TNF-α和nNOS在红霉素预适应大鼠局灶性脑缺血损伤保护作用中变化的研究
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摘要
目的
缺血预适应(Ischemic Preconditioning,IP)是指组织和机体在经受非致死的缺血损伤后对以后长时间的缺血损伤产生显著的耐受性,是机体的一种重要的保护机制。缺血预适应在多种动物得到证实,并且存在于机体的其他众多器官,比如心脏,骨骼肌,脊髓,肺,肝脏,肾脏,胃,小肠等。深入研究预适应现象有助于我们在医疗过程中保护机体功能,具有诱人的临床应用前景。
现在有人根据引发预适应因子的不同将预适应分为缺血预适应和化学预适应,化学预适应是指利用一些可以轻度干扰能量代谢的化学物质模拟缺血的改变从而诱导预适应得保护作用的一种做法。目前在研究中经常用于化学预适应得物质有CoC12,DFO,3-NPA等等,但是这些物质因为它们的毒副作用限制了它们的临床应用。最近Huber等发现临床常用的一些抗生素如红霉素可以诱发机体产生预适应得保护作用,从而减少缺血对神经系统的损害。红霉素可以通过干扰线粒体的能量代谢从而模拟缺血诱发预适应的保护作用。研究还表明预适应除了可以保护脑组织免受缺血损害还可以对其他的损伤起到保护作用,如癫痫,脑水肿等。
关于预适应的保护机制的研究表明很多的因素参与到这一过程中,其中神经型一氧化氮合酶(Neuronal nitric oxide sythases,nNOS)和肿瘤坏死因子(TNF-α)的作用日益受到关注。已有实验证明在一些预适应过程中nNOS通过调控凋亡相关基因如Caspase3,Bc1-2等的表达发挥保护作用,而TNF-α也是目前缺血缺氧预适应研究最多的因子,是哺乳动物不同组织、细胞形成缺氧耐受共同的分子生物学机制。但是在红霉素预适应的过程中nNOS和TNF-α是否起作用,通过什么途径起到作用,现在还没有相关的报道。为此,我们研究TNF-α和nNOS在红霉素预适应大鼠局灶性脑缺血损伤保护作用中的变化,为进一步揭示红霉素预适应对大鼠局灶性脑缺血损伤保护作用的机制提供实验依据。
实验方法
1、实验动物及分组
健康Wistar雄性大鼠,体重270-300g,有中国医科大学动物部提供。将108只雄性大鼠随即分成3大组,每组再随即分成6小组,每小组6只,分别为为对照组和5mg/kg,20mg/kg,35mg/kg,50mg/kg,65mg/kg红霉素预适应的5个实验小组。每大组分别用于TTC染色法脑缺血体积测定,脑水肿测定和RT-PCR检测法及Western blot法测定nNOS及TNF-α的mRNA和蛋白表达。
2、大鼠局灶性脑缺血模型的建立
采用右侧颈内动脉插入线栓法制备大鼠右侧局灶性脑缺血模型。动物苏醒后表现为:①提尾时左前肢内收屈曲;②爬行时项左侧划圈;③站立时向左倾倒。凡具有上述三项体征之一者入选。
3、TTC染色
将缺血后24小时的实验组和对照组大鼠,迅速断头取脑,从前向后每2mm切片,共切6张。将脑片放入1%TTC生理盐水中,37℃避光孵育30分钟,待显色完全(正常脑组织呈红色,损伤组织呈白色)后,置甲醛溶液中固定。
4、脑组织梗死体积测定
应用图像分析软件计算脑梗死面积及各层面积,由公式V=∑(A1+A2)t/2算出梗死体积。其中t为切片厚度,A1和A2分别表示切块嘴、尾侧梗死面积,同样方法计算出同侧大脑半球体积,计算出梗死灶体积占大鼠脑体积的百分比。
5、组织含水量测定
将对照组和5个实验组的大鼠,在缺血后48小时立即断头取脑,立即将脑分别置于电子天平(1/10000)称重后放于烤箱中恒温100℃,持续48小时取出称干重,计算脑含水量=[(湿重－干重)/湿重]×100%。
6、脑组织的取材
将对照组和五个实验组脑缺血模型大鼠饲养24小时后,大鼠麻醉后迅速短头取脑,切取右侧缺血侧大脑半球半暗带区皮质(距嗅球尖端7-11mm,矢状裂至外侧裂上1/3)收集于EP管,-70℃冰箱保存。
7、RT-PCR法测定nNOS和TNF-αmRNA的表达
将组织经过一系列的RT-PCR反应获得的PCR产物用1.5%的琼脂糖凝胶电泳,EB染色,用电泳凝胶成像仪测定扩增带密度来评估其表达。以TNF-α及nNOS和GAPDH的比值作为TNF-α和nNOS表达的标准值。
8、Western blot法检测nNOS和TNF-α蛋白的表达
用ECL试剂盒采用化学发光法显示出TNF-α和nNOS的含量,β-actin作为内参,Western blot结果用软件(Chemi Imager5500 V2.03)扫描,并用图像分析系统定量分析。
9、统计方法
统计学处理用SPSS13.0统计软件包对数据进行处理,实验数据以均数±标准差((?)±s)表示,各组间两两比较用t检验,检验水准a=0.05.
结果
1、脑梗死体积测定结果
经过红霉素预适应的5个实验组大鼠的局灶性脑缺血24小时,其脑梗死体积较对照组脑缺血体积均有不同程度的减少,而且按35mg/kg剂量给予预适应的实验组脑缺血梗死体积减少最为显著。
2、脑水肿测定结果
给予不同剂量红霉素预适应的实验组,在鼠脑急性缺血后48小时的脑水肿程度均比对照组有所减轻,而且给予35mg/kg剂量的实验组效果较其他组更有显著性差异。
3、RT-PCR检测法和Western blot法测定nNOS及TNF-α的mRNA和蛋白的表达结果
实验组较对照组可以上调nNOS mRNA和蛋白的表达和下调TNF-αmRNA和蛋白的表达。
4、图像分析及统计学分析
经红霉素预适应的实验组,脑缺血24小时后,大鼠局灶性脑缺血体积较对照组减小,根据给药剂量不同,减少的程度也不同,35mg/kg剂量组脑缺血梗死体积减少最为显著(P<0.05);经红霉素预适应的实验组,在脑缺血后48小时的脑水肿程度也较对照组有所减轻,35mg/kg剂量组脑水肿程度较对照组减轻尤为显著(P<0.05);经红霉素预适应的实验组,缺血侧半暗袋脑组织中经RT-PCR检测法和Western blot法检测的nNOS及TNF-α的mRNA和蛋白的表达,结果各实验组中nNOS的mRNA和蛋白的表达有所增加,TNF-α的mRNA和蛋白的表达有所减少,35mg/kg剂量组nNOS及TNF-α的mRNA和蛋白的表达量较对照组的差异性最大(P<0.05).
结论
1、红霉素预适应后,对大鼠局灶性急性脑缺血损伤具有保护作用,能明显减少脑缺血后的脑梗死体积及脑水肿的严重程度,并且35mg/kg剂量为最适红霉素预适应剂量。
2、红霉素预适应能提高大鼠脑缺血模型中nNOS mRNA和蛋白的表达,并且也能降低TNF-αmRNA和蛋白的表达,提示红霉素预适应的脑缺血保护机制可能与nNOS和TNF-α有关。
Objective
Ischemic Preconditioning is a phenomenon in which the brain protects itself against future long-time ischemic injury by adapting to sublethal ischemic insults, which is a significant protective mechanism.Ischemic Preconditioning has been acknowledged in various animals and other organs,such as heart,skeletal muscle, spinal cord,lung,liver,kidney,stomach,and intestine.The profoundly research can benefit for organism shielding in medical treatment,and have inviting perspective of clinical application.
In current study,according to the priming factors,preconditioning is generally divided into two sorts:ischemic preconditioning and drug preconditioning.The latter is a stimulant ischemia practice that some chemistry substance is added to interfere with energy metabolism so as to induce the protective effect.Nowadays, preconditioning stimuli include hypoxic exposure,low dose of CoCl2,DFO and 3-NPA among others,whereby those toxic and side-effect restrict their clinical application.Recently,Huber reports some novel findings about this protective effect that was triggered by clinical frequently used antibiotics such as erythromycin,which attenuate the nervous system damage by ischemia.Erythromycin can impact mitochondrial energy metabolism so as to induce the protective effect.What's more, the research is also indicated preconditioning can not only shield the brain from ischemic insults,but also can preserve other injury tissue include epilepsy and cerebral edema.
With regard to the protective mechanism,abundant agents are verified to participate in this procedure,and the Neuronal nitric oxide sythases(nNOS) and tumor necrosis factor-α(TNF-α)have attached increasing attention in the neuroprotective effect.It has been certified that nNOS generate a marked effect by regulating apoptosis related gene such as Caspase3,Bcl-2,and TNF-αis also a frequent researching factor in hypoxic preconditioning and congenerous Molecular Biology mechanism that various tissue and cell mediate hypoxic tolerance in mammalian in present study, however,there is no related report on whether nNOS and TNF-αis effective or which path in the erythromycin-induced Preconditioning.Therefore,we explore the role of nNOS and TNF-αin erythromycin-induced focal cerebral ischemic preconditioning rats,so as to offer actual proof for further illustrating the neuroprotective mechanism in erythromycin-induced focal cerebral ischemic preconditioning rats.
Methods
1、Animal and grouping
Healthy adult Wistar male rat(270-300g) were randomly divided into 6 groups: normal control group,erythromycin-1(EM-1)group,erythromycin-2(EM-2) group, erythromycin-3(EM-3) group,erythromycin-4(EM-4) group,and erythromycin-5(EM-5) group.TTC staining,western-blot and RT-PCR were applied to determine the expression of nNOS and TNF-α
2、Establishment of rat MCAo model
Focal ischemic preconditioning in the brain was accomplished by occlusion of the right internal carotid artery using patching a line.The animals have three phenotypes after palinesthesia:1,Left forelimb show adduction and buckling;2,Drawing a circle to opposite the ipsilateral side while crawling;3,Dumpaging when standing.all the rats that have one of the signs above-mentioned could be selected.
3、TTC(2,3,5-triphenyltetrazolium chlorid) staining
Preconditioned and control rats(ischemia for 24 h) were subjected to decapitation and the brains were isolated at once,the brains were kept 20 min at-20 C and frozen brains were sliced into uniform coronal section about 2-mm-thick.the slices were incubated in 1%TTC(2,3,5-triphenyltetrazolium chlorid) at 37 C in 0.2 M Tris buffer for 30 min away from light Immobilization in the formaldehyde after full coloration.Tissue infarction was identified by the lack of 2,3,5-triphenyltetrazolium chloride staining(total 6 slices).Both sides of each stained coronal slice were photographed by digital camera and then evaluated by digital analysis.
4、Measurement of infarct volume
The brain infarct size was evaluated by image analysis software and calculated according to the formula V=Σ(A1+A2)t/2.T represent slicing depth,A1 and A2 make up infarct size of front and tail,respectively.Similarly,homolateral brain infarct volume and its percentage can be worked out.
5、Evaluation of moisture capacity
Preconditioned and control rats(ischemia for 48 h) were suffered from decapitation and take out of the brain at once.Weight the brain on electronic balance and put into the constant temperature baker(100℃),take out and weight after 48h,computate the moisture capacity[(wet weight-dry weight)/wet weight]×100%
6、Resource of cerebral tissue
Preconditioned and control model rats(ischemia for 24 h) were rendered anesthetic, experienced decapitation and dislodged the brain right away.The right ischemia half-silent cortex(7-11mm to the top of olfactory bulb,lateral fissure sagittal split up to 1/3) were selected and assembled in EP tube,frozen at-70℃。
7、Assay of n NOS and TNF-αby PCR
The PCR products derived from a serious RT-PCR reaction were treatment via electrophoresis in 5%agarose gel and staining with bromphenol blue.The density of amplified bands was evaluated by electrophoresis gelatin image apparatus.The standard value is the expression of proportionality for n NOS and TNF-αto GAPDH
8、Estimation of n NOS and TNF-αprotein by Western blot
The content of n NOS and TNF-αcan be manifest by enhanced chemiluminescence using the ECL system.The result was scanned by Chemi Imager5500 V2.03 and detected by ECL system
9、Statistical Analysis
Statistical analysis was performed using SPSS soft ware.date was expressed as mean±SEM The comparison was done using Student's t test or ANOVA with Fisher's post hoc comparison.Anαof P＜0.05 was considered significant.
Results
1、Measurement of infarct volume
The infarct volume in erythromycin-induced Preconditioning was attenuated at different level compared with control,and the preconditioning yield to 35mg/kg had significant decrease
2、Evaluation of edema
The edema to erythromycin-induced preconditioning was diminished at different level compared with that of control,and the preconditioning yield to 35mg/kg had significant difference
3、Assay of n NOS and TNF-αby PCR and Western blot
The expression of n NOS displayed an up-regulation in Preconditioning rats,but TNF-αdown-regulation,compared with control.
4、Analysis
Local infarct volume in erythromycin-induced preconditioning rats was blunted compared with that of control after 24h ischemia.The degree of decrease was determined by drug dose,while the dose of 35mg/kg had significant difference(P＜0.05).The edema in erythromycin-induced preconditioning was diminished at different level compared with control,and the preconditioning yield to 35mg/kg had significant difference(P＜0.05).The expression of nNOS displayed an up-regulation in Preconditioning rats,but TNF-αdown-regulation,compared with control.The expression of n NOS and TNF-αwith the dose of 35mg/kg had significant difference compared with that of control(P＜0.05)
Conclusion
1、erythromycin-induced Preconditioning in rats can not only defend the brain from acute ischemic focal insults,but also lessen the infarct volume and the degree of brain edema,moreover the dose of 35mg/kg was the most suitable for the preconditioning
2、erythromycin-induced Preconditioning in rats was also enhanced the expression of nNOS in cerebral ischemic model,and then cut down the expression of TNF-α.These findings demonstrate that the protective effect and mechanism of erythromycin-induced Preconditioning may have close relationship with the expression of n NOS and TNF-α
缺血预适应(Ischemic Preconditioning,IP)是指组织和机体在经受非致死的缺血损伤后对以后长时间的缺血损伤产生显著的耐受性,是机体的一种重要的保护机制。缺血预适应在多种动物得到证实,并且存在于机体的其他众多器官,比如心脏,骨骼肌,脊髓,肺,肝脏,肾脏,胃,小肠等。深入研究预适应现象有助于我们在医疗过程中保护机体功能,具有诱人的临床应用前景。
现在有人根据引发预适应因子的不同将预适应分为缺血预适应和化学预适应,化学预适应是指利用一些可以轻度干扰能量代谢的化学物质模拟缺血的改变从而诱导预适应得保护作用的一种做法。目前在研究中经常用于化学预适应得物质有CoC12,DFO,3-NPA等等,但是这些物质因为它们的毒副作用限制了它们的临床应用。最近Huber等发现临床常用的一些抗生素如红霉素可以诱发机体产生预适应得保护作用,从而减少缺血对神经系统的损害。红霉素可以通过干扰线粒体的能量代谢从而模拟缺血诱发预适应的保护作用。研究还表明预适应除了可以保护脑组织免受缺血损害还可以对其他的损伤起到保护作用,如癫痫,脑水肿等。
关于预适应的保护机制的研究表明很多的因素参与到这一过程中,其中神经型一氧化氮合酶(Neuronal nitric oxide sythases,nNOS)和肿瘤坏死因子(TNF-α)的作用日益受到关注。已有实验证明在一些预适应过程中nNOS通过调控凋亡相关基因如Caspase3,Bc1-2等的表达发挥保护作用,而TNF-α也是目前缺血缺氧预适应研究最多的因子,是哺乳动物不同组织、细胞形成缺氧耐受共同的分子生物学机制。但是在红霉素预适应的过程中nNOS和TNF-α是否起作用,通过什么途径起到作用,现在还没有相关的报道。为此,我们研究TNF-α和nNOS在红霉素预适应大鼠局灶性脑缺血损伤保护作用中的变化,为进一步揭示红霉素预适应对大鼠局灶性脑缺血损伤保护作用的机制提供实验依据。
实验方法
1、实验动物及分组
健康Wistar雄性大鼠,体重270-300g,有中国医科大学动物部提供。将108只雄性大鼠随即分成3大组,每组再随即分成6小组,每小组6只,分别为为对照组和5mg/kg,20mg/kg,35mg/kg,50mg/kg,65mg/kg红霉素预适应的5个实验小组。每大组分别用于TTC染色法脑缺血体积测定,脑水肿测定和RT-PCR检测法及Western blot法测定nNOS及TNF-α的mRNA和蛋白表达。
2、大鼠局灶性脑缺血模型的建立
采用右侧颈内动脉插入线栓法制备大鼠右侧局灶性脑缺血模型。动物苏醒后表现为:①提尾时左前肢内收屈曲;②爬行时项左侧划圈;③站立时向左倾倒。凡具有上述三项体征之一者入选。
3、TTC染色
将缺血后24小时的实验组和对照组大鼠,迅速断头取脑,从前向后每2mm切片,共切6张。将脑片放入1%TTC生理盐水中,37℃避光孵育30分钟,待显色完全(正常脑组织呈红色,损伤组织呈白色)后,置甲醛溶液中固定。
4、脑组织梗死体积测定
应用图像分析软件计算脑梗死面积及各层面积,由公式V=∑(A1+A2)t/2算出梗死体积。其中t为切片厚度,A1和A2分别表示切块嘴、尾侧梗死面积,同样方法计算出同侧大脑半球体积,计算出梗死灶体积占大鼠脑体积的百分比。
5、组织含水量测定
将对照组和5个实验组的大鼠,在缺血后48小时立即断头取脑,立即将脑分别置于电子天平(1/10000)称重后放于烤箱中恒温100℃,持续48小时取出称干重,计算脑含水量=[(湿重－干重)/湿重]×100%。
6、脑组织的取材
将对照组和五个实验组脑缺血模型大鼠饲养24小时后,大鼠麻醉后迅速短头取脑,切取右侧缺血侧大脑半球半暗带区皮质(距嗅球尖端7-11mm,矢状裂至外侧裂上1/3)收集于EP管,-70℃冰箱保存。
7、RT-PCR法测定nNOS和TNF-αmRNA的表达
将组织经过一系列的RT-PCR反应获得的PCR产物用1.5%的琼脂糖凝胶电泳,EB染色,用电泳凝胶成像仪测定扩增带密度来评估其表达。以TNF-α及nNOS和GAPDH的比值作为TNF-α和nNOS表达的标准值。
8、Western blot法检测nNOS和TNF-α蛋白的表达
用ECL试剂盒采用化学发光法显示出TNF-α和nNOS的含量,β-actin作为内参,Western blot结果用软件(Chemi Imager5500 V2.03)扫描,并用图像分析系统定量分析。
9、统计方法
统计学处理用SPSS13.0统计软件包对数据进行处理,实验数据以均数±标准差((?)±s)表示,各组间两两比较用t检验,检验水准a=0.05.
结果
1、脑梗死体积测定结果
经过红霉素预适应的5个实验组大鼠的局灶性脑缺血24小时,其脑梗死体积较对照组脑缺血体积均有不同程度的减少,而且按35mg/kg剂量给予预适应的实验组脑缺血梗死体积减少最为显著。
2、脑水肿测定结果
给予不同剂量红霉素预适应的实验组,在鼠脑急性缺血后48小时的脑水肿程度均比对照组有所减轻,而且给予35mg/kg剂量的实验组效果较其他组更有显著性差异。
3、RT-PCR检测法和Western blot法测定nNOS及TNF-α的mRNA和蛋白的表达结果
实验组较对照组可以上调nNOS mRNA和蛋白的表达和下调TNF-αmRNA和蛋白的表达。
4、图像分析及统计学分析
经红霉素预适应的实验组,脑缺血24小时后,大鼠局灶性脑缺血体积较对照组减小,根据给药剂量不同,减少的程度也不同,35mg/kg剂量组脑缺血梗死体积减少最为显著(P<0.05);经红霉素预适应的实验组,在脑缺血后48小时的脑水肿程度也较对照组有所减轻,35mg/kg剂量组脑水肿程度较对照组减轻尤为显著(P<0.05);经红霉素预适应的实验组,缺血侧半暗袋脑组织中经RT-PCR检测法和Western blot法检测的nNOS及TNF-α的mRNA和蛋白的表达,结果各实验组中nNOS的mRNA和蛋白的表达有所增加,TNF-α的mRNA和蛋白的表达有所减少,35mg/kg剂量组nNOS及TNF-α的mRNA和蛋白的表达量较对照组的差异性最大(P<0.05).
结论
1、红霉素预适应后,对大鼠局灶性急性脑缺血损伤具有保护作用,能明显减少脑缺血后的脑梗死体积及脑水肿的严重程度,并且35mg/kg剂量为最适红霉素预适应剂量。
2、红霉素预适应能提高大鼠脑缺血模型中nNOS mRNA和蛋白的表达,并且也能降低TNF-αmRNA和蛋白的表达,提示红霉素预适应的脑缺血保护机制可能与nNOS和TNF-α有关。
Objective
Ischemic Preconditioning is a phenomenon in which the brain protects itself against future long-time ischemic injury by adapting to sublethal ischemic insults, which is a significant protective mechanism.Ischemic Preconditioning has been acknowledged in various animals and other organs,such as heart,skeletal muscle, spinal cord,lung,liver,kidney,stomach,and intestine.The profoundly research can benefit for organism shielding in medical treatment,and have inviting perspective of clinical application.
In current study,according to the priming factors,preconditioning is generally divided into two sorts:ischemic preconditioning and drug preconditioning.The latter is a stimulant ischemia practice that some chemistry substance is added to interfere with energy metabolism so as to induce the protective effect.Nowadays, preconditioning stimuli include hypoxic exposure,low dose of CoCl2,DFO and 3-NPA among others,whereby those toxic and side-effect restrict their clinical application.Recently,Huber reports some novel findings about this protective effect that was triggered by clinical frequently used antibiotics such as erythromycin,which attenuate the nervous system damage by ischemia.Erythromycin can impact mitochondrial energy metabolism so as to induce the protective effect.What's more, the research is also indicated preconditioning can not only shield the brain from ischemic insults,but also can preserve other injury tissue include epilepsy and cerebral edema.
With regard to the protective mechanism,abundant agents are verified to participate in this procedure,and the Neuronal nitric oxide sythases(nNOS) and tumor necrosis factor-α(TNF-α)have attached increasing attention in the neuroprotective effect.It has been certified that nNOS generate a marked effect by regulating apoptosis related gene such as Caspase3,Bcl-2,and TNF-αis also a frequent researching factor in hypoxic preconditioning and congenerous Molecular Biology mechanism that various tissue and cell mediate hypoxic tolerance in mammalian in present study, however,there is no related report on whether nNOS and TNF-αis effective or which path in the erythromycin-induced Preconditioning.Therefore,we explore the role of nNOS and TNF-αin erythromycin-induced focal cerebral ischemic preconditioning rats,so as to offer actual proof for further illustrating the neuroprotective mechanism in erythromycin-induced focal cerebral ischemic preconditioning rats.
Methods
1、Animal and grouping
Healthy adult Wistar male rat(270-300g) were randomly divided into 6 groups: normal control group,erythromycin-1(EM-1)group,erythromycin-2(EM-2) group, erythromycin-3(EM-3) group,erythromycin-4(EM-4) group,and erythromycin-5(EM-5) group.TTC staining,western-blot and RT-PCR were applied to determine the expression of nNOS and TNF-α
2、Establishment of rat MCAo model
Focal ischemic preconditioning in the brain was accomplished by occlusion of the right internal carotid artery using patching a line.The animals have three phenotypes after palinesthesia:1,Left forelimb show adduction and buckling;2,Drawing a circle to opposite the ipsilateral side while crawling;3,Dumpaging when standing.all the rats that have one of the signs above-mentioned could be selected.
3、TTC(2,3,5-triphenyltetrazolium chlorid) staining
Preconditioned and control rats(ischemia for 24 h) were subjected to decapitation and the brains were isolated at once,the brains were kept 20 min at-20 C and frozen brains were sliced into uniform coronal section about 2-mm-thick.the slices were incubated in 1%TTC(2,3,5-triphenyltetrazolium chlorid) at 37 C in 0.2 M Tris buffer for 30 min away from light Immobilization in the formaldehyde after full coloration.Tissue infarction was identified by the lack of 2,3,5-triphenyltetrazolium chloride staining(total 6 slices).Both sides of each stained coronal slice were photographed by digital camera and then evaluated by digital analysis.
4、Measurement of infarct volume
The brain infarct size was evaluated by image analysis software and calculated according to the formula V=Σ(A1+A2)t/2.T represent slicing depth,A1 and A2 make up infarct size of front and tail,respectively.Similarly,homolateral brain infarct volume and its percentage can be worked out.
5、Evaluation of moisture capacity
Preconditioned and control rats(ischemia for 48 h) were suffered from decapitation and take out of the brain at once.Weight the brain on electronic balance and put into the constant temperature baker(100℃),take out and weight after 48h,computate the moisture capacity[(wet weight-dry weight)/wet weight]×100%
6、Resource of cerebral tissue
Preconditioned and control model rats(ischemia for 24 h) were rendered anesthetic, experienced decapitation and dislodged the brain right away.The right ischemia half-silent cortex(7-11mm to the top of olfactory bulb,lateral fissure sagittal split up to 1/3) were selected and assembled in EP tube,frozen at-70℃。
7、Assay of n NOS and TNF-αby PCR
The PCR products derived from a serious RT-PCR reaction were treatment via electrophoresis in 5%agarose gel and staining with bromphenol blue.The density of amplified bands was evaluated by electrophoresis gelatin image apparatus.The standard value is the expression of proportionality for n NOS and TNF-αto GAPDH
8、Estimation of n NOS and TNF-αprotein by Western blot
The content of n NOS and TNF-αcan be manifest by enhanced chemiluminescence using the ECL system.The result was scanned by Chemi Imager5500 V2.03 and detected by ECL system
9、Statistical Analysis
Statistical analysis was performed using SPSS soft ware.date was expressed as mean±SEM The comparison was done using Student's t test or ANOVA with Fisher's post hoc comparison.Anαof P＜0.05 was considered significant.
Results
1、Measurement of infarct volume
The infarct volume in erythromycin-induced Preconditioning was attenuated at different level compared with control,and the preconditioning yield to 35mg/kg had significant decrease
2、Evaluation of edema
The edema to erythromycin-induced preconditioning was diminished at different level compared with that of control,and the preconditioning yield to 35mg/kg had significant difference
3、Assay of n NOS and TNF-αby PCR and Western blot
The expression of n NOS displayed an up-regulation in Preconditioning rats,but TNF-αdown-regulation,compared with control.
4、Analysis
Local infarct volume in erythromycin-induced preconditioning rats was blunted compared with that of control after 24h ischemia.The degree of decrease was determined by drug dose,while the dose of 35mg/kg had significant difference(P＜0.05).The edema in erythromycin-induced preconditioning was diminished at different level compared with control,and the preconditioning yield to 35mg/kg had significant difference(P＜0.05).The expression of nNOS displayed an up-regulation in Preconditioning rats,but TNF-αdown-regulation,compared with control.The expression of n NOS and TNF-αwith the dose of 35mg/kg had significant difference compared with that of control(P＜0.05)
Conclusion
1、erythromycin-induced Preconditioning in rats can not only defend the brain from acute ischemic focal insults,but also lessen the infarct volume and the degree of brain edema,moreover the dose of 35mg/kg was the most suitable for the preconditioning
2、erythromycin-induced Preconditioning in rats was also enhanced the expression of nNOS in cerebral ischemic model,and then cut down the expression of TNF-α.These findings demonstrate that the protective effect and mechanism of erythromycin-induced Preconditioning may have close relationship with the expression of n NOS and TNF-α
引文
1 Huber R,Kasischke K,Ludolph AC,et al.Increase of cellular hypoxic tolerance by erythromycin and other antibiotics.Neuroreport,1999,10(7):1543-1546.
2 Fromenty B,Pessayre D.Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.Pharmacol Ther,1995:67(1).
3 Jiang W,Van Cleemput J,Sheerin AH,et al.Involvement of extracellular regulated kinase and p38 kinase in hippocampal seizure tolerance.J Neurosci Res,2005,15;81(4):581-588.
4 Hua Y,Keep RF,Hoff JT,et al.Thrombin preconditioning attenuates brain edema induced by erythrocytes and iron.J Cereb Blood Flow Metab,2003,23(12):1448-1454.
5 Longa EZ,Weinstein PR,Carison S,et al,Reversivle middle cerebral artery occlusion without craniectomv in rats.Stroke.1989.20:84
6罗勇,董为伟.wistar大鼠插线法局灶性脑缺血/再灌注模型的实验研究[J].重庆医科大学学报,2002.27(1):124,
7 Shi J,Yang SH,Stubley L,et al,Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemicrodent model.Brain Res,2000,853:1-4
8 Liu J,Ginis I,Spam M,et al.Hypoxic preconditioning protects cultured neurons against hypoxic stless via TNF-a and ceramide[J].AmJ Physiol Cell Physiol,2000,278(1):C14-C15
9 Sharp FR,Bergeron M,Bemaudin M.Hypoxia-inducible factor in brain[J].Adv Exp Med Biol,2001,502(3):273-291
10 Bergeron M,Gidday J M,Yu AY,et al.Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain[J].Ann Neurol,2000,48(3):285-296.
11 Wang Q,Xiong L,Chen S,et al.Rapid tolerance to focal cerebral ischemia in rats is induced by preconditioning with electroacupuncture:window of protection and the role of adenosine[J].NeurosciLett,2005,381(1-2):158-162
12 Andrew J.Smith,Trevor W.Stone,Robert Ao Smith.Preconditioning with NMDA protects against toxicity of 3-nitropropionic acid or glutamate in cultured cerebellar granule neurons[J].Neuroscience Letters,2008,440(3):294-298
13 HAN Hong-guang,WANG Zeng-wei,et al.Role of nitric oxide during early phase myocardial ischemic preconditioning in rats[J].Chinese medical journal,2008,121(13):1210-1214
14 Atochin DN,Clark J,Demchenko lT,et al.Rapid cerebral ischemic preconditioning in mice defident in endothelial an d neuronal nitric oxide synthases[J].Stroke,2003,34(5):1299-1303
15 Gonzalez-Zulueta M,Feldman AB,Klesse LJ,et al.Requirement for nitric oxide activation of p21(ras) / extracellular regulated kinases in neuronalischemic preconditioning[J].Proc Natl AcadSci USA,2000,97(1): 436-41
16 Sairanen T ,C arpen O ,Karjalainen-Lindsberg ML,et al.E volution of cerebral tumor necrosis factor production during human ischemic stroke[J].Stroke,2001,32:l 750-1758.
17 Zaremba J.Losy J.Early TNF-alpha levels Core Hate with ischaemic stroke severity.Acta Nenrol Scand.2001.104: 288-295.
18 Vila N.Castillo J.Davalos A,et al.Proinfhmnaatory cytokines and early neurological worsening in ischemic stroke.Stroke,2000,31: 2325-2329
19 Zaremba J.Skrobanski P.Losy J.Tumour necrosis factor-n is increased in the eel"ebrospinal fluid and serum of ischaemic stroke patients and correlates with the volume of evolving brain infarct.Biomed Pharmacother.20ol.55: 258-263.
20 Chan MT,Boet R,Ng SC,et al.Effect of ischemic preconditioning on brain tissue gases and pH during temporary cerebral artery occlusion..Acta Neurochir Suppl.2005;95:93-96.
1 Purcell L,Jeanette E.Strain-dependent response to cerebral ischemic preconditioning: diferences between spontaneously hypertensive and stroke prone spontaneously hypertensive rats[J].Neuroscience Lettem.2003,339(2): 151-155
2 Murry CE,Jennings RB,Reimer KA.Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium.Circulation,1986,74(5): 1124-1136.
3 Baxter GF.The neutrophil as a mediator of my ocardial ischemia-reperfusioin injury: time to move on.Basic Res Cardiol 2002; 97: 268-275
4 Baxter GF,Yellon DM.Current trends and controversies in ischemia-reperfusion research-Meeting report of the Hatter insitute 3 International Workshop on Cardioprotection.Basic Res Cardiol 2003;98(2): 133-136
5 Bolli R,Becker L,Gross G,et al.Myocardial protection at a crossroads: the need for translation into clinical therapy.Circ Res 2004;95(2): 125-134
6 Dimagl U,Simon R P ,Hallenbeck J M.Ischemic tolerance and endogenous neuroprotection.Trends Neurosci,2003,26: 248-254
7 Bolli R.The late phase of preconditioning.Circ Res,2000,87: 972-983
8 Chan MT,Boet R,Ng SC,et al.Effect of ischemic preconditioning on brain tissue gases and pH during temporary cerebral artery occlusion..Acta Neurochir Suppl.2005;95:93-96.
9 Grabb Me,Choi DW.Iscchemic tolerance in muine cortical cell culture: critical role for NMDA receptors[J].J Neurosci ,1999,19(5): 1657-662.
10 Alsbo CW.Wrang ML.Nielsen M.Diemer NH.Ischemic tolerance affecte the adenylstion state of GluR2 mRNA[J].Neuroreport ,2000,11(14): 3279-282.
11 Pugliese AM ,Latini S,Corradetti R,et al.Brief,repeated。oxygen-glucose deprivation episod es protect neurotransmission from a longer ischemic episode in the in vitro hippocampus: role of adenosine receptors.Br J Pharmacol,2003,140(2): 305-314.
12 Nishino K.Nowak TS Jr.Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning.J Cereb Blood Flow Metab,2004,24(2): 167-178.
13 Raval AP,Dave KR,Mochly-Rosen D,et al.Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice.J Neurosci,2003,23(2): 384-391.
14 KatoH,KogureK ArakiT,et al.AS troliat and mcrfogHal reactions in the gerbil hippocampus with induced ischemic tolerance.Brain Res,1994,664: 69-76.
15 Liu J,Bartels M,Lu A,et al.Microglia / macrophages prolife rate in striatum and neocortex but not in hippocampus after brief global ischemia that produces ischemic tolerance in gerbil brain[J].J Cereb Blood Flow Metab,2001,21(4): 361-73.
16 Atochin DN,Clark J,Demchenko IT,et al.Rapid cerebralischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases.Stroke,2003,34(5): 1299-1303.
17 Castellanos M ,Castillo J,Garcia MM ,et al.Inflammation-mediated damage in progressing lacunar infarctions: a potential therapeutic target.Stroke,2002,33(4): 982-987.
18 Pera J,Zawadzka M ,Kaminska B,et al .Influence of chemical and ischemic preconditioning on cytokine expression after focal brain ischemia.J Neurosci Res,2004,78(1): 132-140.
19 Argaud L.GsteauRO.RaiskyO.LoufoustJ.Robert D,OvizeM.Postconditioning inhibits mitochondrial Perrnesbility transition[J].Circulation,2005,111(2): 194-197.
20 Ruscher K,FreyerD,KarschM ,etal.Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model.J Neurosci,2002,22(23): 10291_10301.
21 Chavez JC,Baranova O,Lin J,et al.The transcriptional activator hypoxiainducible factor 2(HIF-2 / EPAS-l)regulates the oxygen-dependent expression of erythropoietin in cortical astrocytes.J Neurosci,2006,26(37): 9471-9481.
22 Siren AL,Fratelli M ,Brines M ,et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress.Proc Natl Acad Se i USA,2001,98(7): 4044—4049.
23 Prass K,Se harffA,RuscherK,et al.Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin.Stroke,2003,34(8): 1981-1986.
24 Wu Ch,Fujihara H,Yao J,Qi SH,Li HP.Shimoji K,et al.Different Expression Patterns of Bcl-2,Bcl-xl,and Bax Proteins After Sublethal Forebrainlschemia in C57Black/Crj6 Mouse Striatnm[J].Stroke,2003,34(7): 1803-808.
25 Soane L,fiskum G.Inhibition of mitochondrial neural cell death pathways by protein transduction bcl-2 family proteins[J].J Bioenerg Biomemb,2005,37(3): 179-190.
26 Cheng T,Liu D,Griffin JH.Fernandez JA,Castellino F,Rosen ED,et al.Activated protein C blocks p53-mediated .Apoptosis in ischemic human hrain endothelitum and is neuroprotective[J].NatMed,2003,9: 338-342.
2 Fromenty B,Pessayre D.Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.Pharmacol Ther,1995:67(1).
3 Jiang W,Van Cleemput J,Sheerin AH,et al.Involvement of extracellular regulated kinase and p38 kinase in hippocampal seizure tolerance.J Neurosci Res,2005,15;81(4):581-588.
4 Hua Y,Keep RF,Hoff JT,et al.Thrombin preconditioning attenuates brain edema induced by erythrocytes and iron.J Cereb Blood Flow Metab,2003,23(12):1448-1454.
5 Longa EZ,Weinstein PR,Carison S,et al,Reversivle middle cerebral artery occlusion without craniectomv in rats.Stroke.1989.20:84
6罗勇,董为伟.wistar大鼠插线法局灶性脑缺血/再灌注模型的实验研究[J].重庆医科大学学报,2002.27(1):124,
7 Shi J,Yang SH,Stubley L,et al,Hypoperfusion induces overexpression of beta-amyloid precursor protein mRNA in a focal ischemicrodent model.Brain Res,2000,853:1-4
8 Liu J,Ginis I,Spam M,et al.Hypoxic preconditioning protects cultured neurons against hypoxic stless via TNF-a and ceramide[J].AmJ Physiol Cell Physiol,2000,278(1):C14-C15
9 Sharp FR,Bergeron M,Bemaudin M.Hypoxia-inducible factor in brain[J].Adv Exp Med Biol,2001,502(3):273-291
10 Bergeron M,Gidday J M,Yu AY,et al.Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain[J].Ann Neurol,2000,48(3):285-296.
11 Wang Q,Xiong L,Chen S,et al.Rapid tolerance to focal cerebral ischemia in rats is induced by preconditioning with electroacupuncture:window of protection and the role of adenosine[J].NeurosciLett,2005,381(1-2):158-162
12 Andrew J.Smith,Trevor W.Stone,Robert Ao Smith.Preconditioning with NMDA protects against toxicity of 3-nitropropionic acid or glutamate in cultured cerebellar granule neurons[J].Neuroscience Letters,2008,440(3):294-298
13 HAN Hong-guang,WANG Zeng-wei,et al.Role of nitric oxide during early phase myocardial ischemic preconditioning in rats[J].Chinese medical journal,2008,121(13):1210-1214
14 Atochin DN,Clark J,Demchenko lT,et al.Rapid cerebral ischemic preconditioning in mice defident in endothelial an d neuronal nitric oxide synthases[J].Stroke,2003,34(5):1299-1303
15 Gonzalez-Zulueta M,Feldman AB,Klesse LJ,et al.Requirement for nitric oxide activation of p21(ras) / extracellular regulated kinases in neuronalischemic preconditioning[J].Proc Natl AcadSci USA,2000,97(1): 436-41
16 Sairanen T ,C arpen O ,Karjalainen-Lindsberg ML,et al.E volution of cerebral tumor necrosis factor production during human ischemic stroke[J].Stroke,2001,32:l 750-1758.
17 Zaremba J.Losy J.Early TNF-alpha levels Core Hate with ischaemic stroke severity.Acta Nenrol Scand.2001.104: 288-295.
18 Vila N.Castillo J.Davalos A,et al.Proinfhmnaatory cytokines and early neurological worsening in ischemic stroke.Stroke,2000,31: 2325-2329
19 Zaremba J.Skrobanski P.Losy J.Tumour necrosis factor-n is increased in the eel"ebrospinal fluid and serum of ischaemic stroke patients and correlates with the volume of evolving brain infarct.Biomed Pharmacother.20ol.55: 258-263.
20 Chan MT,Boet R,Ng SC,et al.Effect of ischemic preconditioning on brain tissue gases and pH during temporary cerebral artery occlusion..Acta Neurochir Suppl.2005;95:93-96.
1 Purcell L,Jeanette E.Strain-dependent response to cerebral ischemic preconditioning: diferences between spontaneously hypertensive and stroke prone spontaneously hypertensive rats[J].Neuroscience Lettem.2003,339(2): 151-155
2 Murry CE,Jennings RB,Reimer KA.Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium.Circulation,1986,74(5): 1124-1136.
3 Baxter GF.The neutrophil as a mediator of my ocardial ischemia-reperfusioin injury: time to move on.Basic Res Cardiol 2002; 97: 268-275
4 Baxter GF,Yellon DM.Current trends and controversies in ischemia-reperfusion research-Meeting report of the Hatter insitute 3 International Workshop on Cardioprotection.Basic Res Cardiol 2003;98(2): 133-136
5 Bolli R,Becker L,Gross G,et al.Myocardial protection at a crossroads: the need for translation into clinical therapy.Circ Res 2004;95(2): 125-134
6 Dimagl U,Simon R P ,Hallenbeck J M.Ischemic tolerance and endogenous neuroprotection.Trends Neurosci,2003,26: 248-254
7 Bolli R.The late phase of preconditioning.Circ Res,2000,87: 972-983
8 Chan MT,Boet R,Ng SC,et al.Effect of ischemic preconditioning on brain tissue gases and pH during temporary cerebral artery occlusion..Acta Neurochir Suppl.2005;95:93-96.
9 Grabb Me,Choi DW.Iscchemic tolerance in muine cortical cell culture: critical role for NMDA receptors[J].J Neurosci ,1999,19(5): 1657-662.
10 Alsbo CW.Wrang ML.Nielsen M.Diemer NH.Ischemic tolerance affecte the adenylstion state of GluR2 mRNA[J].Neuroreport ,2000,11(14): 3279-282.
11 Pugliese AM ,Latini S,Corradetti R,et al.Brief,repeated。oxygen-glucose deprivation episod es protect neurotransmission from a longer ischemic episode in the in vitro hippocampus: role of adenosine receptors.Br J Pharmacol,2003,140(2): 305-314.
12 Nishino K.Nowak TS Jr.Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning.J Cereb Blood Flow Metab,2004,24(2): 167-178.
13 Raval AP,Dave KR,Mochly-Rosen D,et al.Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice.J Neurosci,2003,23(2): 384-391.
14 KatoH,KogureK ArakiT,et al.AS troliat and mcrfogHal reactions in the gerbil hippocampus with induced ischemic tolerance.Brain Res,1994,664: 69-76.
15 Liu J,Bartels M,Lu A,et al.Microglia / macrophages prolife rate in striatum and neocortex but not in hippocampus after brief global ischemia that produces ischemic tolerance in gerbil brain[J].J Cereb Blood Flow Metab,2001,21(4): 361-73.
16 Atochin DN,Clark J,Demchenko IT,et al.Rapid cerebralischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases.Stroke,2003,34(5): 1299-1303.
17 Castellanos M ,Castillo J,Garcia MM ,et al.Inflammation-mediated damage in progressing lacunar infarctions: a potential therapeutic target.Stroke,2002,33(4): 982-987.
18 Pera J,Zawadzka M ,Kaminska B,et al .Influence of chemical and ischemic preconditioning on cytokine expression after focal brain ischemia.J Neurosci Res,2004,78(1): 132-140.
19 Argaud L.GsteauRO.RaiskyO.LoufoustJ.Robert D,OvizeM.Postconditioning inhibits mitochondrial Perrnesbility transition[J].Circulation,2005,111(2): 194-197.
20 Ruscher K,FreyerD,KarschM ,etal.Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model.J Neurosci,2002,22(23): 10291_10301.
21 Chavez JC,Baranova O,Lin J,et al.The transcriptional activator hypoxiainducible factor 2(HIF-2 / EPAS-l)regulates the oxygen-dependent expression of erythropoietin in cortical astrocytes.J Neurosci,2006,26(37): 9471-9481.
22 Siren AL,Fratelli M ,Brines M ,et al.Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress.Proc Natl Acad Se i USA,2001,98(7): 4044—4049.
23 Prass K,Se harffA,RuscherK,et al.Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin.Stroke,2003,34(8): 1981-1986.
24 Wu Ch,Fujihara H,Yao J,Qi SH,Li HP.Shimoji K,et al.Different Expression Patterns of Bcl-2,Bcl-xl,and Bax Proteins After Sublethal Forebrainlschemia in C57Black/Crj6 Mouse Striatnm[J].Stroke,2003,34(7): 1803-808.
25 Soane L,fiskum G.Inhibition of mitochondrial neural cell death pathways by protein transduction bcl-2 family proteins[J].J Bioenerg Biomemb,2005,37(3): 179-190.
26 Cheng T,Liu D,Griffin JH.Fernandez JA,Castellino F,Rosen ED,et al.Activated protein C blocks p53-mediated .Apoptosis in ischemic human hrain endothelitum and is neuroprotective[J].NatMed,2003,9: 338-342.