Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing.

Kidney is one of the most important organs in maintaining the normal life activities. With the high abundance of mitochondria, renal tubular cell plays the vital role in functioning in the reabsorption and secretion of kidney. Reports have shown that mitochondrial dysfunction is of great importance to renal tubular cell senescence and subsequent kidney ageing. However, the underlying mechanisms are not elucidated. Cannabinoid receptor 2 is one of the two receptors responsible for the activation of endocannabinoid system. CB2 is primarily upregulated in renal tubular cells in chronic kidney diseases and mediates fibrogenesis. However, the role of CB2 in tubular mitochondrial dysfunction and kidney ageing has not been clarified. In this study, we found that CB2 was upregulated in kidneys in 24-month-old mice and d-galactose (d-gal)-induced accelerated ageing mice, accompanied by the decrease in mitochondrial mass. Furthermore, gene deletion of CB2 in d-gal-treated mice could greatly inhibit the activation of ß-catenin signalling and restore the mitochondrial integrity and Adenosine triphosphate (ATP) production. In CB2 knockout mice, renal tubular cell senescence and kidney fibrosis were also significantly inhibited. CB2 overexpression or activation by the agonist AM1241 could sufficiently induce the decrease in PGC-1α and a variety of mitochondria-related proteins and trigger cellular senescence in cultured human renal proximal tubular cells. CB2-activated mitochondrial dysfunction and cellular senescence could be blocked by ICG-001, a blocker for ß-catenin signalling. These results show CB2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing. The intrinsic mechanism may be related to its activation in ß-catenin signalling.

Cannabinoid receptor subtype influence on neuritogenesis in human SH-SY5Y cells.

Human SH-SY5Y neuroblastoma cells stably expressing exogenous CB1 (CB1XS) or CB2 (CB2XS) receptors were developed to investigate endocannabinoid signaling in the extension of neuronal projections. Expression of cannabinoid receptors did not alter proliferation rate, viability, or apoptosis relative to parental SH-SY5Y. Transcripts for endogenous cannabinoid system enzymes (diacylglycerol lipase, monoacylglycerol lipase, α/ß-hydrolase domain containing proteins 6 and 12, N-acyl phosphatidylethanolamine-phospholipase D, and fatty acid amide hydrolase) were not altered by CB1 or CB2 expression. Endocannabinoid ligands 2-arachidonoylglycerol (2-AG) and anandamide were quantitated in SH-SY5Y cells, and diacylglycerol lipase inhibitor tetrahydrolipstatin decreased 2-AG abundance by 90% but did not alter anandamide abundance. M3 muscarinic agonist oxotremorine M, and inhibitors of monoacylglycerol lipase and α/ß hydrolase domain containing proteins 6 &12 increased 2-AG abundance. CB1 receptor expression increased lengths of short (<30 µm) and long (>30 µm) projections, and this effect was significantly reduced by tetrahydrolipstatin, indicative of stimulation by endogenously produced 2-AG. Pertussis toxin, Gßγ inhibitor gallein, and ß-arrestin inhibitor barbadin did not significantly alter long projection length in CB1XS, but significantly reduced short projections, with gallein having the greatest inhibition. The rho kinase inhibitor Y27632 increased CB1 receptor-mediated long projection extension, indicative of actin cytoskeleton involvement. CB1 receptor expression increased GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA, whereas CB2 receptor expression increased NCAM and SYT mRNA. We propose that basal endogenous production of 2-AG provides autocrine stimulation of CB1 receptor signaling through Gi/o, Gßγ, and ß-arrestin mechanisms to promote neuritogenesis, and rho kinase influences process extension.

Impact of the endocannabinoid system on murine cranial and alveolar bone phenotype.

PURPOSE: The endocannabionoid signaling system has been demonstrated to be present in the skeleton, with involvement in the regulation of skeletal homeostasis. However, investigations substantiating these findings in cranial and alveolar bones are missing to date. The aim of our study was to investigate a potential impact of the endocannabinoid system on cranial and alveolar bone structures and phenotypes. BASIC PROCEDURES: CB1-/-, CB2-/- and WT mice (n = 5) were scanned via µCT. Reconstructed datasets were processed for analyses. Cranial cephalometric measurements were performed with OnyxCeph3TMsoftware. Alveolar bone densities were determined via mean grey value measurements with Mimics research 18.0. Alveolar bone heights around teeth in upper and lower jaws were morphometrically analyzed. Alveolar osteoclasts were quantified via TRAP staining of paraffin-embedded histologies. Bone-marrow derived macrophages isolated from murine hind legs were analyzed for CD40 and MMR expression via flow cytometry. MAIN FINDINGS: CB2-/- mice exhibited significantly higher bone densities with mean grey values of 138.3 ± 22.6 compared to 121.9 ± 9.3 for WT for upper jaws, and 134.6 ± 22.9 compared to 116.1 ± 12.9 for WT 134.6 ± 22.9. Concurrently, CB2 receptor knockout entailed reduced alveolar bone heights of about 50% compared to WT mice. Antigen-presenting cell marker expression of MMR was significantly diminished in bone-marrow derived macrophages of CB2-/- mice. Cranium dimensions as much as alveolar osteoclasts were unaffected by receptor knockouts.CB1 receptor knockout did not involve statistically significant alterations in the parameters investigated compared to WT mice. PRINCIPAL CONCLUSIONS: The endoncannabinoid system, and particularly CB2 receptor strongly affects murine alveolar bone phenotypes. These observations suggest CB2 as promising target in the modulation of oral bone phenotypes, probably by impact on bone dynamics via osteal immune cells.

The Endocannabinoid System Alleviates Pain in a Murine Model of Cancer-Induced Bone Pain.

Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain. SIGNIFICANCE STATEMENT: Current standard of care for metastatic breast cancer pain is opioid-based therapies with adjunctive chemotherapy, which have highly addictive and other deleterious side effects. The need for effective, non-opioid-based therapies is essential, and harnessing the endogenous cannabinoid system is proving to be a new target to treat various types of pain conditions. We present a novel drug targeting the endogenous cannabinoid system that is effective at reducing pain in a mouse model of metastatic breast cancer to bone.

The endocannabinoid receptors CB1 and CB2 affect the regenerative potential of adipose tissue MSCs.

Human adipose tissue includes large quantities of mesenchymal stromal cells (atMSCs), which represent an abundant cell source for therapeutic applications in the field of regenerative medicine. Adipose tissue secrets various soluble factors including endocannabinoids, and atMSCs express the cannabinoid receptors CB1 and CB2. This indicates that adipose tissue possesses an endocannabinoid system (ECS). The ECS is also ascribed great significance for wound repair, e.g. by modulating inflammation. However, the exact effects of CB1/CB2 activation in human atMSCs have not been investigated, yet. In the present study, we stimulated human atMSCs with increasing concentrations (1-30 µM) of the unspecific cannabinoid receptor ligand WIN55,212-2 and the specific CB2 agonist JWH-133, either alone or co-applied with the receptor antagonist Rimonabant (CB1) or AM 630 (CB2). We investigated the effects on metabolic activity, cell number, differentiation and cytokine release, which are important processes during tissue regeneration. WIN decreased metabolic activity and cell number, which was reversed by Rimonabant. This suggests a CB1 dependent mechanism, whereas the number of atMSCs was increased after CB2 ligation. WIN and JWH increased the release of VEGF, TGF-ß1 and HGF. Adipogenesis was enhanced by WIN, which could be reversed by blocking CB1. There was no effect on osteogenesis, and only WIN increased chondrogenic differentiation. Our results indicate that definite activation of the cannabinoid receptors exerted different effects in atMSCs, which could be of specific value in cell-based therapy for wound regeneration.

Medical cannabis and cannabinoids in rheumatology: where are we now?

Introduction: Clinicians involved in pain management can finally include cannabis or cannabis-related products in their therapeutic armamentarium as a growing number of countries have approved them for pain relief. Despite the several benefits attributed to analgesic, anti-inflammatory and immunomodulatory properties of cannabinoids, there are still significant areas of uncertainty concerning their use in many fields of medicine. The biosynthesis and inactivation of cannabinoids are regulated by a complex signaling system of cannabinoid receptors, endocannabinoids (the endogenous ligands of cannabinoid receptors) and enzymes, with a variety of interactions with neuroendocrinological and immunological systems. Areas covered: A review of studies carried out during clinical development of cannabis and cannabis medical products in systemic rheumatic diseases was performed, highlighting the aspects that we believe to be relevant to clinical practice. Expert opinion: The growing public opinion, pushing toward the legalization of the use of cannabis in chronic pain and various rheumatological conditions, makes it necessary to have educational programs that modify the concerns and widespread preconceptions related to this topic in the medical community by increasing confidence. More extensive basic and clinical research on the mechanisms and clinical utility of cannabis and derivatives in various diseases and their long-term side effects is necessary.

CoCl2 induced hypoxia enhances osteogenesis of rat bone marrow mesenchymal stem cells through cannabinoid receptor 2.

OBJECTIVES: This study aims to investigate the role of Cannabinoid receptor 2 (CB2) on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) under hypoxia. MATERIALS AND METHODS: BMSCs were isolated from Sprague-Dawley rats and cultured in the presence of cobalt chloride (CoCl2) to induce intracellular hypoxia. Cell proliferation was measured with MTT assay. Quantitative real-time PCR and western blot were applied to evaluate the mRNA and protein expressions of CB2 and osteogenic indicators including osteocalcin, RUNX2, collagen-1 and osterix (SP7). The osteogenic differentiation of BMSCs was further examined by ALP assay and alizarin red S (ARS) staining. Moreover, the activation of MAPKs signaling pathways was analyzed by western blot. RESULTS: CoCl2 dose-dependently increased hypoxia inducible factor while higher concentrations (200 and 400 µM) of CoCl2 markedly inhibited cell proliferation. CoCl2 induced hypoxia significantly increased the protein and mRNA expressions of osteocalcin, RUNX2, collagen-1 and osterix, along with enhanced ALP and ARS staining. Interestingly, such effects can be inhibited by the addition of CB2 inhibitor AM630. Moreover, AM630 partially inhibited hypoxia-induced p38 and ERK pathways, which may lead to a decrease in the osteogenic transcripts of RUNX2, collagen-1 and osterix. CONCLUSIONS: CoCl2 induced hypoxia could promote osteogenesis of rat BMSCs possibly through CB2.

Monoacylglycerol lipase inhibition as potential treatment for interstitial cystitis.

Interstitial cystitis is a chronic inflammatory condition of the urinary bladder with an unclear etiology. Currently, there are no widely accepted long-term treatment options available for patients with IC, with the European Association of Urology (EAU, 2017 guidelines), American Urology Association (AUA, 2014 guidelines), and the Royal College of Obstetricians and Gynaecologists (RCOG, 2016 guidelines) all suggesting various different conservative, pharmacological, intravesical, and surgical interventions. The endocannabinoid system represents a potential target for IC treatment and management. Activation of cannabinoid receptor 2 (CBR2) with various agonists has previously been shown to reduce leukocyte differentiation and migration, in addition to inhibiting the release of pro-inflammatory cytokines at the site of inflammation. These receptors have been identified in the detrusor and sensory nerves of the urothelium in various mammalian species, including humans. We hypothesize that by inhibiting the enzymes responsible for the catabolism of endogenous cannabinoids locally, bladder concentrations of CBR2 agonists will increase, particularly 2-arachidonyl glycerol, resulting in a diminished inflammatory response.

The Important Role of the Endocannabinoid System and the Endocannabinoidome in Gut Health.

The endocannabinoid system is an endogenous pathway comprised of the cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands known as endocannabinoids, and the enzymes responsible for their synthesis and degradation. The endocannabinoidome extends this system to include other receptors such as TRPV1, PPARα, GPR55 and 5-HT1A. An extensive amount of research is now linking the endocannabinoidome to intestinal health through fascinating mechanisms that include endocannabinoid receptor expression in the gut and interplay with the intestinal microbiota. A dysregulated endocannabinoid system may lead to inflammatory bowel disease and colon cancer.

Anti-inflammatory activity of cannabinoid receptor 2 ligands in primary hPDL fibroblasts.

OBJECTIVES: Approximately 65 million adults in the US have periodontitis, causing tooth loss and decreased quality of life. Cannabinoids modulate immune responses, and endocannabinoids are prevalent during oral cavity inflammation. Targets for intervention in periodontal inflammation are cannabinoid type 1 and 2 receptors (CB1R, CB2R), particularly CB2R because its levels increase during inflammation. We previously demonstrated that SMM-189 (CB2R inverse agonist) decreased pro-inflammatory cytokine production in primary microglial cells. The hypothesis of this study was that cannabinoids anandamide (AEA), HU-308 (CB2R selective agonist), and SMM-189 decrease pro-inflammatory IL-6 and MCP-1 production by primary human periodontal ligament fibroblasts (hPDLFs) stimulated with P. gingivalis LPS, TNF-α, or IL-1ß. DESIGN: Cytotoxic effects of cannabinoid compounds (10-4-10-6.5 M), LPS (1-1000 ng/ml), TNFα (10 ng/ml) and IL-1ß (1 ng/ml) were assessed by measuring effects on cellular dehydrogenase activity. IL-6 and MCP-1 production were measured using Mesoscale Discovery (MSD) Human Pro-Inflammatory IL-6 and MSD Human Chemokine MCP-1 kits and analyzed using MSD Sector 2400 machine. RESULTS: EC50 values for AEA, SMM-189, and HU-308 were 16 µM, 13 µM, and 7.3 µM respectively. LPS (1 µg/ml), TNF-α (10 ng/ml), and IL-1ß (1 ng/ml) increased IL-6 and MCP-1 production, which were inhibited by AEA, SMM-189, and HU-308. AEA alone significantly increased IL-6, but not MCP-1 levels, but the other cannabinoids alone had no effect. CONCLUSION: The effective inhibition of LPS, TNF-α, IL-1ß stimulated IL-6 and MCP-1 production by CB2R ligands in hPDLFs suggests that targeting the endocannabinoid system may lead to development of novel drugs for periodontal therapy, aiding strategies to improve oral health.

Selective activation of cannabinoid receptor-2 reduces neuroinflammation after traumatic brain injury via alternative macrophage polarization.

Inflammation is an important mediator of secondary neurological injury after traumatic brain injury (TBI). Endocannabinoids, endogenously produced arachidonate based lipids, have recently emerged as powerful anti-inflammatory compounds, yet the molecular and cellular mechanisms underlying these effects are poorly defined. Endocannabinoids are physiological ligands for two known cannabinoid receptors, CB1R and CB2R. In the present study, we hypothesized that selective activation of CB2R attenuates neuroinflammation and reduces neurovascular injury after TBI. Using a murine controlled cortical impact (CCI) model of TBI, we observed a dramatic upregulation of CB2R within infiltrating myeloid cells beginning at 72 h. Administration of the selective CB2R agonist, GP1a (1-5 mg/kg), attenuated pro-inflammatory M1 macrophage polarization, increased anti-inflammatory M2 polarization, reduced edema development, enhanced cerebral blood flow, and improved neurobehavioral outcomes after TBI. In contrast, the CB2R antagonist, AM630, worsened outcomes. Taken together, our findings support the development of selective CB2R agonists as a therapeutic strategy to improve TBI outcomes while avoiding the psychoactive effects of CB1R activation.

Involvement of cannabinoid receptor type 2 in light-induced degeneration of cells from mouse retinal cell line in vitro and mouse photoreceptors in vivo.

Earlier studies showed that the expressions of the agonists of the cannabinoid receptors are reduced in the vitreous humor of patients with age-related macular degeneration (AMD), and the cannabinoid type 2 receptor is present in the retinas of rats and monkeys. The purpose of this study was to determine whether the cannabinoid type 2 receptor is involved in the light-induced death of cultured 661W cells, an immortalized murine retinal cell line, and in the light-induced retinal degeneration in mice. Time-dependent changes in the expression and location of retinal cannabinoid type 2 receptor were determined by Western blot and immunostaining. The cannabinoid type 2 receptor was down-regulated in murine retinae and cone cells. In the in vitro studies, HU-308, a cannabinoid type 2 receptor agonist, had a protective effect on the light-induced death of 661W cells, and this effect was attenuated by SR144528, a cannabinoid type 2 receptor antagonist. Because the cannabinoid type 2 receptor is a G-protein coupled receptor and is coupled with Gi/o protein, we investigated the effects of the cAMP-dependent protein kinase (PKA). HU-308 and H89, a PKA inhibitor, deactivated PKA in retinal cone cells, and H89 also suppressed light-induced cell death. For the in vivo studies, a cannabinoid type 2 receptor agonist, HU-308, or an antagonist, SR144528, was injected intravitreally into mouse eyes before the light exposure. Electroretinography was used to determine the physiological status of the retinas. Injection of HU-308 improved the a- and b-waves of the ERGs and also the thickness of the outer nuclear layer of the murine retina after light exposure. These findings indicate that the cannabinoid type 2 receptor is involved in the light-induced retinal damage through PKA signaling. Thus, activation of cannabinoid type 2 receptor may be a therapeutic approach for light-associated retinal diseases.

Cannabinoid Receptor-2 Ameliorates Inflammation in Murine Model of Crohn's Disease.

BACKGROUND AND AIMS: Cannabinoid receptor stimulation may have positive symptomatic effects on inflammatory bowel disease [IBD] patients through analgesic and anti-inflammatory effects. The cannabinoid 2 receptor [CB2R] is expressed primarily on immune cells, including CD4+ T cells, and is induced by active inflammation in both humans and mice. We therefore investigated the effect of targeting CB2R in a preclinical IBD model. METHODS: Employing a chronic ileitis model [TNFΔARE/+ mice], we assessed expression of the CB2R receptor in ileal tissue and on CD4+ T cells and evaluated the effect of stimulation with CB2R-selective ligand GP-1a both in vitro and in vivo. Additionally, we compared cannabinoid receptor expression in the ilea and colons of healthy human controls with that of Crohn's disease patients. RESULTS: Ileal expression of CB2R and the endocannabinoid anandamide [AEA] was increased in actively inflamed TNF∆ARE/+ mice compared with controls. CB2R mRNA was preferentially induced on regulatory T cells [Tregs] compared with T effector cells, approximately 2.4-fold in wild-type [WT] and 11-fold in TNF∆ARE/+ mice. Furthermore, GP-1a enhanced Treg suppressive function with a concomitant increase in IL-10 secretion. GP-1a attenuated murine ileitis, as demonstrated by improved histological scoring and decreased inflammatory cytokine expression. Lastly, CB2R is downregulated in both chronically inflamed TNF∆ARE/+ mice and in IBD patients. CONCLUSIONS: In summary, the endocannabinoid system is induced in murine ileitis but is downregulated in chronic murine and human intestinal inflammation, and CB2R activation attenuates murine ileitis, establishing an anti-inflammatory role of the endocannabinoid system.

Type-2 cannabinoid receptor regulates proliferation, apoptosis, differentiation, and OPG/RANKL ratio of MC3T3-E1 cells exposed to Titanium particles.

The type-2 cannabinoid receptor (CB2) is expressed in osteoblasts and plays a role in bone metabolism through regulation on bone mass and bone turnover, but the functional importance of CB2 in osteoblasts under Titanium (Ti) stimulation is incompletely understood. This study aimed to investigate the CB2 expression in osteoblasts under Ti stimulation and the effects of CB2 activation on proliferation, apoptosis, differentiation, mineralization, OPG, and RANKL expression of MC3T3-E1 cells exposed to Ti particles. MC3T3-E1 cells were incubated in the presence of Ti particles with or without CB2-specific agonist HU-308 and antagonist SR144528. Ti particles treatment obviously induced the CB2 expression in MC3T3-E1 cells, and reduced the cell survival in a dose- and time-dependent manner (p < 0.05). Addition of HU-308 could dose-dependently alleviate the Ti-induced decrease of cell survival (p < 0.05). The flow cytometry assay showed that comparing with the control group, the apoptosis rate and caspase-3 activity in the Ti group were significantly elevated (p < 0.05), which could be alleviated by HU-308. Moreover, HU-308 effectively attenuated the decrease of cell mineralization capability, alkaline phosphates (ALP) and osteocalcin activity, and increase of OPG/RANKL ratio induced by Ti particles treatment (p < 0.05). These effects were partially counteracted by combined treatment of CB2 antagonist SR144528 (p < 0.05). In conclusion, CB2 activation has a favorable inhibitory effect on Ti-induced reactions in MC3T3-E1 cell through modulating proliferation, apoptosis, differentiation, and RANKL expression. These findings suggest that activation of CB2 might be an effective therapeutic strategy to promote bone formation and reduce bone dissolution.

Cannabinoid receptor-2 and HIV-associated neurocognitive disorders.

Despite the wide spread use of highly active antiretroviral therapy (HAART), mild forms of HIV-associated neuro cognitive disorders (HAND) remain commonplace. HAART treated patients now show low levels of viremia and more subtle yet biologically important signs of brain macrophage and microglial activation. Adjunctive therapeutic strategies are required to eliminate HIV-1 infection and suppress immune activation and its associated neuroinflammation. In this regard, cannabinoid receptor-2(CB2) activation is a promising means to attenuate HAND by inhibiting HIV replication, down regulating inflammation, and suppressing chemokine-like activity of viral neurotoxic proteins (for example, Tat and HIV-1gp120), and thereby prevent neuronal and synaptic loss. Inhibiting even low level HIV replication can attenuate neuronal injury by decreasing the production of neurotoxins. Down regulation of inflammation by CB2 activation is mediated through blunted activation of peri vascular macrophages and microglia; decreased production of tumor necrosis factor-α, chemokines and virotoxins. Down regulated neuroinflammation can decrease blood brain barrier permeability and leukocyte infiltration resulting in reduced neuronal injury. It is suggested that CB2 agonists may further attenuate HAND in HIVinfected patients on HAART. In addition, CB2 activation may also blunt brain injury by attenuating drug addiction.

Cannabinoid type 2 receptor stimulation attenuates brain edema by reducing cerebral leukocyte infiltration following subarachnoid hemorrhage in rats.

Early brain injury (EBI), following subarachnoid hemorrhage (SAH), comprises blood-brain barrier (BBB) disruption and consequent edema formation. Peripheral leukocytes can infiltrate the injured brain, thereby aggravating BBB leakage and neuroinflammation. Thus, anti-inflammatory pharmacotherapies may ameliorate EBI and provide neuroprotection after SAH. Cannabinoid type 2 receptor (CB2R) agonism has been shown to reduce neuroinflammation; however, the precise protective mechanisms remain to be elucidated. This study aimed to evaluate whether the selective CB2R agonist, JWH133 can ameliorate EBI by reducing brain-infiltrated leukocytes after SAH. Adult male Sprague-Dawley rats were randomly assigned to the following groups: sham-operated, SAH with vehicle, SAH with JWH133 (1.0mg/kg), or SAH with a co-administration of JWH133 and selective CB2R antagonist SR144528 (3.0mg/kg). SAH was induced by endovascular perforation, and JWH133 was administered 1h after surgery. Neurological deficits, brain water content, Evans blue dye extravasation, and Western blot assays were evaluated at 24h after surgery. JWH133 improved neurological scores and reduced brain water content; however, SR144528 reversed these treatment effects. JWH133 reduced Evans blue dye extravasation after SAH. Furthermore, JWH133 treatment significantly increased TGF-ß1 expression and prevented an SAH-induced increase in E-selectin and myeloperoxidase. Lastly, SAH resulted in a decreased expression of the tight junction protein zonula occludens-1 (ZO-1); however, JWH133 treatment increased the ZO-1 expression. We suggest that CB2R stimulation attenuates neurological outcome and brain edema, by suppressing leukocyte infiltration into the brain through TGF-ß1 up-regulation and E-selectin reduction, resulting in protection of the BBB after SAH.

Evaluation of selective cannabinoid CB(1) and CB(2) receptor agonists in a mouse model of lipopolysaccharide-induced interstitial cystitis.

Interstitial cystitis is a debilitating bladder inflammation disorder. To date, the understanding of the causes of interstitial cystitis remains largely fragmentary and there is no effective treatment available. Recent experimental results have shown a functional role of the endocannabinoid system in urinary bladder. In this study, we evaluated the anti-inflammatory effect of selective cannabinoid CB1 and CB2 receptor agonists in a mouse model of interstitial cystitis. Bladder inflammation was induced in mice by lipopolysaccharide (LPS) and whole bladders were removed 24h later. LPS induced a significant increase of the contractile amplitude in spontaneous activity and a hypersensitivity to exogenous acetylcholine-induced contraction of whole-isolated bladder. Next, we evaluated the anti-inflammatory activity of cannabinoidergic compounds by pretreating mice with CB1 or CB2 selective agonist compounds, respectively ACEA and JWH015. Interestingly, JWH015, but not ACEA, antagonized LPS-induced bladder inflammation. Additionally, anti-inflammatory activity was studied by evaluation, leukocytes mucosa infiltration, myeloperoxidase activity, and mRNA expression of pro-inflammatory interleukin (IL-1α and IL-1ß), tumor necrosis factor-alpha (TNF-α) and cannabinoid CB1 and CB2 receptors. JWH015 significantly decreased leukocytes infiltration in both submucosa and mucosa, as well as the myeloperoxydase activity, in LPS treated mice. JWH015 reduced mRNA expression of IL-1α, IL-1ß, and TNF-α. LPS treatment increased expression of bladder CB2 but not CB1 mRNA. Taken together, these findings strongly suggest that modulation of the cannabinoid CB2 receptors might be a promising therapeutic strategy for the treatment of bladder diseases and conditions characterized by inflammation, such as interstitial cystitis.

Detailed characterization of the endocannabinoid system in human macrophages and foam cells, and anti-inflammatory role of type-2 cannabinoid receptor.

OBJECTIVE: Cannabinoid receptors are activated in murine macrophages upon exposure to oxidized low-density lipoproteins (oxLDL), and type-1 cannabinoid receptor (CB1R) is considered as a risk factor in atherosclerosis, because it promotes cholesterol accumulation and release of inflammatory mediators. Conversely, accumulated evidence suggests a protective role for type-2 cannabinoid receptor (CB2R). Here, we sought to ascertain whether different elements of the endocannabinoid system (ECS) were activated in human lipid-laden macrophages, and whether CB2R played any role in atherogenesis and inflammation of these cells. METHODS AND RESULTS: Human macrophages were exposed to oxLDL in order to obtain lipid-laden foam cells. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the production of the endocannabinoids in both macrophages and foam cells, and radiometric assays were performed to measure cannabinoid receptor binding and activity of endocannabinoid metabolizing enzymes. OxLDL accumulation was investigated by confocal imaging, and cytokine production and release were measured by means of flow cytometry and ELISA. The results showed that human macrophages possess a fully functional ECS, which was modulated by oxLDL. Selective CB2R activation reduced cellular oxLDL accumulation, which was associated with decreased expression of CD36 scavenger receptor, and decreased production of TNFα, IL-12 and IL-10. These anti-atherogenic and anti-inflammatory effects were reverted by the selective CB2R antagonist SR144528. CONCLUSIONS: A fully active ECS is present in human macrophages and macrophage-derived foam cells. Selective activation of CB2R reduces CD36-dependent oxLDL accumulation and modulates production of inflammatory cytokines, thus representing a potential therapeutic strategy to combat atherosclerosis.

Effects of cannabinoid receptor type 2 on endogenous myocardial regeneration by activating cardiac progenitor cells in mouse infarcted heart.

Cannabinoid receptor type 2 (CB2) activation is recently reported to promote proliferation of some types of resident stem cells (e.g., hematopoietic stem/progenitor cell or neural progenitor cell). Resident cardiac progenitor cell (CPC) activation and proliferation are crucial for endogenous cardiac regeneration and cardiac repair after myocardial infarction (MI). This study aims to explore the role and possible mechanisms of CB2 receptor activation in enhancing myocardial repair. Our results revealed that CB2 receptor agonist AM1241 can significantly increase CPCs by c-kit and Runx1 staining in ischemic myocardium as well as improve cardiomyocyte proliferation. AM1241 also decreased serum levels of MDA, TNF-α and IL-6 after MI. In addition, AM1241 can ameliorate left ventricular ejection fraction and fractional shortening, and reduce fibrosis. Moreover, AM1241 treatment markedly increased p-Akt and HO-1 expression, and promoted Nrf-2 nuclear translocation. However, PI3K inhibitor wortmannin eliminated these cardioprotective roles of AM1241. In conclusion, AM1241 could induce myocardial regeneration and improve cardiac function, which might be associated with PI3K/Akt/Nrf2 signaling pathway activation. Our findings may provide a promising strategy for cardiac endogenous regeneration after MI.