Palmitoylethanolamide dampens neuroinflammation and anxiety-like behavior in obese mice.

High-fat diet (HFD) consumption leads to obesity and a chronic state of low-grade inflammation, named metainflammation. Notably, metainflammation contributes to neuroinflammation due to the increased levels of circulating free fatty acids and cytokines. It indicates a strict interplay between peripheral and central counterparts in the pathogenic mechanisms of obesity-related mood disorders. In this context, the impairment of internal hypothalamic circuitry runs in tandem with the alteration of other brain areas associated with emotional processing (i.e., hippocampus and amygdala). Palmitoylethanolamide (PEA), an endogenous lipid mediator belonging to the N-acylethanolamines family, has been extensively studied for its pleiotropic effects both at central and peripheral level. Our study aimed to elucidate PEA capability in limiting obesity-induced anxiety-like behavior and neuroinflammation-related features in an experimental model of HFD-fed obese mice. PEA treatment promoted an improvement in anxiety-like behavior of obese mice and the systemic inflammation, reducing serum pro-inflammatory mediators (i.e., TNF-α, IL-1ß, MCP-1, LPS). In the amygdala, PEA increased dopamine turnover, as well as GABA levels. PEA also counteracted the overactivation of HPA axis, reducing the expression of hypothalamic corticotropin-releasing hormone and its type 1 receptor. Moreover, PEA attenuated the immunoreactivity of Iba-1 and GFAP and reduced pro-inflammatory pathways and cytokine production in both the hypothalamus and hippocampus. This finding, together with the reduced transcription of mast cell markers (chymase 1 and tryptase ß2) in the hippocampus, indicated the weakening of immune cell activation underlying the neuroprotective effect of PEA. Obesity-driven neuroinflammation was also associated with the disruption of blood-brain barrier (BBB) in the hippocampus. PEA limited the albumin extravasation and restored tight junction transcription modified by HFD. To gain mechanistic insight, we designed an in vitro model of metabolic injury using human neuroblastoma SH-SY5Y cells insulted by a mix of glucosamine and glucose. Here, PEA directly counteracted inflammation and mitochondrial dysfunction in a PPAR-α-dependent manner since the pharmacological blockade of the receptor reverted its effects. Our results strengthen the therapeutic potential of PEA in obesity-related neuropsychiatric comorbidities, controlling neuroinflammation, BBB disruption, and neurotransmitter imbalance involved in behavioral dysfunctions.

Palmitoylethanolamide: A Nutritional Approach to Keep Neuroinflammation within Physiological Boundaries-A Systematic Review.

Neuroinflammation is a physiological response aimed at maintaining the homodynamic balance and providing the body with the fundamental resource of adaptation to endogenous and exogenous stimuli. Although the response is initiated with protective purposes, the effect may be detrimental when not regulated. The physiological control of neuroinflammation is mainly achieved via regulatory mechanisms performed by particular cells of the immune system intimately associated with or within the nervous system and named "non-neuronal cells." In particular, mast cells (within the central nervous system and in the periphery) and microglia (at spinal and supraspinal level) are involved in this control, through a close functional relationship between them and neurons (either centrally, spinal, or peripherally located). Accordingly, neuroinflammation becomes a worsening factor in many disorders whenever the non-neuronal cell supervision is inadequate. It has been shown that the regulation of non-neuronal cells-and therefore the control of neuroinflammation-depends on the local "on demand" synthesis of the endogenous lipid amide Palmitoylethanolamide and related endocannabinoids. When the balance between synthesis and degradation of this bioactive lipid mediator is disrupted in favor of reduced synthesis and/or increased degradation, the behavior of non-neuronal cells may not be appropriately regulated and neuroinflammation exceeds the physiological boundaries. In these conditions, it has been demonstrated that the increase of endogenous Palmitoylethanolamide-either by decreasing its degradation or exogenous administration-is able to keep neuroinflammation within its physiological limits. In this review the large number of studies on the benefits derived from oral administration of micronized and highly bioavailable forms of Palmitoylethanolamide is discussed, with special reference to neuroinflammatory disorders.

N-Palmitoylethanolamine-oxazoline (PEA-OXA): A new therapeutic strategy to reduce neuroinflammation, oxidative stress associated to vascular dementia in an experimental model of repeated bilateral common carotid arteries occlusion.

AIM: Recent studies revealed that pharmacological modulation of NAE-hydrolyzing acid amidase (NAAA) can be achieved with PEA oxazoline (PEA-OXA). Hence, the aim of the present work was to thoroughly evaluate the anti-inflammatory and neuroprotective effects of PEA-OXA in an experimental model of vascular dementia (VaD) induced by bilateral carotid arteries occlusion. At 24 h after VaD induction, animals were orally administered with 10 mg/kg of PEA-OXA daily for 15 days. RESULTS: Brain tissues were handled for histological, immunohistochemical, western blot, and immunofluorescence analysis. PEA-OXA treatment evidently reduced the histological alterations and neuronal death induced by VaD and additionally improved behavioral deficits. Further, PEA-OXA decreased GFAP and Iba-1, markers of astrocytes, and microglia activation, as well as increased MAP-2, a marker of neuron development. Moreover, PEA-OXA reduced oxidative stress, modulated Nrf2-mediated antioxidant response, and inhibited the apoptotic process. INNOVATION: Some drugs may demonstrate their healing potential by regulating neuroinflammation, rather than by their habitually attributed actions only. Palmitoylethanolamide (PEA) is a prototype ALIAmide, well-known for its analgesic, anti-inflammatory, and neuroprotective properties. The inhibition of PEA degradation by targeting NAAA, its catabolic enzyme, is a different approach for treating neuroinflammation. This research offers new insight into the mechanism of PEA-OXA-induced neuroprotection. CONCLUSION: Thus, the modulation of intracellular NAAA by PEA-OXA could offer a novel means of controlling neuroinflammatory conditions associated with VaD.

Palmitoylethanolamide counteracts substance P-induced mast cell activation in vitro by stimulating diacylglycerol lipase activity.

BACKGROUND: Palmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a "dietary food for special medical purposes" against neuropathic pain and neuro-inflammatory conditions. Several mechanisms underlie PEA actions, among which the "entourage" effect, consisting of PEA potentiation of endocannabinoid signaling at either cannabinoid receptors or transient receptor potential vanilloid type-1 (TRPV1) channels. Here, we report novel molecular mechanisms through which PEA controls mast cell degranulation and substance P (SP)-induced histamine release in rat basophilic leukemia (RBL-2H3) cells, a mast cell model. METHODS: RBL-2H3 cells stimulated with SP were treated with PEA in the presence and absence of a cannabinoid type-2 (CB2) receptor antagonist (AM630), or a diacylglycerol lipase (DAGL) enzyme inhibitor (OMDM188) to inhibit the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The release of histamine was measured by ELISA and ß-hexosaminidase release and toluidine blue staining were used as indices of degranulation. 2-AG levels were measured by LC-MS. The mRNA expression of proposed PEA targets (Cnr1, Cnr2, Trpv1, Ppara and Gpr55), and of PEA and endocannabinoid biosynthetic (Napepld, Dagla and Daglb) and catabolic (Faah, Naaa and Mgl) enzymes were also measured. The effects of PEA on the activity of DAGL-α or -ß enzymes were assessed in COS-7 cells overexpressing the human recombinant enzyme or in RBL-2H3 cells, respectively. RESULTS: SP increased the number of degranulated RBL-2H3 cells and triggered the release of histamine. PEA counteracted these effects in a manner antagonized by AM630. PEA concomitantly increased the levels of 2-AG in SP-stimulated RBL-2H3 cells, and this effect was reversed by OMDM188. PEA significantly stimulated DAGL-α and -ß activity and, consequently, 2-AG biosynthesis in cell-free systems. Co-treatment with PEA and 2-AG at per se ineffective concentrations downmodulated SP-induced release of histamine and degranulation, and this effect was reversed by OMDM188. CONCLUSIONS: Activation of CB2 underlies the inhibitory effects on SP-induced RBL-2H3 cell degranulation by PEA alone. We demonstrate for the first time that the effects in RBL-2H3 cells of PEA are due to the stimulation of 2-AG biosynthesis by DAGLs.

Anti-inflammatory Properties of Cannabidiol, a Nonpsychotropic Cannabinoid, in Experimental Allergic Contact Dermatitis.

Phytocannabinoids modulate inflammatory responses by regulating the production of cytokines in several experimental models of inflammation. Cannabinoid type-2 (CB2) receptor activation was shown to reduce the production of the monocyte chemotactic protein-2 (MCP-2) chemokine in polyinosinic-polycytidylic acid [poly-(I:C)]-stimulated human keratinocyte (HaCaT) cells, an in vitro model of allergic contact dermatitis (ACD). We investigated if nonpsychotropic cannabinoids, such as cannabidiol (CBD), produced similar effects in this experimental model of ACD. HaCaT cells were stimulated with poly-(I:C), and the release of chemokines and cytokines was measured in the presence of CBD or other phytocannabinoids (such as cannabidiol acid, cannabidivarin, cannabidivarinic acid, cannabichromene, cannabigerol, cannabigerolic acid, cannabigevarin, tetrahydrocannabivarin, and tetrahydrocannabivarinic acid) and antagonists of CB1, CB2, or transient receptor potential vanilloid type-1 (TRPV1) receptors. HaCaT cell viability following phytocannabinoid treatment was also measured. The cellular levels of endocannabinoids [anandamide (AEA), 2-arachidonoylglycerol] and related molecules (palmitoylethanolamide, oleoylethanolamide) were quantified in poly-(I:C)-stimulated HaCaT cells treated with CBD. We show that in poly-(I:C)-stimulated HaCaT cells, CBD elevates the levels of AEA and dose-dependently inhibits poly-(I:C)-induced release of MCP-2, interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α in a manner reversed by CB2 and TRPV1 antagonists 6-iodopravadoline (AM630) and 5'-iodio-resiniferatoxin (I-RTX), respectively, with no cytotoxic effect. This is the first demonstration of the anti-inflammatory properties of CBD in an experimental model of ACD.

The endocannabinoid 2-AG controls skeletal muscle cell differentiation via CB1 receptor-dependent inhibition of Kv7 channels.

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels.

Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons.

Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A-containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

Anandamide drives cell cycle progression through CB1 receptors in a rat model of synchronized liver regeneration.

The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti-proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation-promoting functions of endocannabinoids through CB1 receptors when pro-growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid-like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals.

Diacerein is a potent and selective inhibitor of palmitoylethanolamide inactivation with analgesic activity in a rat model of acute inflammatory pain.

Palmitoylethanolamide (PEA) is produced by mammalian cells from its biosynthetic precursor, N-palmitoyl-phosphatidyl-ethanolamine, and inactivated by enzymatic hydrolysis to palmitic acid and ethanolamine. Apart from fatty acid amide hydrolase (FAAH), the N-acylethanolamine-hydrolyzing acid amidase (NAAA), a lysosomal enzyme, was also shown to catalyze the hydrolysis of PEA and to limit its analgesic and anti-inflammatory action. Here we report the finding of a new potential inhibitor of NAAA, EPT4900 (4,5-diacetyloxy-9,10-dioxo-anthracene-2-carboxylic acid, diacerein). EPT4900 exhibited a high inhibitory activity on human recombinant NAAA over-expressed in HEK293 cells (HEK-NAAA cells). EPT4900 selectively increased the levels of PEA in intact HEK-NAAA cells, and inhibited inflammation as well as hyperalgesia in rats treated with an intraplantar injection of carrageenan. This latter effect was accompanied by elevation of PEA endogenous levels in the paw skin.

Increased levels of palmitoylethanolamide and other bioactive lipid mediators and enhanced local mast cell proliferation in canine atopic dermatitis.

BACKGROUND: Despite the precise pathogenesis of atopic dermatitis (AD) is unknown, an immune dysregulation that causes Th2-predominant inflammation and an intrinsic defect in skin barrier function are currently the two major hypotheses, according to the so-called outside-inside-outside model. Mast cells (MCs) are involved in AD both by releasing Th2 polarizing cytokines and generating pruritus symptoms through release of histamine and tryptase. A link between MCs and skin barrier defects was recently uncovered, with histamine being found to profoundly contribute to the skin barrier defects.Palmitoylethanolamide and related lipid mediators are endogenous bioactive compounds, considered to play a protective homeostatic role in many tissues: evidence collected so far shows that the anti-inflammatory effect of palmitoylethanolamide depends on the down-modulation of MC degranulation.Based on this background, the purpose of the present study was twofold: (a) to determine if the endogenous levels of palmitoylethanolamide and other bioactive lipid mediators are changed in the skin of AD dogs compared to healthy animals; (b) to examine if MC number is increased in the skin of AD dogs and, if so, whether it depends on MC in-situ proliferation. RESULTS: The amount of lipid extract expressed as percent of biopsy tissue weight was significantly reduced in AD skin while the levels of all analyzed bioactive lipid mediators were significantly elevated, with palmitoylethanolamide showing the highest increase.In dogs with AD, the number of MCs was significantly increased in both the subepidermal and the perifollicular compartments and their granule content was significantly decreased in the latter. Also, in situ proliferation of MCs was documented. CONCLUSIONS: The levels of palmitoylethanolamide and other bioactive lipid mediators were shown to increase in AD skin compared to healthy samples, leading to the hypothesis that they may be part of the body's innate mechanisms to maintain cellular homeostasis when faced with AD-related inflammation. In particular, the increase may be considered a temptative response to down-regulating the observed elevation in the number, functionality and proliferative state of MCs in the skin of AD dogs. Further studies are warranted to confirm the hypothesis.

Corrigendum: Oral ultramicronized palmitoylethanolamide: plasma and tissue levels and spinal antihyperalgesic effect.

[This corrects the article DOI: 10.3389/fphar.2018.00249.].

Pharmacological inhibition of N-Acylethanolamine acid amidase (NAAA) mitigates intestinal fibrosis through modulation of macrophage activity.

BACKGROUND AND AIMS: Intestinal fibrosis, a frequent complication of inflammatory bowel disease, is characterized by stricture formation with no pharmacological treatment to date. N-acylethanolamine acid amidase (NAAA) is responsible of acylethanolamides (AEs, e.g., palmitoylethanolamide and oleoylethanolamide) hydrolysis. Here, we investigated NAAA and AEs signalling in gut fibrosis. METHODS: NAAA and AEs signalling were evaluated in human intestinal specimens from stenotic Crohn's diseases (CD) patients. Gut fibrosis was induced by TNBS, monitored by colonoscopy and unascertained by qRT-PCR, histological analyses, and confocal microscopy. Immune cells were analysed in mesenteric lymph nodes by FACS. Colonic fibroblasts were cultured in conditioned media derived from polarized or not bone marrow-derived macrophages (BMDM). IL-23 signalling was evaluated by qRT-PCR, ELISA, FACS, and western blot in BMDM and in lamina propria CX3CR1+ cells. RESULTS: In ileocolonic human CD strictures, increased transcript expression of NAAA was observed with a decrease of its substrates OEA and PEA. NAAA inhibition reduced intestinal fibrosis in vivo, as revealed by decrease in inflammatory parameters, collagen deposition and fibrosis genes, including epithelial to mesenchymal transition. More in-depth studies revealed modulation of the immune response related to IL-23 following NAAA inhibition. The antifibrotic actions of NAAA inhibition are mediated by Mφ and M2 macrophages that indirectly affect fibroblast collagenogenesis. NAAA inhibitor AM9053 normalized IL-23 signalling in BMDM and in lamina propria CX3CR1+ cells. CONCLUSIONS: Our findings provide new insights into the pathophysiological mechanism of intestinal fibrosis and identify NAAA as a promising target for the development of therapeutic treatments to alleviate CD fibrosis.

Endocannabinoids in amygdala and nucleus accumbens mediate social play reward in adolescent rats.

The brain endocannabinoid system plays a crucial role in emotional processes. We have previously identified an important role for endocannabinoids in social play behavior, a highly rewarding form of social interaction in adolescent rats. Here, we tested the hypothesis that endocannabinoid modulation of social play behavior occurs in brain regions implicated in emotion and motivation. Social play increased levels of the endocannabinoid anandamide in the amygdala and nucleus accumbens (NAc), but not in prefrontal cortex or hippocampus of 4- to 5-week-old male Wistar rats. Furthermore, social play increased phosphorylation of CB1 cannabinoid receptors in the amygdala. Systemic administration of the anandamide hydrolysis inhibitor URB597 increased social play behavior, and augmented the associated elevation in anandamide levels in the amygdala, but not the NAc. Infusion of URB597 into the basolateral amygdala (BLA) increased social play behavior, and blockade of BLA CB1 cannabinoid receptors with the antagonist/inverse agonist SR141716A prevented the play-enhancing effects of systemic administration of URB597. Infusion of URB597 into the NAc also increased social play, but blockade of NAc CB1 cannabinoid receptors did not antagonize the play-enhancing effects of systemic URB597 treatment. Last, SR141716A did not affect social play after infusion into the core and shell subregions of the NAc, while it reduced social play when infused into the BLA. These data show that increased anandamide signaling in the amygdala and NAc augments social play, and identify the BLA as a prominent site of action for endocannabinoids to modulate the rewarding properties of social interactions in adolescent rats.

TRPV1-dependent and -independent alterations in the limbic cortex of neuropathic mice: impact on glial caspases and pain perception.

During neuropathic pain, caspases are activated in the limbic cortex. We investigated the role of TRPV1 channels and glial caspases in the mouse prelimbic and infralimbic (PL-IL) cortex after spared nerve injury (SNI). Reverse transcriptase-polymerase chain reaction, western blots, and immunfluorescence showed overexpression of several caspases in the PL-IL cortex 7 days postinjury. Caspase-3 release and upregulation of AMPA receptors in microglia, caspase-1 and IL-1ß release in astrocytes, and upregulation of Il-1 receptor-1, TRPV1, and VGluT1 in glutamatergic neurons, were also observed. Of these alterations, only those in astrocytes persisted in SNI Trpv1(-/-) mice. A pan-caspase inhibitor, injected into the PL-IL cortex, reduced mechanical allodynia, this effect being reduced but not abolished in Trpv1(-/-) mice. Single-unit extracellular recordings in vivo following electrical stimulation of basolateral amygdala or application of pressure on the hind paw, showed increased excitatory pyramidal neuron activity in the SNI PL-IL cortex, which also contained higher levels of the endocannabinoid 2-arachidonoylglycerol. Intra-PL-IL cortex injection of mGluR5 and NMDA receptor antagonists and AMPA exacerbated, whereas TRPV1 and AMPA receptor antagonists and a CB(1) agonist inhibited, allodynia. We suggest that SNI triggers both TRPV1-dependent and independent glutamate- and caspase-mediated cross-talk among IL-PL cortex neurons and glia, which either participates or counteracts pain.

The endocannabinoid system and pivotal role of the CB2 receptor in mouse spermatogenesis.

The exact role of the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB(2) receptors. In fact, we found that the selective CB(2) receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression toward meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.

Mutual Links between the Endocannabinoidome and the Gut Microbiome, with Special Reference to Companion Animals: A Nutritional Viewpoint.

There is growing evidence that perturbation of the gut microbiome, known as "dysbiosis", is associated with the pathogenesis of human and veterinary diseases that are not restricted to the gastrointestinal tract. In this regard, recent studies have demonstrated that dysbiosis is linked to the pathogenesis of central neuroinflammatory disorders, supporting the existence of the so-called microbiome-gut-brain axis. The endocannabinoid system is a recently recognized lipid signaling system and termed endocannabinoidome monitoring a variety of body responses. Accumulating evidence demonstrates that a profound link exists between the gut microbiome and the endocannabinoidome, with mutual interactions controlling intestinal homeostasis, energy metabolism and neuroinflammatory responses during physiological conditions. In the present review, we summarize the latest data on the microbiome-endocannabinoidome mutual link in health and disease, focalizing the attention on gut dysbiosis and/or altered endocannabinoidome tone that may distort the bidirectional crosstalk between these two complex systems, thus leading to gastrointestinal and metabolic diseases (e.g., idiopathic inflammation, chronic enteropathies and obesity) as well as neuroinflammatory disorders (e.g., neuropathic pain and depression). We also briefly discuss the novel possible dietary interventions based not only on probiotics and/or prebiotics, but also, and most importantly, on endocannabinoid-like modulators (e.g., palmitoylethanolamide) for intestinal health and beyond.

First Evidence of the Protective Effects of 2-Pentadecyl-2-Oxazoline (PEA-OXA) in In Vitro Models of Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) is a serious inflammatory lung disorder and a complication of SARS-CoV-2 infection. In patients with severe SARS-CoV-2 infection, the transition to ARDS is principally due to the occurrence of a cytokine storm and an exacerbated inflammatory response. The effectiveness of ultra-micronized palmitoylethanolamide (PEA-um) during the earliest stage of COVID-19 has already been suggested. In this study, we evaluated its protective effects as well as the effectiveness of its congener, 2-pentadecyl-2-oxazoline (PEA-OXA), using in vitro models of acute lung injury. In detail, human lung epithelial cells (A549) activated by polyinosinic-polycytidylic acid (poly-(I:C)) or Transforming Growth Factor-beta (TGF-ß) were treated with PEA-OXA or PEA. The release of IL-6 and the appearance of Epithelial-Mesenchymal Transition (EMT) were measured by ELISA and immunofluorescence assays, respectively. A possible mechanism of action for PEA-OXA and PEA was also investigated. Our results showed that both PEA-OXA and PEA were able to counteract poly-(I:C)-induced IL-6 release, as well as to revert TGF-ß-induced EMT. In addition, PEA was able to produce an "entourage" effect on the levels of the two endocannabinoids AEA and 2-AG, while PEA-OXA only increased PEA endogenous levels, in poly-(I:C)-stimulated A549 cells. These results evidence for the first time the superiority of PEA-OXA over PEA in exerting protective effects and point to PEA-OXA as a new promising candidate in the management of acute lung injury.

Palmitoylethanolamide counteracts reactive astrogliosis induced by ß-amyloid peptide.

Emerging evidence indicates that astrogliosis is involved in the pathogenesis of neurodegenerative disorders. Our previous findings suggested cannabinoids and Autacoid Local Injury Antagonism Amides (ALIAmides) attenuate glial response in models of neurodegeneration. The present study was aimed at exploring palmitoylethanolamide (PEA) ability to mitigate ß-amyloid (Aß)-induced astrogliosis. Experiments were carried out to investigate PEA's (10(-7) M) effects upon the expression and release of pro-inflammatory molecules in rat primary astrocytes activated by soluble Aß(1-42) (1 µg/ml) as well as to identify mechanisms responsible for such actions. The effects of Aß and exogenous PEA on the astrocyte levels of the endocannabinoidsand of endogenous ALIAmides were also studied. The peroxisome proliferator-activated receptor (PPAR)-α (MK886, 3 µM) or PPAR-γ (GW9662, 9 nM) antagonists were co-administered with PEA. Aß elevated endogenous PEA and d5-2-arachidonoylglycerol (2-AG) levels. Exogenous PEA blunted the Aß-induced expression of pro-inflammatory molecules. This effect was reduced by PPAR-α antagonist. Moreover, this ALIAmide, like Aß, increased 2-AG levels. These results indicate that PEA exhibits anti-inflammatory properties able to counteract Aß-induced astrogliosis, and suggest novel treatment for neuroinflammatory/ neurodegenerative processes.

Cannabinoid CB1 receptor antagonism prevents neurochemical and behavioural deficits induced by chronic phencyclidine.

Clinical and laboratory studies suggest that the endocannabinoid system is involved in schizophrenia disorders. Recent evidence indicates that cannabinoid receptor (CB1) antagonists have a pharmacological profile similar to antipsychotic drugs. We investigated the behavioural and biochemical effects of the CB1 antagonist AM251 in a phencyclidine (PCP) animal paradigm modelling the cognitive deficit and some negative symptoms of schizophrenia. Chronic AM251 (0.5 mg/kg for 3 wk) improved the PCP-altered recognition memory, as indicated by a significant amelioration of the discrimination index compared to chronic PCP alone (2.58 mg/kg for 1 month). AM251 also reversed the PCP-induced increase in immobility in the forced swim test resembling avolition, a negative sign of schizophrenia. In order to analyse the mechanisms underlying these behaviours, we studied the effects of AM251 on the endocannabinoid system (in terms of CB1 receptor density and functional activity and endocannabinoid levels) and c-Fos protein expression. The antagonist counteracted the alterations in CB1 receptor function induced by PCP in selected cerebral regions involved in schizophrenia. In addition, in the prefrontal cortex, the key region in the integration of cognitive and negative functions, AM251 markedly raised anandamide levels and reversed the PCP-induced increase of 2-arachidonoylglycerol concentrations. Finally, chronic AM251 fully reversed the PCP-elicited expression of c-Fos protein in the prefrontal cortical region. These findings suggest an antipsychotic-like profile of the CB1 cannabinoid receptor antagonist which, by restoring the function of the endocannabinoid system, might directly or indirectly normalize some of the neurochemical maladaptations present in this schizophrenia-like animal model.

A Glucuronic Acid-Palmitoylethanolamide Conjugate (GLUPEA) Is an Innovative Drug Delivery System and a Potential Bioregulator.

Palmitoylethanolamide (PEA) is an endogenous anti-inflammatory lipid mediator and a widely used nutraceutical. In this study, we designed, realized, and tested a drug-carrier conjugate between PEA (the active drug) and glucuronic acid (the carrier). The conjugate, named GLUPEA, was characterized for its capability of increasing PEA levels and exerting anti-inflammatory activity both in vitro and in vivo. GLUPEA treatment, compared to the same concentration of PEA, resulted in higher cellular amounts of PEA and the endocannabinoid 2-arachidonoyl glycerol (2-AG), and increased 2-AG-induced transient receptor potential vanilloid type 1 (TRPV1) channel desensitization to capsaicin. GLUPEA inhibited pro-inflammatory monocyte chemoattractant protein 2 (MCP-2) release from stimulated keratinocytes, and it was almost as efficacious as ultra-micronized PEA at reducing colitis in dinitrobenzene sulfonic acid (DNBS)-injected mice when using the same dose. GLUPEA is a novel pro-drug able to efficiently mimic the anti-inflammatory and endocannabinoid enhancing actions of PEA.