Potential Therapeutic Targets to Modulate the Endocannabinoid System in Alzheimer's Disease.

Alzheimer's disease (AD), the most common neurodegenerative disease (NDD), is characterized by chronic neuronal cell death through progressive loss of cognitive function. Amyloid beta (Aß) deposition, neuroinflammation, oxidative stress, and hyperphosphorylated tau proteins are considered the hallmarks of AD pathology. Different therapeutic approaches approved by the Food and Drug Administration can only target a single altered pathway instead of various mechanisms that are involved in AD pathology, resulting in limited symptomatic relief and almost no effect in slowing down the disease progression. Growing evidence on modulating the components of the endocannabinoid system (ECS) proclaimed their neuroprotective effects by reducing neurochemical alterations and preventing cellular dysfunction. Recent studies on AD mouse models have reported that the inhibitors of the fatty acid amide hydrolase (FAAH) and monoacylglycerol (MAGL), hydrolytic enzymes for N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), respectively, might be promising candidates as therapeutical intervention. The FAAH and MAGL inhibitors alone or in combination seem to produce neuroprotection by reversing cognitive deficits along with Aß-induced neuroinflammation, oxidative responses, and neuronal death, delaying AD progression. Their exact signaling mechanisms need to be elucidated for understanding the brain intrinsic repair mechanism. The aim of this review was to shed light on physiology and pathophysiology of AD and to summarize the experimental data on neuroprotective roles of FAAH and MAGL inhibitors. In this review, we have also included CB1R and CB2R modulators with their diverse roles to modulate ECS mediated responses such as anti-nociceptive, anxiolytic, and anti-inflammatory actions in AD. Future research would provide the directions in understanding the molecular mechanisms and development of new therapeutic interventions for the treatment of AD.

Deorphanization of G Protein Coupled Receptors (GPCRs): a historical perspective.

Counting over 800 members, G Protein Coupled Receptors (GPCRs) form the largest family of membrane receptors encoded in the human genome. Since the discovery of G proteins and GPCRs in the late 1970s and early 1980s, a significant portion of the GPCR research has been focused on identifying ligand/receptor pairs in parallel to studies related to their signaling properties. Despite significant advancements, about a fourth of the ~400 non-odorant GPCRs are still considered orphan because their natural or endogenous ligands have yet to be identified. We should consider that every GPCR was once an orphan and that endogenous ligands have often been associated with biological effects without a complete understanding of the molecular identity of their target receptors. Within this framework, this review offers a historical perspective on deorphanization processes for representative GPCRs, including Ghrelin receptor, GABAB receptor, Apelin receptor, Cannabinoid receptors, and GPR15. It explores three main scenarios encountered in deorphanization efforts and discusses key questions and methodologies employed in elucidating ligand-receptor interactions, providing insights for future research endeavors. Significance Statement Understanding how scientists have historically approached the issue of GPCR deorphanization and pairing of biologically active ligands with their cognate receptors are relevant topics in pharmacology. In fact, the biology of each GPCR, including their pathophysiological involvement, has often been uncovered only after their deorphanization, illuminating druggable targets for various diseases. Furthermore, uncovered endogenous ligands have therapeutic value as many ligands - or derivates thereof - are developed into drugs.

Maternal Cannabis Use during Lactation and Potential Effects on Human Milk Composition and Production: A Narrative Review.

Cannabis use has increased sharply in the last 20 y among adults, including reproductive-aged women. Its recent widespread legalization is associated with a decrease in risk perception of cannabis use during breastfeeding. However, the effect of cannabis use (if any) on milk production and milk composition is not known. This narrative review summarizes current knowledge related to maternal cannabis use during breastfeeding and provides an overview of possible pathways whereby cannabis might affect milk composition and production. Several studies have demonstrated that cannabinoids and their metabolites are detectable in human milk produced by mothers who use cannabis. Due to their physicochemical properties, cannabinoids are stored in adipose tissue, can easily reach the mammary gland, and can be secreted in milk. Moreover, cannabinoid receptors are present in adipocytes and mammary epithelial cells. The activation of these receptors directly modulates fatty acid metabolism, potentially causing changes in milk fatty acid profiles. Additionally, the endocannabinoid system is intimately connected to the endocrine system. As such, it is probable that interactions of exogenous cannabinoids with the endocannabinoid system might modify release of critical hormones (e.g., prolactin and dopamine) that regulate milk production and secretion. Nonetheless, few studies have investigated effects of cannabis use (including on milk production and composition) in lactating women. Additional research utilizing robust methodologies are needed to elucidate whether and how cannabis use affects human milk production and composition.

Aerobic Exercise and Endocannabinoids: A Narrative Review of Stress Regulation and Brain Reward Systems.

Aerobic exercise is a widely adopted practice, not solely for enhancing fitness and reducing the risk of various diseases but also for its ability to uplift mood and aid in addressing depression and anxiety disorders. Within the scope of this narrative review, we seek to consolidate current insights into the endocannabinoid-mediated regulation of stress and the brain's reward mechanism resulting from engaging in aerobic exercise. A comprehensive search was conducted across Medline, SPORTDiscus, Pubmed, and Scopus, encompassing data available until November 30, 2023. This review indicates that a bout of aerobic exercise, particularly of moderate intensity, markedly augments circulating levels of endocannabinoids - N-arachidonoyl-ethanolamine (AEA) and 2-acylglycerol (2-AG), that significantly contributes to mood elevation and reducing stress in healthy individuals.  The current understanding of how aerobic exercise impacts mental health and mood improvement is still unclear. Moderate and high-intensity aerobic exercise modulates stress through a negative feedback mechanism targeting both the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system, thereby facilitating stress regulation crucial role in endocannabinoid synthesis, ultimately culminating in the orchestration of negative feedback across multiple tiers of the HPA axis, coupled with its influence over cortical and subcortical brain structures. The endocannabinoid has been observed to govern the release of neurotransmitters from diverse neuronal populations, implying a universal mechanism that fine-tunes neuronal activity and consequently modulates both emotional and stress-related responses. Endocannabinoids further assume a pivotal function within brain reward mechanisms, primarily mediated by CB1 receptors distributed across diverse cerebral centers. Notably, these endocannabinoids partake in natural reward processes, as exemplified in aerobic exercise, by synergizing with the dopaminergic reward system. The genesis of this reward pathway can be traced to the ventral tegmental area, with dopamine neurons predominantly projecting to the nucleus accumbens, thereby inciting dopamine release in response to rewarding stimuli.

The Role of Cannabidiol in Liver Disease: A Systemic Review.

Cannabidiol (CBD), a non-psychoactive phytocannabinoid abundant in Cannabis sativa, has gained considerable attention for its anti-inflammatory, antioxidant, analgesic, and neuroprotective properties. It exhibits the potential to prevent or slow the progression of various diseases, ranging from malignant tumors and viral infections to neurodegenerative disorders and ischemic diseases. Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease, and viral hepatitis stand as prominent causes of morbidity and mortality in chronic liver diseases globally. The literature has substantiated CBD's potential therapeutic effects across diverse liver diseases in in vivo and in vitro models. However, the precise mechanism of action remains elusive, and an absence of evidence hinders its translation into clinical practice. This comprehensive review emphasizes the wealth of data linking CBD to liver diseases. Importantly, we delve into a detailed discussion of the receptors through which CBD might exert its effects, including cannabinoid receptors, CB1 and CB2, peroxisome proliferator-activated receptors (PPARs), G protein-coupled receptor 55 (GPR55), transient receptor potential channels (TRPs), and their intricate connections with liver diseases. In conclusion, we address new questions that warrant further investigation in this evolving field.

Orexin and cannabinoid systems modulate long-term potentiation of the hippocampus CA1 area in anesthetized rats.

Objectives: Long-term potentiation (LTP) is a kind of synaptic plasticity and has a key role in learning and memory. Endocannabinoids and orexins are the endogenous systems that can modulate synaptic plasticity. Given that new studies have shown an interaction between cannabinoid and orexin systems in the brain, we decided to examine this interaction between the two systems on LTP induction in rat's hippocampus. Materials and Methods: Twenty-eight male Wistar rats were used for evaluating the effects of co-administrating of cannabinoid-1 receptor (CB1R) antagonist (AM251) and orexin-2 receptor (OX2R) antagonist (TCS OX2 29) on the induction of LTP in the Schaffer collateral-CA1 synapses of rat hippocampus. The drugs were microinjected into the CA1 area of rat hippocampus 30 min before inducing of LTP. Results: Results showed that sole administration of the antagonists inhibited LTP, with respect to the control group. Also, co-administrating of them reduced LTP as compared to the control group, but not significantly more than that when the antagonists were solely microinjected into the CA1. Nonetheless, the inhibitory effect of concurrent administration of the antagonists on LTP lasted until the end of the recording. Conclusion: These results propose that endogenous cannabinoids and orexins play a role in the expression of LTP, at least by CA1-CB1Rs and CA1-OX2Rs, respectively. Finally, there is no interaction between CB1R and OX2R on the induction of LTP in the Schaffer collateral-CA1 synapses; therefore, these two systems possibly act through common signaling pathways in the hippocampus's CA1 region.

Clinical Importance of Cannabinoid Type 1 Receptor (CB1R) and Cannabinoid Type 2 Receptor (CB2R) Expression in Renal Cell Carcinoma.

Background and objective The purpose of our study was to assess the expression of cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R), including positivity, intensity, percentage, site of distribution, and immunohistochemical score, in renal cell carcinomas (RCCs) and explore their correlation with various clinicopathological aspects. Methodology We retrospectively obtained data and specimens from 87 patients diagnosed with RCC after partial or radical nephrectomy, and the CB1R and CB2R expression was assessed immunohistochemically on paraffin-embedded tissues. The results were statistically analyzed uni- and multi-factorial along with clinicopathological parameters. Results CB1R was not expressed at all, and CB2R was highly expressed in 78 (89.7%) patients with RCC. In unifactorial analysis, no statistical significance was found in any of the analyzed parameters. However, in the multifactorial analysis, we found that patients with a papillary histologic type (P < 0.0005) were associated with a lower likelihood of expression of the CB2R in the membranous compared with those with clear-cell and were also associated with a higher likelihood of moderate or strong expression of CB2R immunohistochemical score compared with those with clear-cell (P = 0.03). Patients with stage T2 (P = 0.010) had more enhanced expression (grade 3 CB2R intensity) compared with those with stage T1. Males (beta coefficient ± standard error [SE] 13.70 ± 7.04; P = 0.056) and patients with chromophobe histologic type (beta coefficient ± SE 23.45 ± 9.86; P = 0.020) were associated with a higher percentage of CB2R expression. Conclusions Our data suggest that although the CB1R was not expressed in RCCs, CB2R was expressed in almost every patient and enhanced expression was noted in correlation with specific clinicopathological aspects of the patients. Thus, following well-designed studies, especially CB2R could be used as a prognostic marker or even as a potential therapeutic target in RCC.

Effects of Repeated Treatment with the Monoacylglycerol Lipase Inhibitor MJN110 on Pain-Related Depression of Nesting and Cannabinoid 1 Receptor Function in Male and Female Mice.

MJN110 inhibits the enzyme monoacylglycerol lipase (MAGL) to increase levels of the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG), an endogenous high-efficacy agonist of cannabinoid 1 and 2 receptors (CB1/2R). MAGL inhibitors are under consideration as candidate analgesics, and we reported previously that acute MJN110 produced partial antinociception in an assay of pain-related behavioral depression in mice. Given the need for repeated analgesic administration in many pain patients and the potential for analgesic tolerance during repeated treatment, this study examined antinociceptive effects of repeated MJN110 on pain-related behavioral depression and CB1R-mediated G-protein function. Male and female ICR mice were treated daily for 7 days in a 2x2 design with (a) 1.0 mg/kg/day MJN110or its vehicle followed by (b) intraperitoneal injection of dilute lactic acid (IP acid) or its vehicle as a visceral noxious stimulus to depress nesting behavior. After behavioral testing, G-protein activity was assessed in lumbar spinal cord andfive brain regions using an assay of CP55,940-stimulated [35S]GTPÉ£S activation. As reported previously, acute MJN110 produced partial but significant relief of IP acid-induced nesting depression on Day 1. After 7 days, MJN110 continued to produce significant but partial antinociception in males, while antinociceptive tolerance developed in females. Repeated MJN110 also produced modest decreases in maximum levels of CP55,940-induced [35S]GTPÉ£S binding in spinal cord and most brain regions. These results indicate that repeated treatment with a relatively low antinociceptive MJN110 dose produces only partial and sex-dependent transient antinociception associated with the emergence of CB1R desensitization in this model of IP acid-induced nesting depression. Significance Statement The drug MJN110 inhibits monoacylglycerol lipase (MAGL) to increase levels of the endogenous cannabinoid 2-arachidonoylglycerol and produce potentially useful therapeutic effects including analgesia. This study used an assay of pain-related behavioral depression in mice to show that repeated MJN110 treatment produced (1) weak but sustained antinociception in male mice, (2) antinociceptive tolerance in females, and (3) modest cannabinoid-receptor desensitization that varied by region and sex. Antinociceptive tolerance may limit the utility of MJN110 for treatment of pain.

The role of cannabinoid receptor 2 in bone remodeling during orthodontic tooth movement.

BACKGROUND: The purpose of this study is to explore the effects of CB2 on bone regulation during orthodontic tooth movement. METHODS: Thirty male mice were allocated into 2 groups (n = 15 in each group): wild type (WT) group and CB2 knockout (CB2-/-) group. Orthodontic tooth movement (OTM) was induced by applying a nickel-titanium coil spring between the maxillary first molar and the central incisors. There are three subgroups within the WT groups (0, 7 and 14 days) and the CB2-/- groups (0, 7 and 14 days). 0-day groups without force application. Tooth displacement, alveolar bone mass and alveolar bone volume were assessed by micro-CT on 0, 7 and 14 days, and the number of osteoclasts was quantified by tartrate-resistant acid phosphatase (TRAP) staining. Moreover, the expression levels of RANKL and OPG in the compression area were measured histomorphometrically. RESULTS: The WT group exhibited the typical pattern of OTM, characterized by narrowed periodontal space and bone resorption on the compression area. In contrast, the accelerated tooth displacement, increased osteoclast number (P < 0.0001) and bone resorption on the compression area in CB2-/- group. Additionally, the expression of RANKL was significantly upregulated, while OPG showed low levels in the compression area of the CB2 - / - group (P < 0.0001). CONCLUSIONS: CB2 modulated OTM and bone remodeling through regulating osteoclast activity and RANKL/OPG balance.

Regulators of G-Protein Signaling (RGS) in Sporadic and Colitis-Associated Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common neoplasms worldwide. Among the risk factors of CRC, inflammatory bowel disease (IBD) is one of the most important ones leading to the development of colitis-associated CRC (CAC). G-protein coupled receptors (GPCR) are transmembrane receptors that orchestrate a multitude of signaling cascades in response to external stimuli. Because of their functionality, they are promising targets in research on new strategies for CRC diagnostics and treatment. Recently, regulators of G-proteins (RGS) have been attracting attention in the field of oncology. Typically, they serve as negative regulators of GPCR responses to both physiological stimuli and medications. RGS activity can lead to both beneficial and harmful effects depending on the nature of the stimulus. However, the atypical RGS-AXIN uses its RGS domain to antagonize key signaling pathways in CRC development through the stabilization of the ß-catenin destruction complex. Since AXIN does not limit the efficiency of medications, it seems to be an even more promising pharmacological target in CRC treatment. In this review, we discuss the current state of knowledge on RGS significance in sporadic CRC and CAC with particular emphasis on the regulation of GPCR involved in IBD-related inflammation comprising opioid, cannabinoid and serotonin receptors.

Function of Presynaptic Inhibitory Cannabinoid CB1 Receptors in Spontaneously Hypertensive Rats and Its Modification by Enhanced Endocannabinoid Tone.

We studied whether the function of presynaptic inhibitory cannabinoid CB1 receptors on the sympathetic nerve fibres innervating resistance vessels is increased in spontaneously hypertensive rats (SHR) like in deoxycorticosterone (DOCA)-salt hypertension. An increase in diastolic blood pressure (DBP) was induced by electrical stimulation of the preganglionic sympathetic neurons or by phenylephrine injection in pithed SHR and normotensive Wistar-Kyoto rats (WKY). The electrically (but not the phenylephrine) induced increase in DBP was inhibited by the cannabinoid receptor agonist CP55940, similarly in both groups, and by the endocannabinoid reuptake inhibitor AM404 in SHR only. The effect of CP55940 was abolished/reduced by the CB1 receptor antagonist AM251 (in both groups) and in WKY by endocannabinoid degradation blockade, i.e., the monoacylglycerol lipase (MAGL) inhibitor MJN110 and the dual fatty acid amide hydrolase (FAAH)/MAGL inhibitor JZL195 but not the FAAH inhibitor URB597. MJN110 and JZL195 tended to enhance the effect of CP55940 in SHR. In conclusion, the function of presynaptic inhibitory CB1 receptors depends on the hypertension model. Although no differences occurred between SHR and WKY under basal experimental conditions, the CB1 receptor function was better preserved in SHR when the endocannabinoid tone was increased by the inhibition of MAGL or the endocannabinoid transporter.

Effects of cannabinoid agonists and antagonists in male rats discriminating the synthetic cannabinoid AM2201.

The synthetic forms of delta-9-tetrahydrocannabinol (Δ9-THC), dronabinol or nabilone, have been approved to treat several indications. However, due to safety concerns their clinical utility remains limited. Consequently, there is a need for developing cannabinoid (CB) ligands that display better behavioral pharmacological profiles than Δ9-THC. Here, we utilized drug discrimination methods to compare the interoceptive effects of CB ligands that vary in potency, efficacy, and selectivity at the CB receptors, including two ligands, AM411 and AM4089, that show CB1 partial agonist-like actions in vitro. Male rats were trained to discriminate 0.1 mg/kg AM2201 from saline under a fixed-ratio (FR) 10 response schedule of food reinforcement. After establishing AM2201's discriminative-stimulus effects, pretreatment tests with the CB1 antagonist/inverse agonist rimonabant blocked AM2201's effects, whereas the peripherally-restricted antagonist AM6545 had no effect. Next, the generalization profiles of AM411 and AM4089 with CB1 full agonists (JWH-018, CP-55,940, AM8936), partial agonist (Δ9-THC), and non-cannabinoids (fentanyl, atropine) were compared. The CBs either fully (AM2201, CP-55,940, JWH-018, AM8936, Δ9-THC) or partially (AM411, AM4089) substituted for AM2201, whereas fentanyl and atropine did not produce AM2201-like effects. All CB drugs were more potent than Δ9-THC and correlation analysis confirmed that the relative behavioral potencies of CBs corresponded strongly with their relative affinities at the CB1 but not CB2 receptors. Together, our results further demonstrate that AM411 and AM4089 exhibit better pharmacological profiles compared to Δ9-THC, in that they are more potent and display in vivo partial agonist-like actions that are centrally mediated via CB1 receptors.

Cannabinoids and the endocannabinoid system in immunotherapy: helpful or harmful?

Numerous studies in various cancer models have demonstrated that ingredients of cannabis can influence tumor growth through the endocannabinoid system (ECS), a network of molecules (mediators, receptors, transporters, enzymes) that maintains homeostasis and protection in many tissues. The main constituents of the ECS are the classical cannabinoid (CB) receptors, such as CB1 and CB2, their endogenous ligands (endocannabinoids), and the endocannabinoids' synthesizing and degrading enzymes. The role of the ECS in cancer is still unclear and its effects often depend on the tumor entity and the expression levels of CB receptors. Many studies have highlighted the tumor cell-killing potential of CB1 agonists. However, cannabis is also known as an immunosuppressant and some data suggest that the use of cannabis during immunotherapy worsens treatment outcomes in cancer patients. CB receptors are widely present in immune cells, and together with monoacylglycerol lipase, the 2-arachidonoylglycerol degrading enzyme, they could be critically involved in the regulation of the immune cell profile of the tumor microenvironment (TME), and hence in tumor progression. So far, data on the impact of the ECS in the immune-TME are still vague. In this review, we discuss the current understanding of the ECS on immunoregulation during tumor growth, and how it might affect the outcome of cancer immunotherapy.

The Interplay between Cannabinoid Receptors and Microglia in the Pathophysiology of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by massive neuronal death, brain atrophy, and loss of neurons and synapses, which all lead to a progressive cognitive decline. Neuroinflammation has been recently identified as one of the main causes of AD progression, and microglia cells are considered to have a central role in this process. Growing evidence suggests that cannabinoids may be used as preventive treatment for AD. An altered expression of the endocannabinoids (eCBs) and their receptors (CBRs) is reported in several neurodegenerative disorders, including AD. Moreover, the modulation of CBRs demonstrated neuroprotective effects in reducing aggregated protein deposition, suggesting the therapeutic potential of natural and synthetic CBR ligands in the treatment of neurodegenerative proteinopathies. Here, we review the current knowledge regarding the involvement of CBRs in the modulation of microglia activation phenotypes, highlighting the role of neuroinflammation in the pathogenesis of neurodegenerative diseases, like AD. We also provide an overview of recently developed candidate drugs targeting CBRs that may afford a new innovative strategy for the treatment and management of AD.

Cannabidiol at Nanomolar Concentrations Negatively Affects Signaling through the Adenosine A2A Receptor.

Cannabidiol (CBD) is a phytocannabinoid with potential as a therapy for a variety of diseases. CBD may act via cannabinoid receptors but also via other G-protein-coupled receptors (GPCRs), including the adenosine A2A receptor. Homogenous binding and signaling assays in Chinese hamster ovary (CHO) cells expressing the human version of the A2A receptor were performed to address the effect of CBD on receptor functionality. CBD was not able to compete for the binding of a SCH 442416 derivative labeled with a red emitting fluorescent probe that is a selective antagonist that binds to the orthosteric site of the receptor. However, CBD reduced the effect of the selective A2A receptor agonist, CGS 21680, on Gs-coupling and on the activation of the mitogen activated kinase signaling pathway. It is suggested that CBD is a negative allosteric modulator of the A2A receptor.

The synthetic cannabinoid 5F-MDMB-PICA enhances the metabolic activity and angiogenesis in human brain microvascular endothelial cells by upregulation of VEGF, ANG-1, and ANG-2.

Brain angiogenesis, the formation of new blood vessels from existing brain vasculature, has been previously associated with neural plasticity and addictive behaviors related to substances. Synthetic cannabinoids (SCs) have become increasingly popular due to their ability to mimic the effects of cannabis, offering high potency and easy accessibility. In the current study, we reveal that the SC 5F-MDMB-PICA, the most common SC in the United States in 2019, increases cell metabolic activity and promotes angiogenesis in human brain microvascular endothelial cells (HBMECs). First, we performed an MTT assay to evaluate the effects of 5F-MDMB-PICA treatment at various concentrations (0.0001 µM, 0.001 µM, 0.01 µM, 0.1 µM, and 1 µM) on HBMECs metabolic activity. The results demonstrated higher concentrations of the SC improved cell metabolic activity. Furthermore, 5F-MDMB-PICA treatment enhanced tube formation and migration of HBMECs in a dosage-dependent manner. Additionally, the mRNA, secreted protein, and intracellular protein levels of vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2, which are involved in the regulation of angiogenesis, as well as the protein levels of cannabinoid receptor type-1, were all increased following treatment with 5F-MDMB-PICA. Notably, the phosphorylation levels at Serine 9 residue of glycogen synthase kinase-3ß were also increased in the 5F-MDMB-PICA treated HBMECs. Collectively, our findings demonstrate that 5F-MDMB-PICA can enhance angiogenesis in HBMECs, suggesting the significant role of angiogenesis in the response to SCs. Manipulating this interaction may pave the way for innovative treatments targeting SC addiction and angiogenesis-related conditions.

Investigating the Effects of Exogenous and Endogenous 2-Arachidonoylglycerol on Retinal CB1 Cannabinoid Receptors and Reactive Microglia in Naive and Diseased Retina.

The endocannabinoid system (ECS) is a new target for the development of retinal disease therapeutics, whose pathophysiology involves neurodegeneration and neuroinflammation. The endocannabinoid 2-arachidonoylglycerol (2-AG) affects neurons and microglia by activating CB1/CB2 cannabinoid receptors (Rs). The aim of this study was to investigate the effects of 2-AG on the CB1R expression/downregulation and retinal neurons/reactive microglia, when administered repeatedly (4 d), in three different paradigms. These involved the 2-AG exogenous administration (a) intraperitoneally (i.p.) and (b) topically and (c) by enhancing the 2-AG endogenous levels via the inhibition (AM11920, i.p.) of its metabolic enzymes (MAGL/ABHD6). Sprague Dawley rats were treated as mentioned above in the presence or absence of CB1/CB2R antagonists and the excitatory amino acid, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Immunohistochemistry, Western blot and a 2-AG level analyses were performed. The 2-AG repeated treatment (i.p.) induced the CB1R downregulation, abolishing its neuroprotective actions. However, 2-AG attenuated the AMPA-induced activation of microglia via the CB2R, as concurred by the AM630 antagonist effect. Topically administered 2-AG was efficacious as a neuroprotectant/antiapoptotic and anti-inflammatory agent. AM11920 increased the 2-AG levels providing neuroprotection against excitotoxicity and reduced microglial activation without affecting the CB1R expression. Our findings show that 2-AG, in the three paradigms studied, displays differential pharmacological profiles in terms of the downregulation of the CB1R and neuroprotection. All treatments, however, attenuated the activation of microglia via the CB2R activation, supporting the anti-inflammatory role of 2-AG in the retina.

Cannabidiol Anticonvulsant Effects Against Lithium-Pilocarpine-Induced Status Epilepticus in Male Rats Are Mediated by Neuroinflammation Modulation and Cannabinoids 1 (CB1), But Not CB2 and GABAA Receptors.

Background: Status epilepticus (SE) is a series of seizures that can lead to serious neurological damages. Cannabidiol (CBD) is extracted from the cannabis plant, which has been approved as an antiseizure medication. This study aimed to determine the efficacy of various doses of CBD on lithium-pilocarpine-induced SE in rats and possible involvement of multiple pharmacological pathways. We hypothesized that cannabinoid receptors type 1 (CB1) and CB2, as well as GABAA receptors, might have important roles in the anticonvulsant effects of CBD against SE by its anti-inflammatory effects. Methods: SE was induced by intraperitoneal (i.p.) injection of lithium (127 mg/kg, i.p.) and pilocarpine (60 mg/kg, i.p., 20 h after lithium). Forty-two male rats were divided into seven groups (including control and sham groups), and the treated groups received different doses of CBD (1, 3, 5, 10, and 25 mg/kg, i.p.). SE score was recorded over the next 2 h following pilocarpine injection. Then, we measured the levels of pro-inflammatory cytokines, including interleukin (IL)-lß and tumor necrosis factor (TNF)-α, using ELISA kits. Also we analyzed the expression of CB1, CB2, and GABAA receptors using the Western blot technique. Results: CBD at 5 mg/kg significantly reduced Racine's scale and duration of seizures, and increased the onset time of seizure. Moreover, CBD 5 mg/kg caused significant reductions in the elevated levels of IL-lß and TNF-α, as well as a significant increase in the decreased level of CB1 receptor expression compared to the control group. In other word, CBD reverted the effects of SE in terms of neuroinflammation and CB1 receptor. Based on the obtained results, CBD was not able to restore the declined levels of CB2 or GABAA receptors. Conclusion: Our study found anticonvulsant effects of CBD on the SE rat model induced by lithium-pilocarpine with probable involvement of CB1 receptors and anti-inflammatory effects by reducing IL-1ß and TNF-α markers independent of CB2 and GABAA receptors.

Mechanisms of weight-loss effect in obese mice by the endogenous cannabinoid receptor 2 agonist beta-caryophyllene.

BACKGROUND AND AIMS: The endogenous cannabinoid system (ECS) is involved in the regulation of a variety of physiological activities in the body, such as metabolism and energy uptake, and cannabinoid receptor 2 (CNR2) is one of these receptors that is predominantly distributed in the periphery. ß-caryophyllene (BCP) is an agonist of CNR2 which is known to possess pharmacological activities such as anti-inflammatory and antioxidant properties. In this study, we wanted to investigate whether BCP possesses pharmacological effects on obese mice and its mechanism. METHODS: Reversed feeding rhythm, propylthiouracil was delivered intraperitoneally, and BCP was gavaged once daily for four weeks to establish a hyperlipidemic obese mouse model. A glucose tolerance test, lipid level measurements, liver, peritoneal, and subcutaneous fat removal, HE and Oil Red O staining of the liver, and immunohistochemistry (IHC) staining with an anti-CNR2 antibody were all carried out. The liver was examined using tools like GO and KEGG databases for differentially expressed genes and signaling pathways linked to medication effectiveness. RESULTS: BCP had significant effects on weight reduction and improvement of dyslipidemia. What's more, it significantly reduced body fat percentage, improved steatosis and ballooning of liver cells, and reduced fat accumulation, while inhibiting the proliferation of peri-abdominal adipocytes. BCP exerted its effects to improve dyslipidemia and reduce body weight probably through circadian regulation and cholesterol metabolic pathways. Finally, and its efficacy in improving dyslipidemia and reducing body weight may be mainly through activating CNR2, activating SIRT1/PGC-1α/PPARγ and SIRT1/AMPK pathways. CONCLUSION: BCP activates the CNR2, SIRT1/PGC-1α/PPARγ signaling pathway, and SIRT1/AMPK signaling pathway to exert dyslipidemia-improving and weight-loss effects.

The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo.

Introduction: Male reproduction is under the control of the hypothalamus-pituitary-gonadal (HPG) axis. The endocannabinoid system (ECS) and the kisspeptin system (KS) are two major signaling systems in the central and peripheral control of reproduction, but their possible interaction has been poorly investigated in mammals. This manuscript analyzes their possible reciprocal modulation in the control of the HPG axis. Materials and methods: Adolescent male rats were treated with kisspeptin-10 (Kp10) and endocannabinoid anandamide (AEA), the latter alone or in combination with the type 1 cannabinoid receptor (CB1) antagonist rimonabant (SR141716A). The hypothalamic KS system and GnRH expression, circulating sex steroids and kisspeptin (Kiss1) levels, and intratesticular KS and ECS were evaluated by immunohistochemical and molecular methods. Non-coding RNAs (i.e., miR145-5p, miR-132-3p, let7a-5p, let7b-5p) were also considered. Results: Circulating hormonal values were not significantly affected by Kp10 or AEA; in the hypothalamus, Kp10 significantly increased GnRH mRNA and aromatase Cyp19, Kiss1, and Kiss1 receptor (Kiss1R) proteins. By contrast, AEA treatment affected the hypothalamic KS at the protein levels, with opposite effects on the ligand and receptor, and SR141716A was capable of attenuating the AEA effects. Among the considered non-coding RNA, only the expression of miR145-5p was positively affected by AEA but not by Kp10 treatment. Localization of Kiss1+/Kiss1R+ neurons in the arcuate nucleus revealed an increase of Kiss1R-expressing neurons in Kp10- and AEA-treated animals associated with enlargement of the lateral ventricles in Kp10-treated animals. In the brain and testis, the selected non-coding RNA was differently modulated by Kp10 or AEA. Lastly, in the testis, AEA treatment affected the KS at the protein levels, whereas Kp10 affected the intragonadal levels of CB1 and FAAH, the main modulator of the AEA tone. Changes in pubertal transition-related miRNAs and the intratesticular distribution of Kiss1, Kiss1R, CB1, and CB2 following KP and AEA treatment corroborate the KS-ECS crosstalk also showing that the CB1 receptor is involved in this interplay. Conclusion: For the first time in mammals, we report the modulation of the KS in both the hypothalamus and testis by AEA and revealed the KP-dependent modulation of CB1 and FAAH in the testis. KP involvement in the progression of spermatogenesis is also suggested.