CB1 Receptor Agonist Research Articles

CB2R induces a protective response against epileptic seizures through ERK and p38 signaling pathways

Background and purpose Epilepsy is a pivotal neurological disorder characterized by the synchronous discharging of neurons to induce momentary brain dysfunction. Temporal lobe epilepsy is the most common type of epilepsy, with seizures originating from the mesial temporal lobe. The hippocampus forms part of the mesial temporal lobe and plays a significant role in epileptogenesis; it also has a vital influence on the mental development of children. In this study, we aimed to explore the effects of CB2 receptor (CB2R) activation on ERK and p38 signaling in nerve cells of a rat epilepsy model. Materials and methods We treated Sprague-Dawley rats with pilocarpine to induce an epilepsy model and treated such animals with a CB2R agonist (JWH133) alone or with a CB2R antagonist (AM630). Nissl’s stain showed the neuron conditon in different groups. Western blot analyzed the level of p-ERK and p-p38. Results JWH133 can increase the latent period of first seizure attack and decrease the Grades IV–V magnitude ratio after the termination of SE. Nissl’s stain showed JWH133 protected neurons in the hippocampus while AM630 inhibited the functioning of CB2R in neurons. Western blot analysis showed that JWH133 decreased levels of p-ERK and p-p38, which is found at increased levels in the hippocampus of our epilepsy model. In contrast, AM630 inhibited the protective function of JWH133 and also enhanced levels of p-ERK and p-p38. Conclusions CB2R activation can induce neurons proliferation and survival through activation of ERK and p38 signaling pathways.

Protective role of neuronal and lymphoid cannabinoid CB2 receptors in neuropathic pain.

Cannabinoid CB2 receptor (CB2) agonists are potential analgesics void of psychotropic effects. Peripheral immune cells, neurons and glia express CB2; however, the involvement of CB2 from these cells in neuropathic pain remains unresolved. We explored spontaneous neuropathic pain through on-demand self-administration of the selective CB2 agonist JWH133 in wild-type and knockout mice lacking CB2 in neurons, monocytes or constitutively. Operant self-administration reflected drug-taking to alleviate spontaneous pain, nociceptive and affective manifestations. While constitutive deletion of CB2 disrupted JWH133-taking behavior, this behavior was not modified in monocyte-specific CB2 knockouts and was increased in mice defective in neuronal CB2 knockouts suggestive of increased spontaneous pain. Interestingly, CB2-positive lymphocytes infiltrated the injured nerve and possible CB2transfer from immune cells to neurons was found. Lymphocyte CB2depletion also exacerbated JWH133 self-administration and inhibited antinociception. This work identifies a simultaneous activity of neuronal and lymphoid CB2that protects against spontaneous and evoked neuropathic pain.

Δ9 -Tetrahydrocannabinolic acid alleviates collagen-induced arthritis: Role of PPARγ and CB1 receptors.

Δ9 -Tetrahydrocannabinolic acid (Δ9 -THCA-A), the precursor of Δ9 -THC, is a non-psychotropic phytocannabinoid that shows PPARγ agonist activity. Here, we investigated the ability of Δ9 -THCA-A to modulate the classic cannabinoid CB1 and CB2 receptors and evaluated its anti-arthritis activity in vitro and in vivo. Cannabinoid receptors binding and intrinsic activity, as well as their downstream signalling, were analysed in vitro and in silico. The anti-arthritis properties of Δ9 -THCA-A were studied in human chondrocytes and in the murine model of collagen-induced arthritis (CIA). Plasma disease biomarkers were identified by LC-MS/MS based on proteomic and elisa assays. Functional and docking analyses showed that Δ9 -THCA-A can act as an orthosteric CB1 receptor agonist and also as a positive allosteric modulator in the presence of CP-55,940. Also, Δ9 -THCA-A seemed to be an inverse agonist for CB2 receptors. In vivo, Δ9 -THCA-A reduced arthritis in CIA mice, preventing the infiltration of inflammatory cells, synovium hyperplasia, and cartilage damage. Furthermore, Δ9 -THCA-A inhibited expression of inflammatory and catabolic genes on knee joints. The anti-arthritic effect of Δ9 -THCA-A was blocked by either SR141716 or T0070907. Analysis of plasma biomarkers, and determination of cytokines and anti-collagen antibodies confirmed that Δ9 -THCA-A mediated its activity mainly through PPARγ and CB1 receptor pathways. Δ9 -THCA-A modulates CB1 receptors through the orthosteric and allosteric binding sites. In addition, Δ9 -THCA-A exerts anti-arthritis activity through CB1 receptors and PPARγ pathways, highlighting its potential for the treatment of chronic inflammatory diseases such as rheumatoid arthritis.

Effects of cannabinoid receptor 2 synthetic agonist, AM1241, on bleomycin induced pulmonary fibrosis

ABSTRACT Bleomycin (BLM) is a chemotherapeutic agent that can cause pulmonary fibrosis. Little is known about the possible protective role of the CB2 receptor agonist, AM1241. We investigated the effects of CB2 receptor activation by AM1241 on BLM induced lung fibrosis in a rat model. BLM was administered via the trachea. Adult female Wistar rats were divided into five groups: saline (control group), BLM (BLM group), CB2 agonist (AM1241) + BLM (BLMA group), CB2 antagonist (AM630) and CB2 agonist (AM1241) + BLM (BLMA + A group), and vehicle (dimethylsulfoxide) + BLM (BLM + vehicle group). Hydroxyproline, collagen type 1, total protein, glutathione (GSH), malondialdehyde (MDA), interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels were measured in lung fibrosis and control tissue using standard methods. We investigated the histopathology of lung tissue to determine the extent of fibrosis. We found significantly higher levels of hydroxyproline, TNF-α, IL-6 and total protein in the BLM group compared to the BLMA group. The level of GSH also was higher in the BLMA group compared to the BLM group. Inflammation and fibrotic changes were significantly reduced in the BLMA group. Our findings suggest that CB2 receptor activation provided protection against BLM induced pulmonary fibrosis by suppressing oxidative stress and increasing cytokines.

Effects of CB2 Receptor Agonist JWH133 on Aβ42-Induced Hippocampal Neurons

Effects of CB2 Receptor Agonist JWH133 on Aβ42-Induced Hippocampal Neurons

Interactions between the endogenous cannabinoid system and the peptides of the Tyr-MIF-1 family modulate heat stress-induced analgesia

The present study aimed at evaluating whether an interaction between the endocannabinoid system (ECS) and peptides from the Tyr-MIF-1 family modulates heat stress-induced analgesia. For this purpose, adult male rats were subjected to 1 hour of heat stress. Pain perception was estimated in vivo by Paw pressure test. Immunohistochemical evaluation of CB1 receptors was also performed in the periaqueductal grey (PAG). Our results showed that the application of CB1-receptor agonist anandamide at the end of the stress led to a tendency of decrease in heat-SIA. We also found that each of the four Tyr-MIF-1 peptides interacted with the ECS after acute heat stress, resulting in changes in the PP-thresholds with different direction, degree, and duration. In particular, the administration of MIF-1 and Tyr-K-MIF-1 after CB1-receptor agonist anandamide increased heat stress-induced analgesia (heat-SIA) after the 10th min, while Tyr-MIF-1 and Tyr-W-MIF-1 produced only short-lasting analgesia. CB1-expression in the PAG was also estimated, showing an increase after Tyr-MIF-1 and Tyr-W-MIF-1 administration with anandamide pretreatment, and a decrease after Tyr-W-MIF-1 administration with the CB1-receptor antagonist AM251- or the opioid receptor antagonist naloxone pretreatment. In summary, it can be inferred that under heat stress conditions the peptides from the Tyr-MIF-1 family, interacting with opioid and non-opioid receptors, differently relate with the cannabinoid system and such an interaction modulates heat stress-induced analgesia. It also seems that Tyr-MIF-1 and Tyr-W-MIF-1 have a direct impact on CB1-expression in the PAG, while MIF-1 and Tyr-K-MIF-1 probably act via second messengers or the activation of additional neurotransmitter system(s).

Beneficial effects of the phytocannabinoid Δ9-THCV in L-DOPA-induced dyskinesia in Parkinson's disease

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.

Evaluation of carboxamide-type synthetic cannabinoids on the functional activities at cannabinoid receptors and biological effects via inhalation exposure test

Three synthetic carboxamide-type cannabinoids (5F-MDMB-PICA, 5F-EMB-PINACA, and AMB-CHMICA) were evaluated in terms of their in vitro activities at the cannabinoid receptors CB1 and CB2 and in vivo biological effects when smoking the synthetic cannabinoids to assess their biological effects. [35S]Guanosine-5′-O-(3-thio)-triphosphate binding assays were performed to investigate the half maximal effective concentration values of the test compounds at the CB1 and CB2 receptors. Additionally, the biological effects were evaluated by observing and scoring the behavior of mice with equipment in which they inhaled smoke from a herbal mixture containing the test compounds. All three synthetic cannabinoids tested in this study activated the CB1 and CB2 receptors in vitro. 5F-MDMB-PICA showed less than 1 nM of the half maximal effective concentration value for both receptors. Therefore, it was suggested that 5F-MDMB-PICA was the strongest CB1 and CB2 receptor agonist in comparison with synthetic cannabinoids evaluated in the past. The degree of the various biological effects, specifically passivity, spontaneous activity, abnormal gait, abnormal position, and grip strength, when smoking the synthetic cannabinoids corresponded to the functional activity at the CB1 receptor. However, some biological effects differed between 5F-MDMB-PICA and 5F-MDMB-PINACA, used as a positive control, and AMB-CHMICA induced some biological effects in contrast to the other tested synthetic cannabinoids. This study provides information regarding the biological effects when smoking synthetic cannabinoids from the functional activities at the CB1 and CB2 receptors, considering their way of inhalation and thermal degradation.

Cannabinoid receptors and the proconvulsant effect of toxoplasmosis in mice

Toxoplasmosis is an infectious disease caused by the intracellular parasite Toxoplasma gondii that harms the brain and increases the risk of epilepsy acquisition. It is well known that cannabinoid (CB) signaling is activated following brain insults and protects the neurons from excitotoxicity and inflammation. We examined the role of CB neurotransmission in the proconvulsant effect of Toxoplasmosis in mice.Toxoplasmosis was established in mice by intraperitoneal injection of T. gondii cysts. The mice with acute and/or chronic Toxoplasma infection were pretreated (through intracerebroventricular injection) with CB1 and CB2 receptor agonists (ACEA and HU308) and antagonists (AM251 and AM630), as well as JZL184 (the irreversible inhibitor of mono acyl glycerol lipase, enzyme degrading the endogenous cannabinoid 2-Acyl glycerol). The seizure threshold was then measured by tail vein infusion of pentylenetetrazole.In healthy uninfected mice JZL184, ACEA, and AM630 increased the seizure threshold in a dose-dependent manner, whereas AM251 and HU308 showed dose-dependent proconvulsant effect. Mice with acute and/or chronic infection had a substantial lower seizure threshold than the uninfected mice. JZL 184, ACEA and AM630 inhibited proconvulsant effect of Toxoplasmosis, while AM251 and HU308 intensified proconvulsant effect of Toxoplasmosis.CB receptors play a role in proconvulsant effect of Toxoplasmosis in mice.

Beta‐caryophyllene, a Volatile Phytocannabinoid, Attenuates Cocaine Self‐administration and Relapse in Rats

Cocaine abuse continues to be a serious health problem worldwide. Despite intense research, there is still no FDA‐approved medication to treat cocaine use disorder. The recent search for effective therapeutics has been focused on the endocannabinoid system because of its critical role in the neurobiology of drug addiction. In the last decade, there has been a growing interest in beta‐caryophyllene (BCP), a volatile phytocannabinoid present in high proportions in cannabis and large number of spice and food plants including black pepper, thyme and cloves. This dietary additive that has a CB2 receptor (CB2R) agonist profile, has shown to produce promising therapeutic effects for multiple neuropsychiatric disorders. Surprisingly, the therapeutic potential of BCP in the treatment of drug abuse and addiction has not been explored. Here, using gold standard animal models of drug abuse, we systematically evaluated the potential utility of BCP against cocaine‐related behaviors in rodents and its mechanisms of action. In a series of experiments we found that BCP attenuated cocaine‐enhanced electrical brain‐stimulation reward and optogenetic intracranial self‐stimulation driven by activation of dopamine (DA) neurons in DAT‐cre mice, both outcomes indicating reduced reward. Intriguingly, when administered systemically or orally, BCP attenuated cocaine self‐administration in rats, suggesting an ability to reduce drug abuse. BCP also reduced drug‐primed reinstatement of cocaine seeking and cocaine conditioned place preference, indicative of its preventative effects against relapse. When BCP was substituted for cocaine, rats ceased responding, suggesting BCP has low liability abuse. In regards to the receptor mechanisms, our pharmacological approach (along with our transgenic approach utilizing with CB1, CB2, GPR55 knockout mice) indicates that BCP produces attenuating effects on cocaine self‐administration that are mediated by non‐cannabinoid receptors. We are now exploring the involvement of peroxisome proliferator‐activated receptor (PPAR) and transient receptor potential vanilloid 1 cation channels (TRPV1) in BCP action against cocaine. Our findings suggest that BCP shows exceptional promise as a therapeutic candidate in the treatment of cocaine use disorder. Importantly, given its good oral bioavailability and the advantage of being an already FDA‐approved non‐toxic dietary additive, BCP is a valuable candidate for drug repurposing programs in translational medicine.

The Sigma1 Receptor Antagonist CM304 Enhances the Antinociceptive Effects of the Cannabinoid Receptor Agonists, but not Mu‐Opioid Receptor Full Agonists in Mice

There is an overdose epidemic associated with the use of illicitly manufactured as well as prescription mu‐opioid receptor agonists. Sigma1 receptor (σ1R) antagonists in combination with other drugs may provide a viable, safer pharmacological option than opioids alone for treating pain. The present study compared pharmacological effects of the σ1R antagonist CM304 alone and in combination with high (fentanyl and morphine) and low (buprenorphine) efficacy opioid agonists and the cannabinoid CB (CP55,940 and THC) receptor agonists in C57BL/6J mice. Rectal temperature, tail withdrawal latency (10‐seconds cutoff) from warm water of various temperatures (45°C, 50°C and 55°C) and counts of unhabituated locomotor activity were measured in this order. Basal latency for tail withdrawal systematically decreased from 10 seconds at 45°C to 1.3 seconds at 55°C. Morphine, fentanyl and buprenorphine dose‐dependently increased maximum possible effects (MPEs) up to 100% at 55°C (ED50 values: 7.50, 0.068, and 1.13 mg/kg, respectively), whereas CP55,940 and THC were less effective at 55°C (Emax values: 56.6% and 58.2%, respectively). CM304 (56 mg/kg, i.p.) produced an upward shift in the CP55,940 dose‐effect function such that 100% MPE was achieved at 3.2 mg/kg CP55,940, while CM304 (56 mg/kg) produced a 3‐fold leftward shift in the dose‐effect curve of THC. On the other hand, CM304 did not enhance the antinociceptive effects of morphine and fentanyl but enhanced the effects of buprenorphine. In contrast to tail withdrawal latency, there were no consistent effects of CM304 on cannabinoid‐induced decreases in rectal temperature or locomotor activity. The present results may support the development of a σ1R antagonist as an adjunct to cannabinoid and low efficacy μ‐opioid receptor agonists for the treatment of acute pain.Support or Funding InformationThis work was supported by National Institute on Drug Abuse grants DA23205 and DA48353.

Evaluation of CB1 antagonists as antidotes for synthetic cannabinoid toxicity in male and female mice

While a plethora of recreational marijuana and cannabis derived products have been legalized across the United States, recent trends also indicate an increase in the number of emergency calls relating to the overdose or accidental exposure to Δ9‐tetrahydrocannabinol (THC) and/or synthetic psychoactive cannabinoids. There are currently no FDA approved medications for treating acute cannabinoid toxicity. To address this public health issue, here we provide key data towards the discovery and development of an effective antidote for treating acute toxicity symptoms that may be associated with cannabis overdose, and by ingestion of certain cannabimimetics such as the potent CB1 receptor agonist JWH‐018. Previously, we established proof‐of‐concept and presented preliminary data showing that the novel CB1 antagonist AM11503 (10 mg/kg, i.v.) effectively reversed the hypothermic effects of an acute dose of JWH‐018 (6 mg/kg, i.p.), effects that were similar to the known CB1 antagonist rimonabant (10 mg/kg, i.v.). Herein, we extend this line of research in male and female CD‐1 mice by: a) examining whether AM11503 induces rimonabant‐like effects on observable behavior; b) assessing the ability of AM11503 to prevent JWH‐018‐mediated tremors and convulsions; and c) comparing the ability of AM11503 and rimonabant to precipitate somatic signs of withdrawal in JWH‐018‐dependent mice. Results show that rimonabant (3.2–32 mg/kg, i.v.) produced a dose‐related increase in scratching and grooming in both male and female mice compared to controls, whereas AM11503 (3.2–32 mg/kg, i.v.) did not alter stereotypic behavior. In interaction studies, administration of 10 mg/kg (i.v.) AM11503 fully blocked tremors and convulsions induced by “suprapharmacological” doses of JWH‐018 (18 mg/kg, i.p.). Finally, rimonabant (10 mg/kg, i.v.) induced significant somatic signs of withdrawal, i.e., head twitches and paw tremors, in male and female mice that were repeatedly exposed to JWH‐018 (3 mg/kg, i.p. once per day for 6 days) compared to vehicle‐treated subjects. In contrast, AM11503 (10 mg/kg, i.v.) did not induce somatic signs of withdrawal in JWH‐018‐dependent mice and effects were similar to vehicle‐treated mice. In conjunction, the inability of AM11503 to produce rimonabant‐like stereotypic behaviors and precipitate somatic signs of withdrawal yet prevent CB1 agonist‐induced acute toxicity allows for the identification and development of safer CB1 antagonist‐based therapies for nullifying synthetic cannabinoid toxicity.Support or Funding InformationFunding Information: Work supported by NIDA/NIH grants R21DA045882 and R41DA044048

The Sigma1 Receptor Antagonist CM304 Potentiates the Antinociceptive but not the Discriminative Stimulus Effects of the Cannabinoid Receptor Agonist THC in Rats

Opioid overdose is one of the leading causes of death in the United States in people under 50 years, which has resulted in a decline in life expectancy in the United States. Thus, there is a need for an alternative to prescription mu‐opioid agonists for pain treatment. Delta (9)‐tetrahydrocannabinol (THC), a cannabinoid (CB) agonist, has been shown to produce antinociceptive effects albeit less effectively than mu‐opioid receptor agonists. Sigma1 receptor (σ1R) antagonists in combination with THC may provide a viable and safe pharmacological alternative to prescription opioids for pain treatment. The present study compared the pharmacological effects of the σ1R antagonist CM304 alone and in combination with THC in Sprague Dawley rats. Hotplate latency was determined at 52°C followed by rectal temperature measurement and the drugs were administered intravenously (i.v.) and in cumulative doses every 5 min. THC dose‐dependently increased maximum possible effects (MPEs) up to 84% which were reversed by 10 mg/kg rimonabant (cannabinoid CB1 receptor antagonist). CM304 alone up to a dose of 10 mg/kg did not produce significant antinociceptive or hypothermic effects, but CM304 (1.0 and 3.2 mg/kg, i.v.) dose‐dependently left‐shifted the dose‐effect functions of the antinociceptive (7.3‐fold at 3.2 mg/kg CM304) and hypothermic effects of THC. In rats discriminating 3.2 mg/kg THC i.p. from vehicle under a fixed ratio (FR) 10 schedule of food delivery, the cannabinoid CB1 receptor agonist CP55,940 fully substituted for THC, whereas CM304 i.p. produced a maximum of 27 % THC‐lever responding at 17.8 mg/kg. CM304, at doses of 32 and 56 mg/kg, decreased response rate to 24 and 9 %, respectively. No dose of BD1063 (σ1R antagonist), rimonabant, SR144528 (cannabinoid CB2 receptor antagonist), morphine (μ‐opioid receptor agonist), or naltrexone (opioid antagonist) produced greater than 30% THC‐lever responding. Pretreatment with rimonabant (1.78 mg/kg) produced a rightward shift in the dose effect curve of THC (4.5‐fold) and CP55940 (4.5‐fold) while pretreatment with CM304, BD1063, SR144528, and naltrexone had no significant effect on the dose effect curve of THC. The present results may support the development of a σ1R antagonist as an adjunct to cannabinoids for treatment of acute pain without enhancing an undesirable side effect of THC.Support or Funding InformationThis work was supported by National Institute on Drug Abuse grants DA23205 and DA48353.

Anxiolytic Effects of Cannabinoid Receptor Agonists in the Zebrafish Species, Danio rerio

As of July 2019, 34 states in the U.S. have legalized medical marijuana and an additional 12 have legalized “low THC, high cannabidiol (CBD)” substances (Hanson & Garcia, 2019). While acute cannabis use has been associated with decreased anxiety levels, users have also experienced adverse side effects including increased anxiety, panic reactions, and psychotic symptoms (Hall & Degenhardt, 2009). Plant‐derived pharmacological agents, like cannabis, can vary in quality and composition (and thus efficacy) due to a variety of environmental factors (Atanasov et. al. 2015). In order to use cannabinoids to treat anxiety safely, a synthetic cannabinoid that can produce consistent therapeutic (anxiolytic) effects without producing psychoactive effects is necessary. Zebrafish are an increasingly popular model organism used due to their high sensitivity to chemicals as absorbed through their gills and quantifiable behavioral phenotypes (Stewart 2014). Humans and zebrafish have comparable endocannabinoid pathways that each contain CB1 and CB2 receptors (Krug and Clark 2015). When observed in a Novel Dive Tank, anxiety levels in zebrafish can be quantified. Like humans, male and female zebrafish differ in hormonal composition and therefore respond to treatments differently. In this study, male and female zebrafish were treated with CB receptor agonists anandamide and WIN 55,212‐2 and CB2 inverse agonist JTE‐907 in order to model the how the endocannabinoid system influences anxiety. The reduced amount of time spent in the upper zone by the JTE + Anandamide treated fish, combined with the increased time classified as highly mobile, suggests that JTE‐907 had an anxiogenic effect. Significant anxiolytic effects were observed the WIN treatment group, particularly in male treated fish. These results suggest that male treated fish are more receptive to the anxiolytic effects of a non‐selective CB agonist such as WIN 55, 212. Future research with a water‐soluble CB1 inverse agonist could provide insight into the differing roles of CB1 and CB2 receptors in cannabis‐based treatments.

Hypoxia training improves hepatic steatosis partly by downregulation of CB1 receptor in obese mice

Hypoxia training (HT) can reduce body weight and improve fatty liver. However, the mechanism is not clear. A previous study indicated that HT-induced weight loss might be associated with the endocannabinoid system (ECS), which has also been reported recently to be involved in the persistent lipid mediators after weight loss. The present study investigated the effects of HT, a new prospective weight-loss method, on nutritionally obese mice and demonstrated that HT significantly reduced body weight, fat mass, transcriptional expression of liver endocannabinoid receptor 1 (CB1), biosynthetic enzyme diacylglycerol lipase α (DAGLα) and improved the transcriptional expression of degrading enzyme monoacylglycerol lipase (MAGL). Liver endocannabinoids 2-arachidonoylglycerol (2-AG) but not anandamide (AEA) was evidently decreased in response to HT. Simultaneously, HT significantly reduced liver index, serum alanine aminotransferase (ALT) and liver fat contents. Western blot showed decreased expression of liver CB1, sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor γ (PPARγ) and increased expression of adipose triglyceride lipase (ATGL) and carnitine palmitoyltransferase-1 (CPT-1) levels after HT. However, intraperitoneal injection of CB1 receptor agonist WIN55212-2 offset the benefits by which HT reduced hepatic fat synthesis, with significant increased protein expression of SREBP-1 and PPARγ. Taken together, these findings reported the alleviation of obesity and hepatic steatosis through HT and provided a putative molecular mechanism by inhibiting the CB1-mediated fat synthesis.

A124 SYNERGISTIC INTERACTION OF CANNABINOIDS AND OPIOIDS REDUCES PAIN SIGNALING IN COLONIC NOCICEPTIVE NERVES

Abstract Background With the recent legalization of recreational marijuana in Canada, increasing numbers of patients with gastrointestinal (GI) disorders are using cannabis to treat their pain, either alone or together with opioids. However, little is known about potential benefits of cannabinoids for treating visceral pain originating within the GI tract and whether the combined use of cannabinoids and opioids could enable the reduction or even discontinuation of opioids. Aims To investigate the effects of cannabinoids alone or in combination with opioids on colonic nociceptive nerves. Methods Extracellular afferent nerve recordings were obtained from ex vivo flat sheet preparations of male C57BL/6 mouse distal colons. Single unit analysis discriminated individual afferent neuron responses to mechanical probing of the colon with a 1g von Frey hair before and after superfusion of HU-210, a selective CB1 receptor agonist, HU-308, a selective CB2 receptor agonist, DAMGO, a selective mu-opioid receptor (MOR) agonist, or a combination. In parallel studies, perforated patch clamp techniques were employed to assess the rheobase as a measure of neuronal excitability in acutely dissociated dorsal root ganglia (DRG) neurons in the presence of one or more of these agonists. Data were analyzed using a one-way ANOVA with Bonferroni multiple comparisons test. Results Superfusion of HU-210 (1 μM), caused significant inhibition in afferent nerve mechanosensitivity compared to control (6.2±1.1 vs. 13.7±2.5 Hz, p=0.005, n=10); lower concentrations (10 nM and 100 nM) had no effect (p>0.99, n=11; p=0.600, n=10 respectively). Conversely, the CB2 agonist HU-308 (1 μM and 10 μM), did not alter mechanosensitivity (p>0.9, n=8 for both concentrations). Superfusion of HU-210 alone (100 nM) or DAMGO (1 nM) alone in the same recording had no effect, but when both agonists were superfused together, there was a significant reduction in mechanosensitivity (8.1±1.7 vs. 14.8±2.3 Hz, p<0.01, n=10). In patch clamp recordings of DRG neurons, HU-210 (1 μM) decreased excitability (i.e. increased rheobase, 94.4±9.4 vs. 62.7±6.4 pA; p=0.031, n=9), whereas a lower concentration (100 nM) had no effect. Similar to afferent nerve recordings, when applied alone, DAMGO (1 nM) and HU-210 (100 nM) did not affect rheobase (DAMGO: p>0.99, n=9; HU-210: p>0.99, n=10), whereas the combination of both agonists significantly decreased excitability (123.0±13.4 vs. 62.7±6.4 pA, p<0.01, n=10). Conclusions Activation of CB1 receptors inhibits mechanosensitivity of colonic afferent nerves while a CB2 agonist had no effect. Interestingly, combination of sub-threshold concentrations of CB1 and MOR agonists inhibited colonic afferent nerves and thus, may suggest that cannabinoids could enable opioid dose reduction or discontinuation. Funding Agencies None

β‐Caryophyllene, a dietary terpenoid, inhibits nicotine taking and nicotine seeking in rodents

β-Caryophyllene (BCP) is a plant-derived terpenoid used as a food additive for many decades. Recent studies indicate that BCP is a cannabinoid CB2 receptor agonist with medical benefits for a number of human diseases. However, little is known about its therapeutic potential for drug abuse and addiction. We used pharmacological, transgenic, and optogenetic approaches to systematically evaluate the effects of BCP on nicotine-taking and nicotine-seeking behaviour in animal models of drug self-administration, electrical, and optical brain-stimulation reward. Systemic administration of BCP dose-dependently inhibited nicotine self-administration and motivation for nicotine seeking in rats and mice. The reduction in nicotine self-administration was blocked by AM630, a selective CB2 receptor antagonist, but not by AM251, a selective CB1 receptor antagonist, suggesting involvement of a CB2 receptor mechanism. Genetic deletion of CB2 receptors in mice blocked the reduction in nicotine self-administration produced only by low doses, but not by high doses, of BCP, suggesting involvement of both CB2 and non-CB2 receptor mechanisms. Furthermore, in the intracranial self-stimulation paradigm, BCP attenuated electrical brain-stimulation reward and nicotine-enhanced brain-stimulation reward in rats. Lastly, BCP also attenuated brain-stimulation reward maintained by optogenetic stimulation of dopaminergic neurons in the ventral tegmental area in DAT-cre mice, suggesting the involvement of a dopamine-dependent mechanism in BCP's action. The present findings suggest that BCP has significant anti-nicotine effects via both CB2 and non-CB2 receptor mechanisms and, therefore, deserves further study as a potential new pharmacotherapy for cigarette smoking cessation.

Insulinotropic and antidiabetic effects of β-caryophyllene with l-arginine in type 2 diabetic rats.

Beta-caryophyllene (BCP) is a flavoring agent, whereas l-arginine (LA) is used as a food supplement. They possess insulinotropic and β cell regeneration activities, respectively. We assessed the antidiabetic potential of BCP, LA, and its combination in RIN-5F cell lines and diabetic rats. Ex vivo studies were carried out for glucose uptake and absorption of the combination of BCP with LA. The results indicated that the combination of BCP with LA showed a significant decrease in glucose absorption and an increase in its uptake in tissues and also an increase in insulin secretion in RIN-5F cells. The combination treatment of BCP with LA showed a significant reduction in glucose, lipid levels, and oxidative stress in pancreatic tissue when compared with the diabetic group. Furthermore, the combination of BCP with LA normalized glucose tolerance and pancreatic cell damage in diabetic rats. In conclusion, the combinational treatment showed significant potentials in the treatment of type 2 diabetes mellitus. PRACTICAL APPLICATIONS: Type 2 diabetes mellitus is the most prevalent chronic metabolic disorder affecting a large population. Beta-caryophyllene is a CB2 receptor agonist shown to have insulinotropic activity. l-Arginine is a food supplement that possesses beta-cell regeneration property. The combination of BCP with LA could work as a potential therapeutic intervention, considering the individual pharmacological activities of each. We evaluated the antidiabetic activity of the combination of BCP with LA in diabetic rats using ex vivo and in vitro experimentations. Results from the study revealed that the combination of BCP with LA showed a significant (p<.001) reduction in glucose and lipid levels as compared to individual treatment. In vitro study also supports the diabetic potential of the combination of BCP with LA in the glucose-induced insulin secretion in RIN-5F cell lines. The study indicates a therapeutic approach to treat T2DM by BCP and LA combination as food and dietary supplement.

Activation of CB2R withAM1241 ameliorates neurodegeneration via the Xist/miR-133b-3p/Pitx3 axis.

Activation of cannabinoid receptor type II (CB2R) by AM1241 has been demonstrated to protect dopaminergic neurons in Parkinson's disease (PD) animals. However, the specific mechanisms of the action of the CB2R agonist AM1241 for PD treatment have not been characterized. Wild-type (WT), CB1R knockout (CB1-KO), and CB2R knockout (CB2-KO) mice were exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 1 week to obtain a PD mouse model. The therapeutic effects of AM1241 were evaluated in each group. Behavioral tests, analysis of neurotransmitters, and immunofluorescence results demonstrated that AM1241 ameliorated PD in WT animals and CB1-KO animals. However, AM1241 did not ameliorate PD symptoms in CB2-KO mice. RNA-seq analysis identified the lncRNA Xist as an important regulator of the protective actions of AM1241. Specifically, AM1241 allowed WT and CB1-KO animals treated with MPTP to maintain normal expression of Xist, which affected the expression of miR-133b-3p and Pitx3. In vitro, overexpression of Xist or AM1241 protected neuronal cells from death induced by 6-hydroxydopamine and increased Pitx3 expression. The CB2 receptor agonist AM1241 alleviated PD via regulation of the Xist/miR-133b-3p/Pitx3 axis, andrevealed a new approach for PD treatment.

Possible interaction between the ventral hippocampal cannabinoid CB2 and muscarinic acetylcholine receptors on the modulation of memory consolidation in mice.

To clarify possible interaction between the ventral hippocampal cannabinoid CB2 receptors and the cholinergic system in control of the memory process, the effects of cannabinoid and acetylcholine receptor agents on memory consolidation have been investigated in mice. Animals implanted with bilateral cannulas at the CA3 region of the ventral hippocampus and microinjected with scopolamine and cannabinergic agents. These animals were tested using a one-trial step-down inhibitory avoidance task. The results indicated impairment of memory consolidation by posttraining intra-CA3 microinjection of scopolamine (1 and 2 µg/mouse). Nevertheless, coinjection of various doses of scopolamine (0.01, 1 and 2 µg/mouse) with an ineffective dose of AM630 (1 µg/mouse) or GP1a (1 µg/mouse) did not show any significant effect on deficiency of memory consolidation produced by scopolamine. Posttraining application of cannabinoid CB2 receptor antagonist, AM630 (1, 10 and 100 µg/mouse; intra-CA3) alone had no significant influence on memory performance, but its coinjection with significant dose of scopolamine (1 µg/mouse) decreased memory consolidation. Moreover, posttraining injection of GP1a, cannabinoid CB2 receptor agonist, (10 and 100 µg/mouse; intra-CA3) decreased memory consolidation. Posttraining coadministration of diverse doses of GP1a (1, 10 and 100 µg/mouse; intra-CA3) with an effective dose of scopolamine (1 µg/mouse) meaningfully increased deficiency of memory consolidation produced by GP1a (100 µg/mouse). In addition, all drugs had no significant effect on locomotion. Consequently, these results propose that a probable interaction between the CA3 cannabinoid CB2 receptors and muscarinic acetylcholine receptors (mAChR) modulates memory consolidation process in mice.