Single nucleotide polymorphisms in the cannabinoid CB2 receptor: Molecular pharmacology and disease associations.

Preclinical evidence implicating cannabinoid receptor 2 (CB2) in various diseases has led researchers to question whether CB2 genetics influence aetiology or progression. Associations between conditions and genetic loci are often studied via single nucleotide polymorphism (SNP) prevalence in case versus control populations. In the CNR2 coding exon, ~36 SNPs have high overall population prevalence (minor allele frequencies [MAF] ~37%), including non-synonymous SNP (ns-SNP) rs2501432 encoding CB2 63Q/R. Interspersed are ~27 lower frequency SNPs, four being ns-SNPs. CNR2 introns also harbour numerous SNPs. This review summarises CB2 ns-SNP molecular pharmacology and evaluates evidence from ~70 studies investigating CB2 genetic variants with proposed linkage to disease. Although CNR2 genetic variation has been associated with a wide variety of conditions, including osteoporosis, immune-related disorders, and mental illnesses, further work is required to robustly validate CNR2 disease links and clarify specific mechanisms linking CNR2 genetic variation to disease pathophysiology and potential drug responses.

Cannabidiol Alleviates Chronic Prostatitis and Chronic Pelvic Pain Syndrome via CB2 Receptor Activation and TRPV1 Desensitization.

PURPOSE: This study elucidates the mechanism of the physiological effect of cannabidiol (CBD) by assessing its impact on lipopolysaccharide (LPS)-induced inflammation in RWPE-1 cells and prostatitis-induced by 17ß-estradiol and dihydrotestosterone in a rat model, focusing on its therapeutic potential for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). MATERIALS AND METHODS: RWPE-1 cells were stratified in vitro into three groups: (1) controls, (2) cells with LPS-induced inflammation, and (3) cells with LPS-induced inflammation and treated with CBD. Enzyme-linked immunosorbent assays and western blots were performed on cellular components and supernatants after administration of CBD. Five groups of six Sprague-Dawley male rats were assigned: (1) control, (2) CP/CPPS, (3) CP/CPPS and treated with 50 mg/kg CBD, (4) CP/CPPS and treated with 100 mg/kg CBD, and (5) CP/CPPS and treated with 150 mg/kg CBD. Prostatitis was induced through administration of 17ß-estradiol and dihydrotestosterone. After four weeks of CBD treatment, a pain index was evaluated, and prostate tissue was collected for subsequent histologic examination and western blot analysis. RESULTS: CBD demonstrated efficacy in vivo for CP/CPPS and in vitro for inflammation. It inhibited the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway by activating the CB2 receptor, reducing expression of interleukin-6, tumor necrosis factor-alpha, and cyclooxygenase-2 (COX2) (p<0.01). CBD exhibited analgesic effects by activating and desensitizing the TRPV1 receptor. CONCLUSIONS: CBD inhibits the TLR4/NF-κB pathway by activating the CB2 receptor, desensitizes the TRPV1 receptor, and decreases the release of COX2. This results in relief of inflammation and pain in patients with CP/CPPS, indicating CBD as a potential treatment for CP/CPPS.

Cannabinoid CB2 receptor orthologues; in vitro function and perspectives for preclinical to clinical translation.

Cannabinoid CB2 receptor agonists are in development as therapeutic agents, including for immune modulation and pain relief. Despite promising results in rodent preclinical studies, efficacy in human clinical trials has been marginal to date. Fundamental differences in ligand engagement and signalling responses between the human CB2 receptor and preclinical model species orthologues may contribute to mismatches in functional outcomes. This is a tangible possibility for the CB2 receptor in that there is a relatively large degree of primary amino acid sequence divergence between human and rodent. Here, we summarise CB2 receptor gene and protein structure, assess comparative molecular pharmacology between CB2 receptor orthologues, and review the current status of preclinical to clinical translation for drugs targeted at the CB2 receptor, focusing on comparisons between human, mouse and rat receptors. We hope that raising wider awareness of, and proposing strategies to address, this additional challenge in drug development will assist in ongoing efforts toward successful therapeutic translation of drugs targeted at the CB2 receptor.

Endocannabinoid System: Chemical Characteristics and Biological Activity.

The endocannabinoid system (eCB) has been studied to identify the molecular structures present in Cannabis sativa. eCB consists of cannabinoid receptors, endogenous ligands, and the associated enzymatic apparatus responsible for maintaining energy homeostasis and cognitive processes. Several physiological effects of cannabinoids are exerted through interactions with various receptors, such as CB1 and CB2 receptors, vanilloid receptors, and the recently discovered G-protein-coupled receptors (GPR55, GPR3, GPR6, GPR12, and GPR19). Anandamide (AEA) and 2-arachidoylglycerol (2-AG), two small lipids derived from arachidonic acid, showed high-affinity binding to both CB1 and CB2 receptors. eCB plays a critical role in chronic pain and mood disorders and has been extensively studied because of its wide therapeutic potential and because it is a promising target for the development of new drugs. Phytocannabinoids and synthetic cannabinoids have shown varied affinities for eCB and are relevant to the treatment of several neurological diseases. This review provides a description of eCB components and discusses how phytocannabinoids and other exogenous compounds may regulate the eCB balance. Furthermore, we show the hypo- or hyperfunctionality of eCB in the body and how eCB is related to chronic pain and mood disorders, even with integrative and complementary health practices (ICHP) harmonizing the eCB.

Tumor necrosis factor α, and agonist and antagonists of cannabinoid receptor type 1 and type 2 alter the immunophenotype of stem cells from human exfoliated deciduous teeth

ABSTRACT Objective To verify the involvement of the endocannabinoid system in the immunomodulatory profile of stem cells from human exfoliated deciduous teeth, in the presence or absence of TNF-α, and agonist and antagonists of CB1 and CB2. Methods Stem cells from human exfoliated deciduous teeth were cultured in the presence or absence of an agonist, anandamide, and two antagonists, AM251 and SR144528, of CB1 and CB2 receptors, with or without TNF-α stimulation. For analysis of immunomodulation, surface molecules linked to immunomodulation, namely human leukocyte antigen-DR isotype (HLA-DR), and programmed death ligands 1 (PD-L1) and 2 (PD-L2) were measured using flow cytometry. Results The inhibition of endocannabinoid receptors together with the proinflammatory effect of TNF-α resulted in increased HLA-DR expression in stem cells from human exfoliated deciduous teeth, as well as, in these cells acquiring an anti-inflammatory profile by enhancing the expression of PD-L1 and PD-L2. Conclusion Stem cells from human exfoliated deciduous teeth respond to the endocannabinoid system and TNF-α by altering key immune response molecules.

Mechanism underlying reduction of cerebral ischemia-reperfusion injury by electroacupuncture preconditioning in rats: relationship between cannabinoid type 1 receptor and NLRP3 inflammasome / 中华麻醉学杂志

Objective:To evaluate the relationship between cannabinoid receptor 1 (CB1R) and the NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) during the reduction of cerebral ischemia-reperfusion (I/R) injury by electroacupuncture (EA) preconditioning in rats.Methods:Forty SPF healthy male Sprague-Dawley rats, aged 7-9 weeks, weighing 250-280 g, were divided into 5 groups ( n=8 each) according to the random number table method: sham operation group (Sham group), cerebral I/R group (I/R group), EA preconditioning group (EA group), CB1R antagonist AM251+ EA preconditioning group (AM251+ EA group), and CB1R agonist WIN 55, 212-2 group (WIN group). Cerebral I/R was induced by middle cerebral artery occlusion (MCAO) in anesthetized animals. In EA group, EA preconditioning was performed, and the acupoint Baihui (GV20) was stimulated for 30 min with disperse-dense waves, the intensity of 1 mA and frequency of 2/15 Hz once a day for 5 consecutive days, and the model of cerebral I/R injury was developed at 24 h after the last EA. In AM251+ EA group, CB1R antagonist AM251 1 mg/kg was intraperitoneally injected at 30 min before each stimulation, and the remaining operations were the same as those previously described in EA group. CB1R agonist WIN 55, 212-2 1.5 mg/kg was intraperitoneally injected for 5 consecutive days, and the model of cerebral I/R injury was prepared at 24 h after the last injection in WIN group. Neurological behavior was assessed and scored at 3 days of reperfusion. Then the rats were sacrificed, and brains were removed, and the infarct volume was measured by TTC staining, and the tissues in the ischemic penumbra were extracted for determination of the expression of NLRP3, caspase-1 and interleukin-1bata (IL-1β) by Western blot. Results:Compared with Sham group, the percentage of cerebral infarct volume was significantly increased, the neurobehavioral score was decreased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in I/R group ( P<0.05). Compared with I/R group, the percentage of cerebral infarct volume was significantly decreased, the neurobehavioral score was increased, and the expression of NLRP3, caspase-1 and IL-1β was down-regulated in EA and WIN groups ( P<0.05). Compared with EA group, the percentage of cerebral infarct volume was significantly increased, the neurobehavioral score was decreased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in AM251+ EA group ( P<0.05). Conclusions:EA preconditioning may inhibit the activation of NLRP3 inflammasomes by activating CB1R, thus alleviating cerebral I/R injury in rats.

Role of cannabinoid type 2 receptor in sevoflurane postconditioning-induced reduction of intestinal ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective:To evaluate the role of cannabinoid type 2 receptor (CB2R) in sevoflurane postconditioning-induced reduction of intestinal ischemia-reperfusion (I/R) injury in rats.Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 220-270 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (group Sham), intestinal I/R group (group I/R), intestinal I/R+ sevoflurane postconditioning group (group Sevo) and intestinal I/R+ sevoflurane postconditioning+ CB2R antagonist AM630 group (group AM). The model of intestinal I/R injury was established by occluding the superior mesenteric artery for 45 min followed by 2 h reperfusion.In the group Sevo, 2% sevoflurane was inhaled immediately at the beginning of reperfusion for 30 min.In the group AM, CB2R antagonist AM630 3 mg/kg was intraperitoneally injected at 1 h before ischemia, and the other treatments were similar to those previously described in group Sevo.At 2 h of reperfusion, the animals were sacrificed after anesthesia, and small intestinal tissues were obtained for microscopic examination of the pathologic changes which was scored according to Chiu and for determination of wet/dry weight ratio (W/D ratio), for detection of malondialdehyde (MDA) content (by thiobarbituric acid colorimetry), for determination of myeloperoxidase (MPO) activity (by MPO assay) and cleaved caspase-3 protein expression (by Western blot). Results:Compared with group Sham, the Chui score, W/D ratio, MDA content and MPO activity in the intestinal tissues were significantly increased, cleaved caspase-3 expression was up-regulated in group I/R ( P<0.05). Compared with group I/R, the Chui score, W/D ratio, MDA content and MPO activity in the intestinal tissues were significantly decreased, cleaved caspase-3 expression was down-regulated in group Sevo ( P<0.05). Compared with group Sevo, the Chui score, W/D ratio, MDA content and MPO activity were significantly increased, cleaved caspase-3 expression was up-regulated in group AM ( P<0.05). Conclusion:CB2R is involved in the process of sevoflurane postconditioning-induced reduction of intestinal I/R injury in rats.

Relationship between over-expression of endocannabinoid receptor 2 and macrophage pyroptosis in mice / 中华麻醉学杂志

Objective:To evaluate the relationship between the over-expression of endocannabinoid receptor 2 (CB2R) and macrophage pyroptosis in mice.Methods:Bone marrow-derived macrophages of mice were transfected by lentivirus vector and successfully screened out two stable cell lines: lentivirus LV5 negative control cells (LV5-NC) and lentivirus LV5CB2R overexpressing cells (OE). Two cell lines were respectively divided into 3 groups ( n=18 each) using a random number table method: control group (LV5-NC-C group, OE-C group), LPS/ATP group (LV5-NC-LPS/ATP group, OE-LPS/ATP group) and CB2R specific agonist HU308 group (LV5-NC-HU308 group, OE-HU308 group). Cells in group C were commonly cultured.In LPS/ATP group, cells were incubated with LPS at a final concentration of 0.5 μg/ml for 5 h, and then incubated with ATP at the final concentration of 5 mmol/L for 1 h. In group LPS/ATP+ HU308, cells were incubated for 5 h with LPS at the final concentration of 0.5 μg/ml and HU308 at the final concentration of 10 μmol/L and then with ATP at the final concentration of 5 mmol/L for 1 h. The expression of CB2R, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) mRNA was detected by real-time polymerase chain reaction, the expression of caspase-1 was detected by Western blot, and the concentrations of interleukin-18 (IL-18) and IL-1β in the culture medium were determined by enzyme-linked immunosorbent assay. Results:In each cell line, compared with group C, the expression of NLRP3, caspase-1 and GSDMD was significantly up-regulated, and the concentrations of IL-18 and IL-1β were increased in group LPS/ATP ( P<0.05). Compared with group LPS/ATP, the expression of NLRP3, caspase-1 and GSDMD was significantly down-regulated, the concentrations of IL-18 and IL-lβ were decreased in group HU308 ( P<0.05). There was no significant differences in the indicators mentioned above between group V5-NC-C and group OE-C, between group LV5-NC-LPS/ATP and group OE-LPS/ATP, and between group LV5-NC-HU308 and OE-HU308 ( P>0.05). Conclusion:Over-expression of CB2R gene cannot effectively inhibit the occurrence of macrophage pyroptosis, and only activation of CB2R can inhibit it in mice.

Progress in research on the role of cannabinoid receptor 1 signaling pathway in epilepsy / 中华神经科杂志

Epilepsy,a group of chronic neurological disorders characterized by spontaneous and recurrent seizures and learning and memory impairments,results in transient brain dysfunction due to sudden abnormal discharge of brain neurons.The pathogenesis of epilepsy is very complicated and has not yet been fully elucidated.The imbalance between excitatory glutamate and inhibitory gamma-aminobutyric acid (GABA) neurotransmitters in the central nervous system and changes in ionic functions of N-methyl-D-aspartate receptors directly induce epileptic seizures.The endocannabinoid system plays an important role in retrograde synaptic transmission and exerts the anti-epileptic effect in cannabinoid receptor 1 (CBR1) dependent manner by regulating the synaptic transmission of glutamatergic and GABAergic neurons and homeostatsis of ionic channel function.Elucidating the specific mechanism of action of CBR1 signaling pathway in epilepsy,can provide an effective theoretical basis and novel drug's target for clinical treatment of epilepsy.

Role of EAAT2 in cannabinoid receptor 2 activation-induced attenuation of microglial injury caused by glutamate / 中华麻醉学杂志

Objective To evaluate the role of excitatory amino acid transporter 2 ( EAAT2) in can-nabinoid receptor 2 ( CB2 receptor) activation-induced attenuation of microglial injury caused by glutamate. Methods N9 microglial cells were divided into 4 groups ( n=26 each) using a random number table meth-od: control group ( group Con) , glutamate group ( group Glu) , CB2 receptor agonist AM1241 plus gluta-mate group (group AM1241+Glu) and AM1241 plus EAAT inhibitor TBOA plus glutamate group (group AM1241+TBOA+Glu) . The cells were routinely cultured for 30 h in group Con. In group Glu, the cells were routinely cultured for 6 h, and then were incubated for 24 h in the culture medium containing gluta-mate 10 mmol∕L. In group AM1241+Glu, the cells were incubated for 4 h in the culture medium containing AM12412 μmol∕L, and then were routinely cultured for 2 h, and then were incubated for 24 h in the cul-ture medium containing glutamate 10 mmol∕L. In group AM1241+TBOA+Glu, the cells were incubated for 4 h in the culture medium containing AM12412 μmol∕L and TBOA 100 μmol∕L, and then were routinely cultured for 2 h, and then were incubated for 24 h in the culture medium containing glutamate 10 mmol∕L. The cell viability was measured by MTT assay, the activity of lactic dehydrogenase ( LDH) in supernatant was determined using colorimetric method, and the expression of EAAT2 was determined by Western blot. Results Compared with group Con, the cell viability was significantly decreased and LDH activity was in-creased in Glu, AM1241+Glu and AM1241+TBOA+Glu groups, and the expression of EAAT2 was signifi-cantly up-regulated in Glu and AM1241+Glu groups ( P<0. 05) . Compared with group Glu, the cell viabil-ity was significantly increased, LDH activity was decreased, and the expression of EAAT2 was up-regulated in group AM1241+Glu ( P<0. 05) , and no significant change was found in the parameters mentioned above in group AM1241+TBOA+Glu ( P>0. 05) . Compared with group AM1241+Glu, the cell viability was sig-nificantly decreased, LDH activity was increased, and the expression of EAAT2 was down-regulated in group AM1241+TBOA+Glu ( P<0. 05) . Conclusion The mechanism by which the activation of CB2 re-ceptor attenuates microglial injury caused by glutamate is related to up-regulating the expression of EAAT2.

Role of hippocampal cannabinoid receptor 2 in postoperative cognitive dysfunction in mice / 中华麻醉学杂志

Objective To investigate the role of hippocampal cannabinoid receptor 2 (CB2R) in postoperative cognitive dysfunction (POCD) in mice.Methods Fifty-four healthy male C57BL/6 mice,aged 12-16 weeks,weighing 20-30 g,were divided into 3 groups (n=18 each) using a random number table:control group (group C),group POCD and POCD plus CB2R agonist JWH133 group (group POCD+J).The mice were subjected to an intramedullary fixation for tibial fracture under 1.5％ isoflurane anesthesia to establish POCD model.In group POCD+J,2 mg/kg JWH133 was injected intraperitoneally at a total volume of 10 ml/kg after emergence from anesthesia and then the injection was repeated once a day until the 7th day after surgery.Open field test was carried out on 1 day before surgery and 1,3 and 7 days after surgery to evaluate the locomotor activity.Fear conditioning test was carried out at 15 min after completion of open field test.Mice were sacrificed at 2 h after the end of behavioral test,and the hippocampus was harvested for determination of the expression of tumor necrosis factor-alpha (TNF-α),interleukin-1beta (IL-1β),monocyte chemotactic protein-1 (MCP-1) and CB2R (by Western blot) and expression of CD11b in hippocampal tissues (by immunofluorescence).Results There was no significant difference in the total exploring distance in open field test,percentage of freezing time in contextual fear conditioning test or percentage of freezing time in tone-fear conditioning text at each time point among the three groups (P＞0.05).Compared with group C,the percentage of freezing time in contextual fear conditioning test was significantly decreased,and the expression of TNF-α,IL-1β,MCP-1,CD11b and CB2R was up-regulated after surgery in group S (P＜0.05).Compared with group S,the percentage of freezing time in contextual fear conditioning test was significantly increased,and the expression of TNF-α,IL-1β,MCP-1,CD11band CB2R was down-regulated after surgery in group S+J (P＜ 0.05).Conclusion Activation of hippocampal CB2R may be involved in the endogenous protective mechanism of POCD in mice,and the mechanism is related to inhibiting activation of hippocampal microglia and attenuating central inflammatory responses.

Effect of activating cannabinoid receptor 2 on sepsis-induced acute lung injury in mice: the role of autophagy / 中华麻醉学杂志

Objective To evaluate the effect of activating cannabinoid receptor 2 (CB2R) on sepsis-induced acute lung injury and the role of autophagy in mice.Methods Twenty-four SPF male C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups (n=6 each) using a random number table method:sham operation group (group Sham),sepsis group (group Sep),sepsis plus CB2R agonist HU308 group (group Sep+HU308) and sepsis plus HU308 plus autophagy inhibitor 3-methyladenine group (group Sep+HU308+3-MA).Sepsis was induced by cecal ligation and puncture in anesthetized mice.HU308 2.5 mg/kg was intraperitoneally injected at 15 min after surgery in Sep+HU308 and Sep+ HU308+3-MA groups,and 15 min later 3-MA 10 mg/kg was intraperitoneally injected in group Sep+ HU308+3-MA.Lung tissues were obtained at 12 h after surgery and stained with haematoxylin and eosin for examination of the pathological changes which were scored and for determination of the expression of tumornecrosis factor-alpha (TNF-α),interleukin-18 (IL-18) and IL-1β mRNA (by real-time polymerase chain reaction),expression of autophagy-related protein 5 (Atg5) (by immuno-histochemistry),and expression of microtubule-associated protein 1 light chain 3 (LC3),Beclin-1 and p62 (by Western blot).The ratio of LC3Ⅱ to LC3Ⅰ (LC3Ⅱ/LC3Ⅰ ratio) was calculated.Results Compared with group Sham,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,and LC3 Ⅱ/LC3 Ⅰ ratio and lung injury score were increased in the other three groups,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep and group Sep+HU308,and the expression of p62 was significantly up-regulated in group Sep+HU308+3-MA (P＜0.05).Compared with group Sep,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly down-regulated,the expression of Atg5 was up-regulated,and lung injury score was decreased in group Sep+ HU308 and group Sep+ HU308 + 3-MA,LC3Ⅱ/LC3Ⅰ ratio was increased,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep+HU308,and the expression of Beclin-1 was down-regulated,and the expression of p62 was up-regulated in group Sep + HU308 + 3-MA (P ＜ 0.05).Compared with group Sep+ HU308,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,the expression of Atg5 and Beclin-1 was down-regulated,LC3Ⅱ/LC3Ⅰ ratio was decreased,the expression of p62 was up-regulated,and lung injury scores were increased in group Sep+HU308+3-MA (P＜0.05).Conclusion Activating CB2R can alleviate acute lung injury in septic mice,and the mechanism may be partially related to enhancing autophagy and reducing inflammatory responses.

Epigenetic Interactions between Alcohol and Cannabinergic Effects: Focus on Histone Modification and DNA Methylation.

Epigenetic studies have led to a more profound understanding of the mechanisms involved in chronic conditions. In the case of alcohol addiction, according to the National Institute on Alcohol Abuse and Alcoholism, 16 million adults suffer from Alcohol Use Disorders (AUDs). Even though therapeutic interventions like behavioral therapy and medications to prevent relapse are currently available, no robust cure exists, which stems from the lack of understanding the mechanisms of action of alcohol and the lack of development of precision medicine approaches to treat AUDs. Another common group of addictive substance, cannabinoids, have been studied extensively to reveal they work through cannabinoid receptors. Therapeutic applications have been found for the cannabinoids and a deeper understanding of the endocannabinoid system has been gained over the years. Recent reports of cannabinergic mechanisms in AUDs has opened an exciting realm of research that seeks to elucidate the molecular mechanisms of alcohol-induced end organ diseases and hopefully provide insight into new therapeutic strategies for the treatment of AUDs. To date, several epigenetic mechanisms have been associated with alcohol and cannabinoids independently. Therefore, the scope of this review is to compile the most recent literature regarding alcohol and cannabinoids in terms of a possible epigenetic connection between the endocannabinoid system and alcohol effects. First, we will provide an overview of epigenetics, followed by an overview of alcohol and epigenetic mechanisms with an emphasis on histone modifications and DNA methylations. Then, we will provide an overview of cannabinoids and epigenetic mechanisms. Lastly, we will discuss evidence of interactions between alcohol and cannabinergic pathways and possible insights into the novel epigenetic mechanisms underlying alcohol-cannabinergic pathway activity. Finalizing the review will be a discussion of future directions and therapeutic applications.

Effect of N-arachidonoyl-l-serine on human cerebromicrovascular endothelium.

N-arachidonoyl-l-serine (ARA-S) is an endogenous lipid, chemically related to the endocannabinoid, N-arachidonoyl ethanolamine (i.e., anandamide) and with similar physiologic and pathophysiologic functions. Reports indicate that ARA-S possesses vasoactive and neuroprotective properties resembling those of cannabinoids. However, in contrast to cannabinoids, ARA-S binds weakly to its known classical receptors, CB1 and CB2, and is therefore considered to be a 'cannabinoid-like' substance. The originally described ARA-S induced-endothelial-dependent vasorelaxation was not abrogated by CB1, CB2 receptor antagonists or TRPV1 competitive inhibitor. The present report demonstrates that ARA-S enhances the fluorescence staining of both cannabinoid receptors (CB1 and CB2) in human brain endothelial cells (HBEC). This reaction is specific since it was reduced by respective selective receptor antagonist (SR141716A and SR141728A). ARA-S alone or in the presence of ET-1 was shown to alter the cytoskeleton (actin). Both ARA-S stimulated phosphorylation of various kinases (MAPK, Akt, JNK and c-JUN) and alteration of cytoskeleton are mediated via CB1, CB2 and TRPV1 receptors. The findings also showed the involvement of Rho/Rock and PI3/Akt/NO pathways in the ARA-S-induced phosphorylation of kinases and actin reorganization in HBEC. All of the above mentioned ARA-S-induced effects were reduced by the treatment with LY294002 (inhibitor of PI3/Akt kinase), except MAPK kinase. In addition, MAPK, JNK, c-JUN phosphorylation were inhibited by H1152 (inhibitor of Rho/ROCK kinase), except Akt kinase. Furthermore, PI3/Akt pathway was inhibited by pretreatment with l-NAME (inhibitor of NOS). The findings suggest that ARA-S is a modulator of Rho kinase and may play a critical role in the regulation of its activity and subsequent effects on the cytoskeleton and its role in supporting essential cell functions like vasodilation, proliferation and movement.

Effects of Rimonabant on Cannabinoid Receptor 1 andα-Smooth Muscle Actin in C57 Mice with Experimental Hepatic Fibrosis / 天津医药

Objective To study the effect of rimonabant, cannabinoid receptor 1(CB1) antagonist, on the expressions of CB1 andα-smooth muscle actin (α-SMA) in C57 mice with experimental hepatic fibrosis, and their mechanisms in liver fibrosis progression thereof. Methods Thirty C57 mice were randomly divided into three groups, normal control group, mod-el control group and model+rimonabant group, 10 mice for each group. The mouse model of experimental hepatic fibrosis was induced by intraperitoneal injection with 10%CCl4 for two weeks. The normal saline was delivered by gavage daily in normal control group and model control group. Rimonabant was given to mice in model+rimonabant group. Mice were sacri-ficed at the end of eight weeks. Samples of liver tissue were collected. The expressions of CB1 andα-SMA in liver tissue of mice were observed by immunohistochemical staining. The score of fibrosis stage (S) in liver tissue was also analyzed. Re-sults The positive expressions of CB1 andα-SMA and the score S were significantly higher in model control group and model+rimonabant group than those in normal control group (P<0.05). The positive expressions of CB1 andα-SMA and the score S were significantly lower in rimonabant group than those in model control group (P<0.05). There were positive corre-lations in CB1,α-SMA and S scores between normal control group, model control group and model+rimonabant group (P<0.05). Conclusion The activation of CB1 can promote the formation of liver fibrosis. The anti-fibrotic effect of rimonabant, CB1 antagonist, related with the inhibiting of the proliferation and activation of hepatic stellate cells (HSC), and the inhibit-ing of the expression of CB1.

Role of cannabinoid receptor 2 in microglial injury induced by glutamate / 中华麻醉学杂志

ObjectlveTo evaluate the role of cannabinoid receptor 2 (CB2 receptor) in microglial injury induced by glutamate.MethodsMicroglia cells were randomly divided into 4 grups:control group (group C),microglial injury group ( group Ⅰ),specific CB2 receptor agonist AM 1241 group ( group AM1241 ) and specific CB2 receptor antagonist AM630 group (group AM630).In group C,the cells were cultured routinely for 26 h.In group Ⅰ,the cells were incubated in the culture medium containing glutamate 10 mmol/L for 24 h.In group AM1241,the cells were incubated in the culture medium containing AM1241 2 μmol/L for 2 h,and then in the culture medium containing glutamate 10 mmol/L for 24 h.In group AM630,the cells were incubated in the culture medium containing AM630 2 μmol/L for 2 h,and then in the culture medium containing glutamate 10 mmol/L for 24 h.The cell viability and release of LDH were measured.Microglial morphology was observed under microscope.Results Compared with group C,the cell viability was significantly decreased,and the release of LDH was significantly increased in groups Ⅰ,AM1241 and AM630 (P ＜ 0.05).Compared with group Ⅰ,the cell viability was significantly increased,and the release of LDH was significantly decreased in group AM1241 ( P ＜ 0.05).There was no significant difference in the cell viability and the release of LDH between groups 1 and AM630 ( P ＞ 0.05).Conclusion Glutamate induces microglial injury through inhibiting the function of CB2 receptor.

The role of peripheral cannabinoid receptors type 1 in rats with visceral hypersensitivity induced by chronic restraint stress.

BACKGROUND/AIMS: This study was designed to investigate the possibility that the enhanced nociceptive responsiveness associated with canabonoid type 1 receptors (CB1Rs) and identify its role in mediating visceral hypersensitivity induced by chronic restraint stress. METHODS: Rats were exposed to daily partial restraint stress or sham partial restraint stress with intraperitoneal injection of the vehicle, CB1R agonist or antagonist for 4 consecutive days. We tested the visceromotor reflex to colorectal distention at day 0 and 5. Reverse-transcription polymerase chain reaction and Western blot were used to assess the expression of CB1Rs. RESULTS: Intraperitoneal CB1 agonist (ACEA) injection significantly diminished (p < 0.05) the enhanced visceromotor reflex to colorectal distention at day 5 in stressed rats. Change in electromyogram response after ACEA over baseline, at pressure of 40 mmHg (+13.3 +/- 2.2), 60 mmHg (+15.3 +/- 2.8) and 80 mmHg (+17.0 +/- 4.0) were much lower than in the control animals, which were +35.9 +/- 5.1, +41.1 +/- 6.3 and +54.1 +/- 9.6, respectively. Whereas, CB1 antagonist (SR141716A) had an opposite effect. Compared with control group, the change in electromyogram response after SR141716A over baseline was significantly enhanced (p < 0.05) for the distending pressure of 40 mmHg (+56.0 +/- 10.3), 60 mmHg (+74.6 +/- 12.3) and 80 mmHg (+82.9 +/- 11.0), respectively. Reverse-transcription polymerase chain reaction and Western blotting demonstrated the stress-induced up-regulation of colon CB1Rs (p < 0.05). CONCLUSIONS: Our results suggest there is a key contribution of peripheral CB1Rs involved in the maintenance of visceral hyperalgesia after repeated restraint stress, providing a novel mechanism for development of peripheral visceral sensitization.

The Role of Peripheral Cannabinoid Receptors Type 1 in Rats With Visceral Hypersensitivity Induced by Chronic Restraint Stress

BACKGROUND/AIMS: This study was designed to investigate the possibility that the enhanced nociceptive responsiveness associated with canabonoid type 1 receptors (CB1Rs) and identify its role in mediating visceral hypersensitivity induced by chronic restraint stress. METHODS: Rats were exposed to daily partial restraint stress or sham partial restraint stress with intraperitoneal injection of the vehicle, CB1R agonist or antagonist for 4 consecutive days. We tested the visceromotor reflex to colorectal distention at day 0 and 5. Reverse-transcription polymerase chain reaction and Western blot were used to assess the expression of CB1Rs. RESULTS: Intraperitoneal CB1 agonist (ACEA) injection significantly diminished (p < 0.05) the enhanced visceromotor reflex to colorectal distention at day 5 in stressed rats. Change in electromyogram response after ACEA over baseline, at pressure of 40 mmHg (+13.3 +/- 2.2), 60 mmHg (+15.3 +/- 2.8) and 80 mmHg (+17.0 +/- 4.0) were much lower than in the control animals, which were +35.9 +/- 5.1, +41.1 +/- 6.3 and +54.1 +/- 9.6, respectively. Whereas, CB1 antagonist (SR141716A) had an opposite effect. Compared with control group, the change in electromyogram response after SR141716A over baseline was significantly enhanced (p < 0.05) for the distending pressure of 40 mmHg (+56.0 +/- 10.3), 60 mmHg (+74.6 +/- 12.3) and 80 mmHg (+82.9 +/- 11.0), respectively. Reverse-transcription polymerase chain reaction and Western blotting demonstrated the stress-induced up-regulation of colon CB1Rs (p < 0.05). CONCLUSIONS: Our results suggest there is a key contribution of peripheral CB1Rs involved in the maintenance of visceral hyperalgesia after repeated restraint stress, providing a novel mechanism for development of peripheral visceral sensitization.

Expression and localization of CB2 (cannabinoid receptor) in rat skin / 解放军医学杂志

Objective To study the expression and distribution of CB2 in rat skin tissue. Method Immunohistochemistrial technics were employed to study the expression and distribution of CB2 in rat skin tissue. Results CB 2 receptors mostly presented in suprabasal layers of the epidermis and hair follicles in dermis by CB2 like immunoreactivity with a N terminal anti CB2 receptor antibody. The positive expression of CB2 appeared in rat spleen and a negative result occured in liver. Conclusion The CB2 receptors distributed mainly in the rats, epidermis and hair follicles, may involve in some of the dermic physiological and pathological process, such as the communication between central neural system and skin.