RESUMO
Post-traumatic stress disorder (PTSD) is a debilitating mental health disease related to traumatic experience, and its treatment outcomes are unsatisfactory. Accumulating research has indicated that cannabidiol (CBD) exhibits anti-PTSD effects, however, the underlying mechanism of CBD remains inadequately investigated. Although many studies pertaining to PTSD have primarily focused on aberrations in neuronal functioning, the present study aimed to elucidate the involvement and functionality of microglia/macrophages in PTSD while also investigated the modulatory effects of CBD on neuroinflammation associated with this condition. We constructed a modified single-prolonged stress (SPS) mice PTSD model and verified the PTSD-related behaviors by various behavioral tests (contextual freezing test, elevated plus maze test, tail suspension test and novel object recognition test). We observed a significant upregulation of Iba-1 and alteration of microglial/macrophage morphology within the prefrontal cortex and hippocampus, but not the amygdala, two weeks after the PTSD-related stress, suggesting a persistent neuroinflammatory phenotype in the PTSD-modeled group. CBD (10 mg/kg, i.p.) inhibited all PTSD-related behaviors and reversed the alterations in both microglial/macrophage quantity and morphology when administered prior to behavioral assessments. We further found increased pro-inflammatory factors, decreased PSD95 expression, and impaired synaptic density in the hippocampus of the modeled group, all of which were also restored by CBD treatment. CBD dramatically increased the level of anandamide, one of the endocannabinoids, and cannabinoid type 2 receptors (CB2Rs) transcripts in the hippocampus compared with PTSD-modeled group. Importantly, we discovered the expression of CB2Rs mRNA in Arg-1-positive cells in vivo and found that the behavioral effects of CBD were diminished by CB2Rs antagonist AM630 (1 mg/kg, i.p.) and both the behavioral and molecular effects of CBD were abolished in CB2Rs knockout mice. These findings suggest that CBD would alleviate PTSD-like behaviors in mice by suppressing PTSD-related neuroinflammation and upregulation and activation of CB2Rs may serve as one of the underlying mechanisms for this therapeutic effect. The present study offers innovative experimental evidence supporting the utilization of CBD in PTSD treatment from the perspective of its regulation of neuroinflammation, and paves the way for leveraging the endocannabinoid system to regulate neuroinflammation as a potential therapeutic approach for psychiatric disorders.
Assuntos
Encéfalo , Canabidiol , Modelos Animais de Doenças , Microglia , Doenças Neuroinflamatórias , Receptor CB2 de Canabinoide , Transtornos de Estresse Pós-Traumáticos , Animais , Canabidiol/farmacologia , Receptor CB2 de Canabinoide/metabolismo , Masculino , Camundongos , Transtornos de Estresse Pós-Traumáticos/metabolismo , Transtornos de Estresse Pós-Traumáticos/tratamento farmacológico , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Endocanabinoides/metabolismo , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologiaRESUMO
The lipid endocannabinoid system has recently emerged as a novel therapeutic target for several inflammatory and tissue-damaging diseases, including those affecting the cardiovascular system. The primary targets of cannabinoids are cannabinoid type 1 (CB1) and 2 (CB2) receptors. The CB2 receptor is expressed in the cardiomyocytes. While the pathological changes in the myocardium upregulate the CB2 receptor, genetic deletion of the receptor aggravates the changes. The CB2 receptor plays a crucial role in attenuating the advancement of myocardial infarction (MI)-associated pathological changes in the myocardium. Activation of CB2 receptors exerts cardioprotection in MI via numerous molecular pathways. For instance, delta-9-tetrahydrocannabinol attenuated the progression of MI via modulation of the CB2 receptor-dependent anti-inflammatory mechanisms, including suppression of pro-inflammatory cytokines like IL-6, TNF-α, and IL-1ß. Through similar mechanisms, natural and synthetic CB2 receptor ligands repair myocardial tissue damage. This review aims to offer an in-depth discussion on the ameliorative potential of CB2 receptors in myocardial injuries induced by a variety of pathogenic mechanisms. Further, the modulation of autophagy, TGF-ß/Smad3 signaling, MPTP opening, and ROS production are discussed. The molecular correlation of CB2 receptors with cardiac injury markers, such as troponin I, LDH1, and CK-MB, is explored. Special attention has been paid to novel insights into the potential therapeutic implications of CB2 receptor activation in MI.
Assuntos
Canabinoides , Infarto do Miocárdio , Receptor CB1 de Canabinoide , Humanos , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Canabinoides/metabolismo , Endocanabinoides/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo , Dronabinol/farmacologiaRESUMO
Pain, especially chronic pain, can strongly affect patients' quality of life. Cannabinoids ponhave been reported to produce potent analgesic effects in different preclinical pain models, where they primarily function as agonists of Gi/o protein-coupled cannabinoid CB1 and CB2 receptors. The CB1 receptors are abundantly expressed in both the peripheral and central nervous systems. The central activation of CB1 receptors is strongly associated with psychotropic adverse effects, thus largely limiting its therapeutic potential. However, the CB2 receptors are promising targets for pain treatment without psychotropic adverse effects, as they are primarily expressed in immune cells. Additionally, as the resident immune cells in the central nervous system, microglia are increasingly recognized as critical players in chronic pain. Accumulating evidence has demonstrated that the expression of CB2 receptors is significantly increased in activated microglia in the spinal cord, which exerts protective consequences within the surrounding neural circuitry by regulating the activity and function of microglia. In this review, we focused on recent advances in understanding the role of microglial CB2 receptors in spinal nociceptive circuitry, highlighting the mechanism of CB2 receptors in modulating microglia function and its implications for CB2 receptor- selective agonist-mediated analgesia.
Assuntos
Canabinoides , Dor Crônica , Humanos , Receptores de Canabinoides/metabolismo , Microglia/metabolismo , Dor Crônica/tratamento farmacológico , Qualidade de Vida , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Canabinoides/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Receptor CB1 de Canabinoide/metabolismoRESUMO
The endocannabinoid system, with its intricate presence in numerous cells, tissues, and organs, offers a compelling avenue for therapeutic interventions. Central to this system are the cannabinoid receptors 1 and 2 (CB1R and CB2R), whose ubiquity can introduce complexities in targeted treatments due to their wide-ranging physiological influence. Injuries to the central nervous system (CNS), including strokes and traumatic brain injuries, induce localized pro-inflammatory immune responses, termed neuroinflammation. Research has shown that compensatory immunodepression usually follows, and these mechanisms might influence immunity, potentially affecting infection risks in patients. As traditional preventive treatments like antibiotics face challenges, the exploration of immunomodulatory therapies offers a promising alternative. This review delves into the potential neuroprotective roles of the cannabinoid receptors: CB1R's involvement in mitigating excitotoxicity and CB2R's dual role in promoting cell survival and anti-inflammatory responses. However, the potential of cannabinoids to reduce neuroinflammation must be weighed against the risk of exacerbating immunodepression. Though the endocannabinoid system promises numerous therapeutic benefits, understanding its multifaceted signaling mechanisms and outcomes remains a challenge.
Assuntos
Endocanabinoides , Acidente Vascular Cerebral , Humanos , Doenças Neuroinflamatórias , Neuroproteção , Receptor CB1 de Canabinoide , Receptor CB2 de Canabinoide , Receptores de CanabinoidesRESUMO
Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-ß-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Sesquiterpenos , Humanos , Lipídeos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , PPAR alfa/metabolismo , PPAR gama/metabolismo , Sesquiterpenos/farmacologia , Receptor CB2 de CanabinoideRESUMO
Visual deficits after ocular blast injury (OBI) are common, but pharmacological approaches to improve long-term outcomes have not been identified. Blast forces frequently damage the retina and optic nerves, and work on experimental animals has shown the pro-inflammatory actions of microglia can further exacerbate such injuries. Cannabinoid type-2 receptor (CB2) inverse agonists specifically target activated microglia, biasing them away from the harmful pro-inflammatory M1 state toward the helpful reparative M2 state. We previously found that treating mice with CB2 inverse agonists after traumatic brain injury, produced by either focal cranial air blast or dorsal cranial impact, greatly attenuated the visual deficits and pathology that otherwise resulted. Here we examined the consequences of single and repeat OBI and the benefit provided by raloxifene, an FDA-approved estrogen receptor drug that possesses noteworthy CB2 inverse agonism. After single OBI, although the amplitudes of the A- and B-waves of the electroretinogram and pupil light response appeared to be normal, the mice showed hints of deficits in contrast sensitivity and visual acuity, a trend toward optic nerve axon loss, and significantly increased light aversion, which were reversed by 2 weeks of daily treatment with raloxifene. Mice subjected to repeat OBI (5 blasts spaced 1 min apart), exhibited more severe visual deficits, including decreases in contrast sensitivity, visual acuity, the amplitudes of the A- and B-waves of the electroretinogram, light aversion, and resting pupil diameter (i.e. hyperconstriction), accompanied by the loss of photoreceptor cells and optic nerve axons, nearly all of which were mitigated by raloxifene. Interestingly, optic nerve axon abundance was strongly correlated with contrast sensitivity and visual acuity across all groups of experimental mice in the repeat OBI study, suggesting optic nerve axon loss with repeat OBI and its attenuation with raloxifene are associated with the extent of these two deficits while photoreceptor abundance was highly correlated with A-wave amplitude and resting pupil size, suggesting a prominent role for photoreceptors in these two deficits. Quantitative PCR (qPCR) showed levels of M1-type microglial markers (e.g. iNOS, IL1ß, TNFα, and CD32) in retina, optic nerve, and thalamus were increased 3 days after repeat OBI. With raloxifene treatment, the overall expression of M1 markers was more similar to that in sham mice. Raloxifene treatment was also associated with the elevation of IL10 transcripts in all three tissues compared to repeat OBI alone, but the results for the three other M2 microglial markers we examined were more varied. Taken together, the qPCR results suggest that raloxifene benefit for visual function and pathology was associated with a lessening of the pro-inflammatory actions of microglia. The benefit we find for raloxifene following OBI provides a strong basis for phase-2 efficacy testing in human clinical trials for treating ocular injury.
Assuntos
Traumatismos por Explosões , Canabinoides , Traumatismos Oculares , Animais , Traumatismos por Explosões/metabolismo , Agonistas de Receptores de Canabinoides , Traumatismos Oculares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Cloridrato de Raloxifeno/metabolismo , Cloridrato de Raloxifeno/farmacologia , Cloridrato de Raloxifeno/uso terapêuticoRESUMO
Cumulative evidence has pointed out cannabinoid CB2 receptors (CB2r) as a potential therapeutic key target for treating alcohol use disorder (AUD). This review provides the most relevant results obtained from rodent and human studies, including an integrative section focused on the involvement of CB2r in the neurobiology of alcohol addiction. A literature search was conducted using the electronic databases Medline and Scopus for articles. The search strategy was as follows: "Receptor, Cannabinoid, CB2" AND "Alcohol-Related Disorders" AND "human/or patients"; "Receptor, Cannabinoid, CB2" AND "Alcohol" OR "Ethanol" AND "rodents/or mice/or rats". Pharmacological approaches demonstrated that the activation or blockade of CB2r modulated different alcohol-addictive behaviors. Rodent models of alcoholism revealed significant alterations of CB2r in brain areas of the reward system. In addition, mice lacking CB2r (CB2KO) show increased alcohol consumption, motivation, and relapse alterations. It has been stressed that the potential neurobiological mechanisms underlying their behavioral effects involve critical elements of the alcohol reward system. Interestingly, recent postmortem studies showed CNR2 alterations in brain areas of alcoholic patients. Moreover, although the number of studies is limited, the results revealed an association between some genetic alterations of the CNR2 and an increased risk for developing AUD. This review provides evidence that CB2r may play a role in alcohol addiction. Clinical studies are necessary to figure out whether CB2r ligands may prove useful for the treatment of AUD in humans.
Assuntos
Alcoolismo , Canabinoides , Alcoolismo/genética , Animais , Canabinoides/farmacologia , Etanol , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Receptor CB1 de Canabinoide , Receptor CB2 de Canabinoide/genética , RecompensaRESUMO
Acute lymphoblastic leukemia type B (B-ALL) is the most common kind of pediatric leukemia, characterized by the clonal proliferation of type B lymphoid stem cells. Important progress in ALL treatments led to improvements in long-term survival; nevertheless, many adverse long-term consequences still concern the medical community. Molecular and cellular target therapies, together with immunotherapy, are promising strategies to overcome these concerns. Cannabinoids, enzymes involved in their metabolism, and cannabinoid receptors type 1 (CB1) and type 2 (CB2) constitute the endocannabinoid system, involved in inflammation, immune response, and cancer. CB2 receptor stimulation exerts anti-proliferative and anti-invasive effects in many tumors. In this study, we evaluated the effects of CB2 stimulation on B-ALL cell lines, SUP-B15, by RNA sequencing, Western blotting, and ELISA. We observe a lower expression of CB2 in SUP-B15 cells compared to lymphocytes from healthy subjects, hypothesizing its involvement in B-ALL pathogenesis. CB2 stimulation reduces the expression of CD9, SEC61G, TBX21, and TMSB4X genes involved in tumor growth and progression, and also negatively affects downstream intracellular pathways. Our findings suggest an antitumor role of CB2 stimulation in B-ALL, and highlight a functional correlation between CB2 receptors and specific anti-tumoral pathways, even though further investigations are needed.
Assuntos
Linfoma de Burkitt , Canabinoides , Leucemia-Linfoma Linfoblástico de Células Precursoras , Western Blotting , Canabinoides/farmacologia , Criança , Expressão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Canais de Translocação SEC/metabolismoRESUMO
Addiction to psychostimulant drugs, such as cocaine, D-amphetamine, and methamphetamine, is a public health issue that substantially contributes to the global burden of disease. Psychostimulant drugs promote an increase in dopamine levels within the mesocorticolimbic system, which is central to the rewarding properties of such drugs. Cannabinoid receptors (CB1R and CB2R) are expressed in the main areas of this system and implicated in the neuronal mechanisms underlying the rewarding effect of psychostimulant drugs. Here, we reviewed studies focusing on pharmacological intervention targeting cannabinoid CB1R and CB2R and their interaction in the modulation of psychostimulant responses.
RESUMO
The fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) enzymes are the predominant catabolic regulators of the major endocannabinoids (eCBs) anadamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively. The expression and roles of eCBs during early embryogenesis remain to be fully investigated. Here, we inhibited FAAH and MAGL in zebrafish embryos during the first 24â h of life and examined motor neuron and locomotor development at 2 and 5â days post fertilization (dpf). Application of the dual FAAH/MAGL inhibitor, JZL195 (2â µmol l-1), resulted in a reduction in primary and secondary motor neuron axonal branching. JZL195 also reduced nicotinic acetylcholine receptor (nAChR) expression at neuromuscular junctions. Application of URB597 (5â µmol l-1), a specific inhibitor of the FAAH enzyme, also decreased primary motor neuron branching but did not affect secondary motor neuron branching and nAChR expression. Interestingly, JZL184 (5â µmol l-1), a specific inhibitor of MAGL, showed no effects on motor neuron branching or nAChR expression. Co-treatment of the enzyme inhibitors with the CB1R inhibitor AM251 confirmed the involvement of CB1R in motor neuron branching. Disruption of FAAH or MAGL reduced larval swimming activity, and AM251 attenuated the JZL195- and URB597-induced locomotor changes, but not the effects of JZL184. Together, these findings indicate that inhibition of FAAH, or augmentation of AEA acting through CB1R during early development, may be responsible for locomotor deficiencies.
Assuntos
Endocanabinoides , Monoacilglicerol Lipases , Amidoidrolases/genética , Animais , Monoacilglicerol Lipases/genética , Peixe-ZebraRESUMO
This review focuses on new aspects of endocannabinoid functions and mechanisms of activity in central and peripheral synapses, different from the general viewpoint that endocannabinoids are retrograde signaling molecules, which inhibit neurotransmitter release by activating specific presynaptic endocannabinoid receptors CB1 and CB2. Biased agonism of the endogenous and synthetic cannabinoids as well as ability of the CB-receptors to couple not only with classical Gi-proteins, but also with Gs- and Gq-proteins and, moreover, with ß-arrestins (thereby triggering additional signaling pathways in synapses) are described here in detail. Examples of noncanonical tonic activity of endocannabinoids and their receptors and their role in synaptic function are also presented. The role of endocannabinoids in short-term and long-term potentiation of neurotransmitter release in central synapses and their facilitating effect on quantal size and other parameters of acetylcholine release in mammalian neuromuscular junctions are highlighted in this review. In conclusion, it is stated that the endocannabinoid system has a wider range of various multidirectional modulating effects (both potentiating and inhibiting) on neurotransmitter release than initially recognized. Re-evaluation of the functions of endocannabinoid system with consideration of its noncanonical features will lead to better understanding of its role in the normal and pathological functioning of the nervous system and other systems of the body, which has an enormous practical value.
Assuntos
Endocanabinoides/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Sinapses/metabolismo , Animais , Humanos , Transdução de Sinais , Sinapses/fisiologia , Transmissão SinápticaRESUMO
In humans, various sites like cannabinoid receptors (CBR) having a binding affinity with cannabinoids are distributed on the surface of different cell types, where endocannabinoids (ECs) and derivatives of fatty acid can bind. The binding of these substance(s) triggers the activation of specific receptors required for various physiological functions, including pain sensation, memory, and appetite. The ECs and CBR perform multiple functions via the cannabinoid receptor 1 (CB1); cannabinoid receptor 2 (CB2), having a key effect in restraining neurotransmitters and the arrangement of cytokines. The role of cannabinoids in the immune system is illustrated because of their immunosuppressive characteristics. These characteristics include inhibition of leucocyte proliferation, T cells apoptosis, and induction of macrophages along with reduced pro-inflammatory cytokines secretion. The review seeks to discuss the functional relationship between the endocannabinoid system (ECS) and anti-tumor characteristics of cannabinoids in various cancers. The therapeutic potential of cannabinoids for cancer-both in vivo and in vitro clinical trials-has also been highlighted and reported to be effective in mice models in arthritis for the inflammation reduction, neuropathic pain, positive effect in multiple sclerosis and type-1 diabetes mellitus, and found beneficial for treating in various cancers. In human models, such studies are limited; thereby, further research is indispensable in this field to get a conclusive outcome. Therefore, in autoimmune disorders, therapeutic cannabinoids can serve as promising immunosuppressive and anti-fibrotic agents.
Assuntos
Doenças Autoimunes do Sistema Nervoso/metabolismo , Endocanabinoides/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Citocinas/metabolismo , Endocanabinoides/farmacologia , Endocanabinoides/uso terapêutico , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Leucócitos/metabolismo , Receptores de Canabinoides/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
The 3-hydroxyquinone derivative of the non-psychotrophic phytocannabinoid cannabigerol, so-called VCE-003.2, and some other derivatives have been recently investigated for neuroprotective properties in experimental models of Parkinson's disease (PD) in mice. The pharmacological effects in those models were related to the activity on the peroxisome proliferator-activated receptor-γ (PPAR-γ) and possibly other pathways. In the present study, we investigated VCE-004.8 (formulated as EHP-101 for oral administration), the 3-hydroxyquinone derivative of cannabidiol (CBD), with agonist activity at the cannabinoid receptor type-2 (CB2) receptor in addition to its activity at the PPAR-γ receptor. Studies were conducted in both in vivo (lesioned-mice) and in vitro (SH-SY5Y cells) models using the classic parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). Our data confirmed that the treatment with VCE-004.8 partially reduced the loss of tyrosine hydroxylase (TH)-positive neurons measured in the substantia nigra of 6-OHDA-lesioned mice, in parallel with an almost complete reversal of the astroglial (GFAP) and microglial (CD68) reactivity occurring in this structure. Such neuroprotective effects attenuated the motor deficiencies shown by 6-OHDA-lesioned mice in the cylinder rearing test, but not in the pole test. Next, we explored the mechanism involved in the beneficial effect of VCE-004.8 in vivo, by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-004.8 at a concentration of 10 µM, which was completely reversed by the addition of antagonists, T0070907 and SR144528, aimed at blocking PPAR-γ and CB2 receptors, respectively. The treatment with T0070907 alone only caused a partial reversal, whereas SR144528 alone had no effect, indicating a major contribution of PPAR-γ receptors in the cytoprotective effect of VCE-004.8 at 10 µM. In summary, our data confirmed the neuroprotective potential of VCE-004.8 in 6-OHDA-lesioned mice, and in vitro studies confirmed a greater relevance for PPAR-γ receptors rather than CB2 receptors in these effects.
Assuntos
Canabidiol/química , Oxidopamina/farmacologia , Doença de Parkinson/tratamento farmacológico , Quinonas/química , Administração Oral , Animais , Benzamidas/farmacologia , Canfanos/farmacologia , Canabinoides/química , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neuroproteção , Oxidopamina/química , PPAR gama/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The antioxidant and CB2 receptor agonist properties of Δ9-tetrahydrocannabivarin (Δ9-THCV) afforded neuroprotection in experimental Parkinson's disease (PD), whereas its CB1 receptor antagonist profile at doses lower than 5 mg/kg caused anti-hypokinetic effects. In the present study, we investigated the anti-dyskinetic potential of Δ9-THCV (administered i.p. at 2 mg/kg for two weeks), which had not been investigated before. This objective was investigated after inducing dyskinesia by repeated administration of L-DOPA (i.p. at 10 mg/kg) in a genetic model of dopaminergic deficiency, Pitx3ak mutant mice, which serves as a useful model for testing anti-dyskinetic agents. The daily treatment of these mice with L-DOPA for two weeks progressively increased the time spent in abnormal involuntary movements (AIMs) and elevated their horizontal and vertical activities (as measured in a computer-aided actimeter), signs that reflected the dyskinetic state of these mice. Interestingly, when combined with L-DOPA from the first injection, Δ9-THCV delayed the appearance of all these signs and decreased their intensity, with a reduction in the levels of FosB protein and the histone pAcH3 (measured by immunohistochemistry), which had previously been found to be elevated in the basal ganglia in L-DOPA-induced dyskinesia. In addition to the anti-dyskinetic effects of Δ9-THCV when administered at the onset of L-DOPA treatment, Δ9-THCV was also effective in attenuating the intensity of dyskinesia when administered for three consecutive days once these signs were already present (two weeks after the onset of L-DOPA treatment). In summary, our data support the anti-dyskinetic potential of Δ9-THCV, both to delay the occurrence and to attenuate the magnitude of dyskinetic signs. Although further studies are clearly required to determine the clinical significance of these data in humans, the results nevertheless situate Δ9-THCV in a promising position for developing a cannabinoid-based therapy for patients with PD.
Assuntos
Antidiscinéticos/administração & dosagem , Dronabinol/análogos & derivados , Discinesia Induzida por Medicamentos/prevenção & controle , Levodopa/administração & dosagem , Doença de Parkinson/complicações , Animais , Modelos Animais de Doenças , Dronabinol/administração & dosagem , Proteínas de Homeodomínio/genética , Masculino , Fatores de Transcrição/genéticaRESUMO
CB2 receptors (CB2 R) are expressed in midbrain neurons. To evidence the control of dopamine release in dorsal striatum by CB2 R, we performed experiments of [3 H]-dopamine release in dorsal striatal slices. We found a paradoxical increase in K+ -induced [3 H]-dopamine release by CB2 R activation with GW 833972A and JWH 133 two selective agonist. To understand the mechanism involved, we tested for a role of the D2 autoreceptor in this effect; because in pallidal structures, the inhibitory effect of CB1 receptors (CB1 R) on GABA release is switched to a stimulatory effect by D2 receptors (D2 R). We found that the blockade of D2 autoreceptors with sulpiride prevented the stimulatory effect of CB2 R activation; in fact, under this condition, CB2 R decreased dopamine release, indicating the role of the D2 autoreceptor in the paradoxical increase. We also found that the effect occurs in nigrostriatal terminals, since lesions with 6-OH dopamine in the middle forebrain bundle prevented CB2 R effects on release. In addition, D2 -CB2 R interaction promoted cAMP accumulation, and the increase in [3 H]-dopamine release was prevented by PKA blockade. D2 -CB2 R coprecipitation and proximity ligation assay studies indicated a close interaction of receptors that could participate in the observed effects. Finally, intrastriatal injection of CB2 R agonist induced contralateral turning in amphetamine-treated rats, which was prevented by sulpiride, indicating the role of the interaction in motor behavior. Thus, these data indicate that the D2 autoreceptor switches, from inhibitory to stimulatory, the CB2 R effects on dopamine release, involving the cAMP â PKA pathway in nigrostriatal terminals.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Receptores de Dopamina D2/metabolismo , Substância Negra/metabolismo , Anfetamina/farmacologia , Animais , Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , AMP Cíclico/metabolismo , Antagonistas dos Receptores de Dopamina D2/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Masculino , Movimento , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos Wistar , Receptor CB2 de Canabinoide/agonistas , Substância Negra/citologia , Substância Negra/efeitos dos fármacos , Sulpirida/farmacologiaRESUMO
A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure-activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 µM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.
Assuntos
Compostos Aza/farmacologia , Indóis/farmacologia , Quinuclidinas/farmacologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/antagonistas & inibidores , Compostos Aza/síntese química , Compostos Aza/química , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Ligantes , Estrutura Molecular , Quinuclidinas/síntese química , Quinuclidinas/química , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Relação Estrutura-AtividadeRESUMO
Neuroprotective M2-skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein-coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A2A (A2A R) and cannabinoid CB2 (CB2 R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2 R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2A R antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A -CB2 receptor heteromer (A2A -CB2 Het). Particularly relevant is the upregulation of A2A -CB2 Het expression in samples from the APPSw ,Ind AD transgenic mice model. The most relevant finding, confirmed in both heterologous cells and in primary cultures of microglia, was that blockade of A2A receptors results in increased CB2 R-mediated signaling. This heteromer-specific feature suggests that A2A R antagonists would potentiate, via microglia, the neuroprotective action of endocannabinoids with implications for AD therapy.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Microglia/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Transdução de Sinais/fisiologia , Animais , Dronabinol/farmacologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Receptor CB2 de Canabinoide/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
Mild TBI is often accompanied by visual system dysfunction and injury, which is at least partly caused by microglial neuroinflammatory processes initiated by the injury. Using our focal cranial blast mouse model of closed-skull mild TBI, we evaluated the ability of the cannabinoid type-2 (CB2) receptor inverse agonist SMM-189, which biases microglia from the harmful M1 state to the beneficial M2 state, to mitigate visual system dysfunction and injury after TBI. Male C57BL/6 or Thy1-EYFP reporter mice received a closed-head blast of either 0-psi (sham) or 50-psi to the left side of the cranium. Blast mice received vehicle or 6 mg/kg SMM-189 daily beginning 2 h after blast. Sham mice received vehicle. In some mice, retina and optic nerve/tract were assessed morphologically at 3-7 days after blast, while other mice were assessed functionally by Optomotry 30 days after blast and morphologically at ≥30 days after blast. Mice sacrificed at 3-7 days were treated daily until sacrificed, while those assessed ≥30 days after blast were treated daily for 2 weeks post blast. Axon damage was evident in the left optic nerve and its continuation as the right optic tract at 3 days post blast in vehicle-treated blast mice in the form of swollen axon bulbs, and was accompanied by a significant increase in the abundance of microglia. Testing at 30 days post blast revealed that the contrast sensitivity function was significantly reduced in both eyes in vehicle-treated blast mice compared to vehicle-treated sham blast mice, and axon counts at ≥30 days after blast revealed a â¼10% loss in left optic nerve in vehicle-treated blast mice. Left optic nerve axon loss was highly correlated with the left eye deficit in contrast sensitivity. Immunolabeling at 30 days post blast showed a significant increase in the abundance of microglia in the retinas of both eyes and in GFAP + Müller cell processes traversing the inner plexiform layer in the left eye of vehicle-treated blast mice. SMM-189 treatment reduced axon injury and microglial abundance at 3 days, and mitigated axon loss, contrast sensitivity deficits, microglial abundance, and Müller cell GFAP upregulation at ≥30 days after blast injury. Analysis of right optic tract microglia at 3 days post blast for M1 versus M2 markers revealed that SMM-189 biased microglia toward the M2 state, with this action of SMM-189 being linked to reduced axonal injury. Taken together, our results show that focal left side cranial blast resulted in impaired contrast sensitivity and retinal pathology bilaterally and optic nerve loss ipsilaterally. The novel cannabinoid drug SMM-189 significantly mitigated the functional deficit and the associated pathologies. Our findings suggest the value of combatting visual system injury after TBI by using CB2 inverse agonists such as SMM-189, which appear to target microglia and bias them away from the pro-inflammatory M1 state, toward the protective M2 state.
Assuntos
Benzofenonas/farmacologia , Lesões Encefálicas Traumáticas/complicações , Microglia/patologia , Nervo Óptico/patologia , Trato Óptico/patologia , Transtornos da Visão/tratamento farmacológico , Acuidade Visual , Animais , Axônios/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Transtornos da Visão/etiologia , Transtornos da Visão/patologiaRESUMO
Cannabinoid CB1 receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB2 receptors (CB2Rs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CB2Rs in the brain. However, this finding is disputed because of the specificity of CB2R antibody signals. We used two strains of currently available partial CB2-knockout (CB2-KO) mice as controls, four anti-rat or anti-mouse CB2R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB2R-like band at ~40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB2-like immunostaining in mouse midbrain dopamine neurons. CB2R deletion in CB2-KO mice may reduce or leave CB2R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB2R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB2-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB2-KO mice. Quantitative RT-PCR assays detected CB2 mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB2-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB2R-specific. Non-specific binding may be related to the expression of mutant or truncated CB2R-like proteins in partial CB2-KO mice and the use of anti-rat CB2 antibodies because the epitopes are different between rat and mouse CB2Rs.
Assuntos
Anticorpos/imunologia , Receptor CB2 de Canabinoide/imunologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Western Blotting , Neurônios Dopaminérgicos/metabolismo , Técnicas de Inativação de Genes , Imuno-Histoquímica , Mesencéfalo/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptor CB2 de Canabinoide/genética , Baço/metabolismoRESUMO
BACKGROUND: The anti-tumor properties of cannabinoids have been investigated in many in vitro and in vivo studies. Many of these anti-tumor effects are mediated via cannabinoid receptor types 1 and 2 (CB1 and CB2), comprising the endocannabinoid system (ECS). In this study, we investigated the ECS based on CB 1 and CB 2 receptor gene and protein expression in renal cell carcinoma (RCC) cell lines. In view of their further use for potential treatments, we thus investigated the roles of CB1 and CB2 receptors in the anti-proliferative action and signal transduction triggered by synthetic cannabinoid agonists [such as JWH-133 and WIN 55,212-2 (WIN-55)] in RCC cell lines. METHODS: Human RCC cell lines were used for this study. The CB 1 and CB 2 gene expression levels were analyzed using real-time PCR. Flow cytometric, immunocytochemical and western blot analyses were performed to confirm CB1 and CB2 receptor protein expression. The anti-proliferative effects of synthetic cannabinoids were investigated on cell viability assay. The CB1 and CB2 receptors were blocked pharmacologically with the antagonists SR141716A and AM-630, respectively, to investigate the effects of the agonists JWH-133 and WIN-55. Cell cycle, apoptosis and LDH-based cytotoxicity were analyzed on cannabinoid-treated RCC cells. RESULTS: The CB1 and CB2 genes expression was shown by real-time PCR and flow cytometric and western blot analysis indicating a higher level of CB2 receptor as compared to CB1 in RCC cells. Immunocytochemical staining also confirmed the expression of the CB1 and CB2 proteins. We also found that the synthetic cannabinoid agonist WIN-55 exerted anti-proliferative and cytotoxic effects by inhibiting the growth of RCC cell lines, while the CB2 agonist JWH-133 did not. Pharmacologically blocking the CB1 and CB2 receptors with their respective antagonists SR141716A and AM-630, followed by the WIN-55 treatment of RCC cells allowed uncovering the involvement of CB2, which led to an arrest in the G0/G1 phase of the cell cycle and apoptosis. CONCLUSIONS: This study elucidated the involvement of CB2 in the in vitro inhibition of RCC cells, and future applications of CB2 agonists in the prevention and management of RCC are discussed.