ABSTRACT
The rostral agranular insular cortex (RAIC) has been associated with pain modulation. Although the endogenous cannabinoid system (eCB) has been shown to regulate chronic pain, the roles of eCBs in the RAIC remain elusive under the neuropathic pain state. Neuropathic pain was induced in C57BL/6 mice by common peroneal nerve (CPN) ligation. The roles of the eCB were tested in the RAIC of ligated CPN C57BL/6J mice, glutamatergic, or GABAergic neuron cannabinoid receptor 1 (CB1R) knockdown mice with the whole-cell patch-clamp and pain behavioral methods. The E/I ratio (amplitude ratio between mEPSCs and mIPSCs) was significantly increased in layer V pyramidal neurons of the RAIC in CPN-ligated mice. Depolarization-induced suppression of inhibition but not depolarization-induced suppression of excitation in RAIC layer V pyramidal neurons were significantly increased in CPN-ligated mice. The analgesic effect of ACEA (a CB1R agonist) was alleviated along with bilateral dorsolateral funiculus lesions, with the administration of AM251 (a CB1R antagonist), and in CB1R knockdown mice in GABAergic neurons, but not glutamatergic neurons of the RAIC. Our results suggest that CB1R activation reinforces the function of the descending pain inhibitory pathway via reducing the inhibition of glutamatergic layer V neurons by GABAergic neurons in the RAIC to induce an analgesic effect in neuropathic pain.
Subject(s)
Mice , Animals , Insular Cortex , Peroneal Nerve , Mice, Inbred C57BL , Neuralgia , GABAergic Neurons , Analgesia , Analgesics , Receptors, CannabinoidABSTRACT
AIM: Recent studies have shown that the cannabinoid receptor 1 (CNR1) gene played an important role in diabetes and its complications development. And liraglutide was of positive therapeutic significance in patients with type 2 diabetes mellitus. This study was to investigate the influence of CNR1 genetic variation on the clinical outcomes of early stage type 2 diabetes mellitus treated with liraglutide. METHODS: From March 2016 to December 2018, a total of 230 patients with early stage type 2 diabetes mellitus were included as the research object in this study. The patients were treated with liraglutide, and the clinical outcomes were evaluated 16 weeks later. Additionally, peripheral blood and part of the patients with fresh peripheral blood specimens of the patients were collected for the genotyping of the genetic variation and CNR1 gene mRNA expression, respectively. The correlation between genetic polymorphism and other baseline characteristics was analyzed by chi square test and non-parametric test. The mRNA expression of CNR1 in different genotypes was analyzed by non-parametric test. RESULTS:All of the 230 diabetes mellitus patients were of available for efficacy evaluation. BMI, FPG, 2H PG and HbA1c of all patients decreased significantly after 16 weeks of treatment, and the difference was statistically significant compared with that before treatment (P<0.05). In terms of the CNR1 gene polymorphism analysis, only rs1049353 was found to be of clinical significance. The prevalence of rs1049353 among the 230 patients were as follows: GG genotype 188 cases (81.74%), GA genotype 39 cases (16.96%), AA genotype 3 cases (1.30%), the minor allele frequency is 0.10, The distribution of three genotypes were in accordance with Hardy-Weinberg Equilibrium (P=0.551). GA genotype and AA genotype patients were merged in the following analysis. The clinical outcomes analysis indicated that BMI index of GA/AA genotype patients decreased (3.4±0.9) kg/m2 on average, FPG index decreased (5.1±0.9) mmol/l on average, HbA1c decreased (2.7±0.5)%. And the BMI index of GG genotype patients decreased (3.0±1.5) kg/m2 on average, FPG index decreased (4.7±1.3) mmol/l on average, HbA1c decreased (2.6±0.5)%, respectively. There were statistically significant differences between the two genotypes. Additionally, of the 125 available specimens for CNR1 gene mRNA analysis, the results showed that the mRNA expression of CNR1 in the patients with GG genotypes were significantly higher than those of the GA/AA genotype patients[(4.2±1.3) vs. (2.8±1.2), P<0.001)]. In terms of adverse reactions, the incidence of adverse reactions during treatment was relatively low, and gradually disappeared after drug withdrawal, and there was no significant correlation with CNR1 gene rs1049353 polymorphism. CONCLUSION:It is safe and effective in early stage type 2 diabetes patients received liraglutide treatment. And the clinical outcomes of the patients received liraglutide treatment may be influenced by CNR1 rs1049353 through mediating the mRNA expression of CNR1.
ABSTRACT
Huntington's disease (HD),a single-gene autosomal dominant neurodegenerative disease,is pathologically characterized by the great loss of striatal neurons in the brain.Clinical manifestations of HD show involuntary dance-like movements,cognitive function and neuropsychiatric symptoms.The pathogenesis of HD is concerned with the protein expression of mutant huntingtin which leads to the selective degeneration of medium spiny neurons in the striatum and the onset of the disease.Cannabinoid receptor (CBR) plays a key role in the release of neurotransmitters,synaptic plasticity,gene expression and modulation of.neuronal activity through the CBR1 and CBR2 signaling pathways.Recent studies have found that CBR-mediated phosphoinositide3-kinases/protein kinase B/mammalian target of rapamycin complex1/brain derived neurotrophic factor,neuropeptide Y/neuronal nitric oxide synthase signaling pathway play a vital role in the regulation.of striatal neuroprotection in HD.This review reveals the research progress of CBR1 related signaling pathways in HD,which provides new theoretical basis and drug targets for the prevention and treatment of HD.
ABSTRACT
BACKGROUND Formation of schistosomal granulomata surrounding the ova can result in schistosomiasis-associated liver fibrosis (SSLF). The current standard of treatment is praziquantel (PZQ), which cannot effectively reverse SSLF. The role of the cannabinoid (CB) receptor family in liver fibrosis has recently been highlighted. OBJECTIVES This study aimed to assess the therapeutic effect of CB1 receptor antagonism in reversing SSLF in a murine model of Schistosoma mansoni infection. METHODS One hundred male Swiss albino mice were divided equally into five groups: healthy uninfected control (group I), infected control (group II), PZQ treated (group III), rimonabant (RIM) (SR141716, a CB1 receptor antagonist)-treated (group IV) and group V was treated with combined PZQ and RIM. Liver sections were obtained for histopathological examination, alpha-1 smooth muscle actin (α-SMA) immunostaining and assessment of CB1 receptor expression using real-time polymerase chain reaction (RT-PCR). FINDINGS The most effective reduction in fibrotic marker levels and granuloma load was achieved by combined treatment with PZQ+RIM (group V): CB1 receptor expression (H = 26.612, p < 0.001), number of α-SMA-positive cells (F = 57.086, p < 0.001), % hepatic portal fibrosis (F = 42.849, p < 0.001) and number of granulomata (F = 69.088, p < 0.001). MAIN CONCLUSIONS Combining PZQ with CB1 receptor antagonists yielded the best results in reversing SSLF. To our knowledge, this is the first study to test this regimen in S. mansoni infection.
Subject(s)
Humans , Fibrosis/diagnosis , Typhus, Endemic Flea-Borne/transmission , Liver/physiopathology , Receptors, CannabinoidABSTRACT
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
Neuropathic pain is a class of pain caused by an injury or diseases of the somatosensory system and characterized by spontaneous pain,allodynia,and hyperalgesia.It is well established that central sensitization is one of the key mechanisms underlying the development and maintenance of neuropathic pain.Cannabinoid receptor 1 (CB1R) of endocannabinoid system modulates synaptic transmission,regulates synaptic plasticity,inhibits central sensitization,and thus attenuates neuropathic pain.Recent studies have shown that activation of CB1R also involves in the relief of neuropathic pain-induced depression.
ABSTRACT
OBJECTIVES: According to previous studies, the cannabinoid receptor 1 (CNR1) gene could be an important candidate gene for schizophrenia. Some studies have linked the (AAT)n trinucleotide repeat polymorphism in CNR1 gene with the risk of schizophrenia. Meanwhile, smooth pursuit eye movement (SPEM) has been regarded as one of the most consistent endophenotypes of schizophrenia. In this study, we investigated the association between the (AAT)n trinucleotide repeats in CNR1 gene and SPEM abnormality in Korean patients with schizophrenia. METHODS: We measured SPEM function in 167 Korean patients with schizophrenia (84 male, 83 female) and they were divided according to SPEM function into two groups, good and poor SPEM function groups. We also investigated allele frequencies of (AAT)n repeat polymorphisms on CNR1 gene in each group. A logistic regression analysis was performed to find the association between SPEM abnormality and the number of (AAT)n trinucleotide repeats. RESULTS: The natural logarithm value of signal/noise ratio (Ln S/N ratio) of the good SPEM function group was 4.34 ± 0.29 and that of the poor SPEM function group was 3.21 ± 0.70. In total, 7 types of trinucleotide repeats were identified, each containing 7, 10, 11, 12, 13, 14, and 15 repeats, respectively. In the patients with (AAT)₇ allele, the distributions of the good and poor SPEM function groups were 18 (11.1%) and 19 (11.0%) respectively. In the patients with (AAT)₁₀ allele, (AAT)₁₁ allele, (AAT)₁₂ allele, (AAT)₁₃ allele, (AAT)₁₄ allele and (AAT)₁₅ allele, the distributions of good and poor SPEM function groups were 13 (8.0%) and 12 (7.0%), 4 (2.5%) and 6 (3.5%), 31 (19.8%) and 35 (20.3%), 51 (31.5%) and 51 (29.7%), 36 (22.2%) and 45 (26.2%), 9 (5.6%) and 4 (2.3%) respectively. As the number of (AAT) n repeat increased, there was no aggravation of abnormality of SPEM function. CONCLUSIONS: There was no significant aggravation of SPEM abnormality along with the increase of number of (AAT)n trinucleotide repeats in the CNR1 gene in Korean patients with schizophrenia.
Subject(s)
Humans , Male , Alleles , Endophenotypes , Eye Movements , Gene Frequency , Logistic Models , Pursuit, Smooth , Receptors, Cannabinoid , Schizophrenia , Trinucleotide RepeatsABSTRACT
Objective To investigate the effect of cannabinoid receptor 1 ( CBR1 ) on spatial learning and memory function of neuropathic pain ( NP ) model rats and the expression of N-methyl-D-aspartic acid receptor 1(NR1) subunit in medial prefrontal cortex (mPFC).Methods Thirty-six healthy male Wistar rats were randomly divided into 4 groups, with 9 rats in each group: the sham operated group (SO group), the neuropathic pain model group (NP group), the NP model group with an mPFC injection of saline ( NS group ) , and the NP model group with an mPFC injection of the CBR 1 antagonist AM251 ( AM251 group).The NP model was prepared using the operation of chronic constriction injury ( CCI) of the right sciatic nerve.The mechanical withdrawal threshold ( MWT ) and the thermal withdrawal latency (TWL) of the rats in each group were detected at 3, 7, 14, 21 and 28 days after the operation.At 29 days after the operation , 18 rats of NP model were randomly selected and given an mPFC injection of saline or AM251 using a three-dimensional brain puncture.At days 30-37 after operation , the eight-arm maze test was performed to detect the spatial learning and memory function of the rats , and the rats were sacrificed immediately after this test.The expression levels of CBR1, NR1 and phosphorylated-N-methyl-D-aspartic acid receptor 1 ( p-NR1 ) ( Ser896 ) in the mPFC brain region were detected by Western blotting , RT-PCR and immunofluorescence.Results Compared with the SO group , the pain thresholds and the spatial learning and memory function of the rats in the NP group were significantly lower ( both P <0.05 ).Compared with the NS group , the rats in the AM251 group showed improvement about spatial learning and memory function ( P<0.05).Compared with the SO group ( the mRNA and protein level of CBR 1:0.23 ± 0.06,0.42 ±0.03), the mRNA(0.43 ±0.12) and protein (0.53 ±0.05) level of CBR1 in NP group increased (both P<0.05).Compared with the NS group (the mRNA and protein level of CBR1:0.42 ± 0.11,0.52 ±0.10), the mRNA (0.53 ±0.05) and protein (0.98 ±0.17) level of CBR1 in AM251 group increased (both P<0.05).Compared with the SO group (the mRNA and protein level of NR1 and the protein level of p-NR1:1.50 ±0.15,0.65 ±0.05,0.79 ±0.15), the mRNA (0.94 ±0.07) and protein (0.24 ±0.05) level of NR1 in NP group decreased (both P<0.05), the protein level of p-NR1 (0.33 ± 0.04) decreased (P<0.05).Compared with the NS group (the mRNA and protein level of NR1 and the protein level of p-NR1:1.09 ±0.14,0.26 ±0.06,0.31 ±0.08), the mRNA(1.58 ±0.10) and protein (1.42 ±0.10) level of NR1 in AM251 group increased (both P<0.05), the protein (0.95 ±0.15) level of p-NR1 increased ( P<0.05).Conclusion CBR1 can decrease the expression level of NR 1 and p-NR1 in the mPFC brain region of NP model rats and induce the spatial learning and memory impairment.
ABSTRACT
In view of the fact that human marijuana users often show dry mouth symptom, the present study was attempted to examine the localization of CB1, which was originally identified in brain, in the submandibular and sublingual salivary glands of postnatal developing male mice by immunohistochemistry. In submandibular gland, CB1-immunoreactivity was positive in a majority of acinar cells in forms of granular appearance in their apical cytoplasm, while it was negative in the ducts at newborn stage. The immunoreactivity decreased in the acinar cells at P1W and no immunoreactivity was detected in the acinar cells at P3W and thereafter. The immunoreactivity was positive in ductal cells at P3W and it remained positive thereafter until P8W stage. The immunoreaction was distinct on the apical plasmalemma of the intercalated ductal cells, while it was distinct on the basal plasmalemma of the granular convoluted ductal cells. The enhanced immunostaining on the lateral plasmalemma of the granular ductal cells was discerned only on P6W. In sublingual gland, CB1-immunoreactivity was detected in the demilune acinar cells and ductal cells only on P4W. Furthermore, CB1-immunoreactivity was shown to occur in the salivary ganglionic neurons, suggesting the CB1-inhibitory action in the saliva secretion through the parasympathetic nervous transmission.
En vista de que los usuarios humanos de la marihuana a menudo presentan síntomas de sequedad oral, en el presente estudio se intentó examinar la localización de CB1, que se identificó originalmente en el cerebro, en las glándulas salivales submandibulares y sublinguales durante el desarrollo postnatal en ratones machos. En la glándula submandibular, la inmunoreactividad CB1 fue positiva en la mayoría de las células acinares de apariencia granular en su citoplasma apical, mientras que fue negativa en los conductos en la etapa de recién nacidos. La inmunorreactividad disminuyó en las células acinares en P1W y no se detectó inmunoreactividad en las células acinares en P3W. La inmunoreactividad fue positiva en las células ductales en P3W y se mantuvo positiva hasta la etapa P8W. La inmunorreacción se observó en el plasmalema apical de las células ductales intercaladas, mientras que fue distinta en el plasmalema basal de las células ductales contorneadas granulares. La inmunotinción mejorada en el plasmalema lateral de las células ductales granulares fue distingible sólo en P6W. En la glándula sublingual, se detectó inmunoreactividad CB1 en las células acinares y se observaron células ductales solamente en P4W. Además, se demostró que la inmunoreactividad CB1 se produce en las neuronas ganglionares salivales, lo que sugiere la acción CB1 inhibitoria en la secreción de saliva a través de la transmisión parasimpática nerviosa.
Subject(s)
Animals , Mice , Salivary Glands/metabolism , Receptor, Cannabinoid, CB1/metabolism , Immunohistochemistry , Animals, NewbornABSTRACT
Objective: LMJ07 is a novel cannabinoid receptorl (CB1R) selective antagonist discovered by our lab. In the present study, its affinity and antagonistic activity against CB1R were evaluated at the molecular and cellular levels by receptor binding experiment, CB1R internalization experiment and by monitoring the change in cytoskeletal and intracellular signal induced by CB1R activation. Methods: With the CB1R selective antagonist rimonabant (SR141716A) as control, the affinity and selectivity of LMJ07 to CB1R and CB2R were assayed by radioligand binding assays, and the G protein-independent antagonistic activity against cannabinoid receptor (CBR) was assayed by enhanced green fluorescein protein (EGFP)-CBR internalization with hierarchiae cluscer anclysis (HCA) analysis. At the same time, we evaluated the changes in cytoskeletal and the intracellular cAMP levels in response to LMJ07 treatment in CHO-CB1 cells by Cellkey label-free assays and homogeneous time-resolved fluorescence (HTRF). Additionally, we also confirmed the CB1R antagonistic efficacy of LMJ07 by detecting the content of Ca2+ in primary cultured hippocampal neuronal cells which could express CB1R with continuous fluorescence detection technology. Results: LMJ07 is a selective CB1R antagonist with high affinity, which can selectively antagonize receptor endocytosis induced by CB1RactivationIt’s affinity and antagonistic efficacy to CB1R were equal to those of rimonabant. In CHO-CB1 cells, LMJ07 (0.01-10μmol/L)could dose-dependently inverse the change in cytoskeletal as well as the increase in intracellular cAMP induced by CBR agonist Win55212-2. In the primary cultured hippocampal neuronal cells, LMJ07 (10 nmol/L-1 μmol/L) could block the increase in [Ca2+] induced by CB1R agonist Win55212-2. Conclusion: LMJ07 is a new selective CB1R antagonist, which shows equal affinity and antagonistic activity against CB1R as the widely accepted CB1R antagonist rimonabant. In addition, the combination of high content analysis (HCS) and Cellkey label-free assay provides a better research tool for rapid and high throughput screening of novel CB1R antagonists.
ABSTRACT
OBJECTIVES: Previous studies suggest that the cannabinoid receptor 1 (CNR1) gene could be an important candidate gene for schizophrenia. According to linkage studies, this gene is located on chromosome 6q14-q15, which is known to harbor the schizophrenia susceptibility locus (locus 5, SCZ5, OMIM 803175). The pharmacological agent delta-9-tetrahydrocannabinol (Delta-9-THC) seems to elicit the symptoms of schizophrenia. The association between CNR1 polymorphisms and schizophrenia is actively being investigated, and some studies have linked the AAT-trinucleotide repeats in CNR1 to the onset of schizophrenia. In this study, we have investigated the association between the AAT-trinucleotide repeats in CNR1 and schizophrenia by studying schizophrenia patients and healthy individuals from Korea. METHODS: DNA was extracted from the blood samples of 394 control subjects and 337 patients diagnosed with schizophrenia (as per the Diagnostic and Statistical Manual of Mental Disorders, fourth edition criteria). After polymerase chain reaction amplification, a logistic regression analysis, with age and gender as the covariates, was performed to study the variations in the AAT-repeat polymorphisms between the two groups. RESULTS: In total, 8 types of trinucleotide repeats were identified, each containing 7, 8, 10, 11, 12, 13, 14, and 15 repeats, respectively. (AAT)13 allele was most frequently observed, with a frequency of 33.6% and 31.6% in the patient and control groups, respectively. The frequency of the other repeat alleles in the patient group (in the decreasing order) was as follows : (AAT)13 33.6%, (AAT)14 21.6%, (AAT)12 18.5%, and (AAT)7 11.1%. The frequency of the repeat alleles in the control group (in the decreasing order) was as follows : (AAT)13 31.6%, (AAT)14 24.5%, (AAT)12 17.2%, and (AAT)7 11.6%. However, there were no significant differences in the AAT-repeat polymorphisms of the CNR1 gene between the patient group and the control group. CONCLUSIONS: Although our study revealed no significant association of the AAT-repeat polymorphism of the CNR1 gene with schizophrenia, it will serve as a good reference for future studies designed to examine the cannabinoid hypothesis of schizophrenia.
ABSTRACT
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.
ABSTRACT
Objective To study the mechanism of brain development delay in rats with intrauterine growth retardation(IUGR) through establishing IUGR animal model by low protein diet during pregnancy and examining the expressions of cannabinoid receptorl (CB1) and L1 cell adhesion molecule (NCAM-L1) in IUGR and normal rats.Methods Thirty-two pregnant rats were randomly fed with normal diet or lower protein diet during pregnancy (16 rats in each group).The offspring rats were divided into IUGR group and control group by birth weight,and sacrificed on day 0,7,14,21 after birth,brain weight was recorded.The expressions of CB1 and NCAM-L1 in the brain were examined by immunohistochemistry staining.Image-Pro Plus 5.1 image processing software was used for semi-quantitative analysis.The integrated optical density of the CB1 and NCAM-L1 of the positive cells was calculated.Results The expressions of CB1 and NCAM-L1 in offspring rats brain were basically in the same area,the changes in both was in accord with the brain weight change.The expression of CB1 in the brain of the pup rats in the IUGR group was significantly lower than that in the control group 0,7,14,21 days after birth,by contrast,the expression of NCAM-L1 was significantly higher (all P ＜ 0.05),and the expressions of CB1 and NCAM-L1 were negatively correlated between both groups(P =0.032,0.010).Conclusions CB1 and NCAM-L1 were involved in the process of rat brain development.The effect of CB1 on rat brain development may be achieved by NCAM-L1.
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
Sleep is a universal experience and a necessary ingredient to life. Young adult humans benefit from spending 8 h a day, every day, sleeping. While the function(s) of sleep is not completely understood, it is known that sleep is critical to the survival of the species. In humans, it restores alertness, helps consolidate memory and «recharge¼ cognitive abilities which are impaired at the end of the activity-phase of the cycle. Humans who do not pay their toll to sleep, for one night, for example, experience difficulties maintaining wakefulness the next day. This condition may put in danger their lives, particularly if they work in the transportation industry (i. e. taxi cabs, truck or trailers-drivers, pilots and/or operating heavy machinery among many other activities). In the past, when humans were more exposed to predators, to be sleepy in the savannah was synonymous with dying. Interestingly enough, the maladaptive strategies exhibited by a sleepy subject (which put at risk his life), are reversed by sleep. It is widely believed that sleep has a restorative function. However, what precisely is being restored during sleep remains a topic of speculation and on-going research. Sleep deprivation in humans results in cognitive deterioration and increased sleepiness, which might compromise survival as aforementioned. It is known that in rats prolonged sleep deprivation leads to death. In humans, it results in sleepiness, decreased attention, compromises memory and learning skills, and may affect motor control. The negative effects of sleep loss are reversed by recovery sleep, which may show features of delta and/or REM-sleep rebound. In this context, insomnia is a condition with known negative consequences to the health of the affected individual and frequently conveys negative effects to the family nucleus and to society in general. It has been estimated that 9% to 15% of the adult population suffers from chronic insomnia. Psychophysiologic insomnia (or primary insomnia) is likely the most prevalent type of insomnia. The patient with insomnia frequently develops an aversive response to sleep and to all aspects related to this activity. The manifestations of insomnia may include difficulty falling (and staying) asleep, frequent awakenings, early morning awakenings and/or un-refreshing quality of sleep. As a result, affected individuals frequently complain of daytime consequences such as decreased concentration, negative effects on memory (and learning), and daytime fatigue. They may also complain of headaches, fuzziness (or grogginess) and might experience manifestations of excessive sleepiness (which might represent a hazard when driving and/or operating machinery). Stress and anxiety frequently represent precipitating and/or perpetuating factors in the development of insomnia. In regards to stress, the role of the hypothalamus-pituitary-adrenal (HPA) axis in preserving homeostasis has been amply studied. The HPA axis involves the participation of peptides such as corticotropin-releasing hormone (CRH), corticotropin itself and cortisol. The autonomic nervous system activates the amygdaloid complex further enhancing the stress response. When patients are unable to control their stress response, the magnified response may be manifested as an anxiety disorder. According to the DSM-IV, the diagnosis of generalized anxiety disorder (GAD) is based on persistent symptoms of excessive anxiety and worry. Patients with GAD as well as those suffering from other anxiety disorders such as PTSD and panic attacks may manifest symptoms of insomnia. Several models of stress have been proposed to better understand these conditions. For example, prenatal stress has been suggested to increase vulnerability to life events and some reports have suggested impaired sleep among some of the animal models that have been studied. Specifically, it has been reported that rats who are deprived of maternal care suffer from dysregulation of the orexinergic system. Consequently, affected rats may have manifestations of sleep-wake dysregulation. There is more. Rats born to a low care maternal provider (which induces an early stress response) have been found to have methylated the gene that encodes the glucocorticoid receptors, which is reflected in a low expression of receptors. As a result, these animals release more corticosterone in response to stressful situations and are less efficient in managing stress. Furthermore, they have a lower expression of the CB1 receptor in several areas of the brain, thus suggesting that the systems responsible for reducing excitability of the brain (and consequently reducing the subjective sensation of fear and anxiety) are shattered. Models of insomnia evaluating the possible role of an inadequate stress-response have not been thoroughly studied. Potential pharmacologic interventions using such a theoretical framework have not been systematically studied and thus offer a venue for novel pharmacological interventions. The addition of new therapies would be particularly useful as the clinical management of patients with chronic insomnia remains a challenging area in medical practice. This despite the availability of multiple approved hypnotic medications in the physician's armamentarium. To date there is no hypnotic medication which can be considered ideal for the treatment of chronic insomnia. Issues of tolerance and dependence remain relevant concerns for those hypnotic medications, which are considered most effective in the treatment of this condition. Research identifying new compounds based on molecules whose physiologic action is to induce sleep may render safer, more efficient pharmacological interventions to treat insomnia. Following this line of thinking, we have tested the effects of endocannabinoids (eCBs). The eCBs represent a family of molecules, lipids in nature, which bind to the same receptors to which marijuana is known to bind. The active metabolite of marijuana (delta-9-tetrahydrocannabinol [THC]) is known to bind to the CB1 receptors and produce a series of effects including relaxation and sleep. Following the discovery of several eCBs by the research groups of Mechoulam and Lerner, we have tested anandamide and oleamide as sleep inducers. Results have indicated that both molecules and a third one, 2-arachidonyl glycerol, induce sleep (mainly REM sleep). In the present review insomnia is speculated to be a consequence of chronic stress. Several animal models of early stress are also discussed to better understand the role of stress in the causation of insomnia. The current limitations in the availability of ideal hypnotic medications prompt us to argue in favor of continued efforts to find additional, novel pharmacologic interventions to treat this condition. In this context, the potential use of endocannabinoid compounds is proposed as a possible new line of hypnotic medications. While eCBs have been used so far only in animal models, they have been amply successful in promoting the expression of non-REM and REM sleep. The endocannabinoid system has the potential to induce sleep and thus suggest that endocannabinoid agonists offer a new research venue for the exploration of novel pharmacologic interventions in the treatment of insomnia.
El sueño es una actividad fundamental para el bienestar y la preservación del la salud. El no dormir resulta en consecuencias potencialmente letales. Por ejemplo, ratas experimentalmente privadas de sueño total (o de sueño MOR) mueren al cabo de algunas semanas de experimentación. Asimismo, en humanos, la privación de tan solo una noche de sueño conlleva consecuencias importantes. Hipersomnolencia y disminución de las habilidades cognoscitivas son consecuencias de la privación de sueño. El individuo privado de sueño corre el riesgo de cometer errores que potencialmente pueden poner en riesgo su vida o integridad física, así como la de otros. Se ha sugerido que el sueño cumple con la muy importante tarea de ofrecer las condiciones para que se lleven a cabo diversos procesos de restauración y de reorganización neuronal así como el procesamiento de información y consolidación de la memoria. La ausencia de sueño interfiere con estos procesos con el consecuente deterioro de la conducta adaptativa del sujeto. El insomnio es un trastorno que deteriora de manera importante la calidad de vida de las personas que lo padecen. Afecta aproximadamente al 10% de la población. El insomnio se presenta en diversas formas. La clasificación internacional de los trastornos del dormir considera 11 tipos de insomnio. Entre ellos, el insomnio psicofisiológico representa el tipo de insomnio que más frecuentemente se manifiesta en la población. Se trata de un padecimiento donde el paciente desarrolla una aversión a dormir y a todo lo que se relacione con ello. Este tipo de insomnio tiene un componente de estrés que precipita la aparición del insomnio y puede contribuir a los elementos que ayudan a perpetuarlo (insomnio crónico). Se han desarrollado diversos modelos animales para el estudio del estrés y sus consecuencias. Por ejemplo, el estrés temprano inducido por privación del cuidado maternal. Asimismo, por inducción de estrés en la madre (rata) gestante. Sin embargo, se han explotado poco para evaluar el insomnio y mejor aún, para ensayar fármacos que puedan beneficiar al paciente insomne. A pesar de contar con una gran variedad de medicamentos hipnóticos, en la actualidad no existe un hipnótico ideal. Los tratamientos más efectivos con los que contamos conllevan riesgos importantes de tolerancia, adicción y potencialmente efectos colaterales. Por ello, la búsqueda de nuevos fármacos con propiedades inductoras de sueño es inaplazable. Especialmente de fármacos que sean capaces de inducir las fases de sueño delta y sueño MOR sin causar sonambulismo, somnolencia residual y/o efectos negativos en la memoria. En este contexto se discute el potencial uso terapéutico de los endocanabinoides (eCBs), ya que son ansiolíticos e inductores de las fases profundas de sueño (delta y sueño MOR). Los eCBs son moléculas endógenas que tienen una actividad semejante a la de la mariguana. Esto es debido a que tanto la mariguana como los eCBs afectan a un mismo receptor, que es el receptor canabinoide 1 (CB1). Los eCBs tienen un potencial terapéutico que hasta ahora no se ha explotado en beneficio de los pacientes que sufren de insomnio y/o ansiedad. Por ello, en esta revisión se analiza el insomnio desde el punto de vista clínico, se detallan sus características para que el médico clínico no experto pueda reconocerlo y potencialmente tratarlo; también se persigue subrayar la influencia potencial del estrés en la fisiopatogénesis de este trastorno. A pesar de que hay cada vez más información acerca de la síntesis y degradación de los eCBs (lo que es muy importante porque estos mecanismos pueden ser afectados por fármacos que inhiban la degradación o la faciliten, dependiendo de las necesidades terapéuticas) no discutiremos estos temas que se vuelven más especializados. Sin embargo, es importante que se conozca y se discuta su posible uso para beneficio del paciente. Esta revisión se centra en discutir los potenciales beneficios causados por la activación del receptor CB1 en el paciente insomne para reducir su dolencia de mal dormir.
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
Objective To observe expression and location of cannabinoid receptor 1 (CB1) in liver tissue of patients with chronic hepatitis B (CHB) ,and analyze the relationship of it with the liver fibrosis score,the serum levels of TGF-β1 and Leptin. Methods Liver biopsies were performed in 118 patients with CHB.The expression of CB1 in liver tissue was observed by immune histochemical staining, and semi-quantitative analysis was carried out to devide the CB1 score into four grades: -, +, + +, + + +. Serum levels of TGF-β1 and Leptin were determined by ABC-ELISA double-antibody sandwich method. Results The expression of CB1 in liver tissue with CHB had significant relationship with the fibrosis score. As the expression of the CB1 increased, the fibrosis score became higher ( F = 23. 369,P = 0. 000). Moreover, the expression of CB1 in liver tissue with CHB had significant relationship with the serum levels of TGF-β1 and Leptin( F values were 8. 762 and 5. 749;P values were 0. 001 and 0. 027, respectively). Conclusion CB1 may play promotive role in the process of hepatic fibrosis through regulation of TGF-β1 and Leptin.