大鼠脑损伤后COX-2、GluR2、PAFR表达变化及其与脑损伤的关系
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摘要
【目的】复制大鼠落体打击模型,同时从m RNA水平和蛋白水平研究环氧化酶-2(cyclooxygenase-2,COX-2)、谷氨酸受体2(glutamate receptor 2,Glu R2)、血小板活化因子受体(platelet activating factor receptor,PAFR)损伤后的时序性变化,探讨它们之间的相互关系以及与脑损伤时序性变化的相关性。【方法】选用体质量350~400 g的雄性SD大鼠,随机分为手术对照组及损伤组,后者于损伤后4、8、12、24、48 h不同时间点取材观察,复制Marmarou's大鼠闭合性颅脑损伤模型,应用RT-PCR技术对海马的COX-2、Glu R2和PAFR的表达情况进行研究,同时应用免疫组化SP法检测它们在海马中的表达情况。【结果】(1)RT-PCR结果显示PAFR和Glu R2在对照组呈高表达,损伤后表达逐渐减弱,12 h达最低值,损伤后各组与对照组相比差别有显著性(P<0.01)。COX-2在对照组呈低表达,损伤后表达逐渐增高,8 h达最高值,损伤后各组与对照组相比差别有显著性(P<0.01)。(2)免疫组化提示COX-2在对照组有少量免疫反应阳性细胞表达,损伤后阳性细胞表达增多,于损伤后8 h达最大值。Glu R2在对照组有大量免疫反应阳性细胞表达,损伤后阳性细胞表达减少,于损伤后24 h达最低值。PAFR在对照组海马部位可见极少量阳性神经元表达。胞浆呈强阳性表达,细胞核不表达,部分细胞有阳性突起,损伤后几乎没见到阳性神经元表达。【结论】COX-2、Glu R2和PAFR三者在损伤前后表达的时序性变化特点可以作为判断脑损伤的客观指标。
【Objective】To establish the falling-attack rat models, study the sequential changes of cyclooxygenase-2(COX-2),glutamate receptor 2(Glu R2)and platelet activating factor receptor(PAFR)in m RNA and protein levels after injury, and explore the relationship between them as well as the correlation with sequential changes of brain injury.【Methods】Male Sprague-Dawley rats weighing 350 to 400 g were randomly divided into control group, sham operation group and injury groups. The rats in the injury groups were subjected to Marmarou's traumatic brain injury and then subdivided into 4 h, 8 h, 12 h, 24 h and 48 h groups according to the time elapsed after injury. The expressions of COX-2, Glu R2 and PAFR in hippocampus on m RNA level were detected by reverse transcription polymerase chain reaction(RT-PCR). The expressions of COX-2, Glu R2 and PAFR in hippocampus on protein level were determined with SP immunohistochemistry.【Results】(1) The results of RT-PCR showed that the expressions of PAFR and Glu R2 were high in the sham operation group. The expressions decreased gradually after injury and reached the minimum value at 12 h. There was significant different between injury groups and sham operation group(P<0.01). The expression of COX-2 was low in the sham operation group. The expression of COX-2 increased gradually after injury and reached the highest at 8 h. It showed significant difference between injury groups and sham operation group(P<0.01).(2) The results of immunohistochemistry indicated that mild expression of COX-2 was observed in the sham operation group. After brain injury, the area of COX-2 positive neurons in hippocampus increased progressively and reached the peak level at 8 h. Much expression of Glu R2 was observed in the sham operation group. After brain injury, the area of Glu R2 positive neurons in hippocampus decreased progressively and reached the lowest level at 24 h. Little expression of PAFR was observed in the sham operation group. Strong positive expression appeared in cytoplasm, no expression in nucleus, and positive neurites in some cells. After brain injury, the area of PAFR positive neurons in hippocampus was hardly observed. 【Conclusion】The temporal characteristics of PAFR, COX-2 and Glu R2 expressions before and after injury can be used as objective indicators for brain injury.
【Objective】To establish the falling-attack rat models, study the sequential changes of cyclooxygenase-2(COX-2),glutamate receptor 2(Glu R2)and platelet activating factor receptor(PAFR)in m RNA and protein levels after injury, and explore the relationship between them as well as the correlation with sequential changes of brain injury.【Methods】Male Sprague-Dawley rats weighing 350 to 400 g were randomly divided into control group, sham operation group and injury groups. The rats in the injury groups were subjected to Marmarou's traumatic brain injury and then subdivided into 4 h, 8 h, 12 h, 24 h and 48 h groups according to the time elapsed after injury. The expressions of COX-2, Glu R2 and PAFR in hippocampus on m RNA level were detected by reverse transcription polymerase chain reaction(RT-PCR). The expressions of COX-2, Glu R2 and PAFR in hippocampus on protein level were determined with SP immunohistochemistry.【Results】(1) The results of RT-PCR showed that the expressions of PAFR and Glu R2 were high in the sham operation group. The expressions decreased gradually after injury and reached the minimum value at 12 h. There was significant different between injury groups and sham operation group(P<0.01). The expression of COX-2 was low in the sham operation group. The expression of COX-2 increased gradually after injury and reached the highest at 8 h. It showed significant difference between injury groups and sham operation group(P<0.01).(2) The results of immunohistochemistry indicated that mild expression of COX-2 was observed in the sham operation group. After brain injury, the area of COX-2 positive neurons in hippocampus increased progressively and reached the peak level at 8 h. Much expression of Glu R2 was observed in the sham operation group. After brain injury, the area of Glu R2 positive neurons in hippocampus decreased progressively and reached the lowest level at 24 h. Little expression of PAFR was observed in the sham operation group. Strong positive expression appeared in cytoplasm, no expression in nucleus, and positive neurites in some cells. After brain injury, the area of PAFR positive neurons in hippocampus was hardly observed. 【Conclusion】The temporal characteristics of PAFR, COX-2 and Glu R2 expressions before and after injury can be used as objective indicators for brain injury.
引文
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[15]Yuruker V,Naziroglu M,Senol N,et al.Reduction in traumatic brain injury-induced oxidative stress,apoptosis and calcium entry in rat hippocampus by melatonin:Possible involvement of TRPM2 channels[J].Metab Brain Dis,2015,30(1):223-231.
[16]Seyhan N,Canseven AG.In vivo effects of ELF MFs on collagen synthesis,free radical processes,natural antioxidant system,respiratory burst system,immune system activities,and electrolytes in the skin,plasma,spleen,lung,kindney,and brian tissues[J].Electromagn Biol Med,2006,25(4):291-305.
[17]Eroglu B,Kimbler DE,Pang J,et al.Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury[J].Neurochem,2014,130(5):626-641.
[18]Lin CJ,Chen TH,Yang LY,et al.Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death[J].Cell Death Dis,2014,5(1):1147.
[19]Zhiqiang Li,Qingming Shu,Lingzhi Li,et al.Sequential expression of cyclooxygenase-2,glutamate receptor-2,and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury[J].Neural Regeneration Research,2014,9(9):978-985.
[20]Harris RE.Cyclooxygenase-2(cox-2)and the infiammo genesis of cancer[J].Subcell Biochem,2007,42(1):93-126.
[2]Conti A,Sanchez-Ruiz Y,Bachi A,et al.Proteome study of human cerebrospinal fluid following traumatic brain injury indicates fibrin(ogen)degradation products as trauma-associated markers[J].Neurotrauma,2004,21(7):854-863.
[3]Blain JF,Paradis E,Gaudreault SB,et al.A role for lipoprotein lipase during synaptic remodeling in the adult mouse brain[J].Neurobiol Dis,2004,15(3):510-519.
[4]Siman R,Mc Intosh TK,Soltesz KM,et al.Proteins released from degenerating neurons are surrogate markers for acute brain damage[J].Neurobiol Dis,2004,16(2):311-320.
[5]Sironi L,Guerrini U,Tremoli E,et al.Analysis of pathological events at the onset of brain damage in stroke-prone rats:a proteomics and magnetic resonance imaging approach[J].Neurosci Res,2004,78(1):115-122.
[6]Anna C,Andrea G,Thomas A,et al.Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex p roteome by 2-D DIGE[J].Proteomics,2008,8(6):1276-1291.
[7]Ding Q,Vaynman S,Souda P,et al.Exercise affects energy metabolism and neural plasticity-related proteins in the Hippocampus as revealed by proteomic analysis[J].Eur J Neurosci,2006,24(5):1265-1276.
[8]Glanzer JG,Enose Y,Wang T,et al.Genomic and proteomic microglial profiling:Pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation[J].Neurochem,2007,102(3):627-645.
[9]Zhan L,Liang L,Shu QM,et al.Distinct proteins in cortex of rats with closed traumatic brain injury detected by a WCX-2protein chip[J].Neural Regen Res,2007,2(6):339-343.
[10]舒清明,李志强,李灵芝,等.闭合性脑损伤后血清和海马的蛋白质表达[J].法医学杂志,2011,27(02):107-111.
[11]Shu QM,Li ZQ,Yang SW,et al.Two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry for detection of protein expression profiles in the hippocampus following closed brain injury[J].Neural Regen Res,2010,5(23):1795-1801.
[12]Nimmo AJ,Cernak I,Heath DL,et al.Neurogenic inflammation is associated with development of edema and functional deficits following traumatic brain injury in rats[J].Neuropeptides,2004,38(1):40-47.
[13]Hellal F,Bonnefont-Rousselot D,Croci N,et al.Pattern of cerebral edema and hemorrhage in a mice model of diffuse brain injury[J].Neurosci Lett,2004,357(1):21-24.
[14]Grond-Ginsbach C,Hummel M,Wiest T,et al.Gene expression in human peripheral blood mononuclear cells upon acute ischemic stroke[J].Neurol,2008,255(5):723-731.
[15]Yuruker V,Naziroglu M,Senol N,et al.Reduction in traumatic brain injury-induced oxidative stress,apoptosis and calcium entry in rat hippocampus by melatonin:Possible involvement of TRPM2 channels[J].Metab Brain Dis,2015,30(1):223-231.
[16]Seyhan N,Canseven AG.In vivo effects of ELF MFs on collagen synthesis,free radical processes,natural antioxidant system,respiratory burst system,immune system activities,and electrolytes in the skin,plasma,spleen,lung,kindney,and brian tissues[J].Electromagn Biol Med,2006,25(4):291-305.
[17]Eroglu B,Kimbler DE,Pang J,et al.Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury[J].Neurochem,2014,130(5):626-641.
[18]Lin CJ,Chen TH,Yang LY,et al.Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death[J].Cell Death Dis,2014,5(1):1147.
[19]Zhiqiang Li,Qingming Shu,Lingzhi Li,et al.Sequential expression of cyclooxygenase-2,glutamate receptor-2,and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury[J].Neural Regeneration Research,2014,9(9):978-985.
[20]Harris RE.Cyclooxygenase-2(cox-2)and the infiammo genesis of cancer[J].Subcell Biochem,2007,42(1):93-126.