ABSTRACT
Objective: To investigate the immunological mechanism of cannabinoid receptor CB2 in ulcerative colitis (UC) by regulating macrophage polarization so as to provide new ideas for the development of new drugs and treatment of the disease. Methods: Human UC specimens were immunohistochemically stained with macrophage mark CD68. Male ICR mice were made into ulcerative colitis model by dextran sulfate sodium DSS. After treatment with CB2 receptor agonist JWH133, the weight of the mice and the inflammatory activity indexes (DAI) (diarrhea and bloody stool) of UC were recorded, and the intestinal tissues were collected. The degree of inflammation and polarity of macrophages of intestinal tract were analyzed with HE staining and Real-time PCR. Peripheral blood-derived macrophages (PBMs) were cultured in vitro and induced into M1 and M2 macrophages by LPS and IL-4, respectively. After intervention with JWH133, the polarity of macrophages was measured by Real-time PCR. Results: There were a large number of macrophages in the colonic tissues of UC patients. JWH133 transformed the polarity of macrophages from M1 to M2 in UC mice, improved the weight loss and DAI of mice, and significantly decreased the degree of intestinal inflammation. In vitro, JWH133 decreased the level of M1-PBM markers (TNF-α, IL-1β and IL-12) and elevated Arg1, Mrc2 and Mgl1 of M2-PBM. Conclusion :CB2 receptor can improve UC by regulating macrophage transformation to M2 type.
ABSTRACT
Objective To investigate the role of activated cannabinoid receptor 2 (CB2R) in lipopolysaccharide (LPS)-induced secretion of RAW264.7 macrophage inflammatory cytokines and its possible mechanism. Methods Macrophages were seeded in 6-well plates (2 mL/well) at the density of 1×105 cells/mL and randomly divided into four groups (n=6 each group): control group (group C), LPS group (group LPS), LPS plus CB2R agonist HU308 group (group LPS+HU308), and LPS plus HU308 plus 3-Methyladenine group (group LPS+HU308+3-MA). LPS with the final concentration of 1 μg/mL were added in group LPS, group LPS+HU308 and group LPS+HU308+3-MA. After incubation for 15 min, 3-MA with a final concentration of 10 mmol/L was added into group LPS+HU308+3-MA . HU308 with the final concentration of 10 μmol/L was added in group LPS+HU308 and group LPS+HU308+3-MA at 15 min after 3-MA intervention, and the cells were then incubated for 24 h. The concentrations of TNF-α, IL-18 and IL-1β in supernatant serum of each group were determined by ELISA. The expressions of ICAM-1 and NLRP3 mRNA were detected by RT-PCR. The expressions of LC3 and Beclin1 were detected by Western blot, and the ratio of LC3-Ⅱ/LC3-Ⅰ was calculated. LSD-t test was used for sample pairwise comparison, and one way ANOVA for inter-group comparison. A P<0.05 was considered statistically significant. Results Compared with group C, the concentrations of TNF-α [(228.86±10.20) pg/mL vs (140.05±5.54) pg/mL], IL-1β [(363.62±8.14) pg/mL vs (244.82±9.11) pg/mL], and IL-18 [(293.28±13.57) pg/mL vs (202.84±9.54) pg/mL] in supernatant serum were increased (all P<0.05), the expressions of ICAM-1 [(5.88±0.32) vs (1.00±0.03)] and NLRP3 [(8.07±0.93) vs (1.01±0.05)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.50±0.03) vs (0.40±0.06)] and Beclin1 [(0.51±0.04) vs (0.16±0.03)] were up-regulated in group LPS (all P<0.05). Compared with group LPS, the concentrations of TNF-α [(165.44±7.07) pg/mL], IL-1β [(272.09±3.35) pg/mL] and IL-18 [(220.41±6.01) pg/mL] in supernatant serum were significantly decreased (all P<0.05), the expressions of ICAM-1 [(3.21±0.35)] and NLRP3 [(1.54±0.30)] mRNA were decreased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.71±0.03)] and Beclin1 [(0.71±0.02)] were up-regulated in group LPS+HU308 (all P<0.05). Compared with group LPS+HU308, the concentrations of TNF-α [(197.06±5.59) pg/mL], IL-1β [(318.98±11.54) pg/mL] and IL-18 [(243.33±8.71) pg/mL] in supernatant serum were significantly increased (all P<0.05), the expressions of ICAM-1 [(4.04±0.21)] and NLRP3 [(5.87±0.77)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.44±0.08)] and Beclin1 [(0.32±0.03)] were down-regulated in group LPS+HU308+3-MA (all P<0.05). Conclusions Activation of cannabinoid receptor 2 can alleviate LPS-induced the secretion of RAW264.7 macrophage inflammatory cytokines, and its mechanism may be related to enhanced autophagy.
ABSTRACT
Abstract Background The endocannabinoid system (eCBs) is one of the modulatory systems widely expressed in the brain. It consists of receptors expressed in the cytoplasmic (CB1 and CB2), the mitochondrial membrane (CB1), and the endogenous ligands known as endocannabinoids, such as anandamide, 2AG and oleamide. CB1 has been found in excitatory and inhibitory neurons in the pre- and post-synaptic membranes. It is expressed in several brain areas such as the hippocampus, dorsal, and ventral striatum, amygdala and prefrontal cortex. The eCBs has been involved in the regulation of learning and memory, mood, energy balance, sleep, and drug addiction. Objective Integrate existing information about the eCBs and its role in brain function and mental health. Method Review of the information of basic and clinical relevance obtained from indexed scientific journals (PubMed/Medline, Scopus). Results Basic and clinical research on eCBs related to central nervous system function is described. Discussion and conclusion At present, the study of eCBs is of importance. The development of drugs that affect this system may be clinically useful to control different debilitating diseases. This is an area of interest to the scientific community and health care providers.
Resumen Antecedentes El sistema de endocannabinoides (eCBs) es uno de los sistemas moduladores más ampliamente expresados en el cerebro. Se compone de receptores expresados en la membrana citoplasmática (CB1 y CB2) y en la membrana mitocondrial (CB1) y ligandos endógenos conocidos como endocannabinoides, como anandamida, 2AG y oleamida. El CB1 se ha encontrado en neuronas excitadoras e inhibidoras, en las membranas pre- y pos-sináptica, en varias áreas cerebrales como el hipocampo, el estriado dorsal y ventral, y en la amígdala y la corteza prefrontal. El eCBs se ha relacionado con la regulación del aprendizaje y la memoria, del estado afectivo, del equilibrio energético, del sueño y del proceso de la adicción a las drogas. Objetivo Integrar la información existente sobre el eCBs y su función sobre los procesos cerebrales y la salud mental. Método Revisión de la información de relevancia básica y clínica obtenida de revistas científicas indexadas (PubMed/Medline, Scopus). Resultados Se describe de manera concisa información de interés básico y clínico de la investigación sobre el eCBs relacionada con la función del sistema nervioso central. Discusión y conclusión En la actualidad, el estudio del eCBs es indispensable debido a su potencial terapéutico. El desarrollo de fármacos que afecten este sistema puede ser clínicamente útil para controlar diferentes enfermedades debilitantes. Ésta es un área de interés para la comunidad científica y los proveedores de salud.
ABSTRACT
Cannabinoid receptor (CBR) has two subtypes,type 1 cannabinoid receptor (CB 1 receptor,CB1R) and type 2 cannabinoid receptor(CB2 receptor,CB2R).CB2R widely is found distributing in the central nervous system and playing important functions.Cannabis can regulate CB2R signal pathway to protect central nervous system in CNS diseases.
ABSTRACT
Objectives To investigate the ameliorative effect of cannabinoid 2 receptor(CB2R)agonist JWH-015 on the cog-nitive impairment of Alzheimer' s disease(AD)model mice and to assess the correlation with microglial phenotype transformation. Methods Twenty adult male C57BL/6J mice were randomly divided into four groups:C57BL/6J solvent group,JWH-015 control group,AD model group,and AD model treated with JWH-015 group. Amyloidβ1-42 oligomers of 4μg and the same volume of saline were intraventricularly administered to construct the AD mouse model and the solvent groups. CB2R agonist JWH-015 or the corre-sponding vehicle at a dose of 0.5 mg/(kg·d)was administered by intraperitoneal injection for 3 weeks. Non-spatial learning and memo-ry was measured using novel object recognition task. Furthermore,the mRNA expression levels of M1 microglia marker inducible ni-tric oxide synthase(iNOS)and M2 microglia marker chitinase-3 like protein(Ym1/2)in brain samples of cortex and hippocampus were evaluated using real-time quantitative PCR(qPCR). In the meantime,fifteen CB2R knockout(CB2RKO)mice and five CB2R wild-type(CB2RWT)littermates were assigned to identify the specificity of CB2R in the research. Based on the genotype and different treatment,the animals were divided into four groups:CB2RKO solvent group,CB2RKO AD model group,CB2RKO AD model treat-ed with JWH-015 group and CB2RWT solvent group. Results Compared with solvent group,there was a significant decrease in nov-el object recognition index in C57BL/6J AD model group(P<0.01). The mRNA expression levels of M1 phenotype microglia marker iNOS in cortex and hippocampus were significantly up-regulated(both P<0.05)and the mRNA expression levels of M2 phenotype mi-croglia marker Ym1/2 were significantly down-regulated(both P<0.01). Interestingly,administration of JWH-015 could reverse the impairment of novel object recognition index(P<0.05);compared with C57BL/6J AD model group,administration of JWH-015 also decreased the iNOS mRNA expression levels(both P<0.05)and increased the Ym1/2 mRNA expression levels(both P<0.05)in cortex and hippocampus;compared with CB2RKO solvent group,the novel object recognition index of CB2RKO AD model group was decreased(P<0.05);the mRNA expression levels of iNOS in cortex and hippocampus were significantly up-regulated(both P<0.05),the mRNA expression level of Ym1/2 in cortex was significantly down-regulated in cortex(P<0.05);compared with CB2RKO AD model group,administration of JWH-015 had no effect on novel object recognition index and the mRNA expression level of M1/M2 in cortex and hippocampus,respectively. Conclusion JWH-015 improves the cognitive impairment of Aβ-induced AD mice by the specific activation of CB2R,the mechanism of which is related to the direct regulation of CB2R on the M1/M2 microglial phenotype transformation and microglia-mediated neuroinflammation in brain.
ABSTRACT
Objective@#To investigate the effect of cannabinoid receptor-2 (CB2) agonist AM1241 on the mRNA and protein expression of platelet-derived growth factor (PDGF) and collagen-III (Col-III) in the liver tissue of mice with experimental liver fibrosis induced by carbon tetrachloride (CCl4).@*Methods@#Totally 38 8-week-old male C57BL/6J mice were randomly divided into control group, model group, 3 mg/kg CB2 receptor agonist (AM1241) group, and 9 mg/kg AM1241 group. All mice, except for the control group, were treated with 30% CCl4 (three times a week, 5 ml/kg body weight, 16 weeks) to establish a liver fibrosis model. Meanwhile, 3 and 9 mg/kg AM1421 was intraperitoneally injected for daily intervention, respectively. The dosage was adjusted according to actual body weight. The same solvent was given in the control group. The serum level of aspartate aminotransferase (AST) was measured by serum enzyme digestion. The liver inflammation and fibrosis were observed by HE staining of tissue slices. The mRNA and protein expression of PDGF and Col-III in hepatic tissue was determined by real-time PCR and immunohistochemistry.@*Results@#Compared with the control group, the mice in model group showed severe liver fibrosis, significantly elevated serum AST level (742 ± 300.8 U/L vs 118.1 ± 31.1 U/L, P < 0.05), and significantly increased mRNA and protein expression of PDGF and Col-III in liver tissue (P < 0.05). Compared with the model group, the mice in 3 mg/kg AM1241 group and 9 mg/kg AM1241 group had less severe liver fibrosis, and significantly reduced serum AST levels (116.6 ± 13.68 U/L vs 742 ± 300.8 U/L, P < 0.05; 113.8 ± 16.01 U/L vs 742 ± 300.8 U/L, P < 0.05) and mRNA and protein expression of PDGF and Col-III in liver tissue (P < 0.05).@*Conclusion@#CB2 receptor agonist AM1241 can inhibit the mRNA and protein expression of PDGF in the liver tissue of mice with hepatic fibrosis, and reduce extracellular matrix synthesis.
ABSTRACT
Objective To observe the role of cannabinoid receptor-2 (CB2) in acute experimental colitis in mice, and to explore its effect on the endoplasmic reticulum stress ( ERS ) in the intestinal mucosa . Methods 32 SPF mice were randomly divided into normal group,model group,CB2 agonist group and CB2 antagonist group. Disease activity index( DAI) and colon histopathological score( HS) were evaluatd. The levels of TNF-α and IL-10 in colon tissue were detected by ELISA. The expression of CB2 and ERS markers GRP78, CHOP in colon tissues were de-tected by immunohistochemical. The mRNA levels of GRP78 and CHOP were detected by RT-PCR. Results Com-pared with the normal group,the level of TNF-αwas significantly higher and the level of IL-10 was significantly low-er(P0. 05 ) . Conclusion Severe endo-plasmic reticulum stress exists in intestinal mucosa of experimental colitis mice. The CB2 agonist activates CB2 ex-pression,reducing colitis in mice. Its mechanism may be related to inhibiting ERS in intestinal mucosa.
ABSTRACT
ObjectiveTo investigate the effects of cannabinoid receptor 2 (cannabinoid receptor 2,CB2R) agonist JWHO15 on the hyperalgesia induced by remifentanil in a rat model of postoperative pain.Methods Sixty SD rats were randomly divided into 10 groups ( n =6 each ):control groups ( C1 and C2 ),incisional pain groups (I1 and I2),incisional pain plus JWHO15 groups (QI and FI),remifentanil groups (R1 and R2),and JWHO15 plus remifentanil groups ( QR and FR).Rats in group QL/QR and FI/FR were intrathecal injection with 10μg JWHO15 ( diluted in 10μl 20％ DMSO solution) and intraperitoneal administration with 100μg JWHO15 ( diluted in 10μl 4％ DMSO solution) respectively 30 min before plantar incision while rats in group C,I and R were received with the same volume of DMSO solution.Plantar incision surgery was operated in rats of group I,R,QI/FI,and QR/FR.In group R and QR/FR,remifentanil (0.04 mg/kg) was infused subcutaneously to rats with a pump for 30 min at the moment of surgical incision.The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) at 24 h before incision and at 2 h,6 h,24 h and 48 h after incision were tested to evaluate the behavioral changes.ResultsCompared with group C and baseline,the level of PWMT and PWTL decreased at 2 h,6 h,24 h and 48h after incision in group Ⅰ (P＜ 0.01 ) ;Compared with group Ⅰ,the significant decrease of PWMT and PWTL were observed after incision in group R (P ＜ 0.05 ) ; Compared with group R,the significant increase of PWMT (7.78 ± 1.09) and PWTL ( 17.28 ± 1.58) were observed from 6 h after incision in group QR(P＜0.05).And the increase of PWMT (7.79 ±0.72,9.50 ± 1.17,7.86 ± 1.16) and PWTL ( 16.23 ± 1.50,19.53 ± 1.63,18.10 ± 0.93) were observed at 6 h,24 h and 48 h after incision in group FR(P＜0.05).ConclusionIntrathecal and intraperitoneal administration of JWHO15 in this investigation dose could relief remifentanil-induced hyperalgesia in a rat model of postoperative pain.
ABSTRACT
Many researchers employed mammalian expression system to artificially express cannabinoid receptors,but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports.In present study,we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system.This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide.In addition,the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95℃),forming a large molecular weight band when analyzed by immuno-blotting.Only denaturing temperatures ≤75℃ yielded a clear band at the predicted molecular weight.Collectively,we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence,and described the requirement for a low sample denaturing temperature in immuno-blot analysis.These findings provide very useful information for efficient mammalian expression and immuno-blotting of mcmbrane receptors.
ABSTRACT
Objective To investigate hepatic expressions and significances of cannabinoid receptor 1 (CB1) and cannabinoid receptor 2(CB2) in C57 mice with experimental cirrhosis.Methods Thirty C57 mice were randomly divided into three groups,i.e.normal control group,model control group and model colchicine group.Hepatic fibrosis model was prepared by intraperitoneal injection of carbon tetrachloride.The expressions of CB1 and CB2 in liver tissue of mice were observed by immunohistochemistry.The scores of inflammation grade (G) and fibrosis stage (S) were simultaneously performed.Results The scores of G and S in model control group and model colchicine group were significantly higher than those in normal control group( F =125.41,P =0.00; F =99.18,P =0.00).The scores of G and S in model control group were significantly higher than those in model colchicine group(P ＜0.01 ).The scores of CB1 and CB2 expressions in model control group and model colchicine group were significantly higher than those in normal control group ( F =29.27,P =0.00; F =36.99,P =0.00).The scores of CB1 and CB2 in model control group were significantly higher than those in model colchicine group( P ＜ 0.05 or P ＜ 0.01 ).There were significant relationships among scores of CB1,CB2,G and S in model control group and model colchicine group(Ps ＜0.05).As the scores of G and S became higher,the expressions of CB1 and CB2 gradually became more intensive.Conclusion The hepatic expressions of CB1 and CB2 in C57 mice with experimental cirrhosis increased significantly and have significant relationship with the grades of liver tissue inflammation and fibrosis.
ABSTRACT
Two types of cannabinoid receptors,named cannabinoid receptor 1(CB1) and cannabinoid receptor 2(CB2),have been cloned.CB2 receptors are expressed predominantly in the peripheral immune tissues,but accumulating evidence has revealed that CB2 receptors are also expressed in CNS.Previous studies showed that CB2 agonists can cure and suppress formation of inflammatory and neurophathic pain without central effects after chronic administration.Therefore,they will have good clinical applications in the treatment of pain and neurodegenerative diseases.In this paper,we will review the tissue distribution of CB2 in CNS and pharmacological characteristics of the CB2 agonists have been reviewed.