Cannabinoid Receptor 1 Expression Research Articles

Electroacupuncture Regulates Cannabinoid Receptor 1 Expression in a Mouse Fibromyalgia Model: Pharmacological and Chemogenetic Modulation

Fibromyalgia is a chronic illness usually accompanied by long-lasting, general pain throughout the body, often accompanied by anxiety, depression, fatigue, and sleep disruption. Meanwhile, doctors and scientists have not entirely discovered detailed mechanisms; patients always have an exaggerated sensation to pervasive pain without satisfied medical service. Given the lack of knowledge on its underlying mechanism, current treatments aim to provide pain and/or symptom relief. The present study aimed to clarify the role of cannabinoid receptor 1 (CB1) signaling in a mouse fibromyalgia pain model. To develop the mouse fibromyalgia model, mice were subjected to intermittent cold stress (ICS). Our results indicated that mechanical (2.09 ± 0.09 g) and thermal hyperalgesia (4.77 ± 0.29 s), which were evaluated by von Frey and Hargraves’ tests, were induced by ICS, suggesting successful modeling. The hurting replies were then provoked by electroacupuncture (EA) but not for sham EA mice. Further, in a Western blot analysis, we found significantly decreased CB1 protein levels in the thalamus, somatosensory cortex, and anterior cingulate cortex. In addition, the levels of pain-related protein kinases and transcription factor were increased. Treatment with EA reliably increased CB1 expression in various brain regions sequentially alleviated by nociceptive mediators. Furthermore, the administration of a CB1 agonist significantly attenuated fibromyalgia pain, reversed EA analgesia by the CB1 antagonist, and further reversed the chemogenetic inhibition of SSC. Our innovative findings evidence the role of CB1 signaling in the interaction of EA and fibromyalgia, suggesting its potential for clinical trials and as a treatment target.

Endocannabinoid remodeling in murine cachexic muscle associates with catabolic and metabolic regulation

Muscle degeneration is a common feature in cancer cachexia that cannot be reversed. Recent advances show that the endocannabinoid system, and more particularly cannabinoid receptor 1 (CB1), regulates muscle processes, including metabolism, anabolism and regenerative capacity. However, it is unclear whether muscle endocannabinoids, their receptors and enzymes are responsive to cachexia and exercise. Therefore, this study investigated whether cachexia and exercise affected muscle endocannabinoid signaling, and whether CB1 expression correlated with markers of muscle anabolism, catabolism and metabolism. Male BALB/c mice were injected with PBS (CON) or C26 colon carcinoma cells (C26) and had access to wheel running (VWR) or remained sedentary (n = 5–6/group). Mice were sacrificed 18 days upon PBS/tumor cell injection. Cachexic mice exhibited a lower muscle CB1 expression (−43 %; p < 0.001) and lower levels of the endocannabinoid anandamide (AEA; −22 %; p = 0.044), as well as a lower expression of the AEA-synthesizing enzyme NAPE-PLD (−37 %; p < 0.001), whereas the expression of the AEA degrading enzyme FAAH was higher (+160 %; p < 0.001). The 2-AG-degrading enzyme MAGL, was lower in cachexic muscle (−34 %; p = 0.007), but 2-AG and its synthetizing enzyme DAGLβ were not different between CON and C26. VWR increased muscle CB1 (+25 %; p = 0.005) and increased MAGL expression (+30 %; p = 0.035). CB1 expression correlated with muscle mass, markers of metabolism (e.g. p-AMPK, PGC1α) and of catabolism (e.g. p-FOXO, LC3b, Atg5). Our findings depict an emerging role of the endocannabinoid system in muscle physiology. Future studies should elaborate how this translates into potential therapies to combat cancer cachexia, and other degenerative conditions.

Cannabinoid receptor 2 alleviates sepsis-associated acute lung injury by modulating maturation of dendritic cells

BackgroundDendritic cells (DCs) play a key role in a variety of inflammatory lung diseases, but their role in sepsis-associated acute lung injury (SA-ALI) is currently not been illuminated. Cannabinoid receptor 2 (CNR2) has been reported to regulate the DCs maturation. However, whether the CNR2 in DCs contributes to therapeutic therapy for SA-ALI remain unclear. In current study, the role of CNR2 on DCs maturation and inflammatory during SA-ALI is to explored. MethodsFirst, the CNR2 level was analyzed in isolated Peripheral Blood Mononuclear Cells (PBMCs) and Bronchoalveolar Lavage Fluid (BALF) from patient with SA-ALI by qRT-PCR and flow cytometry. Subsequently, HU308, a specific agonist of CNR2, and SR144528, a specific antagonist of CNR2, were introduced to explore the function of CNR2 on DCs maturation and inflammatory during SA-ALI. Finally, CNR2 conditional knockout mice were generated to further confirm the function of DCs maturation and Inflammation during SA-ALI. ResultsFirst, we found that the expression of CNR2 on DCs was decreased in patient with SA-ALI. Besides, the result showed HU308 could decrease the maturation of DCs and the level of inflammatory cytokines, simultaneously reduce pulmonary pathological injury after LPS-induced sepsis in mice. In contrast of HU308, SR144528 exhibits opposite function of DCs maturate, inflammatory cytokines and lung pathological injury. Furthermore, comparing with SR144528 treatment, similar results were obtained in DCs specific CNR2 knockout mice after LPS treatment. ConclusionCNR2 could alleviate SA-ALI by modulating maturation of DCs and inflammatory factors levels. Targeting CNR2 signaling specifically in DCs has therapeutic potential for the treatment of SA-ALI.

Overexpression of cannabinoid receptor 2 is associated with human breast cancer proliferation, apoptosis, chemosensitivity and prognosis via the PI3K/Akt/mTOR signaling pathway.

The cannabinoid receptor 2 (CB2) is mainly involved in the immune system. However, although CB2 has been reported to play an anti-tumor function in breast cancer (BC), its specific mechanism in BC remains unclear. We examined the expression and prognostic significance of CB2 in BC tissues by qPCR, second-generation sequencing, western blot, and immunohistochemistry. We assessed the impacts of overexpression and a specific agonist of CB2 on the growth, proliferation, apoptosis, and drug resistance of BC cells in vitro and in vivo using CCK-8, flow cytometry, TUNEL staining, immunofluorescence, tumor xenografts, western blot, and colony formation assays. CB2 expression was significantly lower in BC compared with paracancerous tissues. It was also highly expressed in benign tumors and ductal carcinoma in situ, and its expression was correlated with prognosis in BC patients. CB2 overexpression and treatment of BC cells with a CB2 agonist inhibited proliferation and promoted apoptosis, and these actions were achieved by suppressing the PI3K/Akt/mTOR signaling pathway. Moreover, CB2 expression was increased in MDA-MB-231 cell treated with cisplatin, doxorubicin, and docetaxel, and sensitivity to these anti-tumor drugs was increased in BC cells overexpressing CB2. These findings reveal that CB2 mediates BC via the PI3K/Akt/mTOR signaling pathway. CB2 could be a novel target for the diagnosis and treatment of BC.

Electroacupuncture repairs intestinal barrier by upregulating CB1 through gut microbiota in DSS-induced acute colitis

BackgroundA few studies have reported that electroacupuncture (EA) can repair the intestinal barrier through unknown mechanisms. Cannabinoid receptor 1 (CB1) was shown to play an important role in the protection of the gut barrier in recent studies. Gut microbiota can influence the expression of CB1. In this study, we explored the effect of EA on the gut barrier in acute colitis and its mechanism.MethodsA dextran sulfate sodium (DSS)-induced acute colitis model, CB1 antagonist model and fecal microbiota transplantation (FMT) model were used in this study. The disease activity index (DAI) score, colon length, histological score, and inflammatory factors were detected to evaluate colonic inflammation. Methods for detecting intestinal barrier functions included the expression of tight junction proteins, intestinal permeability, and the number of goblet cells. Moreover, 16S rRNA sequencing was applied to analyze alterations in the gut microbiota. Western blotting and RT-PCR were performed to assess the levels of CB1 and autophagy-related proteins. Autophagosomes were observed by transmission electron microscopy.ResultsEA reduced the DAI score, histological score, levels of inflammatory factors, and restored the colon length. Moreover, EA increased the expression of tight junction proteins and the number of goblet cells, and decreased intestinal permeability. In addition, EA remodeled the community structure of the gut microbiota, increased the expression of CB1, and enhanced the degree of autophagy. However, the therapeutic effects were reversed by CB1 antagonists. In addition, FMT in the EA group exhibited similar effects to EA and upregulated CB1.ConclusionsWe concluded that EA may protect intestinal barrier functions by increasing the expression of CB1 to enhance autophagy through gut microbiota in DSS-induced acute colitis.

A276 CANNABINOID RECEPTOR 1 IS LOCALIZED IN ENTERIC NEURONS AND EPITHELIUM IN FETAL AND EARLY POSTNATAL MOUSE INTESTINE

Abstract Background The endocannabinoid system (ECS) is necessary for gastrointestinal (GI) homeostasis and serves as a target for exogenous medicinal and recreational cannabinoid compounds. While components of the ECS, including receptors, ligands, and biosynthetic/degradative enzymes, have been increasingly characterized in the adult GI tract of both humans and animal models, similar advances in understanding the role of the ECS in GI developmental programming have not yet been made. Purpose We tested the hypothesis that the ECS plays a role in the developmental programming of the GI tract. Method The developmental timeline of ECS component expression was investigated in the GI tract from fetal and early postnatal mice. Samples from timed-pregnant outbred mice were collected from embryonic days 14 (E14), E16, E18 and postnatal days 1 (P1), P7 and P28, representing a timeline of early colonization of the GI tract by enteric neurons (E14) to 1 week after weaning (P28). Localization of cannabinoid receptor 1 (CB1) expression was examined in fetal and postnatal small intestine by immunohistochemistry. Fetal intestine was processed for RNA extraction and relative expression of ECS components determined by RT-qPCR. Result(s) CB1 expression at E14 was observed in the outer wall of the gut and in the developing mucosa. By E18, expression of CB1 became more localized to the enteric plexuses and to mucosal epithelial cells. In postnatal ileum, at least &amp;gt;50% of myenteric ganglia were found to express CB1 from P1 to P28. CB1 expression continued to be present in the mucosal epithelium of ileum from P1 to P28, with a subset of CB1 expression in enterochromaffin cells, as identified by 5-HT immunolabeling. Significant decreases in expression of mRNA encoding CB1 (p&amp;lt;0.0001) and the ECS biosynthetic enzymes NAPE-PLD (p&amp;lt;0.05) and DAGLa (p&amp;lt;0.05) were found between E14 and E18. Conclusion(s) Components of the ECS are present in the fetal and early postnatal GI tract. Further research is needed to better characterize the roles and mechanisms of action of the ECS in GI development. The potential impact of exogenous cannabis exposure in early life, either by in utero exposure or via the use of medicinal cannabinoids in childhood, highlights the importance of identifying the contributions of the ECS to normal development. Disclosure of Interest None Declared

Activation of cannabinoid receptor 2 alleviates acute lung injury in rats with lipopolysaccharide-induced sepsis

To investigate the protective effect of cannabinoid receptor 2 (CB2) activation against acute lung injury in rats with lipopolysaccharide (LPS)-induced sepsis and explore the underlying mechanism. Forty-eight SD rats were randomly assigned into control group, model group, CB2 agonist group and P38 MAPK inhibitor group (n=12). In the latter 3 groups, the rats received intraperitoneal injection of LPS to induce sepsis, and the control rats were given saline injection. In CB2 agonist group, JWH133 (3 mg/kg) was injected intraperitoneally 30 min before LPS injection; in P38 MAPK inhibitor group, the rats received intraperitoneal injection of SB203580 (5 mg/kg) 30 min prior to JWH133 injection. The changes in lung histopathology, water content, fluid clearance rate, inflammatory factors, pulmonary expressions of CB2 and tight junctionrelated genes, and phosphorylation of P38 MAPK in the lung tissues were examined. The rat models of sepsis showed severe damage of alveolar structures with significantly decreased fluid clearance rate, lowered pulmonary expressions of CB2, occludin and ZO-1 mRNA and proteins, increased water content in the lung tissue, and increased phosphorylation level of P38 MAPK and TNF-α and IL-1β levels in lung lavage fluid (all P < 0.05). Treatment with JWH133 improved alveolar pathology in the septic rats, but there was still inflammatory infiltration; lung tissue water content, phosphorylation of P38 MAPK, and TNF-α and IL-1β levels in lung lavage fluid were all significantly decreased, and the fluid clearance rate, pulmonary expressions of CB2, occludin and ZO-1 were significantly increased (all P < 0.05). Additional treatment with SB203580 resulted in further improvements of alveolar pathologies, lowered phosphorylation levels of P38 MAPK in the lung tissue and TNF-α and IL-1β levels in lung lavage fluid, and increased the protein expressions of occludin and ZO-1 (P < 0.05) without causing significant changes in mRNA and protein expression of CB2 (P > 0.05). In rats with LPS-induced sepsis, activation of CB2 can inhibit the p38 MAPK signaling pathway, reduce the release of inflammatory factors in the lung tissues, promote tight junction protein expressions, and thus offer protection against acute lung injury.

Possible Association of Nucleobindin-1 Protein with Depressive Disorder in Patients with HIV Infection.

Background: Mental disorders linked with dysfunction in the temporal cortex, such as anxiety and depression, can increase the morbidity and mortality of people living with HIV (PLWHA). Expressions of both nucleobindin 1 (NUCB1) and cannabinoid receptor 1 (CNR1) in the neurons have been found to alter in patients with depressive disorder, but whether it is involved in the development of depression in the context of HIV infection is unknown. Objectives To investigate the effects of NUCB1 on depressive disorder among PLWHA and preliminarily explore the underlying molecular mechanisms. Methods: Individuals who were newly HIV diagnosed were assessed on the Hospital Anxiety and Depression scale (HADS). Then SHIV-infected rhesus monkeys were used to investigate the possible involvement of the NUCB1 and the CNR1 protein in depression-like behavior. Results: The prevalence rate of depression among PLWHA was 27.33% (41/150). The mechanism results showing elevated NUCB1 levels in cerebrospinal fluid from HIV-infected patients suffering from depression were confirmed compared to those of HIV-infected patients. Moreover, the immunohistochemical analysis indicated the expression of NUCB1 in the temporal cortex neurons of SHIV-infected monkeys was higher than that of the healthy control. Conversely, CNR1 expression was down-regulated at protein levels. Conclusions: Depression symptoms are common among PLWHA and associate with NUCB1 expression increases, and NUCB1 may be a potential target for depression.

Cannabidiol improves thyroid function via modulating vitamin D3 receptor in vitamin D3 deficiency diet-induced rat model.

The study was evaluated the impact of cannabidiol (CBD) on thyroid hormones by modulation cannabinoid receptor-2 (CB2) and vitamin D receptor (VDR) in rats fed with vitamin D3 deficiency diet (VDD). CB2-receptors were analyzed by RT-PCR method and others biomarkers by ELISA. The relative expression of CB2 (thyroid ~ 4 folds), VDR protein (liver, 151.72%), and (kidney, 66%) was significantly increased in CBD-60 compared to VDD. Vitamin D3 metabolites were significantly increased serum (189.42%), kidney (73.84%), and liver (58.11%) in CBD-60 than VDD. Increased thyroxine (59.9%) and calcitonin (213.59%); while decreased thyroid-stimulating hormone (36.15%) and parathyroid hormone (38.64%) was observed CBD treatment in VDD rats. In conclusion, CBD treatment improves CB2 and VDR expression and the level of vitamin D3 metabolites, along with improved thyroid hormones, including calcitonin. This is the first report with an improved CB2 and VDR expression after CBD treatment in VDD induced animals. Thus, CBD can be considered to use in hypothyroidism conditions and to maintain bone health.

Cannabinoid Receptor 2 (CB2) Inverse Agonist SMM-189 Induces Expression of Endogenous CB2 and Protein Kinase A That Differentially Modulates the Immune Response and Suppresses Experimental Colitis.

The causes of Crohn’s disease (CD) and ulcerative colitis (UC), the two most common forms of inflammatory bowel disease (IBD), are multi-factorial and include dysregulation of immune cells in the intestine. Cannabinoids mediate protection against intestinal inflammation by binding to the G-protein coupled cannabinoid receptors 1 and 2 (CB1 and CB2). Here, we investigate the effects of the CB2 inverse agonist SMM-189 on dextran sodium sulfate (DSS)-induced experimental colitis. We observed that SMM-189 effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis, and increased both body weight and colon length. Treatment with SMM-189 also increased the expression of CB2 and protein kinase A (PKA) in colon lamina propria lymphocytes (LPLs). We noticed alterations in the percentage of Th17, neutrophils, and natural killer T (NKT) cells in the spleen, mesenteric lymph nodes (MLNs), and LPLs of mice with DSS-induced colitis after treatment with SMM-189 relative to DSS alone. Further, myeloid-derived suppressor cells (MDSCs) during colitis progression increased with SMM-189 treatment as compared to DSS alone or with control cohorts. These findings suggest that SMM-189 may ameliorate experimental colitis by inducing the expression of endogenous CB2 and PKA in LPLs, increasing numbers of MDSCs in the spleen, and reducing numbers of Th17 cells and neutrophils in the spleen, MLNs, and LPLs. Taken together, these data support the idea that SMM-189 may be developed as a safe novel therapeutic target for IBD.

Modulation of mRNA Expression of Monoacylglycerol Lipase, Diacylglycerol Lipase and Cannabinoid Receptor-1 in Mice Experimentally Infected with T. gondii.

Toxoplasma gondii, an obligate intracellular parasite, infects more than 30% of world's population. This parasite is considered to be neurotropic, and has high tropism for the central nervous system, and potentially induces cryptogenic epilepsy by no clear mechanism. The current study aimed to investigate the alteration of the main components of the endocannabinoid signaling systems in T. gondii-infected mice. For this purpose, the levels of mRNA expression of monoacylglycerol lipase (MAGL), diacylglycerol lipase (DAGL) and cannabinoid receptor-1 (CB1), were measured by quantitative real time PCR.The mRNA expression level of MAGL was increased by ~ 8-fold in the brains of the Toxoplasma-infected group in comparison with non-infected mice (P<0.0001). The mRNA expression of CB1 gene in the brain of the infected mice was ~ 2 times higher than that measured in control group (P<0.01). The mRNA expression level of DAGL remained unchanged in the infected mice. Overall a substantial increase in MAGL and CB1 expression without any changes in DAGL, in the brain of infected mice suggests that T. gondii disturbs the endocannabinoid signaling pathways, which are known as neurotransmitter modulators involved in epilepsy.

Solid lipid nanoparticles loading with curcumin and dexanabinol to treat major depressive disorder.

Dexanabinol (HU-211) is an artificially synthesized cannabinoid derivative that exerts neuroprotective effects through anti-inflammatory and antioxidant effects. Curcumin exhibits antidepressant effects in the treatment of major depressive disorder. To investigate the antidepressant effects of solid lipid nanoparticles loaded with both curcumin and dexanabinol, and the underlying mechanisms associated with this combination, we established wild-type (CBR1+/+) and cannabinoid receptor 1 (CBR1) knockout (CBR1-/-) mouse models of major depressive disorder, through the intraperitoneal injection of corticosterone, for 3 successive days, followed by treatment with intraperitoneal injections of solid lipid nanoparticles loading with curcumin (20 mg/kg) and dexanabinol (0.85 mg/kg), for 2 successive days. Our results revealed that solid lipid nanoparticle loading with curcumin and dexanabinol increased the mRNA and protein expression levels of the mature neuronal markers neuronal nuclei, mitogen-activated protein 2, and neuron-specific beta-tubulin III, promoted the release of dopamine and norepinephrine, and increased the mRNA expression of CBR1 and the downstream genes Rasgef1c and Egr1, and simultaneously improved rat locomotor function. However, solid lipid nanoparticles loaded with curcumin and dexanabinol had no antidepressant effects on the CBR1-/- mouse models of major depressive disorder. This study was approved by the Institutional Ethics Committee of Tongji Hospital of Tongji University, China (approval No. 2017-DW-020) on May 24, 2017.

SUN-LB137 Endocannabinoids Induce Endoplasmic Reticulum Stress in Hepatocytes and Human Coronary Artery Endothelial Cells

Obesity and diabetes are important risk factors for the development of coronary heart disease and stroke. Plasma endocannabinoid (EC) levels are inappropriately elevated in obesity and diabetes, and are hypothesized to play a causal role in central regulation of weight gain. Importantly, it was recently demonstrated that cannabinoid receptor 1 (CNR1) triggers cell stress and induces apoptosis in kidney tubule cells exposed to palmitic acid and high-glucose (HG). HepG2 and human coronary artery endothelial cells (HCAEC) were treated with tunicamycin (TM), thapsigargin (TG), high-glucose (HG), anandamide (AN), and 2-arachondonyl glycerol (2-AG), and endoplasmic reticulum (ER) stress was measured. In cells treated with TM, AM, and 2-AG and the UPR inhibitors 4-phenylbutyrate (4-PB) and taurodeoxycholic acid (TUDCA), both 4-PB and TUDCA prevented AN and 2-AG from promoting ER stress. ER stress in cells treated with AN and 2-AG, but not TM, was inhibited by the CNR1 antagonist rimonabant. Similar results were obtained with HCAEC. Furthermore, treatment with AN and 2-AG induced inositol requiring enzyme 1α and protein kinase R-like endoplasmic reticulum kinase phosphorylation but had no effect on their expression, while activating transcription factor 6 and binding immunoglobulin protein expression were also induced by AN and 2-AG in both HepG2 and HCAEC. Finally, AN and 2-AG treatment induced CNR1 expression in both cell lines. These results strongly suggest that EC’s promote ER stress and potentially induce liver and endothelial cell dysfunction.

Cannabinoid Receptor 1/miR-30b-5p Axis Governs Macrophage NLRP3 Expression and Inflammasome Activation in Liver Inflammatory Disease

Nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) has been regarded as an important initiator or promoter in multiple inflammatory diseases. However, the relationship between cannabinoid receptor 1 (CB1) and macrophage NLRP3 inflammasome and the corresponding molecular mechanism in liver inflammation remain unclear. Mouse liver injury models were induced by carbon tetrachloride (CCl4) or methionine-choline-deficient and high fat (MCDHF) diet. Human liver tissues were obtained from patients with different chronic liver diseases. CB1 expression was increased in liver tissue and macrophages of CCl4- and MCDHF-treated mice, positively correlated with NLRP3. CB1 agonist ACEA (Arachiodonyl-2’-Chloroethylamide) promoted NLRP3 expression and NLRP3 inflammasome activation in macrophages. CB1 blockade with its antagonist AM281 reduced NLRP3 expression, inflammasome activation, and liver inflammation in CCl4- and MCDHF-treated mice. MicroRNA-30b-5p (miR-30b-5p), screened by the intersection of bioinformatics databases and downregulated miRNAs in injured liver, negatively correlated with NLRP3 in mouse and human liver. miR-30b-5p was involved in CB1-mediated activation of NLRP3 inflammasome in macrophages by directly targeting NLRP3. Importantly, administration of miR-30b-5p agomir targeted NLRP3 and attenuated liver inflammation in the injured liver. Altogether, CB1/miR-30b-5p axis modulates NLRP3 expression and NLPR3 inflammasome activation in macrophages during liver inflammation, which provides a potential target for liver disease.

Disability-specific genes GRIN1, GRIN2 and CNR1 show injury-dependent protein expression in the lumbar spinal cord of CCI rats

The sensory changes triggered by peripheral nerve injury result from functional changes in both neurons and glia in the dorsal horn of the spinal cord. Whether the disrupted affective-motivational states often comorbid with injury-evoked changes in sensation are driven directly by these functional changes is a question only recently investigated. Using a combination of GeneChip microarrays and RT-PCR techniques we identified differences in mRNA expression unique to rats with sustained changes to their social behaviour following sciatic nerve chronic constriction injury (CCI). Amongst these changes were the mRNAs encoding several of the NMDA subunits and the CB1 receptor. However, as protein translation is not a necessary consequence of the upregulation or downregulation of genes we decided to evaluate the functional significance of our initial observations using immunohistochemical detection of their translated protein products to determine their location and abundance in the lumbar spinal cord. Spinal cord tissue from rats with (‘Affected’), and without (‘Unaffected’) changes in social behaviour after CCI was compared with tissue from uninjured controls. The expression of NMDA-1 (NR1) subunit, NMDA-2D subunit, Cannabinoid Receptor 1 (CB1), Glucocorticoid Receptor (GR) and Glial Fibrillary Acidic Protein (GFAP) immunoreactivities was quantified for these rats and revealed that nerve injury increased the expression of NMDA-2D, CB1 and GFAP immunoreactivity compared to uninjured controls. However, these changes were not specific to rats whose social behaviours were ‘Affected’ or ‘Unaffected’ by the nerve injury. Our data thus suggest that the development and expression of changes in social behaviour seen in a proportion of rats following CCI are unlikely to be directly related to the spinal changes in NMDA-2D, CB1 and GFAP expression induced by the nerve injury.

Induced Pluripotent Stem Cell‐Derived Neuroprogenitor Cells: A Model for Studying Cannabinoid Induced Cell Signaling

Chronic pain is a growing epidemic and public health issue that continue to affect the aging population in the US. Managing pain is particularly difficult with patients who suffer from cancer‐associated pain, neuropathic pain, as well as central pain states. These conditions are typically treated inadequately with opioids, antidepressants, and anticonvulsive drugs. There is growing evidence that cannabinoids natural products are beneficial for a range of clinical conditions, including pain and inflammations. The use of cannabinoids instead of highly regulated prescription drugs is fast approaching and the development of such cannabinoid drugs requires well planned medical trials as well as very stringent guidelines for contaminants such as heavy metals, pesticides, mycotoxins, etc. However, federal guidelines have not kept up with this emerging industry. Currently, each state is left to manage their own set of regulations, resulting in an industry that has great uncertainty and chaos as it attempts to satisfy the growing demand for its products. A low‐cost cellular model to study the neurological effects of cannabinoids is challenging. Here we show that Neuroprognitor cells (NPs) derived from induced pluripotent stem cells (iPSCs) can be used as a model for studying pathways associated with pain and cannabinoids. NPs derived from iPSCs can be differentiated into multiple lineages of neurons and glial cells. These cells have the Cannabinoid Receptor 1 (CB1) present which, when activated by cannabinoids, inhibits the inflammatory and pain stimulation of Cyclooxygenase 2 (COX2). We show expression and activation of CB1 in correlation to Cannabinoids treatment. Likewise we show how, in iPSC‐derived NPs Cannabinoid treatment leads to the inhibition of Cox‐2, therefore theoretically lessoning the potential for inflammation and pain.Support or Funding InformationOmni International, Inc935 Cobb Place BlvdKennesaw Ga 30144

Both HuR and miR-29s regulate expression of CB1 involved in infiltration of bone marrow monocyte/macrophage in chronic liver injury.

Bone marrow-derived monocytes/macrophages (BMMs) play a vital role in liver inflammation and fibrogenesis. Cannabinoid receptor 1 (CB1) mediates the recruitment of BMMs into the injured liver. In this study, we revealed the molecular mechanisms under CB1-mediated BMM infiltration. Carbon tetrachloride (CCl4 ) was employed to induce mouse liver injury. In vivo, human antigen R (HuR) was upregulated in macrophages of injured liver. HuR messenger RNA (mRNA) expression was positively correlated with CB1 and F4/80 mRNA expression. Furthermore, we detected the binding between HuR and CB1 mRNA in CCl4 -treated livers. In vitro, HuR modulated arachidonyl-2'-chloroethylamide (ACEA, CB1 agonist)-induced BMM migration by regulating CB1 expression. HuR promoted CB1 expression via binding to CB1 mRNA. ACEA promoted the association between HuR and CB1 mRNA via inducing HuR nucleoplasmic transport. In the cytoplasm, HuR competed with the miR-29 family to improve CB1 expression and BMM migration. In conclusion, our results prove that HuR regulates CB1 expression and influences ACEA-induced BMM migration by competing with miR-29 family.

The cannabinoid receptor 1 is involved in renal fibrosis during chronic allograft dysfunction: Proof of concept.

Chronic allograft dysfunction (CAD), defined as the replacement of functional renal tissue by extracellular matrix proteins, remains the first cause of graft loss. The aim of our study was to explore the potential role of the cannabinoid receptor 1 (CB1) during CAD. We retrospectively quantified CB1 expression and correlated it with renal fibrosis in 26 kidney‐transplanted patients who underwent serial routine kidney biopsies. Whereas CB1 expression was low in normal kidney grafts, it was highly expressed during CAD, especially in tubular cells. CB1 expression significantly increased early on after transplantation, from day 0 (D0) to month 3 post‐transplant (M3) (22.5% ± 15.4% vs 33.4% ± 13.8%, P < .01), and it remained stable thereafter. CB1 expression correlated with renal fibrosis at M3 (P = .04). In an in vitro model of tacrolimus‐mediated fibrogenesis by tubular cells, we found that tacrolimus treatment significantly induced mRNA and protein expression of CB1 concomitantly to col3a1 and col4a3 up regulation. Administration of rimonabant, a CB1 antagonist, blunted collagen synthesis by tubular cells (P < .05). Overall, our study strongly suggests an involvement of the cannabinoid system in the progression of fibrosis during CAD and indicates the therapeutic potential of CB1 antagonists in this pathology.

µ-opioid receptor, β-endorphin, and cannabinoid receptor-2 are increased in the colonic mucosa of irritable bowel syndrome patients.

The gut immune, cannabinoid, and opioid systems constitute an integrated network contributing to visceral sensation and pain modulation. We aimed to assess the expression of the µ-opioid receptor (MOR), its ligand β-endorphin (β-END), and cannabinoid receptor-2 (CB2 ) in patients with irritable bowel syndrome (IBS) and asymptomatic controls (AC) and their correlation with sex and symptom perception. Mucosal biopsies were obtained from the left colon of 31 IBS patients (45% women) with predominant constipation (IBS-C, 9) or diarrhea (IBS-D, 10) or with mixed bowel habits (IBS-M, 12) and 32 AC (44% women) and processed for qRT-PCR, Western blotting, and immunohistochemistry. µ-opioid receptor and CB2 mRNA and protein expression and β-END protein levels were increased in patients with IBS compared to AC (all Ps=0.021). A significant sex by IBS interaction was found in relation to CB2 mRNA expression (P=.003) with women showing a markedly higher expression to men (P=.035). In contrast, in AC, men had higher expression than women (P=.033). β-END, MOR, and CB2 immunoreactivities (IR) were localized to CD4+T cells including EMR-1+ eosinophils and CD31+ T cells but not to mast cells. The increased expression of MOR, β-END, and CB2 in the mucosa of IBS patients, where they are localized to immune cells, suggests that opioid and cannabinoid systems play an immune-related compensatory role in visceral pain in IBS patients. Further work is necessary to support this hypothesis.

Identification of Novel Predictive Biomarkers for Endometrial Malignancies: N-Acylethanolamines.

Objective: To identify new biochemical markers for endometrial cancer (EC). Recent evidence suggests that members of the endocannabinoid system (N-acylethanolamines) that bind to and activate receptors that are dysregulated in EC are involved in this tumour's biology. These observations suggest increased N-acylethanolamine levels in the tissue that might appear in plasma and could be used as disease biomarkers.Methods: N-arachidonoylethanolamine (anandamide, AEA) and the N-acylethanolamine substances, N-oleoylethanolamine (OEA), and N-palmitoylethanolamine (PEA) were quantified in plasma and endometrial tissue collected from 31 EC and seven atrophic controls using UHPLC-MS/MS. Receiver-operating characteristics (ROC) and logistic regression were used to determine diagnostic accuracy. Cannabinoid receptor 1 (CB1) and 2 (CB2) protein levels were determined by specific immunohistochemistry and histomorphometric analyses. Correlations between plasma and tissue levels of the three N-acylethanolamines and tissue levels of the three N-acylethanolamines and CB1 and CB2 receptor expression levels were determined using correlation analysis.Results: Plasma and tissue AEA and PEA levels were significantly (p < 0.05) higher in EC than controls whilst OEA levels were significantly elevated in type 1 EC tissues but not in plasma. There were significant positive correlations between plasma and tissue levels of AEA (R2 = 0.302, p = 0.008) and PEA (R2 = 0.182, p = 0.047), but not for OEA (R2 = 0.022, p = 0.506). The diagnostic accuracies for EC were: sensitivity of 53.3%, specificity of 100% for plasma AEA (>1.36 nM); sensitivity of 73.3%, specificity of 100% for plasma PEA (>27.5 nM); and sensitivity of 93.3%, specificity of 28.6% for plasma OEA (>4.97 nM). Logistic regression increased the area under the ROC curve (AUC) from 0.781 for AEA, 0.857 for PEA, and 0.543 for OEA to a combined AUC of 0.933 for EC diagnosis. Significant inverse correlations between tissue AEA (R2 = 0.343, p = 0.003) and PEA (R2 = 0.384, p < 0.0001) levels and CB1 expression were observed. No correlation between tissue levels of OEA and CB1 and tissue levels of any of the three N-acylethanolamines and CB2 protein expression were observed, except in the type 1 EC patients.Conclusion: Since plasma AEA and PEA are significantly elevated in patients with EC and a reflection of production by the endometrial tumour, then these lipids have the potential to be useful biomarkers for the early diagnosis of EC.