Lack of Cannabinoid Type 2 Promoter Activity in Normal or Injured Kidneys Using a Cnr2-GFP Reporter Mouse.

Introduction: Although cannabinoid type 2 (CB2) receptor activity is known to promote diverse biological functions in the kidney, published data regarding CB2 receptor protein levels and cellular distribution within the kidney is inconsistent. The goal of the present study was to investigate the changes of CB2 in the kidney obtained from mice exposed to various forms of kidney injury using a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous cannabinoid receptor 2 (Cnr2) promoter. Materials and Methods: Kidney injury was established in a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous Cnr2 promoter. Kidney injury was initiated by either treatment with different chemicals [cisplatin or lipopolysaccharide (LPS)] or by unilateral ureteral obstruction (UUO). Changes in the detection of GFP were used as a proxy for CB2 levels and localization. Histological changes due to the injury stimuli were observed by time-related, morphological changes in kidney cytoarchitecture and blood parameters, such as serum creatinine levels. Cnr2 mRNA levels were detected by reverse transcription coupled to polymerase chain reaction (RT-PCR) while protein changes in the tissue lysates were measured by Western blot analysis. Cellular localization of GFP was detected by fluorescent microscopy. Results: Our data demonstrated that there was no band or a minimally detectable band for GFP using kidney lysates from vehicle- or cisplatin-treated mice. A similar lack of GFP was detected in the UUO kidney versus the contralateral control kidney. This is consistent with the low, albeit detectable levels of Cnr2 mRNA in the kidney samples from control or cisplatin treatment. In frozen kidney sections from vehicle and cisplatin-treated mice, GFP fluorescence was not detectable in tubular epithelia, glomeruli or blood vessels in the cortex. Instead, GFP was detected in rare cells within the interstitial space. A second chemical injury model using LPS found a similar lack of GFP protein levels and an absence of legitimate GFP fluorescence in the main cell types within the kidney. Conclusion: These findings suggest that Cnr2 promoter activity is minimally active in normal or injured kidneys, and that pharmacological manipulation of CB2 receptors may be associated with receptors being expressed in cells recruited to the kidney.

Cannabinoid type 2 receptor deficiency leads to Aß-induced cognitive impairment through promoting microglial sensitivity to Aß in the prefrontal cortex in mice.

Aims: This study is to investigate the effects of Cannabinoid type 2 receptor (CB2R) deficiency on microglia and cognitive function in both Aß1-42-injected CB2R knockout mice and a transgenic mouse model of Alzheimer's disease (AD) in brain. Methods: After hippocampal injection with Aß1-42 oligomers in CB2R knockout mice with and without CB2R agonist treatment and in transgenic APP/PS1 mice with CB2R deletion, the novel object recognition (NOR) and Morris water maze (MWM) tests were performed to assess the animal behavior performance. Immunofluorescence staining was conducted to detect the microglial morphology and activation status. The expression of proinflammation and anti-inflammation cytokines were determined by qRT-PCR. Results: CB2R deficiency significantly aggravated cognitive impairment in both Aß1-42-induced and transgenic APP/PS1 animal model in NOR. In Aß-injected mice lacking CB2R and transgenic APP/PS1 mice with CB2R deletion, microglia in the prefrontal cortex exhibited enhanced immunoreactivity with altered morphology. Furthermore, transformation of activated microglial phenotype in the prefrontal cortex was reduced in Aß1-42-injected CB2R knockout mice after CB2R agonist treatment. The CB2R deficiency significantly increased the expression of proinflammatory cytokines in the prefrontal cortex, while it was observed in the hippocampus in both Aß1-42-injected and transgenic APP/PS1 AD mouse model. Furthermore, CB2R deficiency increased concentrations of soluble Aß 40 in the prefrontal cortex, but did not affect plaques deposition. Conclusion: CB2R deletion led to enhanced neuroinflammatory responses via direct upregulating microglia activation in the prefrontal cortex during the early symptomatic phase of AD mice. CB2R modulates prefrontal cortical neuroinflammation, which is essential for regulating cognitive functions such as recognition memory at the early stage of AD.

JWH133 attenuates behavior deficits and iron accumulation in 6-OHDA-induced Parkinson's disease model rats.

Growing evidence indicates that activation of cannabinoid type 2 (CB2) receptors protects dopamine neurons in the pathogenesis of Parkinson's disease (PD). However, the mechanisms underlying neuroprotection mediated by CB2 receptors are still elusive. In this study, we investigated the effects of CB2 receptor activation on 6-hydroxydopamine (6-OHDA)-induced dopamine neuron degeneration and iron accumulation in the substantia nigra (SN) of rats. We found that treatment with JWH133, a selective CB2 receptor agonist, significantly improved the apomorphine (APO)-induced rotational behavior in 6-OHDA-treated rats. The decreased numbers of tyrosine hydroxylase (TH)-positive neurons and reduced TH protein expression in the lesioned SN of rats were effectively restored by JWH133. Moreover, we found that JWH133 inhibited the increase of iron-staining cells in the lesioned SN of rats. To explore the protective mechanisms of activation of CB2 receptors on dopamine neurons, we further observed the effect of JWH133 on 1-methyl-4-phenylpyridinium (MPP+)-treated primary cultured ventral mesencephalon (VM) neurons from rats. We found that JWH133 significantly inhibited the increase of intracellular reactive oxygen species (ROS), the activation of Caspase-3, the decrease of mitochondrial transmembrane potential (ΔΨm), and the decrease of Bcl-2/Bax protein expression caused by MPP+ treatment. JWH133 also inhibited the MPP+-induced upregulation of divalent metal transporter-1 (DMT1) and downregulation of ferroportin 1 (FPN1). Furthermore, JWH133 also suppressed the MPP+-accelerated iron influx in the VM neurons. These results suggest that activation of CB2 receptor suppresses MPP+-induced cellular iron accumulation and prevents neurodegeneration.NEW & NOTEWORTHY Expression of cannabinoid type 2 receptors (CB2Rs) was discovered on dopamine neurons in recent years. The role of CB2R expressed on dopamine neurons in the pathogenesis of Parkinson's disease (PD) has not been fully elucidated. The content of iron accumulation in the brain is closely related to the progress of PD. We verified the inhibitory effect of CB2R on iron deposition in dopamine neurons through experiments, which provided a new idea for the treatment of PD.

Discovery and development of macrocyclic peptide modulators of the cannabinoid 2 receptor.

The cannabinoid type 2 receptor (CB2R), a G protein-coupled receptor, is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB2R is typically targeted by small molecules, including endo-, phyto-, and synthetic cannabinoids, peptides-owing to their size-may offer a different interaction space to facilitate differential interactions with the receptor. Here, we explore plant-derived cyclic cystine-knot peptides as ligands of the CB2R. Cyclotides are known for their exceptional biochemical stability. Recently, they gained attention as G protein-coupled receptor modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB2R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics, we identified the cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB2R with an affinity (Ki) of 1 µM and a potency (EC50) of 8 µM. Leveraging deep learning networks, we verified the structural topology of vodo-C1 and modeled its molecular volume in comparison to the CB2R ligand binding pocket. In a fragment-based approach, we designed and characterized vodo-C1-based bicyclic peptides (vBCL1-4), aiming to reduce size and improve potency. Opposite to vodo-C1, the vBCL peptides lacked the ability to activate the receptor but acted as negative allosteric modulators or neutral antagonists of CB2R. This study introduces a macrocyclic peptide phytocannabinoid, which served as a template for the development of synthetic CB2R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.

Activation of Cannabinoid Type 2 Receptor in Microglia Reduces Neuroinflammation through Inhibiting Aerobic Glycolysis to Relieve Hypertension.

BACKGROUND: Studies have shown that the chronic use of cannabis is associated with a decrease in blood pressure. Our previous studies prove that activating the cannabinoid type 2 (CB2) receptor in the brain can effectively reduce blood pressure in spontaneously hypertensive rats; however, the exact mechanism has not been clarified. The objective of this study is to demonstrate that activation of microglial CB2 receptors can effectively reduce the levels of TNF-α, IL-1ß, and IL-6 in the paraventricular nucleus (PVN) through inhibiting aerobic glycolysis, thereby relieving hypertension. METHODS: AngiotensinII (AngII) was administered to BV2 cells and C57 mice to induce hypertension and the release of proinflammatory cytokines. The mRNA and protein expression of the CB2 receptor, TNF-α, IL-1ß, IL-6, and the PFK and LDHa enzymes were detected using RT-qPCR and Western blotting. The Seahorse XF Energy Metabolism Analyzer was used to measure the oxidative phosphorylation and aerobic glycolysis metabolic pathways in BV2 cells. The long-term effects of injecting JWH133, a selective CB2 receptor agonist, intraperitoneally on blood pressure were ascertained. ELISA was used to measure norepinephrine and lactic acid levels while immunofluorescence labeling was used to locate the CB2 receptor and c-Fos. By injecting pAAV-F4/80-GFP-mir30shRNA (AAV2-r-CB2shRNA) into the lateral cerebral ventricle, the CB2 receptor in microglia was specifically knocked down. RESULTS: Activation of CB2 receptors by the agonist JWH133 suppressed TNF-α, IL-1ß, and IL-6 by inhibiting PFK and LDHa enzymes involved in glycolysis, as well as lactic acid accumulation, along with a reduction in glycoPER levels (marks of aerobic glycolysis) in AngII-treated BV2 cells. In AngII-treated mice, the administration of JWH133 specifically activated CB2 receptors on microglia, resulting in decreased expression levels of PFK, LDHa, TNF-α, IL-1ß, and IL-6, subsequently leading to a decrease in c-Fos protein expression within PVN neurons as well as reduced norepinephrine levels in plasma, ultimately contributing to blood pressure reduction. CONCLUSION: The results suggest that activation of the microglia CB2 receptor decreases the neuroinflammation to relieve hypertension; the underlying mechanism is related to inhibiting aerobic glycolysis of microglia.

Beta-Caryophyllene, a Cannabinoid Receptor Type 2 Selective Agonist, in Emotional and Cognitive Disorders.

Mental disorders account for one of the most prevalent categories of the burden of disease worldwide, with depression expected to be the largest contributor by 2030, closely followed by anxiety. The COVID-19 pandemic possibly exacerbated these challenges, especially amongst adolescents, who experienced isolation, disrupted routines, and limited healthcare access. Notably, the pandemic has been associated with long-term neurological effects known as "long-COVID", characterized by both cognitive and psychopathological symptoms. In general, psychiatric disorders, including those related to long-COVID, are supposed to be due to widespread inflammation leading to neuroinflammation. Recently, the endocannabinoid system (ECS) emerged as a potential target for addressing depression and anxiety pathophysiology. Specifically, natural or synthetic cannabinoids, able to selectively interact with cannabinoid type-2 receptor (CB2R), recently revealed new therapeutic potential in neuropsychiatric disorders with limited or absent psychotropic activity. Among the most promising natural CB2R ligands, the bicyclic sesquiterpene ß-caryophyllene (BCP) has emerged as an excellent anti-inflammatory and antioxidant therapeutic agent. This review underscores BCP's immunomodulatory and anti-inflammatory properties, highlighting its therapeutic potential for the management of depression and anxiety.

The effect of cannabinoid type 2 receptor agonist on morphine tolerance.

Pain highly impacts the quality of life of patients. Morphine is used for pain treatment; however, its side effects, especially morphine tolerance, limit its use in the clinic. The problem of morphine tolerance has plagued health workers and patients for years. Unfortunately, the exact mechanism of morphine tolerance has not been fully clarified. The mechanisms of morphine tolerance that are currently being studied may include µ-opioid receptor (MOR) desensitization and internalization, mitogen-activated protein kinase (MAPK) pathway activation and crosstalk, the effects of microglia and the increase in inflammatory factors. Morphine tolerance can be alleviated by improving the pathophysiological changes that lead to morphine tolerance. Previous studies have shown that a cannabinoid type 2 (CB2) receptor agonist could attenuate morphine tolerance in a variety of animal models. Many studies have shown an interaction between the cannabinoid system and the opioid system. The CB2 receptor may modulate the effect of morphine through a pathway that is common to the MOR, since both receptors are G protein-coupled receptors (GPCRs). This study introduces the potential mechanism of morphine tolerance and the effect of CB2 receptor agonists on reducing morphine tolerance, which can provide new ideas for researchers studying morphine and provide beneficial effects for patients suffering from morphine tolerance.

Evaluation of [18F]RoSMA-18-d6 as a CB2 PET Radioligand in Nonhuman Primates.

The cannabinoid type 2 receptor (CB2) has been implicated in a variety of central and peripheral inflammatory diseases, prompting significant interest in the development of CB2-targeted diagnostic and therapeutic agents. A validated positron emission tomography (PET) radioligand for imaging CB2 in the living human brain as well as in peripheral tissues is currently lacking. As part of our research program, we have recently identified the trisubstituted pyridine, [18F]RoSMA-18-d6, which proved to be highly suitable for in vitro and in vivo mapping of CB2 in rodents. The aim of this study was to assess the performance characteristics of [18F]RoSMA-18-d6 in nonhuman primates (NHPs) to pave the way for clinical translation. [18F]RoSMA-18-d6 was synthesized from the respective tosylate precursor according to previously reported procedures. In vitro autoradiograms with NHP spleen tissue sections revealed a high binding of [18F]RoSMA-18-d6 to the CB2-rich NHP spleen, which was significantly blocked by coincubation with the commercially available CB2 ligand, GW405833 (10 µM). In contrast, no specific binding was observed by in vitro autoradiography with NHP brain sections, which was in agreement with the notion of a CB2-deficient healthy mammalian brain. In vitro findings were corroborated by PET imaging experiments in NHPs, where [18F]RoSMA-18-d6 uptake in the spleen was dose-dependently attenuated with 1 and 5 mg/kg GW405833, while no specific brain signal was observed. Remarkably, we observed tracer uptake and retention in the NHP spinal cord, which was reduced by GW405833 blockade, pointing toward a potential utility of [18F]RoSMA-18-d6 in probing CB2-expressing cells in the bone marrow. If these observations are substantiated in NHP models of enhanced leukocyte proliferation in the bone marrow, [18F]RoSMA-18-d6 may serve as a valuable marker for hematopoietic activity in various pathologies. In conclusion, [18F]RoSMA-18-d6 proved to be a suitable PET radioligand for imaging CB2 in NHPs, supporting its translation to humans.

Rational Design, Synthesis, and Evaluation of Fluorescent CB2 Receptor Ligands for Live-Cell Imaging: A Comprehensive Review.

The cannabinoid receptors CB1 and CB2 are class A G protein-coupled receptors (GPCRs) that are activated via endogenous lipids called endocannabinoids. The endocannabinoid system (ECS) plays a critical role in the regulation of several physiological states and a wide range of diseases. In recent years, drug discovery approaches targeting the cannabinoid type 2 receptor (CB2R) have gained prominence. Particular attention has been given to selective agonists targeting the CB2 receptors to circumvent the neuropsychotropic side effects associated with CB1 receptors. The pharmacological modulation of CB2R holds therapeutic promise for various diseases, such as inflammatory disorders and immunological conditions, as well as pain management and cancer treatment. Recently, the utilization of fluorescent probes has emerged as a valuable technique for investigating the interactions between ligands and proteins at an exceptional level of spatial and temporal precision. In this review, we aim to examine the progress made in the development of fluorescent probes targeting CB2 receptors and highlight their significance in facilitating the successful clinical translation of CB2R-based therapies.

Cannabinoid type 2 receptor activation inhibits MPP+-induced M1 differentiation of microglia through activating PI3K/Akt/Nrf2 signal pathway.

BACKGROUND: Growing evidence indicates that cannabinoid type 2 (CB2) receptor activation inhibits neuroinflammation in the pathogenesis of Parkinson's disease (PD). Nonetheless, the precise mechanisms of CB2 receptor-mediated neuroprotection have not been fully elucidated. The differentiation of microglia from the M1 to M2 phenotype plays a vital role in neuroinflammation. METHODS: In the present study, we investigated the effect of CB2 receptor activation on the M1/M2 phenotypic transformation of microglia treated with 1-methyl-4-phenylpyridinium (MPP+). The M1 phenotype microglia markers, including inducible nitric oxide (iNOS), interleukin 6 (IL-6), and CD86, and the M2 phenotype microglia markers, including arginase-1 (Arg-1), IL-10, and CD206, were detected by western blots and flow cytometry. The levels of phosphoinositide-3-kinase (PI3K)/Akt and nuclear factor erythroid 2-related factor 2 (Nrf2) were determined by Western blots. Subsequent addition of Nrf2 inhibitors initially revealed the specific mechanism by which CB2 receptors affect phenotypic changes in microglia. RESULTS: Our results showed that pretreatment with JWH133 significantly inhibited the MPP+-induced up-regulation of M1 phenotype microglia markers. Meanwhile, JWH133 increased the levels of M2 phenotype microglia markers. JWH133-mediated effects were blocked by co-treatment with AM630. Mechanism studies found that MPP+ treatment downregulated PI3K, Akt phosphorylated proteins, and nuclear Nrf2 protein. JWH133 pretreatment promoted PI3K/Akt activation and facilitated nuclear translocation of Nrf2, which was reversed by the PI3K inhibitor. Further studies showed that Nrf2 inhibitors inverted the effect of JWH133 on microglia polarization. CONCLUSION: The results indicate that CB2 receptor activation promotes MPP+-induced microglia transformation from M1 to M2 phenotype through PI3K/Akt/Nrf2 signaling pathway.

[Research progress on anti-inflammatory effects of plant-derived cannabinoid type 2 receptor modulators].

Excessive and persistent inflammatory responses are a potential pathological condition that can lead to diseases of various systems, including nervous, respiratory, digestive, circulatory, and endocrine systems. Cannabinoid type 2 receptor(CB2R) belongs to the G protein-coupled receptor family and is widely distributed in immune cells, peripheral tissues, and the central nervous system. It plays a role in inflammatory responses under various pathological conditions. The down-regulation of CB2R activity is an important marker of inflammation and and CB2R modulators have been shown to have anti-inflammatory effects. This study explored the relationship between CB2R and inflammatory responses, delved into its regulatory mechanisms in inflammatory diseases, and summarized the research progress on CB2R modulators from plants other than cannabis, including plant extracts and monomeric compounds, in exerting anti-inflammatory effects. The aim is to provide new insights into the prevention and treatment of inflammatory diseases.

Evaluation of cannabinoid type 2 receptor expression and pyridine-based radiotracers in brains from a mouse model of Alzheimer's disease.

Neuroinflammation plays an important role in the pathophysiology of Alzheimer's disease. The cannabinoid type 2 receptor (CB2R) is an emerging target for neuroinflammation and therapeutics of Alzheimer's disease. Here, we aim to assess the alterations in brain CB2R levels and evaluate novel CB2R imaging tracers in the arcAß mouse model of Alzheimer's disease amyloidosis. Immunohistochemical staining for amyloid-ß deposits (6E10), microgliosis (anti-Iba1 and anti-CD68 antibodies), astrocytes (GFAP) and the anti-CB2R antibody was performed on brain slices from 17-month-old arcAß mice. Autoradiography using the CB2R imaging probes [18F]RoSMA-18-d6, [11C]RSR-056, and [11C]RS-028 and mRNA analysis were performed in brain tissue from arcAß and non-transgenic littermate (NTL) mice at 6, 17, and 24 months of age. Specific increased CB2R immunofluorescence intensities on the increased number of GFAP-positive astrocytes and Iba1-positive microglia were detected in the hippocampus and cortex of 17-month-old arcAß mice compared to NTL mice. CB2R immunofluorescence was higher in glial cells inside 6E10-positive amyloid-ß deposits than peri-plaque glial cells, which showed low background immunofluorescence in the hippocampus and cortex of 17-month-old arcAß mice. Ex vivo autoradiography showed that the specific binding of [18F]RoSMA-18-d6 and [11C]RSR-056 was comparable in arcAß and NTL mice at 6, 17, and 24 months of age. The level of Cnr2 mRNA expression in the brain was not significantly different between arcAß and NTL mice at 6, 17, or 24 months of age. In conclusion, we demonstrated pronounced specific increases in microglial and astroglial CB2R expression levels in a mouse model of AD-related cerebral amyloidosis, emphasizing CB2R as a suitable target for imaging neuroinflammation.

Cannabinoid type-2 receptors: An emerging target for regulating schizophrenia-relevant brain circuits.

Although the cannabinoid type-2 receptor (CB2) is highly expressed in the immune system, emerging evidence points to CB2 playing a key role in regulating neuronal function in the central nervous system. Recent anatomical studies, combined with electrophysiological studies, indicate that CB2 receptors are expressed in specific dopaminergic and glutamatergic brain circuits that are hyperactive in schizophrenia patients. The ability of CB2 receptors to inhibit dopaminergic and hippocampal circuits, combined with the anti-inflammatory effects of CB2 receptor activation, make this receptor an intriguing target for treating schizophrenia, a disease where novel interventions that move beyond dopamine receptor antagonists are desperately needed. The development of new CB2-related pharmacological and genetic tools, including the first small molecule positive allosteric modulator of CB2 receptors, has greatly advanced our understanding of this receptor. While more work is needed to further elucidate the translational value of selectively targeting CB2 receptors with respect to schizophrenia, the studies discussed below could suggest that CB2 receptors are anatomically located in schizophrenia-relevant circuits, where the physiological consequence of CB2 receptor activation could correct circuit-based deficits commonly associated with positive and cognitive deficits.

CB2R Activation Regulates TFEB-Mediated Autophagy and Affects Lipid Metabolism and Inflammation of Astrocytes in POCD.

Evidence suggests that the accumulation of lipid drots (LDs) accelerates damage to mitochondria and increases the release of inflammatory factors. These have been implicated as a mechanism underlying neurodegenerative diseases or tumors and aging-related diseases such as postoperative cognitive dysfunction (POCD), nevertheless, accumulation of lipid droplets has not been extensively studied in the central nervous system (CNS). Here, we found that after surgery, there was activation of astrocytes and lipid accumulation in the hippocampus. However, cannabinoid receptor type II (CB2R) activation significantly reduced lipid accumulation in astrocytes and change the expression of genes related to lipid metabolism. CB2R reduces the release of the inflammatory factors interleukin-1 beta (IL-1ß) and interleukin 6 (IL-6) in peripheral serum and simultaneously improves cognitive ability in mice with POCD. Further research on mechanisms indicates that CB2R activation promotes the nuclear entry of the bHLH-leucine zipper transcription factor, the transcription factor EB (TFEB), and which is a master transcription factor of the autophagy-lysosomal pathway, also reduces TFEB-S211 phosphorylation. When CB2R promotes TFEB into the nucleus, TFEB binds at two sites within promoter region of PGC1α, promoting PGC1α transcription and accelerating downstream lipid metabolism. The aforementioned process leads to autophagy activation and decreases cellular lipid content. This study uncovers a new mechanism allowing CB2R to regulate lipid metabolism and inflammation in POCD.

Win55,212-2 improves neural injury induced by HIV-1 glycoprotein 120 in rats by exciting CB2R.

The occurrence and development of HIV-associated neurocognitive disorders (HAND) is related to synaptic injury and neuron loss, which gradually reduces the ability of learning and memory, and eventually leads to cognitive dysfunction. Human immunodeficiency virus type 1 (HIV-1) enveloped glycoprotein 120 (GP120) is the principal etiological agent of HIV-1-induced nerve damage and HAND. Our previous study demonstrated that GP120 can induce neuronal damage by increasing N-methyl-D-aspartic acid receptor (NMDAR) mediated excitatory postsynaptic currents (EPSCsNMDAR), In addition to neuroexcitotoxicity, the inflammatory response, oxidative stress, and neuronal apoptosis mediated by NMDAR overactivation are also involved in HAND. Because cannabinoids have known effects against neuroinflammation, stress response, and oxidative effects, we researched the effects of the cannabinoid receptor agonist Win55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3 [(4-morpholinyl) methyl] pyrrolo [1,2,3-de]- 1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt] on synaptic changes induced by GP120. In this study, we discovered that Win55,212-2 prevents GP120-induced neurological injury and cognitive dysfunction, and these effects are consistent with the neuroprotective effect of NMDAR blockers. In the Morris water maze (MWM) test, the results revealed that GP120 could induce learning and memory impairment in rats, while antagonizing NMDARs or activating CB2R could counteract GP120-induced cognitive dysfunction in rats; The results of TUNEL staining were consistent with the above results of MWM behavioral experiments. GP120 damaged hippocampal neurons in the CA1 region, while the NMDAR antagonist and cannabinoid 2 receptor (CB2R) agonist prevented GP120-induced effects. In molecular biology experiments, Our results showed that GP120 significantly upregulated the mRNA expressions of inflammatory factors IL-1ß, IL-6, TNF-α and CXCL10; Furthermore, GP120 significantly upregulated the protein expression level of pro-inflammatory cytokine IL-1ß and decreased the protein expression level of anti-inflammatory cytokine IL-10 as measured by ELISA. Additionally, in the GP120 group, the mRNA expression levels of pro-apoptosis factors such as Bax, CytC, and caspase-3, - 8 and - 9 were significantly increased while the expression level of anti-apoptotic factors Bcl-2 was significantly decreased (P < 0.05). Our studie also demonstrated that the mRNA expression levels of apoptotic pathway factors could be regulated by the p38 JNK MAPK pathway. But pretreatment with NMDAR antagonist memantine or CB2R agonist Win55,212-2 significantly reduced the expression levels of inflammatory factors and apoptotic factors. And the effects aroused by Win55,212-2 could be reversed by the CB2R antagonist AM630. These data suggest that the activation of the CB2R is neuroprotective against GP120-induced neurotoxicity, and CB2R agonist might play a potential therapeutic role in HAND.

Cannabinoid Receptor Type 2 Agonist Reduces Morphine Tolerance via Mitogen Activated Protein Kinase Phosphatase Induction and Mitogen Activated Protein Kinase Dephosphorylation.

Morphine is an opioid drug often used in treating moderate to severe pain. However, morphine tolerance in patients limits its used in clinical settings. Our previous study showed that a cannabinoid type 2 (CB2) receptor agonist attenuated morphine tolerance. However, the exact mechanism by which CB2 agonists reduce morphine tolerance remains unclear. In this study, we investigated the effect of mitogen activated protein kinase (MAPK) and mitogen activated protein kinase phosphatases 1 and 3 (MKP-1 and MKP-3) on the regulation of morphine tolerance by CB2 receptor agonist. Chronic morphine treatments for 7 days reduced the protein expression of MKP-1 and MKP-3 in the spinal cord and increased the phosphorylation of p38, ERK1/2 and the level of proinflammatory mediator, such as IL-1ß, IL-6 and TNF-α. Coadministration of CB2 receptor agonist AM1241 alleviated the inhibition of MKP-1 and MKP-3 by chronic morphine administration and reduced the expression of phosphorylated MAPK and proinflammatory factors. The effect of the CB2 receptor agonist on morphine-induced downregulation of MKP-1 and MKP-3 was reversed by the MKP-1 and MKP-3 antagonist triptolide. Our findings suggested that CB2 receptor agonist may induce the expression of MKP-1 and MKP-3 to promote MAPK dephosphorylation and reduce the production of downstream cytokine, thereby reducing morphine tolerance. This finding suggested that MKPs may serve as a new target for alleviating morphine tolerance.

In vivo Imaging of Cannabinoid Type 2 Receptors: Functional and Structural Alterations in Mouse Model of Cerebral Ischemia by PET and MRI.

PURPOSE: Stroke is one of the most prevalent vascular diseases. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptor (CB2R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [18F]RoSMA-18-d6 and structural imaging by magnetic resonance imaging (MRI). PROCEDURES: Our recently developed CB2R PET tracer [18F]RoSMA-18-d6 was used for imaging neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB2R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [18F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T2-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice. RESULTS: mRNA expressions of inflammatory markers TNF-α, Iba1, MMP9 and GFAP, CNR2 were increased to 1.3-2.5 fold at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced to ca. 50 %. Reduced [18F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [18F]RoSMA-18-d6 in ex vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in vivo specificity of [18F]RoSMA-18-d6 was confirmed only in the CB2R-rich spleen. CONCLUSIONS: This study revealed an increased [18F]RoSMA-18-d6 measure of CB2R and a reduced [18F]FDG measure of CMRglc in the ischemic striatum of tMCAO mice at subacute stage. [18F]RoSMA-18-d6 might be a promising PET tracer for detecting CB2R alterations in animal models of neuroinflammation without neuronal loss.

The Neuroprotective Effects of the CB2 Agonist GW842166x in the 6-OHDA Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder associated with dopamine neuron loss and motor dysfunction. Neuroprotective agents that prevent dopamine neuron death hold great promise for slowing the disease's progression. The activation of cannabinoid (CB) receptors has shown neuroprotective effects in preclinical models of neurodegenerative disease, traumatic brain injury, and stroke, and may provide neuroprotection against PD. Here, we report that the selective CB2 agonist GW842166x exerted protective effects against the 6-hydroxydopamine (6-OHDA)-induced loss of dopamine neurons and its associated motor function deficits in mice, as shown by an improvement in balance beam walking, pole, grip strength, rotarod, and amphetamine-induced rotation tests. The neuroprotective effects of GW842166x were prevented by the CB2 receptor antagonist AM630, suggesting a CB2-dependent mechanism. To investigate potential mechanisms for the neuroprotective effects of GW842166x, we performed electrophysiological recordings from substantia nigra pars compacta (SNc) dopamine neurons in ex vivo midbrain slices prepared from drug-naïve mice. We found that the bath application of GW842166x led to a decrease in action potential firing, likely due to a decrease in hyperpolarization-activated currents (Ih) and a shift of the half-activation potential (V1/2) of Ih to a more hyperpolarized level. Taken together, the CB2 agonist GW842166x may reduce the vulnerability of dopamine neurons to 6-OHDA by decreasing the action potential firing of these neurons and the associated calcium load.

CB2-mediated attenuation of nucleus pulposus degeneration via the amelioration of inflammation and oxidative stress in vivo and in vitro.

BACKGROUND: Nucleus pulposus cell (NPC) degeneration is widely accepted as one of the major causes of intervertebral disc (IVD) degeneration (IVDD). The pathogenesis of IVDD is complex and consists of inflammation, oxidative stress, and the loss of extracellular matrix (ECM). Cannabinoid type 2 receptor (CB2) has been shown to be involved in the pathological mechanism of a variety of diseases due to its anti-inflammatory effects and antioxidative stress capacity. METHOD: In Vitro, H2O2 was used to induce degeneration of nucleus pulposus cells, mRNA and protein expression level was determined by RT-PCR and Western Blot, and Immunocytochemical staining were used to detect expression of collagen II, aggrecan, MMP3/13, superoxide dismutase 2 (SOD2) and inducible nitric oxide synthase (iNOS). In vivo, the potential therapeutic effect of CB2 was detected in the rat acupuncture model. RESULT: In vitro, we found that the CB2 agonist (JWH133) treatment reduced the oxidative stress level in NPCs induced by hydrogen peroxide (H2O2) treatment. Furthermore, the expression of inflammatory cytokines was also decreased by JWH133 treatment. We found that collagen II and aggrecan expression was preserved, whereas matrix metalloproteinase levels were reduced. In vivo, we established a rat model by needle puncture. Imaging assessment revealed that the disc height index (DHI) and morphology of IVD were significantly improved, and the disc degeneration process was delayed by treatment of JWH133. Furthermore, immunohistochemical (IHC) staining revealed that JWH133 could inhibit the degradation of collagen II and decrease the expression of MMP3. CONCLUSIONS: The experiment indicates the oxidative stress and inflammatory response of rat NPCs induced by H2O2 could be inhibited by activating CB2. This study reveals that CB2 activation can effectively delay the development of IVDD, providing an effective therapeutic target for IVDD.

Targeting the endocannabinoid system for management of HIV-associated neuropathic pain: A systematic review.

Human immunodeficiency virus (HIV) infection and antiretroviral therapy can independently induce HIV-associated neuropathic pain (HIV-NP). There is a dearth of drugs or therapeutic modalities that can alleviate HIV-NP. Smoked cannabis has been reported to improve pain measures in patients with neuropathic pain. Cannabis, phytocannabinoids, and the endocannabinoids such N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), produce some of their effects via cannabinoid receptors (CBRs). Endocannabinoids are degraded by various enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase. We searched PubMed, Google Scholar, clinicaltrials.gov and clinicaltrialsregister.eu using various key words and their combinations for published papers that studied HIV-NP and cannabis, cannabinoids, or endocannabinoids up to 27th December 2020. All original research articles that evaluated the efficacy of molecules that modulate the endocannabinoid system (ECS) for the prevention and/or treatment of pain in HIV-NP animal models and patients with HIV-NP were included. The PubMed search produced a total of 117 articles, whereas the Google Scholar search produced a total of 9467 articles. Amongst the 13 articles that fulfilled the inclusion criteria 11 articles were found in both searches whereas 2 articles were found in Google Scholar only. The clinicaltrials.gov and clinicaltrialsregister.eu searches produced five registered trials of which three were completed and with results. Ten preclinical studies found that the endocannabinoids (2-AG and AEA), synthetic mixed CB1R/CB2R agonist WIN 55,212-2, a CB2R-selective phytocannabinoid ß-caryophyllene, synthetic CB2R-selective agonists (AM1710, JWH015, JWH133 and Gp1a, but not HU308); FAAH inhibitors (palmitoylallylamide, URB597 and PF-3845) and a drug combination of indomethacin plus minocycline, which produces its effects in a CBR-dependent manner, either prevented the development of and/or attenuated established HIV-NP. Two clinical trials demonstrated greater efficacy of smoked cannabis over placebo in alleviating HIV-NP, whereas another clinical trial demonstrated that cannabidivarin, a cannabinoid that does not activate CBRs, did not reduce HIV-NP. The available preclinical results suggest that targeting the ECS for prevention and treatment of HIV-NP is a plausible therapeutic option. Clinical evidence shows that smoked cannabis alleviates HIV-NP. Further research is needed to find out if non-psychoactive drugs that target the ECS and are delivered by other routes than smoking could be useful as treatment options for HIV-NP.