电针任脉经穴对脑缺血大鼠内源性生长因子表达的影响
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摘要
缺血性卒中(Cerebral Ischemic Stroke,CIS)又称脑梗死(Cerebral Infarction,CI),是由于脑动脉闭塞导致的脑组织的梗死,伴随着神经元、星形胶质细胞、少突胶质细胞的损伤,是现代社会中导致致死和致残的最重要的中枢神经系统血管事件。针刺任脉经穴治疗缺血性卒中具有一定的临床基础。前期的研究发现,电针任脉经穴能够促进脑缺血再灌注大鼠脑内神经干细胞的增殖、分化,从而有利于脑缺血后的神经修复。而引起这一效应的机制,与电针任脉经穴激活脑缺血再灌注大鼠ERK信号转导通路相关。从而我们设想,电针任脉经穴能够产生一些与ERK通路相关的上游因子,而这些因子在脑内的上调,能够激活脑内神经修复的机制,促进大脑神经元的损伤修复。基于此种设想及参阅大量文献,本课题以脑内源性生长因子为切入点,从文献研究与实验研究两方面,进一步探讨电针任脉经穴治疗缺血性卒中的可能作用途径和机制。
目的:观察脑缺血再灌注大鼠缺血侧侧脑室下区、海马齿状回区的碱性成纤维细胞生长因子、表皮生长因子、神经生长因子的变化规律,以及电针任脉经穴对脑内这三种生长因子表达的影响,以期从生长因子角度揭示电针任脉经穴治疗缺血性卒中的可能作用途径和机制,为针灸治疗缺血性脑病提供理论和实验依据。
方法:SPF级成年健康雄性SD大鼠72只,适应环境1周后开始实验。按随机数字表法分为假手术组、模型组、电针任脉组。每组再分别随机分为7天组、14天组和28天组三个亚组,每亚组8只。术前禁食12小时,采用线栓法建立大鼠大脑中动脉栓塞局灶性脑缺血再灌注模型,观察大鼠脑缺血再灌注后神经功能评分及死亡率的变化。电针任脉组在手术24小时后于承浆、气海、关元三穴行电针治疗,采用疏密波刺激(密波15Hz),强度6～15V,持续时间为20分钟,每日一次至动物处死。假手术组和模型组在手术24小时后固定于针刺操作台上20分钟,不作任何治疗。各组大鼠按实验设计时间处死、取脑。运用HE染色观察MCAO大鼠缺血侧纹状体、海马区的病理形态学改变;运用免疫组化法测定MCAO大鼠缺血侧侧脑室下区、海马齿状回区bFGF、EGF、NGF的蛋白表达;运用原位杂交法测定MCAO大鼠缺血侧侧脑室下区、海马齿状回区bFGF、EGF、NGF的mRNA表达。采用德国Leica DC software数码图像分析系统软件包进行图像分析。200倍光镜下进行阳性细胞计数,并采用SPSS13.0软件进行统计分析,比较各组差异。
结果:1、电针任脉经穴对脑缺血再灌注大鼠的神经功能评分、死亡率的影响不明显。2、缺血再灌注后,纹状体及海马区出现明显的脑组织水肿、神经细胞变性坏死等改变,提示运用线栓法制作大鼠局灶性脑缺血再灌注损伤模型是成功的。电针任脉经穴组较同时相模型组能够减轻缺血损伤程度。3、脑缺血再灌注损伤后,侧脑室下区、海马齿状回区内源性bFGF蛋白及mRNA的表达均可出现反应性上调,但该反应性上调具有一定的时限性,随着灌注时间的延长,bFGF的阳性表达呈现下降趋势。再灌注7天,阳性表达最高,再灌注14天阳性表达减弱,至再灌注28天阳性表达恢复至基线水平;电针任脉经穴至少于再灌注14天后上调两区bFGF蛋白及mRNA的表达。4、脑缺血再灌注损伤后,侧脑室下区、海马齿状回区内源性EGF可出现反应性上调,该上调具有一定的时限性,随着灌注时间的延长,EGF的阳性表达呈现下降的趋势。其中蛋白表达于再灌注14天已恢复至基线水平,而mRNA表达于再灌注28天恢复至基线水平,提示内源性EGF蛋白与mRNA的表达并非同步;电针任脉经穴至少能在再灌注7天后上调两区内源性EGF蛋白的表达,而于再灌注14天后上调EGF mRNA的表达。5、脑缺血损伤后,侧脑室下区、海马齿状回区内源性NGF亦可出现反应性上调,该上调亦具有一定的时限性,随着灌注时间的延长,NGF的阳性表达呈现下降的趋势。再灌注7天,阳性表达最高,再灌注14天阳性表达减弱,至再灌注28天阳性表达恢复至基线水平,而电针任脉经穴至少能在再灌注14天后上调内源性NGF蛋白的表达,再灌注28天后始能上调NGFmRNA的表达。
结论:bFGF、EGF、NGF参与脑缺血再灌注损伤的病理过程,而电针任脉经穴能够上调缺血再灌注大鼠脑内bFGF、NGF、EGF的表达,进而促进脑缺血后的神经保护和损伤修复。
Cerebral Ischemic Stroke(CIS),which is also called Cerebral Infarction(CI), is the infarction of brain tissue duing to Cerebral artery occlusion, accompannied with damage of neuron,astrocyte and oligodendrocyte.It is the most important event of central nervous system causing death and mutilation in modern society.Acupuncturing Ren Meridian has clinic foundation on treating CIS.Research discovered that electroacupuncturing Ren Meridian could promote the proliferation and diffrentiation of Nerve Stem Cells which profit the recovery of nerve after cerebral ischemia.Also,we found the effect was related with the activation of ERK signal transduction path.Thus,we assumed that electroacupuncturing Ren Meridian could generate some upstream element which was ERK path dependency,and the up-regulation of these factor would active mechanism of nerve recovery to promte the recovery of cerebral neuron.Based on the assumption and literature study,we chose the endogenous growth factor in brain as cut-in point.By the way of studying on literature and experiment,we can approach the possible mechanism of electroacupuncturing Ren Meridian on treating CIS.
Objective:To observe the change of bFGF/EGF/NGF in subventricular zone(SVZ) and Denatate Gyrus(DG) after Cerebral ischemia-reperfusion in Rats,And approach the effects of electroacupuncturing Ren Meridian on the three growth factor.By the observation of growth factor,we could find the possible mechanism of electroacupuncturing Ren Meridian on treating CIS,and provide theory and experiment evidence for acupuncturing to treat CIS.
Methods:A total of seventy-two adult SD rats from SPF-grade laboratory were taken into the study.Before the test,they were raised to adapt to the surroundings.According to the random digits table,they were divided into sham operation group(Sham),MCAO group(MCAO) and Electroacupuncture Ren Meridian group(R).Each group was averagely divided into 7 days group,14 days group and 28 days group at random.There were eight rats in each subset group. Before operation,rats were abrosia for twelve hours,and Cerebral ischemia-reperfusion injury model was induced by the method of nylon monofilament embolism.After that,We observed the nerve function score and death rate of MCAO rats.Rats in R group were treated with electroacupuncturing Chengjiang/Qihai/Guanyuan points twenty-four hours after operation.As for electric acupuncture,distant-dense wave was chosen.The frequency was15Hz for dense wave and intensity was 6 to15V.The treatment lasted for twenty minutes,and was done once every day until the rats killed.On the other hand, rats in Sham group and MCAO group were fixed on the operation board without any treatment for twenty minutes twenty-four hours after operation.Rats of each group were killed at the design time,and their brain were taken immediately.Then we observed the pathomorphism of corpora striata and hippocampus in ischemic brain of MACO rats by HE stain.Immunohistochemistry was used to detect the protein expression of bFGF/EGF/NGF in SVZ and DG of MCAO rats.In situ hybridization was used to detect the mRNA expression of bFGF/EGF/NGF in SVZ and DG of MCAO rats.At last,we counted positive cells at light microscope of 200×,analyzed the image by analytical system of Leica DC software from German,and compared the difference between groups by SPSS 13.0 software.
Results:1.Electroacupuncturing Ren meridian had little effection on the Nerve function score and death rate of MCAO rats.2.Corpora striata and hippocampus shew obvious brain edema and nerve cell degeneration after focal cerebral ischemia-reperfusion,which shew the Cerebral ischemia-reperfusion injury model was formed successfully.Compared with MCAO group at the same time after focal cerebral ischemia-reperfusion,R group could relieve the degree of injury.3.After focal cerebral ischemia-reperfusion,protein and mRNA expression of bFGF in SVZ and DG shew an increase which could not last for long time.With the extend of time after focal cerebral ischemia-reperfusion,the positive expression of bFGF shew a tendency of decrease.At the time of 7 days after reperfusion,positive expression was maximum;at the time of 14 days after reperfusion,positive expression decreased;and at the time of 28 days after reperfusion,the positive expression decreased to basic level.Electroacupuncturing Ren meridian could at least increase the protein and mRNA expression of bFGF at the time of 14 days after reperfusion.4.After focal cerebral ischemia-reperfusion,protein and mRNA expression of EGF in SVZ and DG shew an increase which could not last for long time.With the extend of time after focal cerebral ischemia-reperfusion,the positive expression of EGF shew a tendency of decrease.At the time of 14 days after reperfusion,the protein expression decreased to basic level,while the mRNA expression decreased to basic level at the time of 28 days after reperfusion.That is to say,the protein and mRNA expression of EGF was asynchronous.Electroacupuncturing Ren meridian could at least increase the protein expression of EGF at the time of 7 days after reperfusion,and could at least increase the mRNA expression of EGF at the time of 14 days after reperfusion.5.After focal cerebral ischemia-reperfusion,protein and mRNA expression of NGF in SVZ and DG shew an increase which could not last for long time.With the extend of time after focal cerebral ischemia-reperfusion,the positive expression of NGF shew a tendency of decrease.At the time of 7 days after reperfusion,positive expression was maximum;at the time of 14 days after reperfusion,positive expression decreased;and at the time of 28 days after reperfusion,the positive expression decreased to basic level.Electroacupuncturing Ren meridian could at least increase the protein expression of NGF at the time of 14 days after reperfusion,and could at least increase the mRNA expression of NGF at the time of 28 days after reperfusion.
Conclusions:BFGF/EGF/NGF participate the process of pathomorphism after ischemical reperfusion injury.Electroacupuncturing Ren meridian can increase the expression of BFGF/EGF/NGF after focal cerebral ischemia-reperfusion.By this way,nerve can be protected and damage is recovered.
目的:观察脑缺血再灌注大鼠缺血侧侧脑室下区、海马齿状回区的碱性成纤维细胞生长因子、表皮生长因子、神经生长因子的变化规律,以及电针任脉经穴对脑内这三种生长因子表达的影响,以期从生长因子角度揭示电针任脉经穴治疗缺血性卒中的可能作用途径和机制,为针灸治疗缺血性脑病提供理论和实验依据。
方法:SPF级成年健康雄性SD大鼠72只,适应环境1周后开始实验。按随机数字表法分为假手术组、模型组、电针任脉组。每组再分别随机分为7天组、14天组和28天组三个亚组,每亚组8只。术前禁食12小时,采用线栓法建立大鼠大脑中动脉栓塞局灶性脑缺血再灌注模型,观察大鼠脑缺血再灌注后神经功能评分及死亡率的变化。电针任脉组在手术24小时后于承浆、气海、关元三穴行电针治疗,采用疏密波刺激(密波15Hz),强度6～15V,持续时间为20分钟,每日一次至动物处死。假手术组和模型组在手术24小时后固定于针刺操作台上20分钟,不作任何治疗。各组大鼠按实验设计时间处死、取脑。运用HE染色观察MCAO大鼠缺血侧纹状体、海马区的病理形态学改变;运用免疫组化法测定MCAO大鼠缺血侧侧脑室下区、海马齿状回区bFGF、EGF、NGF的蛋白表达;运用原位杂交法测定MCAO大鼠缺血侧侧脑室下区、海马齿状回区bFGF、EGF、NGF的mRNA表达。采用德国Leica DC software数码图像分析系统软件包进行图像分析。200倍光镜下进行阳性细胞计数,并采用SPSS13.0软件进行统计分析,比较各组差异。
结果:1、电针任脉经穴对脑缺血再灌注大鼠的神经功能评分、死亡率的影响不明显。2、缺血再灌注后,纹状体及海马区出现明显的脑组织水肿、神经细胞变性坏死等改变,提示运用线栓法制作大鼠局灶性脑缺血再灌注损伤模型是成功的。电针任脉经穴组较同时相模型组能够减轻缺血损伤程度。3、脑缺血再灌注损伤后,侧脑室下区、海马齿状回区内源性bFGF蛋白及mRNA的表达均可出现反应性上调,但该反应性上调具有一定的时限性,随着灌注时间的延长,bFGF的阳性表达呈现下降趋势。再灌注7天,阳性表达最高,再灌注14天阳性表达减弱,至再灌注28天阳性表达恢复至基线水平;电针任脉经穴至少于再灌注14天后上调两区bFGF蛋白及mRNA的表达。4、脑缺血再灌注损伤后,侧脑室下区、海马齿状回区内源性EGF可出现反应性上调,该上调具有一定的时限性,随着灌注时间的延长,EGF的阳性表达呈现下降的趋势。其中蛋白表达于再灌注14天已恢复至基线水平,而mRNA表达于再灌注28天恢复至基线水平,提示内源性EGF蛋白与mRNA的表达并非同步;电针任脉经穴至少能在再灌注7天后上调两区内源性EGF蛋白的表达,而于再灌注14天后上调EGF mRNA的表达。5、脑缺血损伤后,侧脑室下区、海马齿状回区内源性NGF亦可出现反应性上调,该上调亦具有一定的时限性,随着灌注时间的延长,NGF的阳性表达呈现下降的趋势。再灌注7天,阳性表达最高,再灌注14天阳性表达减弱,至再灌注28天阳性表达恢复至基线水平,而电针任脉经穴至少能在再灌注14天后上调内源性NGF蛋白的表达,再灌注28天后始能上调NGFmRNA的表达。
结论:bFGF、EGF、NGF参与脑缺血再灌注损伤的病理过程,而电针任脉经穴能够上调缺血再灌注大鼠脑内bFGF、NGF、EGF的表达,进而促进脑缺血后的神经保护和损伤修复。
Cerebral Ischemic Stroke(CIS),which is also called Cerebral Infarction(CI), is the infarction of brain tissue duing to Cerebral artery occlusion, accompannied with damage of neuron,astrocyte and oligodendrocyte.It is the most important event of central nervous system causing death and mutilation in modern society.Acupuncturing Ren Meridian has clinic foundation on treating CIS.Research discovered that electroacupuncturing Ren Meridian could promote the proliferation and diffrentiation of Nerve Stem Cells which profit the recovery of nerve after cerebral ischemia.Also,we found the effect was related with the activation of ERK signal transduction path.Thus,we assumed that electroacupuncturing Ren Meridian could generate some upstream element which was ERK path dependency,and the up-regulation of these factor would active mechanism of nerve recovery to promte the recovery of cerebral neuron.Based on the assumption and literature study,we chose the endogenous growth factor in brain as cut-in point.By the way of studying on literature and experiment,we can approach the possible mechanism of electroacupuncturing Ren Meridian on treating CIS.
Objective:To observe the change of bFGF/EGF/NGF in subventricular zone(SVZ) and Denatate Gyrus(DG) after Cerebral ischemia-reperfusion in Rats,And approach the effects of electroacupuncturing Ren Meridian on the three growth factor.By the observation of growth factor,we could find the possible mechanism of electroacupuncturing Ren Meridian on treating CIS,and provide theory and experiment evidence for acupuncturing to treat CIS.
Methods:A total of seventy-two adult SD rats from SPF-grade laboratory were taken into the study.Before the test,they were raised to adapt to the surroundings.According to the random digits table,they were divided into sham operation group(Sham),MCAO group(MCAO) and Electroacupuncture Ren Meridian group(R).Each group was averagely divided into 7 days group,14 days group and 28 days group at random.There were eight rats in each subset group. Before operation,rats were abrosia for twelve hours,and Cerebral ischemia-reperfusion injury model was induced by the method of nylon monofilament embolism.After that,We observed the nerve function score and death rate of MCAO rats.Rats in R group were treated with electroacupuncturing Chengjiang/Qihai/Guanyuan points twenty-four hours after operation.As for electric acupuncture,distant-dense wave was chosen.The frequency was15Hz for dense wave and intensity was 6 to15V.The treatment lasted for twenty minutes,and was done once every day until the rats killed.On the other hand, rats in Sham group and MCAO group were fixed on the operation board without any treatment for twenty minutes twenty-four hours after operation.Rats of each group were killed at the design time,and their brain were taken immediately.Then we observed the pathomorphism of corpora striata and hippocampus in ischemic brain of MACO rats by HE stain.Immunohistochemistry was used to detect the protein expression of bFGF/EGF/NGF in SVZ and DG of MCAO rats.In situ hybridization was used to detect the mRNA expression of bFGF/EGF/NGF in SVZ and DG of MCAO rats.At last,we counted positive cells at light microscope of 200×,analyzed the image by analytical system of Leica DC software from German,and compared the difference between groups by SPSS 13.0 software.
Results:1.Electroacupuncturing Ren meridian had little effection on the Nerve function score and death rate of MCAO rats.2.Corpora striata and hippocampus shew obvious brain edema and nerve cell degeneration after focal cerebral ischemia-reperfusion,which shew the Cerebral ischemia-reperfusion injury model was formed successfully.Compared with MCAO group at the same time after focal cerebral ischemia-reperfusion,R group could relieve the degree of injury.3.After focal cerebral ischemia-reperfusion,protein and mRNA expression of bFGF in SVZ and DG shew an increase which could not last for long time.With the extend of time after focal cerebral ischemia-reperfusion,the positive expression of bFGF shew a tendency of decrease.At the time of 7 days after reperfusion,positive expression was maximum;at the time of 14 days after reperfusion,positive expression decreased;and at the time of 28 days after reperfusion,the positive expression decreased to basic level.Electroacupuncturing Ren meridian could at least increase the protein and mRNA expression of bFGF at the time of 14 days after reperfusion.4.After focal cerebral ischemia-reperfusion,protein and mRNA expression of EGF in SVZ and DG shew an increase which could not last for long time.With the extend of time after focal cerebral ischemia-reperfusion,the positive expression of EGF shew a tendency of decrease.At the time of 14 days after reperfusion,the protein expression decreased to basic level,while the mRNA expression decreased to basic level at the time of 28 days after reperfusion.That is to say,the protein and mRNA expression of EGF was asynchronous.Electroacupuncturing Ren meridian could at least increase the protein expression of EGF at the time of 7 days after reperfusion,and could at least increase the mRNA expression of EGF at the time of 14 days after reperfusion.5.After focal cerebral ischemia-reperfusion,protein and mRNA expression of NGF in SVZ and DG shew an increase which could not last for long time.With the extend of time after focal cerebral ischemia-reperfusion,the positive expression of NGF shew a tendency of decrease.At the time of 7 days after reperfusion,positive expression was maximum;at the time of 14 days after reperfusion,positive expression decreased;and at the time of 28 days after reperfusion,the positive expression decreased to basic level.Electroacupuncturing Ren meridian could at least increase the protein expression of NGF at the time of 14 days after reperfusion,and could at least increase the mRNA expression of NGF at the time of 28 days after reperfusion.
Conclusions:BFGF/EGF/NGF participate the process of pathomorphism after ischemical reperfusion injury.Electroacupuncturing Ren meridian can increase the expression of BFGF/EGF/NGF after focal cerebral ischemia-reperfusion.By this way,nerve can be protected and damage is recovered.
引文
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