OBJECTIVES: To investigate the effects of hyperoxia on the expression of N-methyl-D-aspartate receptor 1 (NMDAR1) and its synapse-associated molecules, including cannabinoid receptor 1 (CB1R), postsynaptic density 95 (PSD95), and synapsin (SYN), in the hippocampus of neonatal rats. METHODS: One-day-old Sprague-Dawley neonatal rats were randomly divided into a hyperoxia group and a control group (n=8 per group). The hyperoxia group was exposed to 80% ± 5% oxygen continuously, while the control group was exposed to room air, for 7 days. At 1, 3, and 7 days after hyperoxia exposure, hematoxylin and eosin (HE) staining was used to observe histopathological changes in the brain. The expression levels of NMDAR1, CB1R, PSD95, and SYN proteins and mRNAs in the hippocampus were detected by immunohistochemistry, Western blotting, and quantitative real-time PCR. RESULTS: After 7 days of hyperoxia exposure, the hyperoxia group showed decreased neuronal density and disordered arrangement in brain tissue. Compared with the control group, after 1 day of hyperoxia exposure, CB1R mRNA and both NMDAR1 and CB1R protein expression in the hyperoxia group were significantly downregulated, while SYN protein expression was significantly upregulated (P<0.05). After 3 days, mRNA expression of NMDAR1, CB1R, and SYN was significantly decreased (P<0.05); NMDAR1 and CB1R protein expression was significantly downregulated (P<0.05), while PSD95 and SYN protein expression was significantly upregulated (P<0.05). After 7 days of hyperoxia, the protein expression of NMDAR1 and CB1R was significantly upregulated (P<0.05). CONCLUSIONS Continuous hyperoxia exposure induces time-dependent changes in the expression levels of NMDAR1 and its synapse-associated molecules in the hippocampus of neonatal rats.
Animals ; Receptors, N-Methyl-D-Aspartate/genetics* ; Rats, Sprague-Dawley ; Hippocampus/pathology* ; Rats ; Animals, Newborn ; Receptor, Cannabinoid, CB1/genetics* ; Hyperoxia/metabolism* ; Disks Large Homolog 4 Protein/genetics* ; Synapsins/genetics* ; Synapses ; Male ; Female ; RNA, Messenger/analysis*

Chronic cerebral hypoperfusion leads to white matter injury (WMI), which plays a significant role in contributing to vascular cognitive impairment. While 13-docosenamide is a type of fatty acid amide, it remains unclear whether it has therapeutic effects on chronic cerebral hypoperfusion. In this study, we conducted bilateral common carotid artery stenosis (BCAS) surgery to simulate chronic cerebral hypoperfusion-induced WMI and cognitive impairment. Our findings showed that 13-docosenamide alleviates WMI and cognitive impairment in BCAS mice. Mechanistically, 13-docosenamide specifically binds to cannabinoid receptor 1 (CNR1) in oligodendrocyte precursor cells (OPCs). This interaction results in an upregulation of ubiquitin-specific peptidase 33 (USP33)-mediated CNR1 deubiquitination, subsequently increasing CNR1 protein expression, activating the phosphorylation of the AKT/mTOR pathway, and promoting the differentiation of OPCs. In conclusion, our study suggests that 13-docosenamide can ameliorate chronic cerebral hypoperfusion-induced WMI and cognitive impairment by enhancing OPC differentiation and could serve as a potential therapeutic drug.
Animals ; Oligodendrocyte Precursor Cells/metabolism* ; Mice ; Cell Differentiation/drug effects* ; Male ; Receptor, Cannabinoid, CB1/metabolism* ; Mice, Inbred C57BL ; Ubiquitin Thiolesterase/metabolism* ; Ubiquitination/drug effects* ; Carotid Stenosis/complications* ; Cognitive Dysfunction/drug therapy*

OBJECTIVETo investigate whether cannabinoid receptor 1 (CB1R) is involved in nerve growth in endometriosis-associated ectopic cyst.

METHODSThe effect of CB1R agonist and antagonist on the expression of pan-neuronal marker protein gene product (PGP) 9.5 in ectopic cyst was examined by immunofluorescence and Western blot in endometriosis model of 18 rats.

RESULTSImmunofluorescence revealed that PGP 9.5 was expressed in the nerve fibers and was mainly distributed in the cyst hilum. Western blot revealed that the protein density of either PGP 9.5 (2 week: 0.38 ± 0.05; 4 week: 0.63 ± 0.03; 8 week: 0.80 ± 0.07, P < 0.01) or CB1R (2 week: 0.48 ± 0.04; 4 week: 0.68 ± 0.01; 8 week: 0.80 ± 0.03, P < 0.01) in the ectopic cyst increased with cyst size. In addition, compared to control group (0.75 ± 0.01), PGP 9.5 expression in the ectopic cyst was promoted by CB1R agonist ACPA (0.81 ± 0.01, P < 0.05), and inhibited by CB1R antagonist AM251 (0.67 ± 0.03, P < 0.01).

CONCLUSIONSCB1R was involved in the nerve growth of ectopic cyst associated with endometriosis.

Animals ; Blotting, Western ; Cysts ; metabolism ; Disease Models, Animal ; Endometriosis ; metabolism ; Female ; Peripheral Nerves ; growth & development ; metabolism ; Piperidines ; pharmacology ; Pyrazoles ; pharmacology ; Rats ; Receptor, Cannabinoid, CB1 ; antagonists & inhibitors ; physiology ; Ubiquitin Thiolesterase ; metabolism

Animals ; Cannabinoid Receptor Antagonists ; therapeutic use ; Cannabinoids ; pharmacology ; Fibrosis ; metabolism ; Humans ; Liver Cirrhosis ; etiology ; metabolism ; therapy ; Piperidines ; therapeutic use ; Pyrazoles ; therapeutic use ; Receptor, Cannabinoid, CB1 ; metabolism ; Receptor, Cannabinoid, CB2 ; metabolism ; Receptors, Cannabinoid ; metabolism ; Scleroderma, Diffuse ; metabolism ; Signal Transduction ; drug effects ; Skin ; metabolism ; Smad Proteins ; metabolism ; Transforming Growth Factor beta1 ; metabolism

PURPOSE: Although the presence of cannabinoid type 1 (CB1) receptor in islets has been reported, the major contributor to the protective effect of rimonabant on islet morphology is unknown. We determined whether the protective effect of rimonabant on pancreatic islet morphology is valid in established diabetes and also whether any effect was independent of decreased food intake. MATERIALS AND METHODS: After diabetes was confirmed, Otsuka Long-Evans Tokushima Fatty rats, aged 32 weeks, were treated with rimonabant (30 mg/kg/d, rimonabant group) for 6 weeks. Metabolic profiles and islet morphology of rats treated with rimonabant were compared with those of controls without treatment (control group), a pair-fed control group, and rats treated with rosiglitazone (4 mg/kg/d, rosiglitazone group). RESULTS: Compared to the control group, rats treated with rimonabant exhibited reduced glycated albumin levels (p<0.001), islet fibrosis (p<0.01), and improved glucose tolerance (p<0.05), with no differences from the pair-fed control group. The retroperitoneal adipose tissue mass was lower in the rimonabant group than those of the pair-fed control and rosiglitazone groups (p<0.05). Rimonabant, pair-fed control, and rosiglitazone groups showed decreased insulin resistance and increased adiponectin, with no differences between the rimonabant and pair-fed control groups. CONCLUSION: Rimonabant had a protective effect on islet morphology in vivo even in established diabetes. However, the protective effect was also reproduced by pair-feeding. Thus, the results of this study did not support the significance of islet CB1 receptors in islet protection with rimonabant in established obesity-associated type 2 diabetes.
Adiponectin/metabolism ; Adiposity/drug effects ; Animals ; Cell Proliferation/drug effects ; Diabetes Mellitus, Type 2/diet therapy/*drug therapy ; Eating/*drug effects ; Glucose Intolerance/diet therapy/*drug therapy ; Insulin Resistance ; Insulin-Secreting Cells/*drug effects/pathology ; Male ; Piperidines/adverse effects/*therapeutic use ; Pyrazoles/adverse effects/*therapeutic use ; Rats ; Rats, Inbred OLETF ; Receptor, Cannabinoid, CB1/physiology ; Thiazolidinediones/*therapeutic use

OBJECTIVETo study the influences of endocannabinoid-anandamide (AEA) on the proliferation and apoptosis of the colorectal cancer cell line (CaCo-2) and to elucidate the effects of CB1 and lipid rafts, and to further elucidate the molecular mechanism and the effect of AEA on the generation and development of colorectal cancer.

METHODSHuman colorectal cancer cell line CaCo-2 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in 5% CO(2) atmosphere at 37°C. CaCo-2 cells were divided into different groups and treated with different concentrations of AEA, AEA + SR141716A, AEA + AM630 and AEA + methyl-β-cyclodextrin (MCD). MTT assay was used to determine the effects of AEA, its putative CB1, CB2 receptor antagonists (SR141716A and AM630) and MCD on the proliferation of CaCo-2 cells. Annexin V-PE/7AAD binding assay was used to detect apoptosis in the CaCo-2 cells. Western-blot was applied to check the expressions of CB1, CB2, p-AKT and caspase-3 proteins in different groups of CaCo-2 cells.

RESULTSAEA inhibited the proliferation of CaCo-2 cells in a concentration-dependent manner and the effect could be antagonized by SR141716A and MCD. The inhibiting rates were (21.52 ± 0.45)%, (42.16 ± 0.21)%, (73.64 ± 0.73)% and (83.28 ± 0.71)%, respectively, at different concentrations of AEA (5, 10, 20 and 40 µmol/L). The three groups (20 µmol/L AEA, 20 µmol/L AEA + 10 µmol/L SR141716A and 20 µmol/L AEA + 1 mmol/L MCD) showed different inhibiting rates [(73.64 ± 0.73)%, (16.15 ± 0.75)% and (12.58 ± 0.63)%], respectively. Annexin V-PE/7AAD binding assay showed that AEA induced apoptosis in the CaCo-2 cells and MCD could antagonize this effect. The apoptosis rates of the three groups (control, 20 µmol/L AEA and 20 µmol/L AEA + 1 mmol/L MCD) were (2.95 ± 0.73)%, (39.61 ± 0.73)% and (14.10 ± 0.64)%, respectively. The expressions of CB1, CB2, p-AKT and Caspase-3 proteins were all observed in the CaCo-2 cells. AEA inhibited p-AKT protein expression and induced caspase-3 protein expression. The two actions were also antagonized by MCD.

CONCLUSIONSAEA can strongly suppress the proliferation of colorectal cancer CaCo-2 cells via the CB1 receptor and membrane cholesterol-LRs and induce apoptosis via lipid rafts. Anandamide plays a very important role in the carcinogenesis and development of colorectal cancer. MCD is a critical member in this system.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arachidonic Acids ; antagonists & inhibitors ; pharmacology ; Caco-2 Cells ; Cannabinoid Receptor Modulators ; antagonists & inhibitors ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Endocannabinoids ; Humans ; Indoles ; pharmacology ; Membrane Microdomains ; metabolism ; Piperidines ; pharmacology ; Polyunsaturated Alkamides ; antagonists & inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Pyrazoles ; pharmacology ; Receptor, Cannabinoid, CB1 ; antagonists & inhibitors ; metabolism ; Receptor, Cannabinoid, CB2 ; antagonists & inhibitors ; metabolism ; beta-Cyclodextrins ; metabolism

OBJECTIVETo study the effect of anandamide (AEA) on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer.

METHODSLocalization of the fatty acid hydrolytic enzyme (FAAH), cannabinoid receptors 1(CB1) and cannabinoid receptors 2 (CB2) proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-beta-cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase (p-P38 MAPK) and c-Jun-NH2-terminal kinase (p-JNK) proteins, were analyzed by Western blot.

RESULTSThe FAAH, CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-beta-cyclodextrin treatment prevented necrosis in HepG2 cells (t = 3.702; 5.274; 3.503, P less than 0.05). The expression patterns of FAAH, CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependent manner in HepG2 cells (F = 11.908; 26.054, P less than 0.05) and the increase can be partially by prevented by MCD (t = 2.801; t = 12.829, P less than 0.05).

CONCLUSIONAEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.

Amidohydrolases ; metabolism ; Arachidonic Acids ; pharmacology ; Cannabinoid Receptor Modulators ; pharmacology ; Cholesterol ; metabolism ; Endocannabinoids ; Hep G2 Cells ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Necrosis ; Polyunsaturated Alkamides ; pharmacology ; Receptor, Cannabinoid, CB1 ; metabolism ; Receptor, Cannabinoid, CB2 ; metabolism ; Signal Transduction ; beta-Cyclodextrins ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVE: To investigate the expression of cannabinoid receptor I (CB1R) during mice skin incised wound healing course and time-dependent changes of CB1R in wound age determination. METHODS: The changes of CBIR expression in skin incised wound were detected by immunohistochemistry and Western blotting. RESULTS: The control group showed a low expression of CB1R detected mainly in epidermis, hair follicles, sebaceous gland and dermomuscular layer. CB1R expression was undetectable in neutrophils in the wound specimens from 6h to 12h post-injury. CB1R positive cells were mostly mononuclear cells (MNCs) and fibroblastic cells (FBCs) from 1 d to 5 d post-injury. CB1R positive cells were mostly FBCs from 7 d to 14d post-injury. The ratio of the CB1R positive cells increased gradually in the wound specimens from 6 h to 3 d post-injury, reached peak level at 5 d, and then decreased gradually from 7d to 14 d post-injury. The positive bands of CB1R were observed in all time points of the wound healing course by Western blotting. The expression peak showed at 5 d post-injury. CONCLUSION CB1R is activated during the wound healing course. The expression of CB1R is found in mononuclear cells, which could be involved in inflammation reaction. CBIR is observed in fibroblastic cells, which could participate in the wound healing. CB1R may be a potentially useful marker for determination of wound healing age.
Animals ; Blotting, Western ; Disease Models, Animal ; Fibroblasts/metabolism* ; Forensic Pathology ; Immunohistochemistry ; Male ; Mice ; Monocytes/metabolism* ; Random Allocation ; Receptor, Cannabinoid, CB1/metabolism* ; Skin/metabolism* ; Staining and Labeling ; Time Factors ; Wound Healing ; Wounds and Injuries/pathology*

BACKGROUNDVascular hyporeactivity, which occurs in the terminal stage of hemorrhagic shock, is believed to be critical for treating hemorrhagic shock. The present study was designed to examine whether the CB1 cannabinoid receptor (CB1R) was involved in the development of vascular hyporeactivity in rats suffering from hemorrhagic shock.

METHODSSixteen animals were randomly divided into two groups (n = 8 in each group): sham-operated (Sham) and hemorrhagic shock (HS) groups. Hemorrhagic shock was induced by bleeding. The mean arterial pressure (MAP) was reduced to and stabilized at (25 +/- 5) mmHg for 2 hours. The vascular reactivity was determined by the response of MAP to norepinephrine (NE). In later experiments another twelve animals were used in which the changes of CB1R mRNA and protein in aorta and superior mesenteric artery (SMA) were analyzed by RT-PCR and Western blotting. In addition, we investigated the effects of a CB1R antagonist on the vascular hyporeactivity and survival rates in rats with hemorrhagic shock. Survival rates were analyzed by the Fisher's exact probability test. The MAP response was analyzed by one-way analysis of variance (ANOVA).

RESULTSVascular hyporeactivity developed in all animals suffering from hemorrhagic shock. The expression of CB1R mRNA and protein in aorta and 2 - 3 branches of the SMA were significantly increased in the HS group after the development of vascular hyporeactivity when compared to those in Sham group. When SR141716A or AM251 was administered, the MAP response to NE was (41.75 +/- 4.08) mmHg or (44.78 +/- 1.80) mmHg respectively, which was higher than that in saline groups with (4.31 +/- 0.36) mmHg (P < 0.01). We also showed an increased 4-hour survival rate in the SR141716A or AM251-treated group with 20% or 30%, but with a statistically significant difference present between the AM251-treated and saline groups (P < 0.05).

CONCLUSIONSCB1R is involved in vascular hyporeactivity resulting from hemorrhagic shock in rats, and CB1R antagonist may be useful in treating patients with traumatic, hemorrhagic shock who need field-rescue or initial treatment.

Animals ; Blotting, Western ; Gene Expression Regulation ; drug effects ; Hypotension ; metabolism ; Male ; Piperidines ; pharmacology ; Pyrazoles ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Cannabinoid, CB1 ; antagonists & inhibitors ; genetics ; metabolism ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Shock, Hemorrhagic ; metabolism ; mortality ; Survival Rate

This study examined endogenous cannabinoid (ECB)-anandamide (AEA) and its cannabinoid receptors (CBR) in mice liver with the development of schistosoma japonicum. Mice were infected with schistosoma by means of pasting the cercaria onto their abdomens. Liver fibrosis was pathologically confirmed nine weeks after the infection. High performance liquid chromatography (HPLC) was employed to determine the concentration of AEA in the plasma of mice. Immunofluorescence was used to detect the expression of CBR1 and CBR2 in liver tissue. Morphological examination showed typical pathological changes, with worm tubercles of schistosoma deposited in the liver tissue, fibrosis around the worm tubercles and infiltration or soakage of inflammatory cells. Also, CBR1 and CBR2 were present in hepatocytes and hepatic sinusoids of the two groups, but they were obviously enhanced in the schistosoma-infected mice. However, the average optical density of CBR1 in the negative control and fibrosis group was 13.28+/-7.32 and 30.55+/-7.78, and CBR2 were 28.13+/-6.42 and 52.29+/-4.24 (P<0.05). The levels of AEA in the fibrosis group were significantly increased as compared with those of the control group. The concentrations of AEA were (0.37+/-0.07) and (5.67+/-1.34) ng/mL (P<0.05). It is concluded that the expression of endocannabinoids AEA and its cannabinoid receptor CBR were significantly increased in schistosoma-infected mice. Endogenous endocannabinoids may be involved in the development of schistosoma-induced liver fibrosis.
Arachidonic Acids/*metabolism ; Endocannabinoids/*metabolism ; Liver Cirrhosis/etiology ; Liver Cirrhosis/*metabolism ; Liver Cirrhosis/parasitology ; Polyunsaturated Alkamides/*metabolism ; Random Allocation ; Receptor, Cannabinoid, CB1/*metabolism ; Receptor, Cannabinoid, CB2/*metabolism ; Schistosomiasis japonica/*complications ; Schistosomiasis japonica/metabolism