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Aim To explore the role and mechanism of nuclear receptor subfamily 1,group D,member 1(NR1D1)in the proliferation and migration of mouse adventitial fibroblasts(AFs). Methods Primary AFs isolated from C57BL/6J mice were cultured. Adenovirus carrying Nr1d1 gene was used to overexpress NR1D1 in AFs. The expression of β-catenin was restored by SKL2001. Proliferating cell nuclear antigen(Ki-67)immunofluorescence staining and CCK-8 staining were used to determine cell proliferation,and scratch test was used to determine cell migration. qPCR was used to determine the mRNA level of Nr1d1. Western blot was used to determine the protein levels of NR1D1 and β-catenin. To investigate the role of NR1D1 in intimal hyperplasia,20 male wild type C57BL/6J mice were randomly divided into sham group,carotid artery endothelial injury,sham+SR9009(NR1D1 agonist)group and carotid artery endothelial injury+SR9009(n=5 in each group). They were treated with DMSO or SR9009(100 mg·kg-1·d-1)via intraperitoneal injection for 14 days after operation,respectively. The degree of carotid intimal hyperplasia was measured by HE staining 28 days after operation. Results NR1D1 overexpression significantly reduced the percentage of Ki-67-positive cells(P<0.01),total cell number(P<0.01)and slowed down the rate of wound-healing(P<0.01). NR1D1 overexpression significantly inhibited the expression of β-catenin(P<0.05). After the expression of β-catenin was restored by SKL2001,the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs were abolished(P<0.01). Enhanced activity of NR1D1 significantly ameliorated intimal hyperplasia after carotid endothelial injury(P<0.01). Conclusion NR1D1 may inhibit the proliferation and migration of AFs via suppressing the expression of β-catenin.
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【Objective】 To investigate and compare the effects and mechanisms of risperidone and haloperidol on early socially isolated mice. 【Methods】 C57BL/6 mice aged 3 weeks were raised in single cages after weaning for social isolation （SI）， and the control （GH） group was raised normally. Eight weeks later （mouse adult）， the mice received intraperitoneal injection of equal volumes of normal saline （NS）， risperidone （RIS） and haloperidol （HA）. Then they were divided into four groups： GH+NS， SI+NS， SI+RIS， and SI+HA. The dose of risperidone and haloperidol was 0.1 mg/kg and 0.2 mg/kg， respectively. After administration for 14 days， through the open field experiment， elevated plus maze experiment， forced swimming experiment， nesting experiment， social interaction experiment， novel object discrimination experiment， and prepulse suppression experiment， the mice’s schizophrenia-like behavior was evaluated in terms of autonomous activities， emotions， cognition， and social behavior. We also detected dopamine type 2 receptor （D2R） and the N-methyl-D-aspartate receptor subunit NR1 in the prefrontal cortex （PFC） and nucleus accumbens （NAc）. 【Results】 Compared with those in GH group， the anxiety-like behavior and depression-like behavior of SI mice were significantly increased （P<0.05）， while the nesting ability was significantly decreased （P<0.05） and social interaction and avoidance behavior were significantly reduced （P<0.05）； the cognitive function of PPI was impaired （P<0.05）. Compared with haloperidol， risperidone improved not only depression-like behavior and PPI impairment， but also anxiety-like behavior， nesting ability， social interaction， and avoidance behavior. In SI+RIS and SI+HAL groups， the content of D2R in NAC decreased significantly， and the difference of NR1 in PFC disappeared compared with the control group. 【Conclusion】 Early SI is a good model for simulating schizophrenia. Risperidone has a better intervention effect than haloperidol； risperidone and haloperidol may exert their effects through D2R and NR1.
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Pregnane X receptor (PXR, NR1I2) is a prototypical member of the nuclear receptor superfamily. PXR can be activated by both endobiotics and xenobiotics. As a key xenobiotic receptor, the cellular function of PXR is mostly exerted by its binding to the regulatory gene sequences in a ligand-dependent manner. Classical downstream target genes of PXR participate in xenobiotic responses, such as detoxification, metabolism and inflammation. Emerging evidence also implicates PXR signaling in the processes of apoptosis, cell cycle arrest, proliferation, angiogenesis and oxidative stress, which are closely related to cancer. Here, we discussed, in addition to the characterization of PXR , the biological function and regulatory mechanism of PXR signaling in cancer, and its potential for the targeted prevention and therapeutics.
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Objective@#To investigate the effect of Lactobacillus casei on depressive behavior and the expression of NR1, NR2A receptor in postpartum depression rats induced by maternal separation.@*Methods@#Twenty-four Sprague-Dawley female rats were randomly divided into control group(CON group) (n=8), postpartum depression group(PPD group) (n=8), postpartum depression with lactobacillus casei group(PPD+ Lactobacillus casei group) (n=8). Rats of the control group were given no interventions.Rats in PPD group and PPD+ Lactobacillus casei group were given maternal separation to establish depression model.And then the rats in PPD+ Lactobacillus casei group were treated with Lactobacillus casei(8×108CFU/(kg·d)) for 4 weeks after 14 days of maternal separation.The forced swimming test (FST) was employed to detect the depressive behaviors.Lactobacillus, Bifidobacterium, Enterococcus faecalis and Escherichia coli in cecum of rats and the expression of NR1 and NR2A mRNA in hippocampus of rats were detected by real-time fluorescence quantitative PCR.@*Results@#Compared with CON group ((157.50±8.13) s), the immobility time of PPD group((200.00±10.35) s) was significantly longer (t=-3.23, P<0.05). Compared with PPD group, the immobility time of PPD+ Lactobacillus casei group ((153.25±7.41) s) was significantly shortened (t=3.67, P<0.05), and the depressive behavior was improved.Compared with CON group, the mRNA expression of Lactobacillus ((1.47±0.08), t=-5.87, P<0.01), Enterococcus faecalis ((1.23±0.08), t=-2.85, P<0.05) and Escherichia coli( (1.33±0.07), t=-4.96, P<0.01) in caecum were significantly increased in PPD group, while that of Bifidobacterium decreased significantly((0.65±0.07), t=5.18, P<0.01). Compared with PPD group, the mRNA expression of Lactobacillus( (1.05±0.05), t=3.67, P<0.01), Enterococcus faecalis ((0.97±0.05), t=2.74, P<0.05) and Escherichia coli ((1.06±0.05), t=3.31, P<0.01) were significantly decreased in PPD+ Lactobacillus casei group, while that of Bifidobacterium increased significantly ((0.98±0.04), t=-4.26, P<0.01). Compared with CON group, the mRNA expression of NR1 receptor ((0.57±0.04), t=9.65, P<0.01) and NR2A receptor ((0.60±0.05), t=8.64, P<0.05) in hippocampus of rats in PPD group were significantly decreased.Compared with PPD group, the expression of NR1 receptor ((1.01±0.05), t=-5.39, P<0.01) and NR2A receptor ((0.98±0.07), t=-3.91, P<0.05) in hippocampus were increased significantly in PPD+ Lactobacillus casei group.@*Conclusion@#Lactobacillus casei can improve the depressive behavior of postpartum depression in rats, and improve the intestinal flora, which affect the expression of NR1 and NR2A in hippocampus.
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Objective To investigate the effect of Lactobacillus casei on depressive behavior and the expression of NR1, NR2A receptor in postpartum depression rats induced by maternal separation. Methods Twenty-four Sprague-Dawley female rats were randomly divided into control group( CON group) (n=8),postpartum depression group(PPD group) (n=8),postpartum depression with lactobacillus casei group(PPD+ Lactobacillus casei group) (n=8). Rats of the control group were given no interventions. Rats in PPD group and PPD+Lactobacillus casei group were given maternal separation to establish depression model. And then the rats in PPD+Lactobacillus casei group were treated with Lactobacillus casei ( 8× 108CFU/(kg·d)) for 4 weeks after 14 days of maternal separation. The forced swimming test ( FST) was employed to detect the depressive behaviors. Lactobacillus,Bifidobacterium,Enterococcus faecalis and Esche-richia coli in cecum of rats and the expression of NR1 and NR2A mRNA in hippocampus of rats were detec-ted by real-time fluorescence quantitative PCR. Results Compared with CON group ((157. 50±8. 13) s), the immobility time of PPD group((200. 00±10. 35) s) was significantly longer (t=-3. 23,P<0. 05). Com-pared with PPD group,the immobility time of PPD+ Lactobacillus casei group ((153. 25±7. 41) s) was sig-nificantly shortened (t=3. 67,P<0. 05),and the depressive behavior was improved. Compared with CON group,the mRNA expression of Lactobacillus ((1. 47± 0. 08),t=-5. 87,P<0. 01),Enterococcus faecalis ((1. 23±0. 08),t=-2. 85,P<0. 05) and Escherichia coli( (1. 33±0. 07),t=-4. 96,P<0. 01) in caecum were significantly increased in PPD group, while that of Bifidobacterium decreased significantly (( 0. 65 ± 0. 07),t=5. 18,P<0. 01). Compared with PPD group,the mRNA expression of Lactobacillus ( ( 1. 05 ± 0. 05),t=3. 67,P<0. 01),Enterococcus faecalis ((0. 97±0. 05),t=2. 74,P<0. 05) and Escherichia coli ((1. 06±0. 05),t=3. 31,P<0. 01) were significantly decreased in PPD+ Lactobacillus casei group,while that of Bifidobacterium increased significantly ((0. 98± 0. 04),t=-4. 26,P<0. 01). Compared with CON group,the mRNA expression of NR1 receptor (( 0. 57 ± 0. 04), t=9. 65, P<0. 01) and NR2A receptor ((0. 60±0. 05),t=8. 64,P<0. 05) in hippocampus of rats in PPD group were significantly decreased. Com-pared with PPD group,the expression of NR1 receptor ((1. 01±0. 05),t=-5. 39,P<0. 01) and NR2A re-ceptor ((0. 98±0. 07),t=-3. 91,P<0. 05) in hippocampus were increased significantly in PPD+ Lactoba-cillus casei group. Conclusion Lactobacillus casei can improve the depressive behavior of postpartum depression in rats,and improve the intestinal flora,which affect the expression of NR1 and NR2A in hippocampus.
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Objective To observe the effect of Kidney-nourishing, Blood-activating, Phlegm-resolving and Resuscitation-inducing Decoction(KBPRD)on the expression of N-methyl-D-aspartate(NMDA)receptor subtype 1,2A,2B(NR1,NR2A,NR2B)in the cochlear spiral ganglion neurons(SGN)of tinnitus rats and to explore its mechanism, thus to provide experimental evidence for the treatment of tinnitus with KBPRD. Methods Sixty rats were randomly divided into normal group,model group,western medicine group,and low-,middle-,and high-dose Chinese medicine groups, 10 rats in each group. Rats were given intraperitoneal injection of sodium salicylate combined with water deprivation to induce tinnitus model. After successful establishment of the model, the rats in low-,middle-,and high-dose Chinese medicine groups were given gastric administration of KBPRD in the dosage of 5.5, 11, 22 g·kg-1·d-1 respectively, the rats in western medicine group were given gastric administration of 5 mg·kg-1·d-1 of carbamazepine,and rats in the model group and normal group were given gastric administration of 2 mL of normal saline,once every day,treatment time covering 8 weeks. The expression levels of NR1,NR2A,and NR2B in the cochlear SGN was detected by immunoblotting and real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)after 8 weeks of treatment. Results Compared with the normal group,the expression levels of NR1, NR2A and NR2B in the model group were increased, the difference being significant (P < 0.05). Compared with the model group,the expression of NR1,NR2A and NR2B in low-,middle-,and high-dose Chinese medicine groups were significantly decreased(P<0.05).Conclusion KBPRD is effective on relieving tinnitus of rats, and its mechanism is correlated with lowering the increased expression of NR1,NR2A and NR2B in SGN of tinnitus rats.
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BACKGROUND The time of progression towards AIDS can vary greatly among seropositive patients, and may be associated with host genetic variation. The NR1I2 (PXR) gene, a ligand-activated transcription factor, regulates the transcription immune pathway genes and can therefore be targets of viral replication mechanisms influencing time of progression to AIDS. OBJECTIVE To verify the association of single nucleotide polymorphisms (SNPs) rs3814057, rs6785049, rs7643645, and rs2461817 in the NR1I2 (PXR) gene with progression to AIDS in HIV-1 infected patients. METHODS Blood samples were obtained from 96 HIV-1 positive individuals following informed consent. DNA was isolated and genotyped through real time polymerase chain reaction (PCR) for the presence of SNPs in the NR1I2. Questionnaires on socio-demographic features and behaviors were answered and time of progression to AIDS was estimated based on medical chart analysis. FINDINGS Patients with the GG genotype for rs7643645 were shown to be related with a more rapid disease progression when compared to GA and AA genotypes. This result was maintained by the Multivariate Cox Regression considering sex, ethnicity, and presence of HLA-B*57, HLA-B*27, and CCR5del32 polymorphisms. MAIN CONCLUSIONS Recent studies reported the expression of the nuclear receptors in T-Lymphocytes, suggesting their possible role in the immune response. In addition, nuclear receptors have been shown to inhibit the HIV replication, although no such mechanism has been thoroughly elucidated to date. This is the first time an association between NR1I2 polymorphism and time of progression to AIDS is reported and supports an apparent relationship between the gene in the immune response and identifies another genetic factor influencing AIDS progression.
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Humans , Male , Female , Adult , Acquired Immunodeficiency Syndrome/genetics , Acquired Immunodeficiency Syndrome/pathology , Disease Progression , Polymorphism, Genetic , GenotypeABSTRACT
This study was aimed to observe the effect ofZi-Bu Pi-Yin Recipe (ZBPYR) on the mRNA expressions of NMDA receptor subunits NR1, NR2A, NR2B in different brain regions of spleen-yin deficiency Alzheimer's Disease (AD) model rats. The levels of NR1, NR2A, NR2B mRNA expressions were detected by using RT-PCR method. The results showed that the levels of NR1, NR2A, NR2B mRNA expressions of AD group and spleen-yin deficiency AD group decreased significantly (P < 0.05). The levels of NR1, NR2A, NR2B mRNA expressions of ZBPYR treatment group increased significantly (P < 0.05). It was concluded that the expression levels of NMDAR mRNA in different brain regions of the ZBPYR treatment group increased significantly, which indicated that ZBPYR may up-regulate the protein expressions of NMDAR by increasing the expression levels of NMDAR mRNA, thereby to play the anti-dementia effect.
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Objective To evaluate the effects of curcumin on the activity of NR2B and NR1 in the spinal dorsal horn in a rat model of diabetic neuropathic pain (DNP).Methods Diabetes mellitus was induced by high-sucrose and high-fat diet and intraperitoneal streptozotocin 35 mg/kg,then confirmed by fasting blood glucose level ≥ 16.7 mmol/L 3 days later in male Sprague-Dawley rats.DNP was confirmed by the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) measured on day 14 after streptozotocin administration ＜ 80％ of the baseline value.The rats were then randomly divided into 3 groups (n =27 each) using a random number table:DNP,DNP+ curcumin group (group DCur)and DNP + solvent control group (group DSC).Curcumin 100 mg· kg-1 · d-1 and corn oil 4 ml· kg-1 · d-1 were injected intraperitoneally for 14 consecutive days starting from 14 days after administration of streptozotocin in DCur and DSC groups,respectively.Another 27 normal male Sprague-Dawley rats served as control group (group C) and were fed with normal forage.At 3,7 and 14 days after curcumin injection,MWT and TWL were measured and the lumbar segments (L4-6) of the spinal cord were removed.The expression of pTyr1472-NR2B and pSer896-NR1 in the spinal dorsal horn was determined by immunohistochemistry and Western blot.Results Compared with group C,MWT was significantly decreased,TWL was shortened,and the expression of pTyr1472-NR2B was up-regulated at each time point in group DNP.Compared with group DNP,MWT was significantly increased,and TWL was prolonged at 7 days after curcumin injection,and the expression of pTyr1472-NR2B was down-regulated at 3 days after curcumin injection in group DCur.There was no significant difference in each parameter between DNP and DSC groups,and in the expression of pSer896-NR1 between the four groups.Conclusion The mechanism by which curcumin mitigates neuropathic pain in type 2 diabetic rats may be related to inhibition of up-regulation of pTyr1472-NR2B expression,while unrelated to pSer896-NR1 expression in the spinal dorsal horn.
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Objective To screen for the proteins interacting with CXCR4 during nuclear localization in renal cell carcinoma (RCC) A498 cells. Methods Specific band in co-immunoprecipitation (Co-IP) experiments was sent for mass spectrometry. With the results of Co-IP experiments and mass spectrometry, the proteins interacting with CXCR4 were determined by bioinformatics analyses. Results Three specific bands were found after Co-IP with anti-CXCR4 antibody, and the results of mass spectrometry of the three specific bands showed 36 proteins possibly interacting with CXCR4. Bioinformatics analyses showed that NR1D2, c-src and HSPA8 might interact with CXCR4 and participate in CXCR4 nuclear localization. Conclusion NR1D2, c-src and HSPA8 might have participated in CXCR4 nuclear localization in RCC A498 cells.
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Producing polyclonal antibodies (IgY) in chickens has advantages over those obtained in other animal models, since they have been used as a tool for studying different proteins (NMDA glutamate receptor in our case, specifically the NR1 subunit). We produced specific antibodies against expression products by the alternative splicing of the gene encoding NMDA receptor NR1 subunit in adult rat brain. Three peptides corresponding to the splicing sites (N1, C1 and C2' cassettes) were designed, synthesised and used individually as antigens in hens. Specific immunoglobulins were purified from yolks. The antibodies were then used for purifying the NMDA receptor NR1 subunit using affinity chromatography coupling the three antibodies to the support.
La producción de anticuerpos policlonales en gallinas (IgY) tiene ventajas sobre anticuerpos obtenidos en otros modelos animales y se han empleado como una nueva herramienta para estudiar diferentes proteínas (el receptor de glutamate tipo NMDA en nuestro caso, específicamente la subunidad NR1). Produjimos anticuerpos específicos contra productos de expresión por splicing alternativo del gen que codifica la subunidad NR1 del receptor tipo NMDA en el cerebro de rata adulta. Se diseñaron 3 péptidos correspondientes a los sitios de splicing del gen (conocidos como casetes N1, C1 y C2'), se sintetizaron y se usaron individualmente como antígenos en gallinas. Inmunoglobulinas específicas se purificaron de las yemas. Los anticuerpos se usaron para purificar la subunidad NR1 del receptor tipo NMDA usando cromatografía de afinidad, a través del acople de los tres anticuerpos al soporte.
A produção de anticorpos policlonais (IgY) em galinhas tem vantagens sobre os obtidos em outros modelos animais e eles têm sido usados como uma ferramenta para o estudo de proteínas diferentes (NMDA receptor de glutamato no nosso caso, especificamente a subunidade NR1). Nós produzimos anticorpos específicos contra produtos de expressão pela splicing alternativo do gene que codifica receptor NMDA subunidade NR1 no cérebro de ratos adultos. Três peptídeos correspondentes aos locais de splicing (N1, C1 e C2' cassetes) foram concebidos, sintetizados e utilizados individualmente como antígenos em galinhas. Imunoglobulinas específicas foram purificadas a partir de gemas. Os anticorpos foram então usados para purificar o receptor NMDA subunidade NR1 utilizando cromatografia de afinidade, por meio da junção dos três anticorpos ao suporte.
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Objective To evaluate the effects of electromagnetic irradiation of 2000 μW/cm2 exposure on mRNA and protein expression levels of immunoreactive protein and mRNA of NMDAR1 in rats hippocampal,and to explore the impaired mechanism of electromagnetic irradiation on learning and memory.Methods Rats were randomly divided into normal control group, sham-radiated group, and 1 h/d, 2 h/d, and 3 h/d radiation groups.The rats in the radiation groups were fixed and recieved microwave exposure of 2000 μW/cm2, then their learning and memory abilities were tested by Morris water maze experiment, the change of NR1 protein in hippocampal neurons of each group of rats was measured with immunohistochmistry and western blot techniques, and the expression of NR1 mRNA in hippocampus was determined by RT-PCR.Results In the water maze test,compared with the normal control group (8.8 ± 1.66 ), the escape latency of three radiated groups rats ( 1 h/d ( 12.29 ±1.36) s,2 h/d ( 17.99 ±2.25) s,and 3 h/d (24.66 ±5.56) s) were significantly longer (P＜0.05).In the radiation group,the hippocampal neurons of rats showed evident reduction in the ratio of NR1 positive cells,irregular,and arrayed in disorder.Moreover,compared with the normal control group ( (0.70 ±0.11 ), (0.68 ±0.11 ) ) ,the expession of NR1 protein ( 1 h/d (0.122 ±0.026) ,2 h/d (0.102 ±0.023) ,and 3 h/d (0.060 ± 0.009) ) and its mRNA ( 1 h/d (0.46 ±0.07) ,2 h/d (0.35 ±0.05) ,and 3 h/d (0.12 ±0.02) ) in hippocampal neurons was significantly decreased (P＜0.05).Among the indicators, there was no significant difference between sham-radiated group and normal control group.Conclusions Electromagnetic irradiation of 2000 μW/cm2 exposure can impair the learning and memory abilities of rats possibly through a mechanism correlated with the lower expression of NR1 protein and its mRNA in hippocampus.
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Objetivo. Identificar la influencia de los cambios respecto a la estructura secundaria y a la relación evolutiva de los receptores NMDA, AMPA Y KAINATO en las especies Homo sapiens, Pan troglodytes, Pongo pygmaeus, y Macaca mulata. Materiales y métodos. Se recopilaron 91 secuencias correspondientes a los receptores NMDA, AMPA y Kainato y se sometieron a los programas de predicción de estructura secundaria, sitios de fosforilación, alineamientos múltiples, selección del modelo de evolución y predicción filogenética. Resultados. Se encontró que las subunidades GLUR5, NR2A, NR2C y NR3A presentaron cambios de estructura en la región C-terminal y aparición o pérdida de sitios de fosforilación en esta zona. Adicionalmente la predicción filogenética nos propone que las subunidades NR2 de NMDA son las más cercanas al nodo ancestral que da origen a los demás subunidades. Conclusiones. Los cambios de estructura y sitios de fosforilación en las subunidades GLUR5, NR2A, NR2C y NR3A nos sugieren variaciones en la interacción de la región C-terminal con proteínas quinasas y con proteínas con dominios PDZ lo cual podría afectar el tráfico y anclaje de las subunidades. Por otra parte la predicción filogenética nos propone que los cambios que se presentaron en las subunidades NR2 dieron origen a las demás subunidades de los receptores ionotrópicos de glutamato, básicamente porque son las subunidades de NMDA y en particular NR2D las que se encuentran más estrechamente relacionadas con el nodo ancestral que posiblemente dio origen a los iGluRs.
Objective. To identify the influence of changes on the secondary structure and evolutionary relationship of NMDA, AMPA and kainate receptors in Homo sapiens, Pan troglodytes, Pongo pygmaeus and Macaca mulatta. Materials and methods. We identified 91 sequences for NMDA, AMPA and kainate receptors and analyzed with software for predicting secondary structure, phosphorylation sites, multiple alignments, selection of protein evolution models and phylogenetic prediction. Results. We found that subunits GLUR5, NR2A, NR2C and NR3A showed structural changes in the C-terminal region and formation or loss of phosphorylation sites in this zone. Additionally the phylogenetic prediction suggests that the NMDA NR2 subunits are the closest to the ancestral node that gives rise to the other subunits. Conclusions. Changes in structure and phosphorylation sites in GLUR5, NR2A, NR2C and NR3A subunits suggest variations in the interaction of the C-terminal region with kinase proteins and with proteins with PDZ domains, which could affect the trafficking and anchoring of the subunits. On the other hand, the phylogenetic prediction suggests that the changes that occurred in the NR2 subunits gave rise to the other subunits of glutamate ionotropic receptors, primarily because the NMDA and particularly the NR2D subunits are the most closely related to the ancestral node that possibly gave rise to the iGluRs.
Objetivo. Identificar a influência das mudanças da estrutura secundária e da relação evolutiva dos receptores NMDA, AMPA e cainato nas espécies Homo sapiens, Pan troglodytes, Pongo pygmaeus e Macaca mulatta. Materiais e métodos. Foram recopiladas 91 seqüências correspondentes aos receptores NMDA, AMPA e cainato e foram submetidos a programas de predição de estrutura secundária, sítios de fosforilação, alinhamentos múltiplos, seleção do modelo de evolução e predição da filogenia. Resultados. Descobrimos que as subunidades GLUR5, NR2A, NR2C e NR3A apresentaram alterações estruturais na região C-terminal e aparecimento ou perda de sítios de fosforilação nesta área. Além disso, a predição filogenética sugere ainda que as subunidades NR2 de NMDA são as mais próximas ao nó ancestral que dá origem às demais subunidades. Conclusões. As mudanças na estrutura e nos sítios de fosforilação nas subunidades GLUR5, NR2A, NR2C e NR3A sugerem variações na interação da região C-terminal com proteínas quinase e com proteínas de domínios PDZ que poderia afetar o tráfego e fixação das subunidades. Além disso, a predição filogenética sugere que as mudanças ocorridas nas subunidades NR2 deram origem às outras subunidades de receptores ionotrópicos de glutamato, principalmente porque são subunidade NMDA e particularmente NR2D aquelas que são mais estreitamente relacionadas com o nó ancestral que provavelmente deu origem aos iGluRs.
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Objective To study the effect of Tongbiding injection on expression of N-methyl-D-aspartate (NMDA)receptor 1 mRNA in the spinal cord of chronic constriction injury(CCI)rats.Methods Thirty male Spragru-Dawley rats were randomly divided into control group(group A),CCI group(group B)and Tongbiding group(group C),ten rat for eah group.The changes in NR1 mRNA expression in the spinal cord was detected by reverse transcription-polymerase chain reaction(RT-PCR)techniques.Results The expression of NR1 mRNA in the spinal cord was minimal in control group,CCI induced significant increase in the expression of NR1 mRNA in the spinal cord and Tongbiding significantly inhibited the increase in NR1 mRNA expression.There was significant difference between CCI and Tongbiding group(P
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Objective To investigate the mechanisms of the inhibitory effects of peripheral electrical stimu- lation(PES)on chronic central pain(CCP)after spinal cord injury(SCI).Methods Twenty-four male Sprague- Dawley rats with CCP following SCI were randomly divided into three groups:a group without stainless steel needles implanted (NSSN group,n=8),a group with a stainless steel needle implanted but no peripheral electrical stimula- tion applied(NPES group,n=8)and a PES group(PES group,n=8).The rats' CCP was evaluated through ob- serving their response to nociceptive stimulation by means of the paw withdrawal pressure threshold(PWPT)and the paw withdrawal latency(PWL).Spontaneous pain behaviors including autophagia and scratching were observed at the same time.PES was applied via stainless steel needles inserted into standard acupoints on the hind limps and the back.The expression of the NMDA receptor 1(NR-1)subunit in the spinal cord horn was measured using immuno- chemical methods.Results Compared with the NSSN and NPES groups,CCP in the PES group was alleviated, PWPT and PWL were dramatically increased(P＜0.01)and the expression of NR-1 was obviously decreased (P＜0.01).Conclusion Peripheral electrical stimulation may alleviate chronic central pain after spinal cord injury in rats.
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Objective To observe the expression and phosphorylation of N-methyl-D-aspartate (NMDA) receptor NR1 subunit in rat cortex and hippocampus under high altitude hypoxia. Methods The adult SD rats were exposed to hypobaric hypoxia imitating 5 500-meter high altitude for 8 h daily for 3, 7, 14, 21 d. Immunohistochemical staining, Western blotting and co-immunoprecipitation were used to detect the expression and phosphorylation of NR1 in rat cortex and hippocampus. Results Both immunohistochemistry and Western blotting showed NR1 expression in rat cortex and hippocampus was increased under hypoxia in a time-dependent manner. The tyrosine phosphorylation of hypoxia groups was increased, and reached the peak on day 14 after hypoxia, then decreased, still higher than that of control groups till day 21 (P
ABSTRACT
PURPOSE: Excessive amounts of glutamate are released into the extracelluar space during hypoxia/ischemia and contribute to neuronal injury through overactivation of the NMDA receptors. It will be expected that the changes of the NMDA receptors to decrease the injury are developed as a kind of defense mechanism. The NMDA receptors are composed of NR1 and NR2, and there are four NR2 subunits; NR2A, NR2B, NR2C, and NR2D. The NR1 is essential for the function of the NMDA receptors and multiple NR2 subunits potentiate and differentiate the function of the NMDA receptors. The NR2 subunits alone show no ability to respond to several agonists, however when coexpressed with the NR1, the NR2 subunits markedly potentiated the NR1 activity and produced functional variability of the NMDA receptors. Compared with the NR2C and the NR2D, the NR2A and the NR2B show prominent expression in the hippocampus. In an in vitro model of rat hippocampal slices after hypoxia, we investigated the changes of the NR2A and the NR2B amounts through immunoblot with anti-NR2A and anti-NR2B antisera. METHODS: Hippocampi from adult rats(Sprague Dawley) were isolated and sliced into a 500 m section in a glucose containing artificial CSF medium on ice. In order to induce a hypoxia, sample slices were treated with 95% N2/5% CO2 for 10 min, 20 min, 30 min, and 60 min, respectively and control slices were treated with 95% O2/5% CO2 to supply sufficient oxygen as the same way. And then the control and the sample slices were homogenated and 40 g of each homogenates were electrophoresed in a 6% SDS-gel, transfered to nitrocellulose, and immunostained with anti-NR2A and anti-NR2B antisera. RESULTS: 1) Anti-NR2A antisera were produced by recombinant DNA technology. 2) The NR2A and the NR2B were enriched in the order of PSD, synaptosome and brain homogenate. 3) There was no difference of the NR2A and the NR2B expression in both control and experimental group. CONCLUSIONS: At least up to one hour after hypoxic damage, it is likely that there is no prominent changes of the NR2A and the NR2B amounts. Considering that the changes could occur locally or microscopically in this experimental protocol, the relative amounts of the NR2A and the NR2B in the hippocampal homogenates are too small to be detected by immunoblot analyses. And we can not exclude the possibility of no changes in one hour after hypoxia, if these changes evolve with a extremely slow progression.
Subject(s)
Adult , Animals , Humans , Infant, Newborn , Rats , Hypoxia , Brain , Collodion , DNA, Recombinant , Glucose , Glutamic Acid , Hippocampus , Ice , Immune Sera , N-Methylaspartate , Neurons , Oxygen , Receptors, N-Methyl-D-Aspartate , SynaptosomesABSTRACT
PURPOSE: Excessive amounts of glutamate are released into the extracelluar space during hypoxia/ischemia and contribute to neuronal injury through overactivation of the NMDA receptors. It will be expected that the changes of the NMDA receptors to decrease the injury are developed as a kind of defense mechanism. The NMDA receptors are composed of NR1 and NR2, and there are four NR2 subunits; NR2A, NR2B, NR2C, and NR2D. The NR1 is essential for the function of the NMDA receptors and multiple NR2 subunits potentiate and differentiate the function of the NMDA receptors. The NR2 subunits alone show no ability to respond to several agonists, however when coexpressed with the NR1, the NR2 subunits markedly potentiated the NR1 activity and produced functional variability of the NMDA receptors. Compared with the NR2C and the NR2D, the NR2A and the NR2B show prominent expression in the hippocampus. In an in vitro model of rat hippocampal slices after hypoxia, we investigated the changes of the NR2A and the NR2B amounts through immunoblot with anti-NR2A and anti-NR2B antisera. METHODS: Hippocampi from adult rats(Sprague Dawley) were isolated and sliced into a 500 m section in a glucose containing artificial CSF medium on ice. In order to induce a hypoxia, sample slices were treated with 95% N2/5% CO2 for 10 min, 20 min, 30 min, and 60 min, respectively and control slices were treated with 95% O2/5% CO2 to supply sufficient oxygen as the same way. And then the control and the sample slices were homogenated and 40 g of each homogenates were electrophoresed in a 6% SDS-gel, transfered to nitrocellulose, and immunostained with anti-NR2A and anti-NR2B antisera. RESULTS: 1) Anti-NR2A antisera were produced by recombinant DNA technology. 2) The NR2A and the NR2B were enriched in the order of PSD, synaptosome and brain homogenate. 3) There was no difference of the NR2A and the NR2B expression in both control and experimental group. CONCLUSIONS: At least up to one hour after hypoxic damage, it is likely that there is no prominent changes of the NR2A and the NR2B amounts. Considering that the changes could occur locally or microscopically in this experimental protocol, the relative amounts of the NR2A and the NR2B in the hippocampal homogenates are too small to be detected by immunoblot analyses. And we can not exclude the possibility of no changes in one hour after hypoxia, if these changes evolve with a extremely slow progression.