Cannabinoid Receptor 1 Receptor Research Articles

Chitin-glucan improves important pathophysiological features of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is one of the most frequent and debilitating conditions leading to gastroenterological referrals. However, recommended treatments remain limited, yielding only limited therapeutic gains. Chitin-glucan (CG) is a novel dietary prebiotic classically used in humans at a dosage of 1.5-3.0 g/d and is considered a safe food ingredient by the European Food Safety Authority. To provide an alternative approach to managing patients with IBS, we performed preclinical molecular, cellular, and animal studies to evaluate the role of chitin-glucan in the main pathophysiological mechanisms involved in IBS. To evaluate the roles of CG in visceral analgesia, intestinal inflammation, barrier function, and to develop computational molecular models. Visceral pain was recorded through colorectal distension (CRD) in a model of long-lasting colon hypersensitivity induced by an intra-rectal administration of TNBS [15 milligrams (mg)/kilogram (kg)] in 33 Sprague-Dawley rats. Intracolonic pressure was regularly assessed during the 9 wk-experiment (weeks 0, 3, 5, and 7) in animals receiving CG (n = 14) at a human equivalent dose (HED) of 1.5 g/d or 3.0 g/d and compared to negative control (tap water, n = 11) and positive control (phloroglucinol at 1.5 g/d HED, n = 8) groups. The anti-inflammatory effect of CG was evaluated using clinical and histological scores in 30 C57bl6 male mice with colitis induced by dextran sodium sulfate (DSS) administered in their drinking water during 14 d. HT-29 cells under basal conditions and after stimulation with lipopolysaccharide (LPS) were treated with CG to evaluate changes in pathways related to analgesia (µ-opioid receptor (MOR), cannabinoid receptor 2 (CB2), peroxisome proliferator-activated receptor alpha, inflammation [interleukin (IL)-10, IL-1b, and IL-8] and barrier function [mucin 2-5AC, claudin-2, zonula occludens (ZO)-1, ZO-2] using the real-time PCR method. Molecular modelling of CG, LPS, lipoteichoic acid (LTA), and phospholipomannan (PLM) was developed, and the ability of CG to chelate microbial pathogenic lipids was evaluated by docking and molecular dynamics simulations. Data were expressed as the mean ± SEM. Daily CG orally-administered to rats or mice was well tolerated without including diarrhea, visceral hypersensitivity, or inflammation, as evaluated at histological and molecular levels. In a model of CRD, CG at a dosage of 3 g/d HED significantly decreased visceral pain perception by 14% after 2 wk of administration (P < 0.01) and reduced inflammation intensity by 50%, resulting in complete regeneration of the colonic mucosa in mice with DSS-induced colitis. To better reproduce the characteristics of visceral pain in patients with IBS, we then measured the therapeutic impact of CG in rats with TNBS-induced inflammation to long-lasting visceral hypersensitivity. CG at a dosage of 1.5 g/d HED decreased visceral pain perception by 20% five weeks after colitis induction (P < 0.01). When the CG dosage was increased to 3.0 g/d HED, this analgesic effect surpassed that of the spasmolytic agent phloroglucinol, manifesting more rapidly within 3 wk and leading to a 50% inhibition of pain perception (P < 0.0001). The underlying molecular mechanisms contributing to these analgesic and anti-inflammatory effects of CG involved, at least in part, a significant induction of MOR, CB2 receptor, and IL-10, as well as a significant decrease in pro-inflammatory cytokines IL-1b and IL-8. CG also significantly upregulated barrier-related genes including muc5AC, claudin-2, and ZO-2. Molecular modelling of CG revealed a new property of the molecule as a chelator of microbial pathogenic lipids, sequestering gram-negative LPS and gram-positive LTA bacterial toxins, as well as PLM in fungi at the lowesr energy conformations. CG decreased visceral perception and intestinal inflammation through master gene regulation and direct binding of microbial products, suggesting that CG may constitute a new therapeutic strategy for patients with IBS or IBS-like symptoms.

Beyond prediction: unveiling the prognostic power of μ-opioid and cannabinoid receptors, alongside immune mediators, in assessing the severity of SARS-CoV-2 infection

BackgroundThis study aims to explore the potential of utilizing the expression levels of cannabinoid receptor 2 (CB2), μ-opioid receptor (MOR), MCP-1, IL-17, IFN-γ, and osteopontin as predictors for the severity of SARS-CoV-2 infection. The overarching goal is to delineate the pathogenic mechanisms associated with SARS-CoV-2.MethodsUsing quantitative Real-time PCR, we analyzed the gene expression levels of CB2 and MOR in nasopharynx specimens obtained from patients diagnosed with SARS-CoV-2 infection, with 46 individuals classified as having severe symptoms and 46 as non-severe. Additionally, we measured the circulating levels of MCP-1, IL-17, IFN-γ, and osteopontin using an ELISA assay. We examined the predictive capabilities of these variables and explored their correlations across all patient groups.ResultsOur results demonstrated a significant increase in MOR gene expression in the epithelium of patients with severe infection. The expression of CB2 receptor was also elevated in both male and female patients with severe symptoms. Furthermore, we observed concurrent rises in MCP-1, IL-17, IFN-γ, and osteopontin levels in patients, which were linked to disease severity. CB2, MOR, MCP-1, IL-17, IFN-γ, and osteopontin showed strong predictive abilities in distinguishing between patients with varying degrees of SARS-CoV-2 severity. Moreover, we identified a significant correlation between CB2 expression and the levels of MOR, MCP-1, osteopontin, and IFN-γ.ConclusionsThese results underline the interconnected nature of molecular mediators in a sequential manner, suggesting that their overexpression may play a role in the development of SARS-CoV-2 infections.Graphical

A Novel Non-Psychoactive Fatty Acid from a Marine Snail, Conus inscriptus, Signals Cannabinoid Receptor 1 (CB1) to Accumulate Apoptotic C16:0 and C18:0 Ceramides in Teratocarcinoma Cell Line PA1.

The cannabinoid-type I (CB1) receptor functions as a double-edged sword to decide cell fate: apoptosis/survival. Elevated CB1 receptor expression is shown to cause acute ceramide accumulation to meet the energy requirements of fast-growing cancers. However, the flip side of continual CB1 activation is the initiation of a second ceramide peak that leads to cell death. In this study, we used ovarian cancer cells, PA1, which expressed CB1, which increased threefold when treated with a natural compound, bis(palmitoleic acid) ester of a glycerol (C2). This novel compound is isolated from a marine snail, Conus inscriptus, using hexane and the structural details are available in the public domain PubChem database (ID: 14275348). The compound induced two acute ceramide pools to cause G0/G1 arrest and killed cells by apoptosis. The compound increased intracellular ceramides (C:16 to 7 times and C:18 to 10 times), both of which are apoptotic inducers in response to CB1 signaling and thus the compound is a potent CB1 agonist. The compound is not genotoxic because it did not induce micronuclei formation in non-cancerous Chinese hamster ovarian (CHO) cells. Since the compound induced the cannabinoid pathway, we tested if there was a psychotropic effect in zebrafish models, however, it was evident that there were no observable neurobehavioral changes in the treatment groups. With the available data, we propose that this marine compound is safe to be used in non-cancerous cells as well as zebrafish. Thus, this anticancer compound is non-toxic and triggers the CB1 pathway without causing psychotropic effects.

Increasing CB2 Receptor Activity after Early Life Stress Prevents Depressive Behavior in Female Rats.

Early adversity, the loss of the inhibitory GABAergic interneuron parvalbumin, and elevated neuroinflammation are associated with depression. Individuals with a maltreatment history initiate medicinal cannabis use earlier in life than non-maltreated individuals, suggesting self-medication. Female rats underwent maternal separation (MS) between 2 and 20 days of age to model early adversity or served as colony controls. The prelimbic cortex and behavior were examined to determine whether MS alters the cannabinoid receptor 2 (CB2), which has anti-inflammatory properties. A reduction in the CB2-associated regulatory enzyme MARCH7 leading to increased NLRP3 was observed with Western immunoblots in MS females. Immunohistochemistry with stereology quantified numbers of parvalbumin-immunoreactive cells and CB2 at 25, 40, and 100 days of age, revealing that the CB2 receptor associated with PV neurons initially increases at P25 and subsequently decreases by P40 in MS animals, with no change in controls. Confocal and triple-label microscopy suggest colocalization of these CB2 receptors to microglia wrapped around the parvalbumin neuron. Depressive-like behavior in MS animals was elevated at P40 and reduced with the CB2 agonist HU-308 or a CB2-overexpressing lentivirus microinjected into the prelimbic cortex. These results suggest that increasing CB2 expression by P40 in the prelimbic cortex prevents depressive behavior in MS female rats.

Cannabinoid Receptor 2 Gene Polymorphism and the Risk of Developing Rheumatoid Arthritis in Lebanese Patients.

Objective: Evidence supports a role of cannabinoid receptor 2 (CB2) in regulating the immune response. Some variations in the CB2 receptor gene (CB2) were linked to the susceptibility of developing rheumatoid arthritis (RA). The aim of this study is to assess the relationship between CNR2 rs2501431 and the risk of developing RA in Lebanese patients. Methods: A total of one hundred five Lebanese RA patients and one hundred five controls participated in the study. CNR2 was genotyped and analyzed. Results: Using χ2 test, our results show that the CC genotype was the most common (47.6%, p<0.00001) and that the C allele highly predominated (64%, p<0.00001) in the RA group compared to the control group. The relative odds ratio show that carriers of the CC genotype have more than 13-fold risk of developing RA as compared to TT. Conclusion: Our results suggest that the rs2501431 variant of CNR2 gene can be considered as a risk factor for RA development, and thus implicate the potential targeting of CB2 receptor for the treatment of RA.

Deciphering the Molecular Association of Human CRIP1a with an Agonist-Bound Cannabinoid Receptor 1.

Cannabinoid receptor 1 (CB1) is a class A G-protein-coupled receptor that plays important roles in several physiological and pathophysiological processes. Therefore, targeted regulation of CB1 activity is a potential therapeutic strategy for several diseases, including neurological disorders. Apart from cannabinoid ligands, CB1 signaling can also be regulated by different CB1-associated proteins. In particular, the cannabinoid receptor interacting protein 1a (CRIP1a) associates with an activated CB1 receptor and alters the G-protein selectivity, thereby reducing the agonist-mediated signal transduction of the CB1 receptor. Experimental evidence suggests that two peptides corresponding to the distal and central C-terminal segments of CB1 could interact with CRIP1a. However, our knowledge of the molecular basis of CB1-CRIP1a recognition is still limited. In this work, we use an extensive combination of computational methods to build the first comprehensive atomistic model human CB1-CRIP1a complex. Our model provides novel structural insights into the interactions of CRIP1a with a membrane-embedded, complete, agonist-bound CB1 receptor in humans. Our results highlight the key residues that stabilize the CB1-CRIP1a complex, which will be useful to guide in vitro mutagenesis experiments. Furthermore, our human CB1-CRIP1a complex presents a model system for structure-based drug design to target this physiologically important complex for modulating CB1 activity.

Cannabinoid receptor 2 (CB2) agonists and L-arginine ameliorate diabetic nephropathy in rats by suppressing inflammation and fibrosis through NF-κβ pathway.

Diabetic nephropathy (DN) is a condition that leads to end-stage chronic kidney disease characterized by inflammation and a deficiency ofnitric oxide (NO).Cannabinoid receptor (CB2) activation by specific agonist reduces nuclear factor kappa beta (NF-κβ) expression. Beta caryophyllene (BCP), a natural CB2 receptor activator, protects kidney function in several diseases. L-Arginine (LA) modulates several physiological processes by donating nitric oxide (NO). Hence, we tested a novel BCP-LA combination to treat DN and investigated its molecular mechanisms. BCP, LA, and combinations of both were evaluated in LPS-induced RAW 264.7 macrophage inflammation as well as in streptozotocin (55mg/kg)-induced diabetes in SD rats. Diabetic rats were administered200 mg/kg of BCP, 100mg/kg of LA, and combination of both orally for 28days. Biochemical markers and inflammatory cytokines were assessed in plasma; also, kidney tissue was examined for renal oxidative stress injury, NF-κβ expression, and histology. After 28days of treatment, BCP and LA combination significantly lowered plasma glucose levels than the disease control group. BCP and LA also normalized renal markers and oxidative stress of diabetic rats. Plasma and RAW macrophage cell lines showed reduced levels of IL-6 and TNF-α (P < 0.001). Histopathological evaluations revealed that BCP and LA together decreased renal fibrosis and collagen deposition also improved nephrotic indices. Meanwhile, the effect of BCP and LA together significantly reduced the NF-κβ (P < 0.01) against diabetic rats. These results indicate that the innovative regimen BCP with LA may be a therapeutic treatment for DN, as it protects kidney tissue from diabetes via NF-κβ inhibition.

Pharmacological blockade of cannabinoid receptor 2 signaling does not affect LPS/IFN-γ-induced microglial activation

Cannabinoid receptor 2 (CB2) signaling modulates microglial responses to inflammatory stimuli. Our previous studies demonstrated that genetic deletion of CB2 inhibits microglial activation during inflammatory stimulation of toll-like receptors (TLRs) or in neurodegenerative conditions. However, we cannot exclude developmental effects of the constitutive CB2 knockout (CB2−/−), which could mediate compensatory outcomes in CB2−/− mice. In the present study, we therefore tested whether acute pharmacological inhibition of CB2 receptor has a similar effect on microglial activation as in CB2−/− in response to inflammatory stimulation. Our findings suggest that the CB2-specific antagonist SR144528 has little or no effect on LPS/IFN-γ-induced activation in primary microglia or organotypic hippocampal slice cultures at nanomolar concentrations. We show that SR144528 did not alter LPS/IFN-γ-mediated microglial cytokine secretion, Iba1 and CD68 staining intensity or morphology at 1 and 10 nM. Although SR144528 suppressed LPS/IFN-γ-induced microglial activation at 1 µM, this anti-inflammatory effect was not dependent on CB2 receptors and exceeded the Ki on CB2 receptors by more than a thousand-fold. Thus, SR144528 does not mimic the anti-inflammatory effects observed in the CB2−/− microglia after LPS/IFN-γ stimulation. Therefore, we propose that the deletion of CB2 probably triggered an adaptive mechanism, making microglia less responsive to inflammatory stimulation.

Inhibition of synaptic transmission by anandamide precursor 20:4-NAPE is mediated by TRPV1 receptors under inflammatory conditions.

Transient receptor potential ion channel, vanilloid subfamily, type 1 (TRPV1) cation channel, and cannabinoid receptor 1 (CB1) are essential in the modulation of nociceptive signaling in the spinal cord dorsal horn that underlies different pathological pain states. TRPV1 and CB1 receptors share the endogenous agonist anandamide (AEA), produced from N-arachidonoylphosphatidylethanolamine (20:4-NAPE). We investigated the effect of the anandamide precursor 20:4-NAPE on synaptic activity in naive and inflammatory conditions. Patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) from superficial dorsal horn neurons in rat acute spinal cord slices were used. Peripheral inflammation was induced by subcutaneous injection of carrageenan. Under naive conditions, mEPSCs frequency (0.96 ± 0.11 Hz) was significantly decreased after 20 μM 20:4-NAPE application (55.3 ± 7.4%). This 20:4-NAPE-induced inhibition was blocked by anandamide-synthesizing enzyme N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor LEI-401. In addition, the inhibition was prevented by the CB1 receptor antagonist PF 514273 (0.2 μM) but not by the TRPV1 receptor antagonist SB 366791 (10 μM). Under inflammatory conditions, 20:4-NAPE (20 μM) also exhibited a significant inhibitory effect (74.5 ± 8.9%) on the mEPSCs frequency that was prevented by the TRPV1 receptor antagonist SB 366791 but not by PF 514273 application. Our results show that 20:4-NAPE application has a significant modulatory effect on spinal cord nociceptive signaling that is mediated by both TRPV1 and CB1 presynaptic receptors, whereas peripheral inflammation changes the underlying mechanism. The switch between TRPV1 and CB1 receptor activation by the AEA precursor 20:4-NAPE during inflammation may play an important role in nociceptive processing, hence the development of pathological pain.

In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies

G protein-coupled receptors (GPCRs) are important signal transducers that are phosphorylated upon activation at intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has not been possible. Here, we show that phosphorylation of receptors is highly unstable during routine immunohistochemical procedures, requiring the use of appropriate phosphatase inhibitors particular during tissue perfusion, post-fixation, and cryoprotection but not during immunostaining of tissue sections. We provide proof of concept using phosphorylation state-specific μ-opioid receptor (MOP) and cannabinoid receptor 1 (CB1) antibodies. Indeed, three of four well-characterized phosphosite-specific MOP antibodies, including pS375-MOP, pT376-MOP, and pT379-MOP, showed robust neuronal immunostaining in brain and spinal cord sections of opioid-treated mice only after inclusion of phosphatase inhibitors. We then extended this approach to the CB1 receptor and demonstrated that one of three newly-generated phosphosite-specific CB1 antibodies, namely pS425-CB1, showed striking staining of fibers and varicosities in brain slices from cannabinoid-treated mice. Although subsequent experiments showed that phospho-CB1 immunostaining was less sensitive to phosphatases, we conclude that the use of phosphatase inhibitors should always be considered in the development of immunohistochemical procedures for new phosphosite-specific GPCR antibodies. In summary, we anticipate that this improved protocol will facilitate the widespread use of phosphorylation state-specific antibodies to monitor the activation of endogenous GPCRs under physiological and pharmacological conditions. Our approach may also prove useful to confirm target engagement of GPCR drug candidates in native tissues.

The impact of changes in membrane lipid composition on the CB1 receptor.

A number of conditions have a higher prevalence among people who are obese or old, and this includes chronic pain. These two demographics are increasing, with obesity nearly tripling since 1975 and the number and fraction of older people are increasing faster than ever before. These changes lead to alterations of the lipid composition of cell membranes. When the lipid composition changes it influences the biophysical properties of the membrane and is thought to be affecting the dynamics of membrane proteins. The cannabinoid receptor 1 (CB1) is a pain receptor which is found in high abundance in neuronal and plasma membranes. The CB1 is an interesting target to investigate when it comes to chronic pain as it is a non-opioid target. We found that the composition of the local lipid environment influences the protein dynamics. The global biophysical properties indirectly play a role by modulating the local lipid environment around the protein.

Global cannabinoid receptor 1 deficiency affects disuse-induced bone loss in a site-specific and sex-dependent manner

Bone loss during mechanical unloading increases fracture risk and is a major concern for the general population and astronauts during spaceflight. The endocannabinoid system (ECS) plays an important role in bone metabolism. One of the main ECS receptors, cannabinoid receptor 1 (CB1), has been studied in regards to basic bone metabolism; however, little is known as to how CB1 and the ECS affect bone in different mechanical environments. In this study, we analyzed the influence of global CB1 deficiency and sex on mice during disuse caused by single limb immobilization. Female mice were more sensitive to disuse-induced BV/TV loss than males in both the femoral metaphysis and tibial epiphysis. Genotype also affected bone loss in a sex-dependent manner, with male mice deficient in CB1 receptors (CB1KO) and female wildtype (WT) mice experiencing increased bone loss in both the tibial metaphysis and femoral epiphysis. Genotype affected the response to disuse as CB1KO mice displayed greater changes in femoral ultimate force, along with lower tibial ultimate stress, compared to WT mice. Female mice had a significantly higher femoral, and lower tibial ultimate force compared to male mice. These results reveal that disuse-induced bone loss due to CB1 deficiency is sex-dependent. CB1 deficiency in male mice exacerbated bone loss, while in females CB1 deficiency appeared to protect against disuse-induced bone loss. Regardless of genotype, female mice were more sensitive than males to disuse. These results suggest that CB1 receptors may represent a potential therapeutic target for mitigation of disuse-induced bone loss.

The Impact of JWH-133 on Articular Cartilage Regeneration in Osteoarthritis Via Metalloproteinase 13-Dependent Mechanism.

Objective: Osteoarthritis (OA) is common degenerative joint disease, mostly characterized by gradual cartilage breakdown. Currently there are no disease-modifying drugs available, therefore, there is an increasing need for basic research to focus on cartilage function in OA. Changes in cannabinoid receptor 2 (CB2) expression were observed in the OA-affected joints, although its action on cartilage chondrocytes remain unclear. We studied the action of dimethylbutyl-deoxy-delta-8-THC (JWH-133), selective CB2 agonist, on chondrocytes metabolism using both in vitro and in vivo studies. Design: Intraarticular (i.a.) injection of monoiodoacetate (MIA) was used to induce OA in rats. OA-related pain symptoms were assessed by pressure application measurements (PAMs). Primary human chondrocytes treated with MIA were used to investigate action of JWH-133 on chondrocytes viability, proliferation, and motility. Cannabinoid system components, inflammatory cytokines and metalloproteinases (MMPs) expression was measured on messenger RNA and protein levels in chondrocytes and animal cartilage. Results: Repeated, i.a. administration of JWH-133 showed antinociceptive potential in PAM, as well as decreased levels of MMPs, which suggests that CB2 agonism may modify degradation of cartilage. JWH-133 administration partially reduced toxicity, increased proliferation, and chondrocytes' migration. Moreover, our data suggest that CB2 agonism leads to alleviation of MMPs expression both in vitro and in vivo. Conclusions: In this study, we demonstrate modifying effect of JWH-133 local administration on cartilage metabolism and MMP13 expression that was shown to be involved in cartilage degradation. CB2 receptors' activation may be of benefit for chondrocytes' proliferation, therefore delaying disease progression. Our results propose direction of studies on OA-modifying treatment that can benefit in management of human OA.

CB1 Cannabinoid Receptor is a Target for Neuroprotection in Light Induced Retinal Degeneration.

In the last few years, an increasing interest in the neuroprotective effect of cannabinoids has taken place. The aim of the present work was to study the effects of modulating cannabinoid receptor 1 (CB1) in the context of light induced retinal degeneration (LIRD), using an animal model that resembles many characteristics of human age-related macular degeneration (AMD) and other degenerative diseases of the outer retina. Sprague Dawley rats (n = 28) were intravitreally injected in the right eye with either a CB1 agonist (ACEA), or an antagonist (AM251). Contralateral eyes were injected with respective vehicles as controls. Then, rats were subjected to continuous illumination (12,000 lux) for 24h. Retinas from 28 animals were processed by GFAP-immunohistochemistry (IHC), TUNEL technique, Western blotting (WB), or qRT-PCR. ACEA-treated retinas showed a significantly lower number of apoptotic nuclei in the outer nuclear layer (ONL), lower levels of activated Caspase-3 by WB, and lower levels of glial reactivity by both GFAP-IHC and WB. qRT-PCR revealed that ACEA significantly decreased the expression of Bcl-2 and CYP1A1. Conversely, AM251-treated retinas showed a higher number of apoptotic nuclei in the ONL, higher levels of activated Caspase-3 by WB, and higher levels of glial reactivity as determined by GFAP-IHC and WB. AM251 increased the expression of Bcl-2, Bad, Bax, Aryl hydrocarbon Receptor (AhR), GFAP, and TNFα. In summary, the stimulation of the CB1 receptor, previous to the start of the pathogenic process, improved the survival of photoreceptors exposed to LIRD. The modulation of CB1 activity may be used as a neuroprotective strategy in retinal degeneration and deserves further studies.

N-linoleyltyrosine ameliorates high-fat diet-induced obesity in C57BL/6 mice via cannabinoid receptor regulation.

ObjectivesN-linoleyltyrosine (NITyr) showed mild effects in preclinical studies. The research discussed the effect of NITyr on a high-fat diet (HFD) induced obese (DIO) mice, and preliminarily explored its mechanism.MethodsThe DIO mice were established by feeding an HFD for 12 weeks and subsequently administrated orally with NITyr (30, 60 and 100 mg/kg) for four weeks. The indexes of serum and liver samples were determined by ELISA kit. The pathological status of adipose and liver were detected by HE staining. The factors related to energy and lipid metabolism were measured via western blot.ResultsNITyr at 60 and 100 mg/kg/day suppressed the weight gain without affecting water and food intake. Accordingly, NITyr reduced adipose weight and the area of individual adipocytes and increased the number of adipocytes. Moreover, NITyr didn’t affect the appetite-related indexes such as ghrelin, peptide YY and brain-derived neurotrophic factor. Besides, NITyr didn’t affect other organ coefficients except for the liver. Correspondingly, NITyr reduced alanine aminotransferase and aspartate aminotransferase levels, yet didn’t influence IL-1β and TNF-α levels, and the liver injury. The levels of triacylglycerol (TG), total cholesterol (TC), glucose, insulin, adiponectin and leptin in serum were assessed to evaluate the effect of NITyr on glucose and lipid metabolism. NITyr decreased the levels of TG, TC and glucose, and didn’t affect insulin, adiponectin and leptin levels. Meanwhile, NITyr up-regulated p-AMPK and the cannabinoid receptor 2 (CB2) expressions, and down-regulated PPAR, FAS and cannabinoid receptor 1 (CB1) expressions.Overall, NITyr suppressed lipid accumulation via improving lipid and glucose metabolism involving CB1 and CB2 receptors.

Yin and yang of cannabinoid CB1 receptor: CB1 deletion in immune cells causes exacerbation while deletion in non-immune cells attenuates obesity

SummaryWhile blockade of cannabinoid receptor 1 (CB1) has been shown to attenuate diet-induced obesity (DIO), its relative role in different cell types has not been tested. The current study investigated the role of CB1 in immune vs non-immune cells during DIO by generating radiation-induced bone marrow chimeric mice that expressed functional CB1 in all cells except the immune cells or expressed CB1 only in immune cells. CB1−/− recipient hosts were resistant to DIO, indicating that CB1 in non-immune cells is necessary for induction of DIO. Interestingly, chimeras with CB1−/− in immune cells showed exacerbation in DIO combined with infiltration of bone-marrow-derived macrophages to the brain and visceral adipose tissue, elevated food intake, and increased glucose intolerance. These results demonstrate the opposing role of CB1 in hematopoietic versus non-hematopoietic cells during DIO and suggests that targeting immune CB1 receptors provides a new pathway to ameliorate obesity and related metabolic disorders.

Synthetic cannabimimetics with cyclobutyl methyl and norbornyl methyl side chains – Pharmacological data and legislative implications

Structural elucidation and analytical characterization of six newly emerged synthetic cannabimimetics (SCs) carrying a cyclobutyl methyl (CBM) or a norbornyl methyl (NBM) side chain were performed within the ADEBAR project. Pharmacological evaluation allowed for estimating in vivo cannabimimetic activities as required in many countries for the amendment of laws controlling these substances. SCs, one of the largest subgroups of new psychoactive substances (NPS), possess a unique dynamic in Germany, where new variants have been observed to circumvent national legislation. In 2019–2020, the CBM moiety was identified as a new side chain in three SCs, leading to substances not covered by the German new psychoactive substances act (NpSG) at that time. In the following year, indole, indazole, and γ-carbolinone SCs featuring a NBM side chain were detected shortly after an amendment of the NpSG covering the CBM side chain. The introduction of these moieties that were not described in the synthetic cannabinoid research literature prior to their appearance on the drug market underpins the creativity of drug designers, obviously motivated by a recent amendment of the German NpSG. Analytical characterization of six SCs carrying a CBM or a NBM side chain was performed using GC-MS, ATR-FT-(N)IR, GC-sIR, NMR, LC-(HR)MS, and Raman spectroscopy. Binding affinities and functional activities were studied at the cannabinoid receptor 1 (CB1) using filtration-based radioligand assays. All six SCs showed full agonist properties at the CB1 receptor compared to Δ9-THC and CP-55940. The three SCs carrying a CBM group showed binding affinities from 29.4 to 0.65 nM (CBMICA >> CBMINACA > CBMeGaClone). The potencies of the compounds increased in the same order (EC50: 483–40.1 nM), which also reflects the order of their occurrence on the drug market. The norbornyl derivatives exhibit higher affinities (Ki: 1.87–0.25 nM), with the indazole derivative exhibiting the highest affinity. Functional activity data confirmed the indazole being the most potent and efficacious of these three NBM SCs (EC50: 169-–1.78 nM). The binding affinities and potencies were significantly increased by the introduction of the relatively voluminous norbornyl side chain. Only the γ-carbolinone analog exhibited a slight decrease of the binding affinity from 0.65 to 2.4 nM when exchanging the CBM group for the NBM group. Still, potency increased by factor 4 (indole: factor 2, indazole: factor 31). Cumyl-CBMICA showed the lowest binding affinity and functional activity of the six compounds. Interestingly, the average concentration of Cumyl-CBMICA increased from 5 mg/g to 71 mg/g in commercially available ‘herbal blends’ between July 2019 and December 2019 ( n = 14 blends analyzed) and the herbal blend product with the highest concentration was advertised as “strong” in the online shop. The concentrations were likely increased in the herbal blends due to the low potency of Cumyl-CBMICA in vivo, which has also been reported by user feedback given in online forums. New CBM and NBM SCs have been detected by the ADEBAR project in Germany and the six investigated SCs showed to act as full agonists at the CB1 receptor. The pharmacological data underpin their abuse potential and facilitated the legislative amendments to the German NpSG.

Endogenous Cannabinoid Receptors Modulate Plasticity at Immature Synapses

To explore the mechanism of endocannabinoid cannabinoid receptor 1 (CB1) receptor pathway that regulates synaptic plasticity in the dorsal horn of the spinal cord of rats with neuropathic pain at different ages. Neonatal, juvenile, and adult male sprague dawley (SD) rats were divided into the spinal nerve preservation injury (SNI), SNI + Anandamide (AEA), SNI + D-AP5, SNI + CNQX, SNI + D-AP5 + AEA, SNI + CNQX + AEA, sham SNI, sham SNI + AEA, sham SNI + D-AP5, sham SNI + CNQX, sham SNI + D-AP5 + AEA, and sham SNI + CNQX + AEA groups, respectively. Paw withdrawal threshold (PWT) and long-term potentiation (LTP) of the spinal dorsal horn PS (field potential) were assessed to judge the spinal cord's functional state. Immunohistochemical staining and Western blot were conducted to detect CB1 protein levels in the spinal dorsal horn. The LTP response in the spinal cord was alleviated in the SNI + AEA group. After treatment with the N-methyl-D-aspartate (NMDA) receptor blocker D-AP5, the LTP of neonatal A nerve was relieved further. After treatment with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker CNQX, LTP change in the A nerve was not obvious. The LTP of the A and C nerves were relieved after D-AP5 or CNQX treatment in young and adult animals; however, the blocking effect of CNQX was obvious. The altered levels of PWT and CB1 support these results. The CB1 receptor activation produces analgesia in neonatal rats through NMDA receptor formation for PS inhibitory activity. In juvenile and adult rats, this phenomenon was effectuated through NMDA and AMPA receptors. This difference could be attributed to the varied number of NMDA and/or AMPA receptors activated during development and changes in the NMDA/AMPA receptor ratio.

A mechanistic review on immunomodulatory effects of selective type two cannabinoid receptor β-caryophyllene.

β-Caryophyllene (BCP) is a plant-derived compound and occurs naturally in various foods and spices, including cinnamon, citrus, fruits, clove, curry, and pepper. BCP showed different pharmacological effects, such as antioxidant and antimicrobial properties. This article tried to gather updated knowledge of the anti-inflammatory, antioxidant, and immunomodulatory effects of BCP and searched using various databases and appropriate keywords until April 2022. Several studies showed that the anti-inflammatory effects of BCP are mainly provided through cannabinoid receptor 2 (CB2 ) receptor activation and the peroxisome proliferator-activated receptor (PPAR) γ pathway. It has also been demonstrated that BCP suppresses both protein and mRNA expression levels of interleukin (IL)-6 and reduces relevant proinflammatory cytokines but increases the anti-inflammatory cytokine IL-13. Previous results indicated that the antioxidant effects of β-caryophyllene were suggested through different pathways, including activation of nuclear factor erythroid 2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1)/antioxidant axis and inhibition of the HMG-CoA reductase activity, and oxidative stress biomarkers levels. Furthermore, various results showed immunomodulatory effects of BCP through inhibiting microglial cells, CD4+ and CD8+ T lymphocytes, modulated Th1 /Treg immune balance through the activation of the CB2 receptor, and reducing mitogen-activated protein kinases (p38MAPK) and NF-kB activation and increased ionized calcium-binding adaptor molecule-1 (Iba-1) and IL-1β.

Inverse-agonist (SMM-189) suppresses colitis by inducing endogenous cannabinoids and attenuating Th17, neutrophils, natural killer cells

Abstract The cause of inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are multi-factorial and include activated immune cells, chronic inflammation, genetics, and environmental exposure. It is well known that endocannabinoids mediate protection against intestinal inflammation and other autoimmune diseases. However, the effect of cannabinoid receptors induction by inverse-agonist during experimental colitis has not been investigated. Here, we investigate the activation of the cannabinoid receptor-2 (CB2) by SMM-189, a potent CB2 inverse agonist, on the inhibition of dextran sodium sulfate (DSS) induced colitis. In the present study, we found that SMM-189 effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis, increased body weight and colon length while increasing the expression of CB2 receptors and protein kinase A (PKA) in the colon lamina propria (LPs). We also measured a decrease in the percentage and number of Th17 cells in the spleen, mesenteric lymph nodes (MLNs), and LPs treated with SMM-189 compared to DSS controls. Similarly, the percentage and number of natural killer T (NKT) cells and neutrophils are decreased after SMM-189 treatment. SMM-189 also induces the myeloid-derived suppressor cells (MDSCs) during chronic colitis progression as compared to DSS alone. These findings suggest that SMM-189 ameliorates experimental colitis by inducing CB2 receptors and PKA in the LPs, MDSCs and reduces Th17, NKT cells, and neutrophils from the spleen and LPs. Taken together, these data support the idea that the CB2 inverse agonists, such as SMM-189, may be developed as a novel therapeutic target for IBD. Supported by NIH grant R01 AI140405