jhw-015 and Inflammation

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.

Topics: Arachidonic Acids; Biological Transport; Caco-2 Cells; Cannabinoid Receptor Agonists; Colitis; Colon; Colorectal Neoplasms; Dioxoles; Endocannabinoids; Epithelial Cells; Epithelium; Female; Humans; Hydrocortisone; Indoles; Inflammation; Interleukin-1beta; Intestinal Mucosa; Lymphocyte Count; Male; Permeability; Polyunsaturated Alkamides; Quinolones; Receptor, Cannabinoid, CB2; Tumor Necrosis Factor-alpha

Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB(2) and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.. We examined the effects of µ-opioid and CB(2) receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.. Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB(2) receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.. Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB(2) receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids.

Topics: Animals; CHO Cells; Cricetinae; Cytokines; Gene Expression Regulation; Humans; Indoles; Inflammation; Lipopolysaccharides; MAP Kinase Kinase 1; Mice; Mice, Inbred BALB C; Microglia; Morphine; Naphthols; Nitric Oxide; Nitrites; Receptor, Cannabinoid, CB2; Receptors, Opioid, mu; RNA Interference; RNA, Small Interfering; Signal Transduction; Triazines

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Activation of cannabinoid CB1 and CB2 receptors is known to attenuate nociception and hyperalgesia in somatic inflammatory conditions. The aim of this study was to determine whether cannabinoids modulate colonic sensitivity in basal and inflammatory conditions. The effects of CB1 and CB2 receptor agonists and antagonists on the abdominal contractile response to colorectal distension (CRD) in basal conditions and after 2,4,6-trinitrobenzenesulphonic acid-induced colitis were investigated. As previously described, colitis triggered a hypersensitivity to CRD. In basal conditions, both CB1 (WIN 55212-2) and CB2 (JWH 015) agonists reduced the abdominal response to CRD at a dose of 1 mg kg(-1), i.p. Both compounds were active at a lower dose (0.1 mg kg(-1)) abolishing the hypersensitivity induced by colitis. Administered alone, CB1 (Rimonabant) and CB2 (SR 144528) receptor antagonists (10 mg kg(-1)) had no effect on basal sensitivity. In contrast, the CB1, but not the CB2, receptor antagonist enhanced colitis-induced hyperalgesia. It is concluded that colonic inflammation enhances the antinociceptive action of CB1 and CB2 receptor agonists, and activates an endogenous, CB1 receptor mediated, antinociceptive pathway.

Topics: Animals; Benzoxazines; Calcium Channel Blockers; Camphanes; Colitis; Colon; Dose-Response Relationship, Drug; Indoles; Inflammation; Male; Manometry; Morpholines; Muscle Contraction; Muscle, Smooth; Naphthalenes; Nociceptors; Pain; Piperidines; Pressure; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Rimonabant; Trinitrobenzenesulfonic Acid