JWH133 Treatment Research Articles

Quantitative Proteomics Reveal That CB2R Agonist JWH-133 Downregulates NF-κB Activation, Oxidative Stress, and Lysosomal Exocytosis from HIV-Infected Macrophages.

HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.

Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages

HIV-associated neurocognitive disorders (HAND) are prevalent despite combined antiretroviral therapy (cART), affecting 52% of people living with HIV. Our laboratory has demonstrated increased expression of cathepsin B (CATB) in postmortem brain tissue with HAND. Increased secretion of CATB from in vitro HIV-infected monocyte-derived macrophages (MDM) induces neurotoxicity. Activation of cannabinoid receptor type 2 (CB2R) inhibits HIV-1 replication in macrophages and the neurotoxicity induced by viral proteins. However, it is unknown if CB2R agonists affect CATB secretion and neurotoxicity in HIV-infected MDM. We hypothesized that HIV-infected MDM exposed to CB2R agonists decrease CATB secretion and neurotoxicity. Primary MDM were inoculated with HIV-1ADA and treated with selective CB2R agonists JWH-133 and HU-308. HIV-1 p24 and CATB levels were determined from supernatants using ELISA. MDM were pre-treated with a selective CB2R antagonist SR144528 before JWH-133 treatment to determine if CB2R activation is responsible for the effects. Neuronal apoptosis was assessed using a TUNEL assay. Results show that both agonists reduce HIV-1 replication and CATB secretion from MDM in a time and dose-dependent manner and that CB2R activation is responsible for these effects. Finally, JWH-133 decreased HIV/MDM-CATB induced neuronal apoptosis. Our results suggest that agonists of CB2R represent a potential therapeutic strategy against HIV/MDM-induced neurotoxicity.

CB2-mediated attenuation of nucleus pulposus degeneration via the amelioration of inflammation and oxidative stress in vivo and in vitro

BackgroundNucleus pulposus cell (NPC) degeneration is widely accepted as one of the major causes of intervertebral disc (IVD) degeneration (IVDD). The pathogenesis of IVDD is complex and consists of inflammation, oxidative stress, and the loss of extracellular matrix (ECM). Cannabinoid type 2 receptor (CB2) has been shown to be involved in the pathological mechanism of a variety of diseases due to its anti-inflammatory effects and antioxidative stress capacity.MethodIn Vitro, H2O2 was used to induce degeneration of nucleus pulposus cells, mRNA and protein expression level was determined by RT-PCR and Western Blot, and Immunocytochemical staining were used to detect expression of collagen II, aggrecan, MMP3/13, superoxide dismutase 2 (SOD2) and inducible nitric oxide synthase (iNOS). In vivo, the potential therapeutic effect of CB2 was detected in the rat acupuncture model.ResultIn vitro, we found that the CB2 agonist (JWH133) treatment reduced the oxidative stress level in NPCs induced by hydrogen peroxide (H2O2) treatment. Furthermore, the expression of inflammatory cytokines was also decreased by JWH133 treatment. We found that collagen II and aggrecan expression was preserved, whereas matrix metalloproteinase levels were reduced. In vivo, we established a rat model by needle puncture. Imaging assessment revealed that the disc height index (DHI) and morphology of IVD were significantly improved, and the disc degeneration process was delayed by treatment of JWH133. Furthermore, immunohistochemical (IHC) staining revealed that JWH133 could inhibit the degradation of collagen II and decrease the expression of MMP3.ConclusionsThe experiment indicates the oxidative stress and inflammatory response of rat NPCs induced by H2O2 could be inhibited by activating CB2. This study reveals that CB2 activation can effectively delay the development of IVDD, providing an effective therapeutic target for IVDD.

Targeting CYP2J2 to Enhance the Anti-Glioma Efficacy of Cannabinoid Receptor 2 Stimulation by Inhibiting the Pro-Angiogenesis Function of M2 Microglia.

Enhancing the therapeutic efficacy of anti-tumor drugs is essential for cancer management. Although cannabinoid receptor 2 (CB2R) stimulation exerts anti-tumor action in glioma cells by regulating cellular proliferation, differentiation, or apoptosis, selective CB2R agonist alone does not achieve a satisfactory therapeutic outcome. Herein, we aimed to evaluate the possible strategy for enhancing the anti-glioma efficacy of JWH133, a selective CB2R agonist. In this study, immunofluorescence and qRT-PCR were used to investigate microglia polarization. Tumor growth was monitored via bioluminescent imaging using the IVIS Spectrum System. The angiogenesis of human brain microvascular endothelial cells (HBMECs) was detected by the tube formation assay. qRT-PCR was used to investigate cytochrome P450 2J2 (CYP2J2) and 11,12-epoxyeicosatrienoic acid (11,12-EET) expression. Our results showed that administration of JWH133 significantly promoted microglial M2 polarization both in vitro and in vivo. The medium supernatant of M2 microglia induced by JWH133 treatment facilitated angiogenesis of HBMECs. CYP2J2 expression and 11,12-EET release in the supernatant of JWH133-induced M2 microglia were significantly upregulated. Treatment with 11,12-EET prompted HBMEC angiogenesis and glioma growth. CYP2J2 knockdown restrained the release of 11,12-EET and significantly enhanced the anti-tumor effect of JWH133 on glioma. This study showed that targeting CYP2J2 might be a beneficial strategy to enhance the anti-glioma efficacy of JWH133 by inhibiting the pro-angiogenesis function of M2 microglia.

Adjuvant Cannabinoid Receptor Type 2 Agonist Modulates the Polarization of Microglia Towards a Non-Inflammatory Phenotype in Experimental Pneumococcal Meningitis.

BackgroundMicroglia initiates and sustains the inflammatory reaction that drives the pathogenesis of pneumococcal meningitis. The expression of the G-protein cannabinoid receptor type 2 (CB2) in the brain is low, but is upregulated in glial cells during infection. Its activation down-regulates pro-inflammatory processes, driving microglia towards an anti-inflammatory phenotype. CB2 agonists are therefore therapeutic candidates in inflammatory conditions like pneumococcal meningitis. We evaluated the effects of JWH-133, a specific CB2 agonist on microglial cells, inflammation, and damage driven by S. pneumoniae in vitro and in experimental pneumococcal meningitis.Materials/methodsPrimary mixed glial cultures were stimulated with live or heat-inactivated S. pneumoniae, or lipopolysaccharide and treated with JWH-133 or vehicle. Nitric oxide and cytokines levels were measured in the supernatant. In vivo, pneumococcal meningitis was induced by intracisternal injection of live S. pneumoniae in 11 days old Wistar rats. Animals were treated with antibiotics (Ceftriaxone, 100 mg/kg, s.c. bid) and JWH-133 (1 mg/kg, i.p. daily) or vehicle (10% Ethanol in saline, 100 µl/25g body weight) at 18 h after infection. Brains were harvested at 24 and 42 h post infection (hpi) for histological assessment of hippocampal apoptosis and cortical damage and determination of cyto/chemokines in tissue homogenates. Microglia were characterized using Iba-1 immunostaining. Inflammation in brain homogenates was determined using membrane-based antibody arrays.Results In vitro, nitric oxide and cytokines levels were significantly lowered by JWH-133 treatment. In vivo, clinical parameters were not affected by the treatment. JWH-133 significantly lowered microglia activation assessed by quantification of cell process length and endpoints per microglia. Animals treated with JWH-133 demonstrated significantly lower parenchymal levels of chemokines (CINC-1, CINC-2α/β, and MIP-3α), TIMP-1, and IL-6 at 24 hpi, and CINC-1, MIP-1α, and IL-1α at 42 hpi. Quantitative analysis of brain damage did not reveal an effect of JWH-133.ConclusionsJWH-133 attenuates microglial activation and downregulates the concentrations of pro-inflammatory mediators in pneumococcal infection in vitro and in vivo. However, we didn’t observe a reduction in cortical or hippocampal injury. This data provides evidence that inhibition of microglia by adjuvant CB2 agonists therapy effectively downmodulates neuroinflammation but does not reduce brain damage in experimental pneumococcal meningitis

The ameliorating effect of cannabinoid type 2 receptor activation on brain, lung, liver and heart damage in cecal ligation and puncture-induced sepsis model in rats

Uncontrolled infection and increased inflammatory mediators might cause systemic inflammatory response. It is already known that Cannabinoid Type 2 (CB2) receptors, which are commonly expressed in immune cells and in many other tissues, have an effect on the regulation of immune response. In the present study of ours, the effects of CB2 receptor agonist JWH-133 was investigated on cecal ligation and puncture (CLP)-induced polymicrobial sepsis model in rats. In the present study, Sprague-Dawley rats were divided into 5 groups (i.e. the Sham, CLP, JWH-133 0.2 mg/kg, JWH-133 1 mg/kg, and JWH-133 5 mg/kg Groups). Except for the Sham Group, the CLP-induced sepsis model was applied to all groups. The histopathological damage in brain, lung, liver and, heart was examined and the caspase-3, p-NF-κB, TNF-α, IL-1β and IL-6 levels were determined immunohistochemically. The serum TNF-α, IL-1β, IL-6, IL-10 levels were examined with the ELISA Method. The JWH-133 treatment decreased the histopathological damage in brain, heart, lung, and liver and reduced the caspase-3, p-NF-κB, TNF-α, IL-1β, IL-6 levels in these tissues. In addition to this, JWH-133 treatment also decreased the serum TNF-α, IL-1β, IL-6 levels, which were increased due to CLP, and increased the anti-inflammatory cytokine IL-10 levels. In the present study, it was determined that the CB2 receptor agonist JWH-133 decreases the CLP-induced inflammation, and reduces the damage in brain, lung, liver and heart. Our findings show the therapeutic potential of the activation of CB2 receptors with JWH-133 in sepsis.

Liver X Receptor α-Induced Cannabinoid Receptor 2 Inhibits Ubiquitin-Specific Peptidase 4 Through miR-27b, Protecting Hepatocytes From TGF-β.

Liver X receptor‐alpha (LXRα) acts as a double‐edged sword in different biological situations. Given the elusive role of LXRα in hepatocyte viability, this study investigated whether LXRα protects hepatocytes from injurious stimuli and the underlying basis. LXRα activation prevented hepatocyte apoptosis from CCl4 challenges in mice. Consistently, LXRα protected hepatocytes specifically from transforming growth factor‐beta (TGF‐β), whereas LXRα deficiency aggravated TGF‐β‐induced hepatocyte injury. In the Gene Expression Omnibus database analysis for LXR−/− mice, TGF‐β receptors were placed in the core network. Hierarchical clustering and correlation analyses enabled us to find cannabinoid receptor 2 (CB2) as a gene relevant to LXRα. In human fibrotic liver samples, both LXRα and CB2 were lower in patients with septal fibrosis and cirrhosis than those with portal fibrosis. LXRα transcriptionally induced CB2; CB2 then defended hepatocytes from TGF‐β. In a macrophage depletion model, JWH133 (a CB2 agonist) treatment prevented toxicant‐induced liver injury. MicroRNA 27b (miR‐27b) was identified as an inhibitor of ubiquitin‐specific peptidase 4 (USP4), deubiquitylating TGF‐β receptor 1 (TβRI), downstream from CB2. Liver‐specific overexpression of LXRα protected hepatocytes from injurious stimuli and attenuated hepatic inflammation and fibrosis. Conclusion: LXRα exerts a cytoprotective effect against TGF‐β by transcriptionally regulating the CB2 gene in hepatocytes, and CB2 then inhibits USP4‐stabilizing TβRI through miR‐27b. Our data provide targets for the treatment of acute liver injury.

Cannabinoid Receptor 2 Agonist Prevents Local and Systemic Inflammatory Bone Destruction in Rheumatoid Arthritis.

Cannabinoid receptor 2 (CB2) has been implicated as an important clinical regulator of inflammation and malignant osteolysis. Here, we observed that CB2 expression was markedly higher in the collagen-induced arthritis (CIA) mice synovium and bone tissues than in the noninflamed synovium and bone tissues. The CB2 selective agonist (JWH133) but not antagonist (SR144528) suppressed CIA in mice without toxic effects, as demonstrated by the decreased synovial hyperplasia, inflammatory responses, cartilage damage, and periarticular and systemic bone destruction. JWH133 treatment decreased the infiltration of pro-inflammatory M1-like macrophages and repolarized macrophages from the M1 to M2 phenotype. Similarly, activation of CB2 increased the expression of anti-inflammatory cytokine interleukin (IL)-10 and reduced the expression of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-1β, and IL-6. In addition, JWH133 treatment attenuated osteoclast formation and osteoclastic bone resorption, and reduced the expression of receptor activators of the nuclear factor-κB (NF-κB) ligand (RANKL), matrix metallopeptidase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), and nuclear factor of activated T-cells 1 (NFAT-1) in CIA mice and osteoclast precursors, which were obviously blocked by pretreatment with SR144528. Mechanistically, JWH133 inhibited RANKL-induced NF-κB activation in the osteoclast precursors. We found that JWH133 ameliorates pathologic bone destruction in CIA mice via the inhibition of osteoclastogenesis and modulation of inflammatory responses, thereby highlighting its potential as a treatment for human rheumatoid arthritis. © 2018 American Society for Bone and Mineral Research.

CB2R induces a protective response for epileptic seizure via the PI3K 110α-AKT signaling pathway.

Epilepsy is a chronic brain disease caused by abnormal discharging in the brain, which induces momentary brain dysfunction. Cannabinoid 2 receptor (CB2R) is expressed in central nervous system (CNS) and serves an important role in the pathogenesis of CNS diseases. The aim of the present study was to explore the effects of CB2R activation on phosphoinositide 3-kinase (PI3K) 110α-protein kinase B (AKT) signaling in an astrocyte model of epilepsy. Rat CTX TNA2 astrocytes were treated with Mg free solution to establish a cell model of epilepsy and were subsequently treated with a CB2R agonist (JWH133) and antagonist (AM630). Cell cycle analysis revealed that treatment using Mg free solution inhibited cell cycle transition. JWH133 facilitated cell cycle progression while AM630 inhibited it. Western blotting results demonstrated that treatment with Mg free solution downregulated the expression of cyclin D1, cyclin E, phosphorylated Retinoblastoma (p-Rb), B-cell lymphoma 2 (Bcl-2), PI3K 110α, p-AKT and p-mammalian target of rapamycin, whereas JWH133 treatment upregulated these proteins. AM630 ameliorated the JWH133-induced upregulation of these proteins. To confirm the involvement of AKT signaling, the AKT inhibitor wortmannin was used. The results revealed that wortmannin inhibited the effect of JWH133 on p-AKT, cyclin D1, p-Rb and Bcl-2 expression. In addition, the effects of JWH133 and AM630 on PI3K 110α-AKT signaling were verified using a rat model of epilepsy. In conclusion, the present study demonstrates that CB2R activation induces astrocyte proliferation and survival via activation of the PI3K 110α-AKT signaling pathway.

Cannabinoid receptor 2 agonist attenuates blood‑brain barrier damage in a rat model of intracerebral hemorrhage by activating the Rac1 pathway

Blood‑brain barrier (BBB) disruption and consequent edema formation are the most common brain injuries following intracerebral hemorrhage (ICH). Endocannabinoid receptors can alter the permeability of various epithelial barriers and have potential neuroprotective effects. The present study aimed to explore whether the selective cannabinoid receptor2 (CNR2) agonist, JWH133, can ameliorate BBB integrity and behavioral outcome by activating Ras‑related C3 botulinum toxin substrate1 (Rac1) following ICH. Autologous arterial blood was injected into the basal ganglia of rats to induce ICH. Animals were randomly divided into the following groups: Sham‑operated, ICH+vehicle, ICH+JWH133, ICH+JWH13+vehicle, ICH+JWH133+AM630 (a selective CNR2 antagonist), ICH+AM630, ICH+JWH133 +NSC23766 (a Rac1 antagonist) and ICH+NSC23766. JWH133 and AM630 were independently intraperitoneally administrated at 1h prior to ICH. NSC23766 was intracerebroventricularly (ICV) administered 30min prior to ICH. A modified Garcia test, corner test, Evans blue extravasation and brain water content analysis were performed at 24 and 72h following ICH. Western blotting and pull‑down assays were performed at 24h following ICH. The results demonstrated that JWH133 treatment improved neurofunctional deficits, reduced perihematomal brain edema and alleviated BBB damage at 24 and 72h following ICH. In addition, JWH133 treatment increased the protein expression levels of guanosine‑5'‑triphosphate‑Rac1 and of the adherens junction proteins occludin, zonula occludens‑1 and claudin‑5. However, these effects were reversed by AM630 and NSC23766 treatment. In conclusion, the present findings revealed that JWH133 treatment attenuated brain injury in a rat model of ICH via activation of the Rac1 signaling pathway, thus preserving BBB integrity.

Pharmacological regulation of cannabinoid CB2 receptor modulates the reinforcing and motivational actions of ethanol

The endocannabinoid system plays a pivotal role in the regulation of ethanol reinforcing and motivational actions. The pharmacological manipulation of cannabinoid CB1 receptor (CB1r) in alcoholic patients provided discouraging results, so researchers have recently turned their attention to the cannabinoid CB2 receptor (CB2r). In this regard, the present study aims to characterise CB2r-mediated effects on ethanol self-administration and to describe the neurochemical mechanisms that may be involved.We performed an oral ethanol self-administration (OEA) experiment to analyse the effects of repeated administration of AM630 (1 mg kg−1, i.p.) or JWH133 (1 mg kg−1, i.p.) on the number of reinforced responses, the 8% ethanol intake and the breaking point values in male C57BL/6J mice. By means of real-time polymerase chain reaction (PCR), we also analysed relative gene expression of tyrosine hydroxylase (TH) in the ventral tegmental area (VTA), and of mu-opioid receptor (OPRM1), CNR1 and CNR2 in the nucleus accumbens (NAcc).AM630-induced blockade of CB2r significantly increased the number of reinforced responses, 8% ethanol consumption and breaking point, whereas JWH133 treatment induced the opposite effect. Interestingly, the administration of AM630 significantly increased TH gene expression in the VTA, as well as OPRM1 and CNR1 gene expression in the NAcc, whereas administration with JWH133 decreased gene expression of these targets. In addition, CNR2 gene expression was unchanged with AM630 treatment but increased in animals treated with JWH133.The therapeutic implications of our results hold promise for CB2r as a means to manage alcohol use disorder and significantly contribute to improving understanding of the underlying neurobiological mechanisms involved.

Activation of cannabinoid receptor type 2 by JWH133 alleviates bleomycin-induced pulmonary fibrosis in mice.

Activation of cannabinoid receptor type 2 has been shown to have anti-fibrosis function in skin and heart. However, whether activating cannabinoid receptor type 2 inhibits pulmonary fibrosis remains elusive. Lung fibroblasts and TGF-β1 are key players in the pathogenesis of pulmonary fibrosis. In this research, we aimed to investigate the role of cannabinoid receptor type 2 in pulmonary fibrosis in vitro and in vivo. In lung fibroblasts stimulated by TGF-β1, preincubated by cannabinoid receptor type 2 agonist JWH133 not only reduced the elevated levels of collagen I and α-SMA, but also inhibited fibroblasts’ proliferation and migration. The dosage of JWH133 had no clear cytotoxic activity, and all these JWH133 effects were partially abrogated by cannabinoid receptor type 2 antagonist SR144528. In bleomycin-induced mice pulmonary fibrosis model, CT images of the lung tissue revealed an extensive ground-glass opacity, reticular pattern and fibrosis stranding. Notably, JWH133 treatment controlled the ongoing fibrotic process (showed by decreased lung density and fibrosis score). Meanwhile, lung histological results revealed that JWH133 treatment suppressed both the inflammatory response and extracellular collagen deposition. SR144528 may increase the pulmonary fibrosis, but no statistically significant difference was proved. Importantly, JWH133 reduced serum profibrotic cytokines levels of TGF-β1 and inhibited TGF-β1/Smad2 pathway in vitro and in vivo. Our research indicated that activating cannabinoid receptor type 2 by a pharmacological method might be a potential strategy for pulmonary fibrosis.

Activation of cannabinoid receptor type 2 attenuates surgery-induced cognitive impairment in mice through anti-inflammatory activity

BackgroundNeuroinflammation plays a major role in postoperative cognitive dysfunction (POCD). Accumulated evidence indicates that cannabinoid receptor type 2 (CB2R) can mediate anti-inflammatory and immunomodulatory effects in part by controlling microglial activity. However, the impact of CB2R on postoperative cognition has not been investigated. We hypothesized that CB2R is involved in surgery-induced cognitive impairment in adult mice.MethodsAdult C57BL/6 mice were subjected to intramedullary fixation surgery for tibial fracture under isoflurane anesthesia and CB2R agonist (JWH133) or CB2R antagonist (AM630) treatment. The mice were trained 24 h prior to surgery using a fear conditioning protocol and assessed in a novel context on postoperative days 1, 3, and 7 to evaluate cognitive function. Open-field testing was performed to evaluate the locomotor activity of the mice. The expression levels of IL-1β, TNF-α, MCP-1, and CB2R in the hippocampus and prefrontal cortex were assessed by Western blotting; the expression of microglial marker CD11b in the CA1 area of the hippocampus and medial prefrontal cortex was assessed by immunostaining.ResultsThe mice displayed no changes in locomotor activity after surgery and drug treatments. The mice exhibited impaired hippocampal-dependent memory accompanied by an increased expression of proinflammatory factors in the hippocampus and prefrontal cortex 1, 3, and 7 days after surgery, while hippocampal-independent memory remained unaffected at the same time points. JWH133 treatment attenuated surgery-induced memory loss, while AM630 treatment aggravated surgery-induced memory loss, paralleled by a decreased or increased expression of proinflammatory factors in the hippocampus and prefrontal cortex. The expression of CB2R in the hippocampus and prefrontal cortex was upregulated following surgery; however, it was downregulated by postoperative treatment with JWH133. Similarly, the expression of CD11b in the CA1 area of the hippocampus and medial prefrontal cortex was upregulated following surgery and downregulated by postoperative treatment with JWH133.ConclusionsThese findings indicate that CB2R may modulate the neuroinflammatory and cognitive impairment in a mouse model of orthopedic surgery, and the activation of CB2R may effectively ameliorate the hippocampal-dependent memory loss of mice in the early postoperative stage.

Cannabinoid receptor 2 attenuates microglial accumulation and brain injury following germinal matrix hemorrhage via ERK dephosphorylation in vivo and in vitro

Microglia accumulation plays detrimental roles in the pathology of germinal matrix hemorrhage (GMH) in the immature preterm brain. However, the underlying mechanisms remain poorly defined. Here, we investigated the effects of a cannabinoid receptor 2 (CB2R) agonist on microglia proliferation and the possible involvement of the mitogen-activated protein kinase (MAPK) family pathway in a collagenase-induced GMH rat model and in thrombin-induced rat microglia cells. We demonstrated that activation of CB2R played a key role in attenuating brain edema, neuronal degeneration, microglial accumulation and the phosphorylated extracellular signal-regulated kinase (p-ERK) protein level 24 h following GMH. In vitro, Western blot analysis and immunostaining indicated that ERK and P38 phosphorylation levels in microglia stimulated by thrombin were decreased after JWH-133 (CB2R selective agonist) treatment in a concentration-dependent manner. Microglia proliferation (EDU + microglia) and inflammatory and oxidative stress responses were attenuated by UO126 (ERK pathway inhibitor) 24 h after thrombin stimulation, an activity that was prevented by AM630 (CB2R selective antagonist). Overall, these findings suggest that activation of the endocannabinoid system might attenuate inflammation-induced secondary brain injury after GMH in rats by reducing microglia accumulation through a mechanism involving ERK dephosphorylation. Enhancing CB2R activation is a potential treatment to slow down the course of GMH in preterm newborns.

Protective Role of Cannabinoid Receptor 2 Activation in Galactosamine/Lipopolysaccharide-Induced Acute Liver Failure through Regulation of Macrophage Polarization and MicroRNAs

Acute liver failure (ALF) is a potentially life-threatening disorder without any effective treatment strategies. d-Galactosamine (GalN)/lipopolysaccharide (LPS)-induced ALF is a widely used animal model to identify novel hepato-protective agents. In the present study, we investigated the potential of a cannabinoid receptor 2 (CB2) agonist, JWH-133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran], in the amelioration of GalN/LPS-induced ALF. JWH-133 treatment protected the mice from ALF-associated mortality, mitigated alanine transaminase and proinflammatory cytokines, suppressed histopathological and apoptotic liver damage, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, JWH-133 pretreatment of M1/M2-polarized macrophages significantly increased the secretion of anti-inflammatory cytokine interleukin-10 (IL-10) in M1 macrophages and potentiated the expression of M2 markers in M2-polarized macrophages. In vivo, JWH-133 treatment also suppressed ALF-triggered expression of M1 markers in liver MNCs, while increasing the expression of M2 markers such as Arg1 and IL-10. microRNA (miR) microarray analysis revealed that JWH-133 treatment altered the expression of only a few miRs in the liver MNCs. Gene ontology analysis of the targets of miRs suggested that Toll-like receptor (TLR) signaling was among the most significantly targeted cellular pathways. Among the altered miRs, miR-145 was found to be the most significantly decreased. This finding correlated with concurrent upregulated expression of its predicted target gene, interleukin-1 receptor-associated kinase 3, a negative regulator of TLR4 signaling. Together, these data are the first to demonstrate that CB2 activation attenuates GalN/LPS-induced ALF by inducing an M1 to M2 shift in macrophages and by regulating the expression of unique miRs that target key molecules involved in the TLR4 pathway.

Spinal Cord is the Primary Site of Action of the Cannabinoid CB2 Receptor Agonist JWH133 that Suppresses Neuropathic Pain: Possible Involvement of Microglia

Neuropathic pain, a highly debilitating condition that commonly occurs after damage to the nervous system, is often resistant to commonly used analgesic agents such as non-steroidal anti-inflammatory drugs and even opioids.Several studies using rodent models reported that cannabinoid CB2 receptor (CB2R) agonists are effective for treating chronic pain. However, the analgesic mechanism of CB2R agonists in neuropathic pain states is not fully understood. In this study, we investigated the role of CB2Rs in the development and maintenance phases of neuropathic pain, and the mechanism of the CB2R-mediated analgesic effect on neuropathic pain. In a rat model of neuropathic pain, systemic administration of JWH133, a CB2R agonist, markedly improved tactile allodynia, and this effect was prevented by intrathecal pretreatment with AM630, a CB2R antagonist. The antiallodynic effect of intrathecally administered JWH133 was inhibited by intrathecal pretreatment with pertussis toxin or forskolin. In the spinal cord, CB2R expression was significantly increased on post-operative day 3, and persisted for 2 weeks. Furthermore, repeated intrathecal administration of JWH133 notably attenuated the development of tactile allodynia after peripheral nerve injury. In a culture of microglia activated by overexpressing interferon regulatory factor 8, a transcription factor crucial for neuropathic pain, JWH133 treatment suppressed the increased expression of interleukin-1β. Our findings suggest that activation of CB2Rs upregulated in the spinal cord after nerve injury alleviates existing tactile allodynia through the Gi/oadenylate cyclase signaling pathway and suppresses the development of allodynia. This process may reduce the inflammatory response of microglia. Therefore, spinal CB2Rs may be a therapeutic target for the treatment of neuropathic pain.

Cannabinoid receptor type 2 agonist attenuates apoptosis by activation of phosphorylated CREB–Bcl-2 pathway after subarachnoid hemorrhage in rats

Early brain injury (EBI) which comprises of vasogenic edema and apoptotic cell death is an important component of subarachnoid hemorrhage (SAH) pathophysiology. This study evaluated whether cannabinoid receptor type 2 (CB2R) agonist, JWH133, attenuates EBI after SAH and whether CB2R stimulation reduces pro-apoptotic caspase-3 via up-regulation of cAMP response element-binding protein (CREB)-Bcl-2 signaling pathway. Male Sprague–Dawley rats (n=123) were subjected to SAH by endovascular perforation. Rats received vehicle or JWH133 at 1h after SAH. Neurological deficits and brain water content were evaluated at 24h after SAH. Western blot was performed to quantify phosphorylated CREB (pCREB), Bcl-2, and cleaved caspase-3 levels. Neuronal cell death was evaluated with terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling staining. Additionally, CREB siRNA was administered to manipulate the proposed pathway. JWH133 (1.0mg/kg) improved neurological deficits and reduced brain water content in left hemisphere 24h after SAH. JWH133 significantly increased activated CREB (pCREB) and Bcl-2 levels and significantly decreased cleaved caspase-3 levels in left hemisphere 24h after SAH. CREB siRNA reversed the effects of treatment. TUNEL positive neurons in the cortex were reduced with JWH133 treatment. Thus, CB2R stimulation attenuated EBI after SAH possibly through activation of pCREB–Bcl-2 pathway.

Long-term cannabinoid type 2 receptor agonist therapy decreases bacterial translocation in rats with cirrhosis and ascites

Intestinal hyperpermeability, impaired peritoneal macrophages (PMs) phagocytosis, and bacterial translocation (BT), resulting in increased systemic and local infection/inflammation such as spontaneous bacterial peritonitis (SBP) together with increased tumor necrosis factor-α (TNFα) levels, are all implicated in the pathogenesis of cirrhosis-related complications. Manipulation of the cannabinoid receptors (CB1R and CB2R), which are expressed on the gut mucosa and PMs, has been reported to modulate intestinal inflammation and systemic inflammatory cytokine release. Our study aims to explore the effects of chronic CB1R/CB2R agonist/antagonist treatments on relevant abnormalities in cirrhotic ascitic rats. Vehicle, archidonyl-2-chloroethylamide (ACEA, CB1R agonist), JWH133 (CB2R agonist), and AM630 (CB2R antagonist) were given to thioacetamide (TAA) and common bile duct ligation (BDL) cirrhotic rats with ascites for two weeks and various measurement were performed. Compared to sham rats, CB2Rs were downregulated in cirrhotic rat intestines and PMs. The two-week JWH133 treatment significantly decreased systemic/intestinal oxidative stress, TNFα and inflammatory mediators, infection, intestinal mucosal damage and hyperpermeability; the JWH133 treatment also decreased bacterial overgrowth/adhesion, BT and SBP, upregulated intestinal tight junctions and downregulated the PM TNFα receptor/NFκBp65 protein expression in cirrhotic rats. Acute and chronic JWH133 treatment corrected the TNFα-induced suppression of phagocytosis of cirrhotic rat PMs, which then could be reversed by concomitant AM630 treatment. Our study suggests that CB2R agonists have the potential to treat BT and various relevant abnormalities through inhibition of systemic/intestinal oxidative stress, inflammatory cytokines and TNFα release in cirrhosis. Overall, the chronic CB2R agonist treatment affects multiple approach mechanisms, and its direct effect on the hyperdynamic circulation is only minor.

Regulatory role of the cannabinoid CB2 receptor in stress‐induced neuroinflammation in mice

Stress exposure produces excitotoxicity and neuroinflammation, contributing to the cellular damage observed in stress-related neuropathologies. The endocannabinoids provide a homeostatic system, present in stress-responsive neural circuits. Here, we have assessed the possible regulatory role of cannabinoid CB2 receptors in stress-induced excitotoxicity and neuroinflammation. We used wild type (WT), transgenic overexpressing CB2 receptors (CB2xP) and CB2 receptor knockout (CB2-KO) mice exposed to immobilization and acoustic stress (2 h·day(-1) for 4 days). The CB2 receptor agonist JWH-133 was administered daily (2 mg·kg(-1), i.p.) to WT and CB2-KO animals. Glutamate uptake was measured in synaptosomes from frontal cortex; Western blots and RT-PCR were used to measure proinflammatory cytokines, enzymes and mediators in homogenates of frontal cortex. Increased plasma corticosterone induced by stress was not modified by manipulating CB2 receptors. JWH-133 treatment or overexpression of CB2 receptors increased control levels of glutamate uptake, which were reduced by stress back to control levels. JWH-133 prevented the stress-induced increase in proinflammatory cytokines (TNF-α and CCL2), in NF-κB, and in NOS-2 and COX-2 and in the consequent cellular oxidative and nitrosative damage (lipid peroxidation). CB2xP mice exhibited anti-inflammatory or neuroprotective actions similar to those in JWH-133 pretreated animals. Conversely, lack of CB2 receptors (CB2-KO mice) exacerbated stress-induced neuroinflammatory responses and confirmed that effects of JWH-133 were mediated through CB2 receptors. Pharmacological manipulation of CB2 receptors is a potential therapeutic strategy for the treatment of stress-related pathologies with a neuroinflammatory component, such as depression.

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-β1 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-β1 up-regulation and E-selectin reduction, resulting in protection of the BBB after SAH.