Cannabinoid Receptor 2 Receptor Activation Research Articles

AM-1241 CB2 Receptor Agonist Attenuates Inflammation, Apoptosis and Stimulate Progenitor Cells in Bile Duct Ligated Rats.

BACKGROUND:The cannabinoid receptor 2 (CB2) plays a pleiotropic role in the innate immunity and is considered a crucial mediator of liver disease. Cannabinoid CB2 receptor activation has been reported to attenuate liver fibrosis in CCl4 exposed mice and also plays a potential role in liver regeneration in a mouse model of I/R and protection against alcohol-induced liver injury.AIM:In this study, we investigated the impact of CB2 receptors on the antifibrotic and regenerative process associated with cholestatic liver injury.METHODS:Twenty-six rats had bile duct ligation co-treated with silymarin and AM1241 for 3 consecutive weeks. Serum hepatotoxicity markers were determined, and histopathological evaluation was performed.RESULTS:Following bile duct ligation (BDL) for 3 weeks, there was increased aminotransferase levels, marked inflammatory infiltration and hepatocyte apoptosis with induced oxidative stress, as reflected by increased lipid peroxidation. Conversely, following treatment with the CB2 agonist, AM-1241, BDL rats displayed a reduction in liver injury and attenuation of fibrosis as reflected by expression of hydroxyproline and α-smooth muscle actin. AM1241 treatment also significantly attenuated lipid peroxidation end-products, p53-dependent apoptosis and also attenuated inflammatory process by stimulating IL-10 production. Moreover, AM1241 treated rats were associated with significant expression of hepatic progenitor/oval cell markers.CONCLUSION:In conclusion, this study points out that CB2 receptors reduce liver injury and promote liver regeneration via distinct mechanisms including IL-10 dependent inhibition of inflammation, reduction of p53-reliant apoptosis and through stimulation of oval/progenitor cells. These results suggest that CB2 agonists display potent hepatoregenrative properties, in addition to their antifibrogenic effects.

Abstract P2-06-24: Cannabinoid receptors agonist induces lysosome-mediated cell death in TNBC

Abstract Breast cancer is one of the leading causes of death among women in the United States. Among all the breast cancer subtypes, triple-negative breast cancer (TNBC) is denoted as having the highest rate of mortality due to inefficacious therapy, drug resistance, and high recurrences rate. Despite many therapeutic advances over the past decades, novel therapeutic approaches are required to treat TNBC. Although lysosomes have recently been identified as novel targets in a variety of cancers, not much known about their role in breast cancer especially in TNBC. Due to the altered features of lysosomes in cancerous cells, its functional ability affects cell proliferation, metabolism, adaptation, and autophagy in tumor cells. Thus, the induction of lysosomal membrane permeabilization (LMP) may be used as a novel approach in controlling the aberrant cellular functions in TNBC. Cannabinoid receptor 2 (CB2) is expressed in breast cancer and its agonist JWH-015 has been shown to inhibit TNBC growth and metastasis. However, not much is known about the mechanisms by which JWH015 inhibit growth and metastasis. To investigate JWH015-mediated therapeutic mechanisms, we examined the lysosome-mediated cell death in TNBC cells. We found that JWH015 induces cell death in TNBC cells through lysosomal disruption mechanism. We also showed that JWH-015 treatment increases releases of lysosomal proteins, cathepsin D/B, and autophagy marker LC3B and that leads to activation of apoptotic cell death signaling in TNBC. These alterations result in the formation of autolysosomes to promote LMP. Our findings elucidate the mechanism by which JWH-015 promotes LMP induced autophagy and regulates TNBC cell proliferation. These studies elucidate novel lysosomal-mediated cell death mechanisms through activation of CB2 receptors in TNBC. Citation Format: Chatterjee N, Das S, Ahiwar D, Mishra S, Kaul K, Ganju R. Cannabinoid receptors agonist induces lysosome-mediated cell death in TNBC [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-24.

Activation of the cannabinoid receptor 2 increases renal perfusion.

Acute kidney injury (AKI) is an increasing clinical problem that is associated with chronic kidney disease progression. Cannabinoid receptor 2 (CB2) activation has been shown to mitigate some of the deleterious tubular effects due to AKI, but its role on the renal vasculature has not been fully described. In this study, we investigated the effects of our novel CB2 receptor agonist, SMM-295, on renal vasculature by assessing cortical perfusion with laser Doppler flowmetry and changes in luminal diameter with isolated afferent arterioles. In this study, intravenously infused SMM-295 (6 mg/kg) significantly increased cortical renal perfusion (13.8 ± 0.6%; P < 0.0001; n = 7) compared with vehicle (0.1 ± 1.5%; n = 10) normalized to baseline values in anesthetized C57BL/6J mice. This effect was not dependent upon activation of the CB1 receptor (met-anandamide; 6 mg/kg iv) and was predominantly abolished in Cnr2 knockout mice with SMM-295 (6 mg/kg iv). Ablation of the renal afferent nerves with capsaicin blocked the SMM-295-dependent increase in renal cortical perfusion, and the increased renal blood flow was not dependent upon products synthesized by cyclooxygenase or nitric oxide synthase. The increased renal perfusion by CB2 receptor activation is also attributed to a direct vascular effect, since SMM-295 (5 μM) engendered a significant 37 ± 7% increase ( P < 0.0001; n = 4) in luminal diameters of norepinephrine-preconstricted afferent arterioles. These data provide new insight into the potential benefit of SMM-295 by activating vascular and nonvascular CB2 receptors to promote renal vasodilation, and provide a new therapeutic target to treat renal injuries that impact renal blood flow dynamics.

Modulating the endocannabinoid pathway as treatment for peripheral neuropathic pain: a selected review of preclinical studies.

Chemotherapy-induced neuropathic pain is a distressing and commonly occurring side effect of many commonly used chemotherapeutic agents, which in some cases may prevent cancer patients from being able to complete their treatment. Cannabinoid based therapies have the potential to manage or even prevent pain associated with this syndrome. Pre-clinical animal studies that investigate the modulation of the endocannabinoid system (endogenous cannabinoid pathway) are being conducted to better understand the mechanisms behind this phenomenon. Five recent pre-clinical studies identified from Medline published between 2013 and 2016 were selected for review. All studies evaluated the effect of small-molecule agonists or antagonists on components of the endocannabinoid system in rats or mice, using cisplatin or paclitax-el-induced allodynia as a model of chemotherapy-induced neuropathic pain. Activation of the cannabinoid receptor-2 (CB-2) receptor by AM1710 blocked paclitaxel-induced mechanical and cold allodynia in one study. Four studies investigating the activation of both cannabinoid receptor-1 (CB-1) and CB-2 receptors by dual-agonists (WIN55,21 and CP55,940), or by the introduction of inhibitors of endocannabinoid metabolisers (URB597, URB937, JZL184, and SA-57) showed reduction of chemotherapy-induced al-lodynia. In addition, their results suggest that anti-allodynic effects may also be mediated by additional receptors, including TRPV1 and 5-hydroxytryptamine (5-HT1A). Pre-clinical studies demon-strate that the activation of endocannabinoid CB-1 or CB-2 receptors produces physiological effects in animal models, namely the reduction of chemotherapy-induced allodynia. These studies also provide in-sight into the biological mechanism behind the therapeutic utility of cannabis compounds in managing chemotherapy-induced neuropathic pain, and provide a basis for the conduct of future clinical studies in patients of this population.

Selective Cannabinoid 2 Receptor Stimulation Reduces Tubular Epithelial Cell Damage after Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), which is an increasing problem in the clinic and has been associated with elevated rates of mortality. Therapies to treat AKI are currently not available, so identification of new targets that can be modulated to ameliorate renal damage upon diagnosis of AKI is essential. In this study, a novel cannabinoid receptor 2 (CB2) agonist, SMM-295 [3'-methyl-4-(2-(thiophen-2-yl)propan-2-yl)biphenyl-2,6-diol], was designed, synthesized, and tested in vitro and in silico. Molecular docking of SMM-295 into a CB2 active-state homology model showed that SMM-295 interacts well with key amino acids to stabilize the active state. In human embryonic kidney 293 cells, SMM-295 was capable of reducing cAMP production with 66-fold selectivity for CB2 versus cannabinoid receptor 1 and dose-dependently increased mitogen-activated protein kinase and Akt phosphorylation. In vivo testing of the CB2 agonist was performed using a mouse model of bilateral IRI, which is a common model to mimic human AKI, where SMM-295 was immediately administered upon reperfusion of the kidneys after the ischemia episode. Histologic damage assessment 48 hours after reperfusion demonstrated reduced tubular damage in the presence of SMM-295. This was consistent with reduced plasma markers of renal dysfunction (i.e., creatinine and neutrophil gelatinase-associated lipocalin) in SMM-295-treated mice. Mechanistically, kidneys treated with SMM-295 were shown to have elevated activation of Akt with reduced terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL)-positive cells compared with vehicle-treated kidneys after IRI. These data suggest that selective CB2 receptor activation could be a potential therapeutic target in the treatment of AKI.

ACPA and JWH-133 modulate the vascular tone of superior mesenteric arteries through cannabinoid receptors, BKCa channels, and nitric oxide dependent mechanisms.

Some cannabinoids, a family of compounds derived from Cannabis sativa (marijuana), have previously shown vasodilator effects in several studies, a feature that makes them suitable for the generation of a potential treatment for hypertension. The mechanism underlying this vasodilator effect in arteries is still controversial. In this report, we explored how the synthetic cannabinoids ACPA (CB1-selective agonist) and JWH-133 (CB2-selective agonist) regulate the vascular tone of rat superior mesenteric arteries. To screen the expression of CB1 (Cannabinoid receptor 1) and CB2 (Cannabinoid receptor 2) receptors in arterial rings or isolated smooth muscle cells obtained from the artery, immunocytochemistry, immunohistochemistry, and confocal microscopy were performed. In addition, the effects on vascular tone induced by the two cannabinoids were tested in isometric tension experiments in rings obtained from superior mesenteric arteries. The participation of voltage and calcium-activated potassium channel of big conductance (BKCa) and the role of nitric oxide (NO) release on the vascular effects induced by ACPA and JWH-133 were tested. CB1 and CB2 receptors were highly expressed in the rat superior mesenteric artery, in both smooth muscle and endothelium. The vasodilation effect shown by ACPA was endothelium-dependent through a mechanism involving CB1 receptors, BKCa channel activation, and NO release; meanwhile, the vasodilator effect of JWH-133 was induced by the activation of CB2 receptors located in smooth muscle and by a CB2 receptor-independent mechanism inducing NO release. CB1 and CB2 receptor activation in superior mesenteric artery causes vasorelaxation by mechanisms involving BKCa channels and NO release.

Activation of cannabinoid receptor 2 attenuates mechanical allodynia and neuroinflammatory responses in a chronic post-ischemic pain model of complex regional pain syndrome type I in rats.

Complex regional pain syndrome type 1 (CRPS-I) remains one of the most clinically challenging neuropathic pain syndromes and its mechanism has not been fully characterized. Cannabinoid receptor 2 (CB2) has emerged as a promising target for treating different neuropathic pain syndromes. In neuropathic pain models, activated microglia expressing CB2 receptors are seen in the spinal cord. Chemokine fractalkine receptor (CX3CR1) plays a substantial role in microglial activation and neuroinflammation. We hypothesized that a CB2 agonist could modulate neuroinflammation and neuropathic pain in an ischemia model of CRPS by regulating CB2 and CX3CR1 signaling. We used chronic post-ischemia pain (CPIP) as a model of CRPS-I. Rats in the CPIP group exhibited significant hyperemia and edema of the ischemic hindpaw and spontaneous pain behaviors (hindpaw shaking and licking). Intraperitoneal administration of MDA7 (a selective CB2 agonist) attenuated mechanical allodynia induced by CPIP. MDA7 treatment was found to interfere with early events in the CRPS-I neuroinflammatory response by suppressing peripheral edema, spinal microglial activation and expression of CX3CR1 and CB2 receptors on the microglia in the spinal cord. MDA7 also mitigated the loss of intraepidermal nerve fibers induced by CPIP. Neuroprotective effects of MDA7 were blocked by a CB2 antagonist, AM630. Our findings suggest that MDA7, a novel CB2 agonist, may offer an innovative therapeutic approach for treating neuropathic symptoms and neuroinflammatory responses induced by CRPS-I in the setting of ischemia and reperfusion injury.

Novel Triazolopyrimidine-Derived Cannabinoid Receptor 2 Agonists as Potential Treatment for Inflammatory Kidney Diseases.

The cannabinoid receptor 2 (CB2) system is described to modulate various pathological conditions, including inflammation and fibrosis. A series of new heterocyclic small-molecule CB2 receptor agonists were identified from a high-throughput screen. Lead optimization gave access to novel, highly potent, and selective (over CB1) triazolopyrimidine derivatives. A preliminary structure-activity relationship was established, and physicochemical properties in this compound class were significantly improved toward better solubility, lipophilicity, and microsomal stability. An optimized triazolopyrimidine derivative, (3S)-1-[5-tert-butyl-3-[(1-cyclopropyltetrazol-5-yl)methyl]triazolo[4,5-d]pyrimidin-7-yl]pyrrolidin-3-ol (39), was tested in a kidney ischemia-reperfusion model, in which it showed efficacy at a dose of 10 mg kg(-1) (p.o.). A significant depletion of the three measured kidney markers indicated a protective role of CB2 receptor activation toward inflammatory kidney damage. Compound 39 was also protective in a model of renal fibrosis. Oral treatment with 39 at 3 mg kg(-1) per day significantly decreased the amount of fibrosis by ∼ 40% which was induced by unilateral ureter obstruction.

Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release.

Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists.

The cannabinoid receptor 2 agonist β-caryophyllene reduces voluntary alcohol intake and attenuated ethanol-induced place preference and sensitivity in mice

Introduction: Several recent studies have suggested that brain cannabinoid receptor 2 (CB2) plays a major role in alcohol reward. In fact, the implication of cannabinoid neurotransmission in the reinforcing effects of ethanol (EtOH) is becoming increasingly evident. Objectives: The CB2 receptor agonist β -caryophyllene (BCP) was used to investigate the role of the CB2 receptors in mediating alcohol intake and ethanol-induced conditioned place preference (EtOH-CPP) and sensitivity in mice. Material and methods: The effect of BCP on alcohol intake was evaluated using the standard two-bottle-choice drinking method. The mice were presented with increasing EtOH concentrations and its consumption was measured daily. Consumption of saccharin and quinine solutions were measured following the EtOH preference tests. Finally, the effect of BCP on alcohol reward and sensitivity was tested using an unbiased EtOH-CPP and loss of righting reflex (LORR) procedures, respectively. Results: BCP dose-dependently decreased alcohol consumption and preference. In addition, BCP-injected mice did not show any difference from vehicle mice in total fluid intake in a 24-hour paradigm or in their intake of graded concentrations of saccharin or quinine, suggesting that the CB2 receptor activation did not alter taste function. More importantly, BCP inhibited EtOH-CPP acquisition and exacerbated LORR duration. Interestingly, these effects were abrogated when mice were preinjected with a selective CB2 receptor antagonist, AM630. Conclusions: The CB2 receptor system appears to be involved in alcohol dependence and sensitivity and may represent a potential pharmacological target for the treatment of alcoholism. Acknowledgements: Dr Amine Bahi for supervising this project.

β-Caryophyllene Ameliorates the Alzheimer-Like Phenotype in APP/PS1 Mice through CB2 Receptor Activation and the PPARγ Pathway

Background/Aims: The activation of cannabinoid receptor 2 (CB2) has the beneficial effect of reducing neuroinflammatory response in the treatment of Alzheimer's disease (AD) and is suggested to trigger the peroxisome proliferator-activated receptor-γ (PPARγ) pathway; agonists of both receptors improve AD. Recently, the plant metabolite β-caryophyllene was shown to selectively bind to CB2 receptor and act as a full agonist. Methods: In this study, we examined the anti-inflammatory effect of β-caryophyllene in a transgenic APP/PS1 AD model and analyzed whether this effect was mediated by CB2 and PPARγ. Results: β-Caryophyllene, given orally, prevented cognitive impairment in APP/PS1 mice, and this positive cognitive effect was associated with reduced β-amyloid burden in both the hippocampus and the cerebral cortex. Moreover, β-caryophyllene reduced astrogliosis and microglial activation as well as the levels of COX-2 protein and the mRNA levels of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1β in the cerebral cortex. The use of the CB2 antagonist AM630 or the PPARγ antagonist GW9662 significantly reversed the protective effects of β-caryophyllene on APP/PS1 mice. Conclusion: These results demonstrate that the anti-inflammatory effect of the sesquiterpene β-caryophyllene involves CB2 receptor activation and the PPARγ pathway and suggest β-caryophyllene as an attractive molecule for the development of new drugs with therapeutic potential for the treatment of AD.

Cannabinoid receptor 2 agonist attenuates pain related behavior in rats with chronic alcohol/high fat diet induced pancreatitis.

BackgroundChronic Pancreatitis (CP) is a complex and multifactorial syndrome. Many contributing factors result in development of dysfunctional pain in a significant number of patients. Drugs developed to treat a variety of pain states fall short of providing effective analgesia for patients with chronic pancreatitis, often providing minimal to partial pain relief over time with significant side effects. Recently, availability of selective pharmacological tools has enabled great advances in our knowledge of the role of the cannabinoid receptors in pathophysiology. In particular, cannabinoid receptor 2 (CB2) has emerged as an attractive target for management of chronic pain, as demonstrated in several studies with inflammatory and neuropathic preclinical pain models. In this study, the analgesic efficacy of a novel, highly selective CB2 receptor agonist, LY3038404 HCl, is investigated in a chronic pancreatitis pain model, induced with an alcohol/high fat (AHF) diet.ResultsRats fed the AHF diet developed visceral pain-like behaviors detectable by week 3 and reached a maximum at week 5 that persists as long as the diet is maintained. Rats with AHF induced chronic pancreatitis were treated with LY3038404 HCl (10 mg/kg, orally, twice a day for 9 days). The treated animals demonstrated significantly alleviated pain related behaviors after 3 days of dosing, including increased paw withdrawal thresholds (PWT), prolonged abdominal withdrawal latencies (ABWL), and decreased nocifensive responses to noxious 44°C hotplate stimuli. Terminal histological analysis of pancreatic tissue sections from the AHF chronic pancreatitis animals demonstrated extensive injury, including a global pancreatic gland degeneration (cellular atrophy), vacuolization (fat deposition), and fibrosis. After the LY3038404 HCl treatment, pancreatic tissue was significantly protected from severe damage and fibrosis. LY3038404 HCl affected neither open field exploratory behaviors nor dark/light box preferences as measures of higher brain and motor functions.ConclusionLY3038404 HCl, a potent CB2 receptor agonist, possesses tissue protective and analgesic properties without effects on higher brain function. Thus, activation of CB2 receptors is suggested as a potential therapeutic target for visceral inflammation and pain management.

CB2 receptor activation ameliorates the proinflammatory activity in acute lung injury induced by paraquat.

Paraquat, a widely used herbicide, is well known to exhibit oxidative stress and lung injury. In the present study, we investigated the possible underlying mechanisms of cannabinoid receptor-2 (CB2) activation to ameliorate the proinflammatory activity induced by PQ in rats. JWH133, a CB2 agonist, was administered by intraperitoneal injection 1 h prior to PQ exposure. After PQ exposure for 4, 8, 24, and 72 h, the bronchoalveolar lavage fluid was collected to determine levels of TNF-α and IL-1β, and the arterial blood samples were collected for detection of PaO2 level. At 72 h after PQ exposure, lung tissues were collected to determine the lung wet-to-dry weight ratios, myeloperoxidase activity, lung histopathology, the protein expression level of CB2, MAPKs (ERK1/2, p38MAPK, and JNK1/2), and NF-κBp65. After rats were pretreated with JWH133, PQ-induced lung edema and lung histopathological changes were significantly attenuated. PQ-induced TNF-α and IL-1β secretion in BALF, increases of PaO2 in arterial blood, and MPO levels in the lung tissue were significantly reduced. JWH133 could efficiently activate CB2, while inhibiting MAPKs and NF-κB activation. The results suggested that activating CB2 receptor exerted protective activity against PQ-induced ALI, and it potentially contributed to the suppression of the activation of MAPKs and NF-κB pathways.

Cannabinoid receptor 2 counteracts interleukin-17-induced immune and fibrogenic responses in mouse liver

Interleukin (IL)-17 is a proinflammatory and fibrogenic cytokine mainly produced by T-helper (Th)17 lymphocytes, together with the hepatoprotective and antifibrogenic cytokine, IL-22. Cannabinoid receptor 2 (CB2) is predominantly expressed in immune cells and displays anti-inflammatory and antifibrogenic effects. In the present study, we further investigated the mechanism underlying antifibrogenic properties of CB2 receptor and explored its effect on the profibrogenic properties of IL-17. After bile duct ligation (BDL), the hepatic expression of Th17 markers and IL-17 production were enhanced in CB2(-/-) mice, as compared to wild-type (WT) counterparts, and correlated with increased fibrosis in these animals. In contrast, IL-22-induced expression was similar in both animal groups. Inhibition of Th17 differentiation by digoxin lowered Th17 marker gene expression and IL-17 production and strongly reduced liver fibrosis in CB2(-/-) BDL mice. In vitro, differentiation of CD4(+) naïve T cells into Th17 lymphocytes was decreased by the CB2 agonist, JWH-133, and was associated with reduced Th17 marker messenger RNA expression and IL-17 production, without modification of IL-22 release. The inhibitory effect of JWH-133 on IL-17 production relied on signal transducer and activator of transcription (STAT)5 phosphorylation. Indeed, STAT5 phosphorylation and translocation into the nucleus was enhanced in JWH133-treated Th17 lymphocytes, and the addition of a STAT5 inhibitor reversed the inhibitory effect of the CB2 agonist on IL-17 production, without affecting IL-22 levels. Finally, in vitro studies also demonstrated that CB2 receptor activation in macrophages and hepatic myofibroblasts blunts IL-17-induced proinflammatory gene expression. These data demonstrate that CB2 receptor activation decreases liver fibrosis by selectively reducing IL-17 production by Th17 lymphocytes via a STAT5-dependent pathway, and by blunting the proinflammatory effects of IL-17 on its target cells, while preserving IL-22 production.

Cannabinoid CB2 receptor activation attenuates cytokine-evoked mucosal damage in a human colonic explant model without changing epithelial permeability

Cannabinoid receptor activation is protective in animal colitis models. We sought to investigate if cannabinoids attenuated colitis-like tissue damage in human colonic specimens, with the hypothesis that cannabinoids would be protective in a cytokine-driven model of human colonic mucosal damage. Healthy human colonic mucosa was incubated with pro-inflammatory cytokines TNF-α and IL-1β to elicit colitis-like tissue damage. The cytokine-driven increase in scored crypt and mucosal damage and lymphocyte density was attenuated with concomitant hydrocortisone pretreatment. The cannabinoid receptor 2 (CB2) receptor-selective agonist JWH-015 significantly reduced colitis scores following cytokine incubation, as evidenced by a reduction in mucosal crypt and luminal epithelial damage and lymphocyte density in the lamina propria. The effect of JWH-015 was reversed in the presence of the CB2 receptor inverse agonist JTE-907. Anandamide was also protective in the cytokine-incubated explant colitis model in a manner reversible with JTE-907, while CB1 receptor agonism with ACEA was without effect. TNF-α and IL-1β together evoked an increase in paracellular epithelial permeability in Caco-2 cell monolayers over 48h of incubation. However, neither CB2 nor CB1 receptor activation altered the cytokine-evoked increase in permeability. These findings support a discrete role for CB2 receptors in the attenuation of detrimental pro-inflammatory cytokine-mediated mucosal damage in the human colon without directly affecting mucosal epithelial barrier function.

Inhibitory effects of AM630 on inflammatory osteolysis induced by titanium particles

Objective To explore the effect of cannabinoid receptor 2 (CB2) selective antagonist (AM630) on the regulation of osteoclast differentiation from a murine macrophage cell line (RAW264.7)stimulated with titanium(Ti) particles and the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL).Methods The effect of AM630 and Ti particles on RAW264.7 cell viability was examined by using the methyl thiazol tetrazolium (MTT) assay.Osteoclast formation was measured by tartrate resistant acid phosphatase (TRAP) staining using a commercial kit.The mRNA levels of CB2,creatine phosphokinase(CPK) and NF-κB receptor activation factor(RANK) were detected by using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR).Enzyme-linked immunosorbent assay (ELISA)was performed to determine interleukin (IL)-1β and tumor necrosis factor (TNF)-t.Results AM630 (0-200 nmol/L) did not affect viability of RAW264.7 cells cultured with Ti particles.The mature osteoclasts were obtained from RAW264.7 cells stimulated with RANKL and Ti particles for 6 days and detected by TRAP staining.AM630 at a doses of ≥ 100 nmol/L significantly reduced the number of TRAP-positive cells as compared with controls.After culture for 6 days,mRNA levels of CB2,RANK and CPK were significantly higher in the media with RANKL and Ti particles than in those without RANKL and Ti particles.When 100 nmol/L AM630 in combination with RANKL and Ti particles was added to the culture system,mRNA levels of CB2,RANK and CPK were significantly decreased.The concentrations of IL-1β and TNF-α in RAW264.7 cell cultures were measured by using ELISA.After culture for 24 h,IL-13 and TNF-α concentrations were significantly increased in the media with Ti particles as compared with those in the media withou Ti particles.The cytokine concentration was further increased with culture time.Conclusion AM630 could significantly inhibit Ti particle-induced inflammatory osteoclastogenesis. Key words: Osteoclasts ; Inflammation ; Titanium particles ; Cannabinoid receptor 2

β-Caryophyllene Inhibits Dextran Sulfate Sodium-Induced Colitis in Mice through CB2 Receptor Activation and PPARγ Pathway

Cannabinoid receptor 2 (CB2) activation is suggested to trigger the peroxisome proliferator-activated receptor-γ (PPARγ) pathway, and agonists of both receptors improve colitis. Recently, the plant metabolite (E)-β-caryophyllene (BCP) was shown to bind to and activate CB2. In this study, we examined the anti-inflammatory effect of BCP in dextran sulfate sodium (DSS)-induced colitis and analyzed whether this effect was mediated by CB2 and PPARγ. Oral treatment with BCP reduced disease activity, colonic macro- and microscopic damage, myeloperoxidase and N-acetylglucosaminidase activities, and levels and mRNA expression of colonic tumor necrosis factor-α, IL-1β, interferon-γ, and keratinocyte-derived chemokine. BCP treatment also inhibited the activation of extracellular signal-regulated kinase 1/2, nuclear factor κB, IκB-kinase α/β, cAMP response element binding and the expression of caspase-3 and Ki-67. Moreover, BCP enhanced IL-4 levels and forkhead box P3 mRNA expression in the mouse colon and reduced cytokine levels (tumor necrosis factor-α, keratinocyte-derived chemokine, and macrophage-inflammatory protein-2) in a culture of macrophages stimulated with lipopolysaccharide. The use of the CB2 antagonist AM630 or the PPARγ antagonist GW9662 significantly reversed the protective effect of BCP. Confirming our results, AM630 reversed the beneficial effect of BCP on pro-inflammatory cytokine expression in IEC-6 cells. These results demonstrate that the anti-inflammatory effect of BCP involves CB2 and the PPARγ pathway and suggest BCP as a possible therapy for the treatment of inflammatory bowel disease.