巴曲酶用药次数对脑缺血再灌注沙土鼠脑保护作用的影响
摘要
脑卒中(中风)是中老年人发病率较高的疾病。急性脑卒中的早期可表现为运动、感觉、认知方面功能障碍及生活不能自理,如不能得到及时有效的干预,将遗留神经功能缺陷,严重影响患者的生活质量,给其家庭和社会造成极大的负担。目前对脑卒中的治疗方法主要包括急性期常规药物治疗及康复治疗,其中超早期溶栓治疗是治疗缺血性脑卒中的最有效方法,但溶栓治疗受时间、技术、设备的影响,使其应用受到限制,因而探讨针对缺血性脑卒中巴曲酶的最佳脑保护作用总量的研究及其安全性显得较为重要。
巴曲酶可直接作用于血浆纤维蛋白元,形成易于被纤溶系统清除的非交联的纤维蛋白单体或多聚体,还可促进内皮细胞释放t-pA,且增强t-pA的作用,降低纤溶酶原活化剂抑制剂的活性,降低血浆纤维蛋白原,降低血小板黏附力。
巴曲酶对脑缺血后抗细胞凋亡的最佳总量实验研究尚未有报道,因而探讨巴曲酶对沙土鼠前脑缺血再灌注后抗细胞凋亡的最佳总量,可以为巴曲酶在临床上的合理应用提供参考依据。
本课题选用不同总剂量的巴曲酶沙土鼠腹腔注射观察脑保护作用的强弱来探讨其最佳总给药剂量。在缺血再灌注模型的基础上通过沙土鼠腹腔注射(8BU/Kg·次)巴曲酶,观察不同用药次数的海马CA_1区的细胞超微结构变化以及流式细胞仪来检测凋亡细胞比例来了解巴曲酶的抗细胞凋亡作用。
研究已表明,Bcl-2家族中Bcl-2和Bax是与细胞凋亡关系密切的两个基因。Bcl-2为抗凋亡基因,可抑制各种因素诱导的细胞凋亡,保护并延长细胞寿命;Bax为促凋亡基因,一方面它自己有促进凋亡的作用,另一方面它还能抑制Bcl-2的表达。通过统计不同次数用药组的海马CA_1区Bcl-2、Bax阳性细胞数的情况来阐明巴曲酶的脑保护作用和Bcl-2、Bax的表达的关系,揭示巴曲酶脑保护作用的机制。
课题分为三个部分。第一部分:用流式细胞仪检测方法来探讨巴曲酶对沙土鼠脑缺血后海马CA_1区抗细胞凋亡作用的最佳总剂量;第二部分:通过电镜观察比较不同巴曲酶用药总量对IR沙土鼠脑海马CA_1区超微结构改变的影响;第三部分:巴曲酶对脑缺血沙土鼠脑保护作用的可能机制的研究。
研究结果:
(一)IR后,模型组细胞凋亡数为10.36±2.58%,3次组为7.27±1.10%,4次用药组细胞凋亡数为5.64±0.27%,5次用药组细胞凋亡数为4.34±0.93%,6次组细胞凋亡数为4.68±1.29%,正常组细胞凋亡数为0.35±0.12%。经统计分析,提示:IR组凋亡细胞数显著高于各用药组(p＜0.05),4次用药组细胞凋亡率要低于3次用药组,组间比较差异有统计学意义(p＜0.05),5次用药组细胞凋亡数显著低于4次用药组(p＜0.05),而5次用药组与6次用药组比较差异无统计学意义(p＞0.05)。这说明脑IR后5次用药组脑保护作用效果好于3次和4次组,但是＞5次的用药脑保护作用并不好于5次用药。
(二)超微结构观察显示IR组细胞凋亡显著,胞质内空泡明显,线粒体肿胀,部分膜崩解,粗面内质网腔扩大,核膜部分皱折,染色体聚集靠边,附于核膜碎裂。3次及4次用药组细胞超微结构改变轻于IR组,但仍可见到胞质内细胞器空泡样变,核周空泡,染色质仍有断裂靠近核膜的现象,可见到凋亡小体。5次用药组的海马CA_1区细胞结构损伤要明显轻于3次及4次用药组,胞质内空泡较少,核膜相对完整。而6次组和5次组超微结构改变不明显。这也说明5次的用药效果要好于3次及4次用药。
根据(一)(二)结果,我们认为在脑IR后巴曲酶(8 BU/Kg·次)5次用药为沙土鼠脑保护的最佳总剂量。
(三)然后将沙土鼠随机分为手术对照组,巴曲酶3次用药组,4次用药组,5次用药组,免疫组化检测各组海马CA_1区Bcl-2、Bax阳性细胞数表达情况,结果显示:随着巴曲酶用药次数增加,Bcl-2阳性细胞数逐渐增加,5次用药组阳性细胞数最高,组间比较差异有统计学意义。而Bax阳性细胞数随着巴曲酶的用药次数的增加而逐渐减少,3次用药组Bax阳性细胞数最多(45.82±4.27)而五次用药组Bax阳性细胞数最少(35.85±2.49),组间比较差异有统计学意义。结合(一)(二)的试验结果我们认为巴曲酶是通过增加Bcl-2表达而降低Bax表达,减少了细胞的凋亡,产生了脑保护作用。
Stroke is one of the most threatening diseases for human health. The patients are always afflicted with functional disorder of movement, sense,cognition and self-care in early stage after stroke.If the functional disorder cannot be effectively treated in time,it will influence the quality of patients′life and become a burden to society and patients′families.At present,the remedy and rehabilitation therapy constitute the methods in treatment of the patients after stroke.Thrombolytic therapy is regarded as an effective way to treat ischemic stroke nowadays.Studies have found that batroxobin does something good in neuroprotection and it has some effect on thrombolysis.
Batroxobin could directly affect plasma fibrinogen to form non-bind mono or multiple fibrin which could be easily cleaned by fibrinolysis system,and could promote the t-pA releasing from the endothelial cells,enhance the effect of t-pA and lower the activity of plasminogen activator inhibitor.It could decline the plasma fibrinogen levels and the blood viscosity.
The total dose of batroxobin on protection of forebrain ischemia-reperfusion injury in experiment hasn't been reported,thus,this study may give the optimal dose according to corresponding animal experiment.
The previous results had showed that Bcl-2 have the role of reducing cell apoptosis but Bax have the opposite effect.There are many animal experiments about brain protection of batroxobin.Hence we examined the expressions of Bcl-2,Bax in different times by intraperitoneally injection,and explain probable mechanism and relationship between times and efficiency of batroxobin to protect brain neurons.
There are three sections in this project:1.To study the total dose of batroxobin to protect forebrain ischemia in gerbils 2.The changes of ultrastructure in hippocampal pyramidal in cerebral ischemic reperfusion in gerbils.3.The probable mechanism of batroxobin to protect brain neurons.
The results manifested that the quantity of apoptotic neurons in IR group was significantly more than that in other groups,and the quantity of apoptotic neurons in five-time group is significantly less than that in three-time group and in four-time group(P＜0.05),but no significant difference was found between five-time group and six-time group (P＞0.05).The ultrastrctural changes in five-time group were lighter than that in three-time group and four-time group,but no significant difference was found between five-time group and six-time group.From the above results,we reckon here that the optimal times of batroxobin to protect forebrain ischemia-reperfusion injury in gerbils is 5 times.
Thereafter gerbils were divided into four groups:normal group,IR group,three-time group,four-time group,five-time group.We used immunohistochemistry technology to accumulate the positive cells of Bcl-2,Bax after forebrain ischemia-reperfusion injury in different group. The result is that with the times of batroxobin used increased,the number of bcl-2 positive cell was gradually increased.but the number of Bax positive cell was gradually decreased with the times of batroxobin increased.Reasonably we think that the neuroprotective effect of batroxobin is achieved by alleviating Bax expression and upgrading the Bcl-2 expression.
巴曲酶可直接作用于血浆纤维蛋白元,形成易于被纤溶系统清除的非交联的纤维蛋白单体或多聚体,还可促进内皮细胞释放t-pA,且增强t-pA的作用,降低纤溶酶原活化剂抑制剂的活性,降低血浆纤维蛋白原,降低血小板黏附力。
巴曲酶对脑缺血后抗细胞凋亡的最佳总量实验研究尚未有报道,因而探讨巴曲酶对沙土鼠前脑缺血再灌注后抗细胞凋亡的最佳总量,可以为巴曲酶在临床上的合理应用提供参考依据。
本课题选用不同总剂量的巴曲酶沙土鼠腹腔注射观察脑保护作用的强弱来探讨其最佳总给药剂量。在缺血再灌注模型的基础上通过沙土鼠腹腔注射(8BU/Kg·次)巴曲酶,观察不同用药次数的海马CA_1区的细胞超微结构变化以及流式细胞仪来检测凋亡细胞比例来了解巴曲酶的抗细胞凋亡作用。
研究已表明,Bcl-2家族中Bcl-2和Bax是与细胞凋亡关系密切的两个基因。Bcl-2为抗凋亡基因,可抑制各种因素诱导的细胞凋亡,保护并延长细胞寿命;Bax为促凋亡基因,一方面它自己有促进凋亡的作用,另一方面它还能抑制Bcl-2的表达。通过统计不同次数用药组的海马CA_1区Bcl-2、Bax阳性细胞数的情况来阐明巴曲酶的脑保护作用和Bcl-2、Bax的表达的关系,揭示巴曲酶脑保护作用的机制。
课题分为三个部分。第一部分:用流式细胞仪检测方法来探讨巴曲酶对沙土鼠脑缺血后海马CA_1区抗细胞凋亡作用的最佳总剂量;第二部分:通过电镜观察比较不同巴曲酶用药总量对IR沙土鼠脑海马CA_1区超微结构改变的影响;第三部分:巴曲酶对脑缺血沙土鼠脑保护作用的可能机制的研究。
研究结果:
(一)IR后,模型组细胞凋亡数为10.36±2.58%,3次组为7.27±1.10%,4次用药组细胞凋亡数为5.64±0.27%,5次用药组细胞凋亡数为4.34±0.93%,6次组细胞凋亡数为4.68±1.29%,正常组细胞凋亡数为0.35±0.12%。经统计分析,提示:IR组凋亡细胞数显著高于各用药组(p＜0.05),4次用药组细胞凋亡率要低于3次用药组,组间比较差异有统计学意义(p＜0.05),5次用药组细胞凋亡数显著低于4次用药组(p＜0.05),而5次用药组与6次用药组比较差异无统计学意义(p＞0.05)。这说明脑IR后5次用药组脑保护作用效果好于3次和4次组,但是＞5次的用药脑保护作用并不好于5次用药。
(二)超微结构观察显示IR组细胞凋亡显著,胞质内空泡明显,线粒体肿胀,部分膜崩解,粗面内质网腔扩大,核膜部分皱折,染色体聚集靠边,附于核膜碎裂。3次及4次用药组细胞超微结构改变轻于IR组,但仍可见到胞质内细胞器空泡样变,核周空泡,染色质仍有断裂靠近核膜的现象,可见到凋亡小体。5次用药组的海马CA_1区细胞结构损伤要明显轻于3次及4次用药组,胞质内空泡较少,核膜相对完整。而6次组和5次组超微结构改变不明显。这也说明5次的用药效果要好于3次及4次用药。
根据(一)(二)结果,我们认为在脑IR后巴曲酶(8 BU/Kg·次)5次用药为沙土鼠脑保护的最佳总剂量。
(三)然后将沙土鼠随机分为手术对照组,巴曲酶3次用药组,4次用药组,5次用药组,免疫组化检测各组海马CA_1区Bcl-2、Bax阳性细胞数表达情况,结果显示:随着巴曲酶用药次数增加,Bcl-2阳性细胞数逐渐增加,5次用药组阳性细胞数最高,组间比较差异有统计学意义。而Bax阳性细胞数随着巴曲酶的用药次数的增加而逐渐减少,3次用药组Bax阳性细胞数最多(45.82±4.27)而五次用药组Bax阳性细胞数最少(35.85±2.49),组间比较差异有统计学意义。结合(一)(二)的试验结果我们认为巴曲酶是通过增加Bcl-2表达而降低Bax表达,减少了细胞的凋亡,产生了脑保护作用。
Stroke is one of the most threatening diseases for human health. The patients are always afflicted with functional disorder of movement, sense,cognition and self-care in early stage after stroke.If the functional disorder cannot be effectively treated in time,it will influence the quality of patients′life and become a burden to society and patients′families.At present,the remedy and rehabilitation therapy constitute the methods in treatment of the patients after stroke.Thrombolytic therapy is regarded as an effective way to treat ischemic stroke nowadays.Studies have found that batroxobin does something good in neuroprotection and it has some effect on thrombolysis.
Batroxobin could directly affect plasma fibrinogen to form non-bind mono or multiple fibrin which could be easily cleaned by fibrinolysis system,and could promote the t-pA releasing from the endothelial cells,enhance the effect of t-pA and lower the activity of plasminogen activator inhibitor.It could decline the plasma fibrinogen levels and the blood viscosity.
The total dose of batroxobin on protection of forebrain ischemia-reperfusion injury in experiment hasn't been reported,thus,this study may give the optimal dose according to corresponding animal experiment.
The previous results had showed that Bcl-2 have the role of reducing cell apoptosis but Bax have the opposite effect.There are many animal experiments about brain protection of batroxobin.Hence we examined the expressions of Bcl-2,Bax in different times by intraperitoneally injection,and explain probable mechanism and relationship between times and efficiency of batroxobin to protect brain neurons.
There are three sections in this project:1.To study the total dose of batroxobin to protect forebrain ischemia in gerbils 2.The changes of ultrastructure in hippocampal pyramidal in cerebral ischemic reperfusion in gerbils.3.The probable mechanism of batroxobin to protect brain neurons.
The results manifested that the quantity of apoptotic neurons in IR group was significantly more than that in other groups,and the quantity of apoptotic neurons in five-time group is significantly less than that in three-time group and in four-time group(P＜0.05),but no significant difference was found between five-time group and six-time group (P＞0.05).The ultrastrctural changes in five-time group were lighter than that in three-time group and four-time group,but no significant difference was found between five-time group and six-time group.From the above results,we reckon here that the optimal times of batroxobin to protect forebrain ischemia-reperfusion injury in gerbils is 5 times.
Thereafter gerbils were divided into four groups:normal group,IR group,three-time group,four-time group,five-time group.We used immunohistochemistry technology to accumulate the positive cells of Bcl-2,Bax after forebrain ischemia-reperfusion injury in different group. The result is that with the times of batroxobin used increased,the number of bcl-2 positive cell was gradually increased.but the number of Bax positive cell was gradually decreased with the times of batroxobin increased.Reasonably we think that the neuroprotective effect of batroxobin is achieved by alleviating Bax expression and upgrading the Bcl-2 expression.
引文
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12 Niwa M, Hara A, Iwai T, et al. Caspase activation as an apoptotic evidence in the gerbil hippocampal CA1 pyramidal cells following transient forebrain ischemia[J]. Neurosci Lett, 2001, 300: 103-106.
13 Strasser A, O'Connor L, Dixit VM, Apoptosis signaling[J]. Annu Rev Biochem,2000,69:217-245.
14 Cao G, Pei W, Lan J, et al. Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures [J]. Neurosci, 2001,21:4678-4690.
15 Luo Y, Cao G, Pei W, et al. Induction of caspase-activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion[J]. Cereb Blood Flow Metab, 2002.22: 15- 20.
16 Harrison DC, Davis RP, Bond BC, et al. Caspase mRNA expression in a rat model of focal cerebral ischemia[J]. Mol Brain Res, 2001, 89: 133-146.
17 Schulz JB, Waller M, Moskowitz MA. Caspases as treatment targets in stroke and neurodegenerative disease[J]. Ann Neurol,1999, 45:921-429
18 Love S, Barber R,Wilcock GK. Neuronal death in brain infarcts in man[J]. Neuropathol Appl Neurobiol, 2000, 26: 55- 66.
19 Love S, Barber R, Srinivasan A, et al. Activation of caspase-3 in permanent and transient brain ischaemia in man[J]. Neuro Report, 2000, 11:2495-2499.
20 Baeuerle PA. I k B-NF- k B structures: at the interface of inflammation control[J].Cell, 1998,95:729-731.
21 Li Y, Chopp M, Zhang Z, et al. P53-immunoreactive protein and P53 mRNA expression after transient middle cerebral artery occlusion in rats[J]. Stroke, 1994,25:849-856
22 Martin LJ, Kaiser A, Yu JW, et al. Injury-induced apoptosis of neurons in adult brain is mediated by p53- dependent and p53-independent pathways and requires Bax[J]. Comp Neurol, 2001, 433:299-311.
23 Maeda K, Hata R, Gillardon F et al. Aggravation of brain injury after transient focal ischemia in p53-deficient mice[J]. Mol Brain Res, 2001,88:54-61.
24 Motioto RI, Sarge KD, Abacavaga K, et al. Transcriptional regulation of heat shock genes paradigm for inducible genomice response[J]. Biol Chem,1992 ,267(31):21987-21990
25 Plumlic JC, Krueger AM, Curie RW, et al. Transgenic mice expression the human induible HSP70 hippocampus neurons resistant to ischemia injury[J]. Cell Stress Chapperones, 1997,2(3 ): 192-197
26 Estus S, Zaks WJ, Freeman RS, el al. Altered gene expression in neurons during programmed cell death: indentification of c-jun as necessary for neuronal apoptosis[J]. J Cell Biol,1994,127:171 7-17279
27 Whitfield J, Neame SJ, Paquet L, et al. Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[J]. Neuron,2001, 29:629-643.
28 Sorinno MA, Ferrer 2, Rodriguez FE, et al. Expression of c-fos and inducible hsp70 mRNA following a transient epiesode of focal ischmia that had non-lethal effeces on the rat brain[J]. Brain Rev, 1995,670:317,
29 Abbas AK. Eliominating dangerous lymphocytes [J]. Cell, 1996,84:655-557
30 Ranaka M, Suda T, Yatomi T, et al. Lethal effect of recombinant human Fas ligand in mice pretreated with propionibacterium acnes[J]. Immunol, 1997,158:2303-2309
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3 Harris MH., Thompson CB. The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability [J]. Cell Death Differ, 2000,7: 1182-1191.
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5 Antonawich FJ, Krajewski S, Reed JC, et al. Bcl-xl Bax interaction after transient global ischemia[J]. Cereb Blood Flow Metab, 1998, 18: 882- 886.
6 Cao G, Minami M, Pei W, et al. Intracellular bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death[J]. Cereb Blood Flow Metab, 2001, 21: 321- 333.
7 Kitagawa K, Matsumoto M, Tsujimoto Y, et al. Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of BCL-2 in transgenic mice[J]. Stroke ,1998,29: 2616-2621.
8 Wang HD, Fukuda T, Suzuki T, et al. Differential effects of Bcl-2 overexpression on hippocampal CA1 neurons and dentate granule cells following hypoxic ischemia in adult mice[J]. Neurosci, 1999 Res. 57: 1 -12.
9 Hata R, Gillardon F, Michaelidis TM, et al. Targeted disruption of the bcl-2 gene in mice exacerbates focal ischemic brain injury[J].Metab Brain Dis, 1999,14: 117-124.
10 Love S. Apoptosis and brain ischemia[J]. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 2003,27: 267-269.
11 Hermann DM, Kilic E, Hata R, et al. Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice[J]. Neuroscience, 2001,104: 947- 955.
12 Niwa M, Hara A, Iwai T, et al. Caspase activation as an apoptotic evidence in the gerbil hippocampal CA1 pyramidal cells following transient forebrain ischemia[J]. Neurosci Lett, 2001, 300: 103-106.
13 Strasser A, O'Connor L, Dixit VM, Apoptosis signaling[J]. Annu Rev Biochem,2000,69:217-245.
14 Cao G, Pei W, Lan J, et al. Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures [J]. Neurosci, 2001,21:4678-4690.
15 Luo Y, Cao G, Pei W, et al. Induction of caspase-activated deoxyribonuclease activity after focal cerebral ischemia and reperfusion[J]. Cereb Blood Flow Metab, 2002.22: 15- 20.
16 Harrison DC, Davis RP, Bond BC, et al. Caspase mRNA expression in a rat model of focal cerebral ischemia[J]. Mol Brain Res, 2001, 89: 133-146.
17 Schulz JB, Waller M, Moskowitz MA. Caspases as treatment targets in stroke and neurodegenerative disease[J]. Ann Neurol,1999, 45:921-429
18 Love S, Barber R,Wilcock GK. Neuronal death in brain infarcts in man[J]. Neuropathol Appl Neurobiol, 2000, 26: 55- 66.
19 Love S, Barber R, Srinivasan A, et al. Activation of caspase-3 in permanent and transient brain ischaemia in man[J]. Neuro Report, 2000, 11:2495-2499.
20 Baeuerle PA. I k B-NF- k B structures: at the interface of inflammation control[J].Cell, 1998,95:729-731.
21 Li Y, Chopp M, Zhang Z, et al. P53-immunoreactive protein and P53 mRNA expression after transient middle cerebral artery occlusion in rats[J]. Stroke, 1994,25:849-856
22 Martin LJ, Kaiser A, Yu JW, et al. Injury-induced apoptosis of neurons in adult brain is mediated by p53- dependent and p53-independent pathways and requires Bax[J]. Comp Neurol, 2001, 433:299-311.
23 Maeda K, Hata R, Gillardon F et al. Aggravation of brain injury after transient focal ischemia in p53-deficient mice[J]. Mol Brain Res, 2001,88:54-61.
24 Motioto RI, Sarge KD, Abacavaga K, et al. Transcriptional regulation of heat shock genes paradigm for inducible genomice response[J]. Biol Chem,1992 ,267(31):21987-21990
25 Plumlic JC, Krueger AM, Curie RW, et al. Transgenic mice expression the human induible HSP70 hippocampus neurons resistant to ischemia injury[J]. Cell Stress Chapperones, 1997,2(3 ): 192-197
26 Estus S, Zaks WJ, Freeman RS, el al. Altered gene expression in neurons during programmed cell death: indentification of c-jun as necessary for neuronal apoptosis[J]. J Cell Biol,1994,127:171 7-17279
27 Whitfield J, Neame SJ, Paquet L, et al. Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[J]. Neuron,2001, 29:629-643.
28 Sorinno MA, Ferrer 2, Rodriguez FE, et al. Expression of c-fos and inducible hsp70 mRNA following a transient epiesode of focal ischmia that had non-lethal effeces on the rat brain[J]. Brain Rev, 1995,670:317,
29 Abbas AK. Eliominating dangerous lymphocytes [J]. Cell, 1996,84:655-557
30 Ranaka M, Suda T, Yatomi T, et al. Lethal effect of recombinant human Fas ligand in mice pretreated with propionibacterium acnes[J]. Immunol, 1997,158:2303-2309