RESUMO
The pterocarpan fraction from aerial parts of Bituminaria bituminosa was investigated for both chemical characterization and biological evaluation. Chemical studies were in accordance with the literature data on Bituminaria genus resulting in the identification of typical 4,8-prenyl pterocarpans. Three new members, bituminarins A-C (1-3), were isolated along with main bitucarpin A (4), erybraedin C (5) and erybraedin D (6) already reported from this plant. Further, biological studies evidenced antiproliferative properties of the most abundant pterocarpans 4 and 5 on neuroblastoma SH-SY5Y cell line, in agreement with previously described antiproliferative activity of these compounds against cancer cell lines other than neuroblastoma. The structure and the stereochemistry of the new molecules was determined by extensive spectroscopic analysis and chemical derivatization methods. The biological investigation was carried out by using an assay platform based on a live-cell imaging system revealing an apoptotic cell death induction.
Assuntos
Antineoplásicos Fitogênicos , Neuroblastoma , Pterocarpanos , Humanos , Neuroblastoma/patologia , Neuroblastoma/tratamento farmacológico , Linhagem Celular Tumoral , Pterocarpanos/química , Pterocarpanos/farmacologia , Pterocarpanos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacosRESUMO
Cannabinoid receptor subtype 2 (CB2R) is emerging as a pivotal biomarker to identify the first steps of inflammation-based diseases such as cancer and neurodegeneration. There is an urgent need to find specific probes that may result in green and safe alternatives to the commonly used radiative technologies, to deepen the knowledge of the CB2R pathways impacting the onset of the above-mentioned pathologies. Therefore, based on one of the CB2R pharmacophores, we developed a class of fluorescent N-adamantyl-1-alkyl-4-oxo-1,4-dihydroquinoline-3-carboxamide derivatives spanning from the green to the near-infrared (NIR) regions of the light spectrum. Among the synthesized fluorescent ligands, the green-emitting compound 55 exhibited a favorable binding profile (strong CB2R affinity and high selectivity). Notably, this ligand demonstrated versatility as its use was validated in different experimental settings such as flow cytometry saturation, competitive fluorescence assays, and in vitro microglia cells mimicking inflammation states where CB2R are overexpressed.
Assuntos
Corantes Fluorescentes , Microglia , Receptor CB2 de Canabinoide , Receptor CB2 de Canabinoide/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Microglia/metabolismo , Humanos , Animais , Quinolinas/química , Quinolinas/síntese química , Adamantano/análogos & derivados , Adamantano/química , Adamantano/síntese química , Adamantano/farmacologia , Ligantes , Relação Estrutura-AtividadeRESUMO
The short-chain fatty acids (SCFAs) acetate, propionate and butyrate, the major products of intestinal microbial fermentation of dietary fibres, are involved in fine-tuning brain functions via the gut-brain axis. However, the effects of SCFAs in the hypothalamic neuronal network regulating several autonomic-brain functions are still unknown. Using NMR spectroscopy, we detected a reduction in brain acetate concentrations in the hypothalamus of obese leptin knockout ob/ob mice compared to lean wild-type littermates. Therefore, we investigated the effect of acetate on orexin/hypocretin neurons (hereafter referred as OX or OX-A neurons), a subset of hypothalamic neurons regulating energy homeostasis, which we have characterized in previous studies to be over-activated by the lack of leptin and enhancement of endocannabinoid tone in the hypothalamus of ob/ob mice. We found that acetate reduces food-intake in concomitance with a reduction of orexin neuronal activity in ob/ob mice. This was demonstrated by evaluating food-intake behaviour and orexin-A/c-FOS immunoreactivity coupled with patch-clamp recordings in Hcrt-eGFP neurons, quantification of prepro-orexin mRNA, and immunolabeling of GPR-43, the main acetate receptor. Our data provide new insights into the mechanisms of the effects of chronic dietary supplementation with acetate, or complex carbohydrates, on energy intake and body weight, which may be partly mediated by inhibition of orexinergic neuron activity.
Assuntos
Acetatos , Eixo Encéfalo-Intestino , Metabolismo Energético , Homeostase , Neurônios , Orexinas , Animais , Orexinas/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Camundongos , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Acetatos/farmacologia , Acetatos/metabolismo , Eixo Encéfalo-Intestino/efeitos dos fármacos , Eixo Encéfalo-Intestino/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácidos Graxos Voláteis/metabolismo , Ácidos Graxos Voláteis/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Hipotálamo/metabolismo , Hipotálamo/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologiaRESUMO
The mechanism underlying the transition from the pre-symptomatic to the symptomatic state is a crucial aspect of epileptogenesis. SYN2 is a member of a multigene family of synaptic vesicle phosphoproteins playing a fundamental role in controlling neurotransmitter release. Human SYN2 gene mutations are associated with epilepsy and autism spectrum disorder. Mice knocked out for synapsin II (SynII KO) are prone to epileptic seizures that appear after 2 months of age. However, the involvement of the endocannabinoid system, known to regulate seizure development and propagation, in the modulation of the excitatory/inhibitory balance in the epileptic hippocampal network of SynII KO mice has not been explored. In this study, we investigated the impact of endocannabinoids on glutamatergic and GABAergic synapses at hippocampal dentate gyrus granule cells in young pre-symptomatic (1-2 months old) and adult symptomatic (5-8 months old) SynII KO mice. We observed an increase in endocannabinoid-mediated depolarization-induced suppression of excitation in young SynII KO mice, compared to age-matched wild-type controls. In contrast, the endocannabinoid-mediated depolarization-induced suppression of inhibition remained unchanged in SynII KO mice at both ages. This selective alteration of excitatory synaptic transmission was accompanied by changes in hippocampal endocannabinoid levels and cannabinoid receptor type 1 distribution among glutamatergic and GABAergic synaptic terminals contacting the granule cells of the dentate gyrus. Finally, inhibition of type-1 cannabinoid receptors in young pre-symptomatic SynII KO mice induced seizures during a tail suspension test. Our results suggest that endocannabinoids contribute to maintaining network stability in a genetic mouse model of human epilepsy.
Assuntos
Transtorno do Espectro Autista , Epilepsia , Sinapsinas , Animais , Camundongos , Endocanabinoides , Camundongos Knockout , Fenótipo , Convulsões , Sinapses , Sinapsinas/genéticaRESUMO
N-oleoylglycine (OlGly), a lipid derived from the basic component of olive oil, oleic acid, and N-oleoylalanine (OlAla) are endocannabinoid-like mediators. We report that OlGly and OlAla, by activating the peroxisome proliferator-activated receptor alpha (PPARα), reduce the rewarding properties of a highly palatable food, dopamine neuron firing in the ventral tegmental area, and the obesogenic effect of a high-fat diet rich in lard (HFD-L). An isocaloric olive oil HFD (HFD-O) reduced body weight gain compared to the HFD-L, in a manner reversed by PPARα antagonism, and enhanced brain and intestinal OlGly levels and gut microbial diversity. OlGly or OlAla treatment of HFD-L mice resulted in gut microbiota taxonomic changes partly similar to those induced by HFD-O. We suggest that OlGly and OlAla control body weight by counteracting highly palatable food overconsumption, and possibly rebalancing the gut microbiota, and provide a potential new mechanism of action for the obeso-preventive effects of olive oil-rich diets.
Assuntos
Endocanabinoides , PPAR alfa , Animais , Camundongos , Azeite de Oliva/farmacologia , Obesidade/etiologia , Obesidade/prevenção & controle , Peso CorporalRESUMO
Cannabis sativa L. is a plant that humankind has been using for millennia. The basis of its widespread utilization is its adaptability to so many different climatic conditions, with easy cultivability in numerous diverse environments. Because of its variegate phytochemistry, C. sativa has been used in many sectors, although the discovery of the presence in the plant of several psychotropic substances (e.g., Δ9-tetrahydrocannabinol, THC) caused a drastic reduction of its cultivation and use together with its official ban from pharmacopeias. Fortunately, the discovery of Cannabis varieties with low content of THC as well as the biotechnological development of new clones rich in many phytochemical components endorsed with peculiar and many important bioactivities has demanded the reassessment of these species, the study and use of which are currently experiencing new and important developments. In this review we focus our attention on the phytochemistry, new matrices, suitable agronomic techniques, and new biological activities developed in the five last years.
Assuntos
Canabinoides , Cannabis , Alucinógenos , Cannabis/química , Canabinoides/química , Agonistas de Receptores de Canabinoides , Encéfalo , Dronabinol/farmacologiaRESUMO
Orexins (also known as hypocretins) are neuropeptides located exclusively in hypothalamic neurons that have extensive projections throughout the central nervous system and bind two different G protein-coupled receptors (OX1R and OX2R). Since its discovery in 1998, the orexin system has gained the interest of the scientific community as a potential therapeutic target for the treatment of different pathological conditions. Considering previous basic science research, a dual orexin receptor antagonist, suvorexant, was the first orexin agent to be approved by the US Food and Drug Administration to treat insomnia. In this review, we discuss and update the main preclinical and human studies involving the orexin system with several psychiatric and neurodegenerative diseases. This system constitutes a nice example of how basic scientific research driven by curiosity can be the best route to the generation of new and powerful pharmacological treatments.
Assuntos
Doenças Neurodegenerativas , Neuropeptídeos , Animais , Humanos , Orexinas/metabolismo , Receptores de Orexina/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Receptores Acoplados a Proteínas GRESUMO
OBJECTIVE: Orexin-A (OX-A) is a neuropeptide produced selectively by neurons of the lateral hypothalamus. It exerts powerful control over brain function and physiology by regulating energy homeostasis and complex behaviors linked to arousal. Under conditions of chronic or acute brain leptin signaling deficiency, such as in obesity or short-term food deprivation, respectively, OX-A neurons become hyperactive and promote hyperarousal and food seeking. However, this leptin-dependent mechanism is still mostly unexplored. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) is known to be implicated in food consumption by promoting hyperphagia and obesity, and we and others demonstrated that OX-A is a strong inducer of 2-AG biosynthesis. Here, we investigated the hypothesis that, under acute (6 h fasting in wt mice) or chronic (in ob/ob mice) hypothalamic leptin signaling reduction, OX-A-induced enhancement of 2-AG levels leads to the production of the 2-AG-derived 2-arachidonoyl-sn-glycerol-3-phosphate (2-AGP), a bioactive lipid belonging to the class of lysophosphatidic acids (LPAs), which then regulates hypothalamic synaptic plasticity by disassembling α-MSH anorexigenic inputs via GSK-3ß-mediated Tau phosphorylation, ultimately affecting food intake. METHODS: We combined cell-type-specific morphological (CLEM and confocal microscopy), biochemical, pharmacological, and electrophysiological techniques to dissect the leptin- and OX-A/2-AGP-mediated molecular pathways regulating GSK-3ß-controlled pT231-Tau production at POMC neurons of obese ob/ob and wild-type (wt) lean littermate mice and in an in vitro model of POMC neurons such as mHypoN41 neurons (N41). RESULTS: 2-AGP is overproduced in the hypothalamus of obese leptin-deficient, or lean 6 h food-deprived mice, and promotes food intake by reducing α-MSH-expressing synaptic inputs to OX-A neurons via lysophosphatidic acid type-1 receptor (LPA1-R) activation, and pT231-Tau accumulation in α-MSH projections. This effect is due to the activation of the Pyk2-mediated pTyr216-GSK3ß pathway and contributes to further elevating OX-A release in obesity. Accordingly, we found a strong correlation between OX-A and 2-AGP levels in the serum of obese mice and of human subjects. CONCLUSIONS: Hypothalamic feeding pathways are endowed with 2-AGP-mediated synaptic plasticity according to their inherent functional activities and the necessity to adapt to changes in the nutritional status. These findings reveal a new molecular pathway involved in energy homeostasis regulation, which could be targeted to treat obesity and related disturbances.
Assuntos
Endocanabinoides , Leptina , Camundongos , Humanos , Animais , Orexinas/metabolismo , Leptina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Endocanabinoides/metabolismo , alfa-MSH/metabolismo , Pró-Opiomelanocortina/metabolismo , Hipotálamo/metabolismo , Obesidade/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos EndogâmicosRESUMO
Conventional techniques to reveal the neuroanatomical distribution of type 1 cannabinoid receptor (CB1) in the brain, at the cellular and subcellular level, are mainly represented by light, confocal, and electron microscopy. By using immunoperoxidase and immunofluorescence methods, it is possible to reveal CB1 distribution and localization in the brain and its changes under pathological conditions. Moreover, by using electron microscopy, we can define the ultrastructural localization at the level of subcellular structures and organelles. Here, we describe immunoperoxidase, immunofluorescence, and electron microscopy protocols used to get information about CB1 spatial distribution and localization in the brain. Preparation of reagents, resin embedding, preparation for an endogenous activity-blocking step, and background counterstaining and revelation of CB1 by using specific labeled secondary antibodies will be presented. The methods here discussed are highly sensitive and specific multistep processes, where each step is critical to finally obtain an optimum signal.
Assuntos
Encéfalo , Encéfalo/metabolismo , Imunofluorescência , Técnicas Imunoenzimáticas , Microscopia Confocal , Microscopia Eletrônica , Receptores de Canabinoides/metabolismoRESUMO
A regular sleep-wake cycle plays a positive function that preserves synaptic plasticity and brain activity from neuropathological injuries. The hypothalamic neuropeptide orexin-A (OX-A) is central in sleep-wake regulation and has been found to be over-expressed in the cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) suffering from sleep disturbances. OX-A promotes the biosynthesis of 2-arachidonoylglycerol (2-AG), which, in turn, could be phosphorylated to 2-arachidonoyl lysophosphatidic acid (2-AGP). The reorganization of the actin cytoskeleton during neurite retraction is one of the best-characterized effects of lysophosphatidic acids. However, less information is available regarding the reorganization of the neuronal microtubule network in response to OX-A-induced 2-AG and, possibly consequent, 2-AGP production in AD patients. This is of special relevance also considering that higher 2-AG levels are reported in the CSF of AD patients. Here, we found a positive correlation between OX-A and 2-AGP concentrations in the plasma, and an increase of 2-AGP levels in the CSF of AD patients. Furthermore, a negative correlation between the plasmatic 2-AGP levels and the mini-mental state examination score is also revealed in AD patients. By moving from the human patients to in vitro and in vivo models of AD we investigated the molecular pathway linking OX-A, 2-AG and 2-AGP to the phosphorylation of pT231-Tau, which is a specific early plasma biomarker of this disorder. By LC-MS analysis we show that OX-A, via OX-1R, induces 2-AG biosynthesis via DAGLα, and in turn 2-AG is converted to 2-AGP in primary hippocampal neurons. By confocal microscopy and western blotting assay we found an OX-A- or 2-AGP-mediated phosphorylation of Tau at threonine 231 residue, in a manner prevented by LPA1R (2-AGP receptor) or OX1R (OX-A receptor) antagonism with AM095 or SB334867, respectively. Finally, by patch-clamp recording we documented that 2-AGP-mediated pT231-Tau phosphorylation impairs glutamatergic transmission in the mouse hippocampus. Although further additional research is still required to clarify the potential role of orexin signaling in neurodegeneration, this study provides evidence that counteraction of aberrant OX-A signaling, also via LPA-1R antagonism, may be beneficial in the mild-to-moderate age-related cognitive decline associated with sleep disturbances.
RESUMO
Anxiety and stress disorders are often characterized by an inability to extinguish learned fear responses. Orexins/hypocretins are involved in the modulation of aversive memories, and dysregulation of this system may contribute to the aetiology of anxiety disorders characterized by pathological fear. The mechanisms by which orexins regulate fear are unknown. Here we investigated the role of the endogenous cannabinoid system in the impaired fear extinction induced by orexin-A (OXA) in male mice. The selective inhibitor of 2-arachidonoylglycerol (2-AG) biosynthesis O7460 abolished the fear extinction deficits induced by OXA. Accordingly, increased 2-AG levels were observed in the amygdala and hippocampus of mice treated with OXA that do not extinguish fear, suggesting that high levels of this endocannabinoid are related to poor extinction. Impairment of fear extinction induced by OXA was associated with increased expression of CB2 cannabinoid receptor (CB2R) in microglial cells of the basolateral amygdala. Consistently, the intra-amygdala infusion of the CB2R antagonist AM630 completely blocked the impaired extinction promoted by OXA. Microglial and CB2R expression depletion in the amygdala with PLX5622 chow also prevented these extinction deficits. These results show that overactivation of the orexin system leads to impaired fear extinction through 2-AG and amygdalar CB2R. This novel mechanism could be of relevance for the development of novel potential approaches to treat diseases associated with inappropriate retention of fear, such as post-traumatic stress disorder, panic anxiety and phobias.
Assuntos
Canabinoides , Medo , Tonsila do Cerebelo , Animais , Extinção Psicológica , Masculino , Camundongos , Orexinas/farmacologia , Receptor CB2 de CanabinoideRESUMO
The mammalian brain stores and distinguishes among episodic memories, i.e. memories formed during the personal experience, through a mechanism of pattern separation computed in the hippocampal dentate gyrus. Decision-making for food-related behaviors, such as the choice and intake of food, might be affected in obese subjects by alterations in the retrieval of episodic memories. Adult neurogenesis in the dentate gyrus regulates the pattern separation. Several molecular factors affect adult neurogenesis and exert a critical role in the development and plasticity of newborn neurons. Orexin-A/hypocretin-1 and downstream endocannabinoid 2-arachidonoylglycerol signaling are altered in obese mice. Here, we show that excessive orexin-A/2-arachidonoylglycerol/cannabinoid receptor type-1 signaling leads to the dysfunction of adult hippocampal neurogenesis and the subsequent inhibition of plasticity and impairment of pattern separation. By inhibiting orexin-A action at orexin-1 receptors we rescued both plasticity and pattern separation impairment in obese mice, thus providing a molecular and functional mechanism to explain alterations in episodic memory in obesity.
Assuntos
Endocanabinoides/metabolismo , Hipocampo/crescimento & desenvolvimento , Neurogênese , Plasticidade Neuronal , Obesidade/metabolismo , Obesidade/psicologia , Orexinas/metabolismo , Animais , Feminino , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Masculino , Memória Episódica , Camundongos , Camundongos Obesos , Neurônios/citologia , Neurônios/metabolismo , Obesidade/genética , Obesidade/fisiopatologia , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Transdução de SinaisRESUMO
The potential, multifaceted therapeutic profile of cannabidiol (CBD), a major constituent derived from the Cannabis sativa plant, covers a wide range of neurological and psychiatric disorders, ranging from anxiety to pediatric epilepsy and drug addiction. However, the molecular targets responsible for these effects have been only partially identified. In this view, the involvement of the orexin system, the key regulator in arousal and the sleep/wake cycle, and in motivation and reward processes, including drug addiction, prompted us to explore, using computational and experimental approaches, the possibility that CBD could act as a ligand of orexin receptors, orexin 1 receptor of type 1 (OX1R) and type 2 (OX2R). Ligand-binding assays showed that CBD is a selective ligand of OX1R in the low micromolar range (Ki 1.58 ± 0.2 µM) while in vitro functional assays, carried out by intracellular calcium imaging and mobilization assays, showed that CBD acts as an antagonist at this receptor. Finally, the putative binding mode of CBD has been inferred by molecular docking and molecular dynamics simulations and its selectivity toward the OX1R subtype rationalized at the molecular level. This study provides the first evidence that CBD acts as an OX1R antagonist, supporting its potential use in addictive disorders and/or body weight regulation.
Assuntos
Ansiolíticos/farmacologia , Anticonvulsivantes/farmacologia , Canabidiol/farmacologia , Receptores de Orexina/química , Orexinas/química , Animais , Ansiolíticos/química , Ansiolíticos/metabolismo , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Sítios de Ligação , Células CHO , Cálcio/metabolismo , Canabidiol/química , Canabidiol/metabolismo , Cricetulus , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Imagem Molecular , Antagonistas dos Receptores de Orexina , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ensaio Radioligante , TransgenesRESUMO
Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.
Assuntos
Transtorno do Espectro Autista/metabolismo , Dopamina/metabolismo , Heparitina Sulfato/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Animais , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/patologia , Benzazepinas/uso terapêutico , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Antagonistas de Dopamina/uso terapêutico , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Heparitina Sulfato/farmacologia , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/patologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/embriologia , Mesencéfalo/patologia , Camundongos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/metabolismoRESUMO
Disinhibition of orexin-A/hypocretin-1 (OX-A) release occurs to several output areas of the lateral hypothalamus (LH) in the brain of leptin knockout obese ob/ob mice. In this study, we have investigated whether a similar increase of OX-A release occurs to the ventral tegmental area (VTA), an orexinergic LH output area with functional effects on dopaminergic signaling at the mesolimbic circuit. By confocal and correlative light and electron microscopy (CLEM) morphological studies coupled to molecular, biochemical, and pharmacological approaches, we investigated OX-A-mediated dopaminergic signaling at the LH-VTA-nucleus accumbens (NAc) pathway in obese ob/ob mice compared to wild-type (wt) lean littermates. We found an elevation of OX-A trafficking and release to the VTA of ob/ob mice and consequent orexin receptor-1 (OX1R)-mediated over-activation of dopaminergic (DA) neurons via phospholipase C (PLC)/diacylglycerol lipase (DAGL-α)-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). In fact, by retrograde signaling to cannabinoid receptor type 1 (CB1R) at inhibitory inputs to DA neurons, 2-AG inhibited GABA release thus inducing an increase in DA concentration in the VTA and NAc of ob/ob mice. This effect was prevented by the OX1R antagonist SB-334867 (30 mg/Kg, i.p.), or the CB1R antagonist AM251 (10 mg/Kg, i.p.) and mimicked by OX-A injection (40 µg/Kg, i.p.) in wt lean mice. Enhanced DA signaling to the NAc in ob/ob mice, or in OX-A-injected wt mice, was accompanied by ß-arrestin2-mediated desensitization of dopamine D2 receptor (D2R) in a manner prevented by SB-334867 or the D2R antagonist L741 (1.5 mg/Kg, i.p.). These results further support the role of OX-A signaling in the control of neuroadaptive responses, such as compulsive reward-seeking behavior or binge-like consumption of high palatable food, and suggest that aberrant OX-A trafficking to the DA neurons in the VTA of ob/ob mice influences the D2R response at NAc, a main target area of the mesolimbic pathway, via 2-AG/CB1-mediated retrograde signaling.
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Toll-like receptors (TLRs) are key receptors through which infectious and non-infectious challenges act with consequent activation of the inflammatory cascade that plays a critical function in various acute and chronic diseases, behaving as amplification and chronicization factors of the inflammatory response. Previous studies have shown that synthetic analogues of lipid A based on glucosamine with few chains of unsaturated and saturated fatty acids, bind MD-2 and inhibit TLR4 receptors. These synthetic compounds showed antagonistic activity against TLR4 activation in vitro by LPS, but little or no activity in vivo. This study aimed to show the potential use of N-palmitoyl-D-glucosamine (PGA), a bacterial molecule with structural similarity to the lipid A component of LPS, which could be useful for preventing LPS-induced tissue damage or even peripheral neuropathies. Molecular docking and molecular dynamics simulations showed that PGA stably binds MD-2 with a MD-2/(PGA)3 stoichiometry. Treatment with PGA resulted in the following effects: (i) it prevented the NF-kB activation in LPS stimulated RAW264.7 cells; (ii) it decreased LPS-induced keratitis and corneal pro-inflammatory cytokines, whilst increasing anti-inflammatory cytokines; (iii) it normalized LPS-induced miR-20a-5p and miR-106a-5p upregulation and increased miR-27a-3p levels in the inflamed corneas; (iv) it decreased allodynia in peripheral neuropathy induced by oxaliplatin or formalin, but not following spared nerve injury of the sciatic nerve (SNI); (v) it prevented the formalin- or oxaliplatin-induced myelino-axonal degeneration of sciatic nerve. SIGNIFICANCE STATEMENT We report that PGA acts as a TLR4 antagonist and this may be the basis of its potent anti-inflammatory activity. Being unique because of its potency and stability, as compared to other similar congeners, PGA can represent a tool for the optimization of new TLR4 modulating drugs directed against the cytokine storm and the chronization of inflammation.
Assuntos
Analgésicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Glicolipídeos/uso terapêutico , Hiperalgesia/prevenção & controle , Ceratite/tratamento farmacológico , Neuralgia/tratamento farmacológico , Receptor 4 Toll-Like/antagonistas & inibidores , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Glicolipídeos/farmacologia , Células HEK293 , Humanos , Hiperalgesia/etiologia , Ceratite/induzido quimicamente , Ceratite/patologia , Lipopolissacarídeos/toxicidade , Antígeno 96 de Linfócito/metabolismo , Masculino , Camundongos , MicroRNAs/genética , Modelos Moleculares , Nociceptores/efeitos dos fármacos , Nociceptores/fisiologia , Conformação Proteica , Células RAW 264.7 , Distribuição Aleatória , Nervo Isquiático/lesões , Canal de Cátion TRPA1/metabolismoRESUMO
The 3,5-diiodo-L-thyronine (3,5-T2) is an endogenous metabolite of thyroid hormones, whose administration to rodents fed high-fat diet (HFD) prevents body weight increase and reverts the expression pattern of pro-inflammatory factors associated to HFD. The diet-induced obese (D.I.O.) zebrafish (Danio rerio) has been recently used as an experimental model to investigate fundamental processes underlying central and peripheral obesity-driven inflammation. Herein, we aim to understand the role of 3,5-T2 in regulating central and peripheral inflammation in D.I.O. model of zebrafish. 3,5-T2 (10 nM and 100 nM) was administered with the obesity-inducing diet (D.I.O. with 3,5-T2) or after 4 weeks of obesity-inducing diet (D.I.O. flw 3,5-T2). 3,5-T2 significantly increased the body weight and serum triglyceride levels in D.I.O. zebrafish in both conditions. Moreover, 3,5-T2 sustained or increased inflammation in the anterior (AI) and mid (MI) intestine when administered with the obesity-inducing diet, as indicated by the immunoexpression of the inflammatory markers tumor-necrosis factor-α (TNFα), cyclooxygenase 2 (COX2), calnexin, caspase 3, and proliferating cell nuclear antigen (PCNA). On the contrary, when 3,5-T2 was administered after the obesity-inducing diet, partly reverted the intestinal alteration induced by D.I.O. In addition, brain inflammation, as indicated by the increase in the activation of microglia, was detected in D.I.O. zebrafish and D.I.O. treated with 3,5-T2. These findings reveal that the effects of 3,5-T2 on fish intestine and brain can deviate from those shown in obese mammals, opening new avenues to the investigation of the potential impact of this thyroid metabolite in different diseases including obesity.
RESUMO
Orexin-A (OX-A) protects the brain against oxidative stress-mediated ischemic injury. Since the endocannabinoid 2-arachidonoylglycerol (2-AG) and cannabinoid type-1 (CB1) receptors were previously shown to mediate some of the effects of OX-A exerted through the orexin-1 receptor (OX-1R), we investigated the involvement of 2-AG in OX-A-induced neuroprotection following oxygen and glucose deprivation (OGD) in mouse cortical neurons. OGD-induced reactive oxygen species (ROS) accumulation and neuronal death were prevented by both OX-A and arachidonyl-2'-chloroethylamide (ACEA), a synthetic CB1 receptor agonist, in a manner sensitive to OX-1R and CB1 receptor antagonists, SB334867 and AM251. OX-A stimulated 2-AG biosynthesis in cortical neurons. In neurons isolated from monoacylglycerol lipase (MAGL, a 2-AG hydrolyzing enzyme) null mice, 10-fold higher 2-AG concentrations were found and OGD failed to induce ROS production and cell death, whereas AM251 restored these noxious effects. OX-A-induced neuroprotection was mediated by the phosphoinositide-3-kinase/Akt (PI3K/Akt) survival pathway since both OX-A and ACEA induced phosphorylation of Akt and prevented OGD-induced cytochrome c release from the mitochondria, in a manner counteracted by SB334867 or AM251. Administration of OX-A reduced infarct volume and elevated brain 2-AG levels in a mouse model of transient ischemia. These results suggest that 2-AG and CB1 receptor mediate OX-A prevention of ischemia-induced neuronal apoptosis.
Assuntos
Ácidos Araquidônicos/metabolismo , Endocanabinoides/metabolismo , Glucose/metabolismo , Glicerídeos/metabolismo , Neurônios/metabolismo , Orexinas/metabolismo , Oxigênio/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Humanos , CamundongosRESUMO
The hypothalamus regulates energy homeostasis by integrating environmental and internal signals to produce behavioral responses to start or stop eating. Many satiation signals are mediated by microbiota-derived metabolites coming from the gastrointestinal tract and acting also in the brain through a complex bidirectional communication system, the microbiota-gut-brain axis. In recent years, the intestinal microbiota has emerged as a critical regulator of hypothalamic appetite-related neuronal networks. Obesogenic high-fat diets (HFDs) enhance endocannabinoid levels, both in the brain and peripheral tissues. HFDs change the gut microbiota composition by altering the Firmicutes:Bacteroidetes ratio and causing endotoxemia mainly by rising the levels of lipopolysaccharide (LPS), the most potent immunogenic component of Gram-negative bacteria. Endotoxemia induces the collapse of the gut and brain barriers, interleukin 1ß (IL1ß)- and tumor necrosis factor α (TNFα)-mediated neuroinflammatory responses and gliosis, which alter the appetite-regulatory circuits of the brain mediobasal hypothalamic area delimited by the median eminence. This review summarizes the emerging state-of-the-art evidence on the function of the "expanded endocannabinoid (eCB) system" or endocannabinoidome at the crossroads between intestinal microbiota, gut-brain communication and host metabolism; and highlights the critical role of this intersection in the onset of obesity.