salmonella-induced autophagy in IECs. By using methyl-beta-cyclodextrin (MBCD), it was demonstrated that disruption of membrane cholesterol by MBCD enhanced NOD2 and Atg16L1 proteins expression in membrane, and autophagic LC3II proteins expression and LC3 punctae in Salmonella-infected Caco-2 cells, which was counteracted by Atg16L1 siRNA. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) siRNA enhanced the Salmonella-induced activation of Akt in Caco-2 cells. However, inhibitors of Akt or extracellular signal-regulated kinases (ERK) had no significant effect on Salmonella-induced autophagy Beclin 1 or LC3 proteins expression. In conclusion, our study suggests that cholesterol accumulation in the plasma membrane at the entry site of Salmonella results in the formation of Salmonella-containing vacuole (SCV) and decreased autophagy. Our results offer mechanistic insights on the critical role of membrane cholesterol in the pathogenesis of Salmonella infection in intestinal epithelial cells and the therapeutic potential of its antagonists.     

Antioxidant,Anti-Tyrosinase and Anti-Inflammatory Activities of Oil Production Residues from Camellia tenuifloria

Camellia tenuifloria is an indigenous Camellia species used for the production of camellia oil in Taiwan. This study investigated for the first time the potential antioxidant, anti-tyrosinase and anti-inflammatory activities of oil production byproducts, specifically those of the fruit shell, seed shell, and seed pomace from C. tenuifloria. It was found that the crude ethanol extract of the seed shell had the strongest DPPH scavenging and mushroom tyrosinase inhibitory activities, followed by the fruit shell, while seed pomace was the weakest. The IC₅₀ values of crude extracts and fractions on monophenolase were smaller than diphenolase. The phenolic-rich methanol fraction of seed shell (SM) reduced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. It also repressed the expression of IL-1β, and secretion of prostaglandin E₂ (PGE₂) and IL-6 in response to LPS. SM strongly stimulated heme oxygenase 1 (HO-1) expression and addition of zinc protoporphyrin (ZnPP), a HO-1 competitive inhibitor, reversed the inhibition of NO production, indicating the involvement of HO-1 in its anti-inflammatory activity. The effects observed in this study provide evidence for the reuse of residues from C. tenuifloria in the food additive, medicine and cosmetic industries.     

Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes

The decriminalization and legalization of cannabis has paved the way for investigations into the potential of the use of phytocannabinoids (pCBs) as natural therapeutics for the treatment of human diseases. This growing interest has recently focused on rare (less abundant) pCBs that are non-psychotropic compounds, such as cannabigerol (CBG), cannabichromene (CBC), Δ⁹-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). Notably, pCBs can act via the endocannabinoid system (ECS), which is involved in the regulation of key pathophysiological processes, and also in the skin. In this study, we used human keratinocytes (HaCaT cells) as an in vitro model that expresses all major ECS elements in order to systematically investigate the effects of CBG, CBC, THCV and CBGA. To this end, we analyzed the gene and protein expression of ECS components (receptors: CB₁, CB₂, GPR55, TRPV1 and PPARα/γ/δ; enzymes: NAPE-PLD, FAAH, DAGLα/β and MAGL) using qRT-PCR and Western blotting, along with assessments of their functionality using radioligand binding and activity assays. In addition, we quantified the content of endocannabinoid(-like) compounds (AEA, 2-AG, PEA, etc.) using UHPLC-MS/MS. Our results demonstrated that rare pCBs modulate the gene and protein expression of distinct ECS elements differently, as well as the content of endocannabinoid(-like) compounds. Notably, they all increased CB_1/2 binding, TRPV1 channel stimulation and FAAH and MAGL catalytic activity. These unprecedented observations should be considered when exploring the therapeutic potential of cannabis extracts for the treatment of human skin diseases.     

Development of a pilot process for the production of alfalfa peptide isolate

Enzymatic membrane reactors are widely used to produce protein hydrolysates. During the past few years, leaf extracts have been recognised as a good source of high quality protein. The interest in alfalfa protein concentrate (APC), a hydrophobic protein with excellent functional and nutritional properties, is due to its abundance, to its amino acid composition and to its ribulose 1,5‐biphosphate carboxylase–oxygenase content. In order to use this potential protein source in various fields (food, pharmaceutical and cosmetic industries), a pilot process for APC hydrolysis in a continuous stirred tank membrane reactor (CSTMR) was carried out. At pH 9.5 and 40 °C, the hydrolysis of APC (30.6 g dm^?3) by Delvolase (2.4 g dm^?3) with a residence time of 180 min, gave a conversion of 759 g kg^?1 protein at steady state. Coupling the reactor with an inorganic ultrafiltration membrane (Carbosep M5) with a 10 kDa nominal molecular weight cut‐off (NMWCO), allowed production of a soluble and reproducible peptide permeate with 23 g dm^?3 of hydrolysed protein. Phenolic compounds, responsible for the brown colour of the permeate, were removed at pH 5.0 and room temperature by anion‐exchange chromatography using Amberlite IRA900Cl, with a yield of 920 g kg^?1. After electrodialysis and spray‐drying of the decolorised permeate, an alfalfa peptide isolate (API) was obtained. It was soluble over the full pH range and its amino acid composition was comparable to that of the FAO/WHO standard. It could be used as a protein supplement in human diets and other fields. Copyright © 2003 Society of Chemical Industry     

Anti-Inflammatory and Analgesic Activities of a Novel Biflavonoid from Shells of Camellia oleifera

Shells are by-products of oil production from Camellia oleifera which have not been harnessed effectively. The purpose of this research is to isolate flavonoid from shells of Camellia oleifera and evaluate its anti-inflammatory and analgesic effects. The flavonoid was identified as bimolecular kaempferol structure by UV, MS, ¹H NMR and ¹³C NMR spectra, which is a new biflavonoid and first found in Camellia oleifera. It showed dose-dependent anti-inflammatory activity by carrageenin-induced paw oedema in rats and croton oil induced ear inflammation in mice, and analgesic activity by hot plate test and acetic acid induced writhing. The mechanism of anti-inflammation of biflavonoid is related to both bradykinin and prostaglandins synthesis inhibition. The biflavonoid showed both central and peripheral analgesic effects different from aspirin, inhibition of the synthesis or action of prostaglandins may contribute to analgesic effect of biflavonoid. The biflavonoid significantly decreased malonaldehyde (MDA) and increased superoxidase dismutase (SOD) and Glutathione peroxidase (GSH-Px) activity in serum (p < 0.01), revealed strong free radical scavenging activity in vivo. It indicates the biflavonoid can control inflammation and pain by eliminating free radical so as to inhibit the mediators and decrease the prostaglandins. The biflavonoid can be used as a prospective medicine for inflammation and pain.     

Fatty Acid Amide Hydrolase (FAAH) Inhibition Modulates Amyloid-Beta-Induced Microglia Polarization

The ability of endocannabinoid (eCB) to change functional microglial phenotype can be explored as a possible target for therapeutic intervention. Since the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), may provide beneficial effects in mice model of Alzheimer’s disease (AD)-like pathology, we aimed at determining whether the FAAH inhibitor URB597 might target microglia polarization and alter the cytoskeleton reorganization induced by the amyloid-β peptide (Aβ). The morphological evaluation showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. URB597 treatment partially rescued the control phenotype of BV-2 cells when co-incubated with Aβ. Moreover, URB597 reduced both the increase of Rho protein activation in Aβ-treated BV-2 cells and the Aβ-induced migration of BV-2 cells, while an increase of Cdc42 protein activation was observed in all samples. URB597 also increased the number of BV-2 cells involved in phagocytosis. URB597 treatment induced the polarization of microglial cells towards an anti-inflammatory phenotype, as demonstrated by the decreased expression of iNOS and pro-inflammatory cytokines along with the parallel increase of Arg-1 and anti-inflammatory cytokines. Taken together, these data suggest that FAAH inhibition promotes cytoskeleton reorganization, regulates phagocytosis and cell migration processes, thus driving microglial polarization towards an anti-inflammatory phenotype.     

Carboxymethyl chitosan-graft-phosphatidylethanolamine: Amphiphilic matrices for controlled drug delivery

Modified carboxymethyl chitosan (CMC) containing phosphatidylethanolamine (PEA) groups were synthesized by a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated coupling reaction. The structure of the modified CMC exhibiting an amphiphilic character was analysed by FT-IR and ¹H NMR. CMC-g-PEA beads were prepared with sodium tripolyphosphate (TPP) by ionic-crosslinking. The beads sizes were in range from 800 to 1200 μm and encapsulation efficiencies of drug were more than 68%. The morphologies of CMC-g-PEA beads were examined with scanning electron microscopy (SEM). The release experiments were performed using ketoprofen as an hydrophobic model drug. The drug dissolution kinetics showed longer release times for CMC-g-PEA beads: 20 h (at pH 1.4) and 45 h (at pH 7.4). The amount of the drug release was much higher in acidic solution than in basic solution due to the swelling properties of the matrix at acidic pH. These results suggest that modified CMC with PEA may become a potential delivery system to control the release of hydrophobic drugs.