大鼠缺血脑区Caspase3表达与活化及抑制其活化对半暗带神经元生存和凋亡的影响
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摘要
本实验利用大鼠大脑中动脉梗塞及再灌流模型为观察对象，结合缺血前30min同侧侧脑室注射Caspase3抑制剂z-DEVD．fmk或安慰剂DMSO，采用Western Blotting、免疫组化及原位细胞凋亡检测等方法，观察了大脑中动脉梗塞2小时后再灌注不同时间点，半暗带区神经元凋亡程度的动态变化，Caspase3表达和活化水平的变化，PARP表达和切割速度的变化，以及这些变化之间的相互关系。同时观察了Caspase3抑制剂是否能有效地阻断凋亡信号传导通路，减少半暗带区神经元的凋亡。并同期观察了胶质细胞的损伤情况。旨在探讨缺血脑区Caspase3表达与活化及抑制其活化对半暗带神经元生存和凋亡的影响，以便为探索缺血性脑血管病的新治疗途径奠定理论基础。
TUNEL及Western Blotting检测结果显示，脑缺血再灌注损伤后，各组动物半暗带区内神经元的凋亡程度均随脑缺血再灌注损伤时间的延长而逐渐加重，Caspase3的表达(前体)和活化水平(切割片段)及PARP切割片段均相应增加。经免疫组化染色结果表明PARP阳性神经元密度及提示胶质细胞损伤的GFAP阳性细胞密度亦随损伤时间延长和细胞凋亡程度加重而增加。经直线相关分析证实，各组Caspase3切割片段、PARP切割片段、PARP阳性细胞密度与凋亡细胞密度彼此间均呈显著性正相关。GFAP阳性细胞密度亦与凋亡细胞密度间呈显著性正相关。
缺血前30min同侧侧脑室给予安慰剂DMSO对半暗带神经元凋亡程度无明显影响，上述检测指标亦无明显变化。而缺血前30min同侧侧脑室给予
C卿ase3抑制剂zAIEVD．ink后，半暗带区内神经元的凋亡明显减少，
Caspase3切割片段、PARP切割片段的含量降低；PAny阳性细胞密度、
GFAP阳性细胞密度减少。四者的变化趋势始终与凋亡变化趋势一致，故可以
说明caspase3活化及其介导的下游信号传导通路在启动缺血脑区半暗带神经
元损伤性凋亡过程中起了关键性作用。C卿ase3切割片段和PARP切割片段
多少能客观反映半暗带神经元的凋亡程度，且C呷ase3抑制剂可减少GFAP
阳性细胞密度，减轻胶质细胞损伤。可能由于本实验所用动物模型造成的脑
缺血损伤过强，未见到胶质细胞先于神经元损伤的病理改变。
同时实验结果显示，随损伤时间延长，RMIP蛋白含量逐渐增高，提示
细胞的修复能力逐渐增强。抑制剂组Caspase3前体和I？Alip蛋白增加尤其显
著。我们认为，一方面这是由于细胞凋亡和修复机能同时增强，使蛋白表达
增加所致；另一方面是由于z－DEVD．l？ill，’的应用抑制了Caspase3的切割激活，
进而使PABJ，切割失活减少所致。
本课题在动物实班实，0闷的％抑制剂可以有效删制O呸阳d活化，
减少PAllP切割失活，缓解半暗带神经元的凋亡程度；同时为细胞保存了能
量，增强了神经元的修复和存活。故Caspase3抑制剂有明显且持续的神经元
保护作用。本实验结果为应用C呷ase3抑制剂治疗缺血性脑血管病的可行性
及研制开发前景提供了客观的实验依据。
Ischemic cerebral vascular disease is still a serious disease which threats human health and life quality. Now it is generally agreed that apoptosis is the chief death pattern of neurons in penumbra after ischemia and reperfusion injury of brain. During apoptosis progress, firstly, caspaseS is activated by proteolysis as two fiagments. Then, poly(ADP-ribose)polymerase(PARP), a 116 KDa DNA repairing enzyme , served as CaspaseS's substrate, is cleaved in a 89KDa C -fiagment and a 24KDa N -fragment. This is the definit characterization of apoptosis. hi present study, apoptosis degree was observed by TUNEL as well as changes of expression and activity levels of Caspase3, and expression and cleavage of PARP in penumbra were detected the used rat model of middle cerebral artery filament occlusion and reperfusion (MCAO), Western Blotting and immunohistochemistry. Besides, we also investigated the protecting effect of z-DEVD.fink, a caspaseS inhibitor, on the neurons in penumbra. At the same time, we detected GFAP p
ositive cells density by immunohistochemistry which is a indicator of astrocytes ischemic injury. Especially, we analysed the correlation among above parameters one another. The results showed that , as a function of time intervals , the levels of expression and activity of CaspaseS, and quantities of expression and cleavage of PARP increased after MCAO, in concordance with the deterioration of apoptosis. After using z-DEVD.fink, the activity of CaspaseS and cleavage of PARP were decreased, but the expression were still increased The results were consistent with our previous expectation and suggested that caspaseS inhibitor could effectively protecte ischemic neurons and astrocytes and prevent them from apoptosis. We have not observed astrocytes injury before neurons pathological changes after cerebral ischemia.
TUNEL及Western Blotting检测结果显示，脑缺血再灌注损伤后，各组动物半暗带区内神经元的凋亡程度均随脑缺血再灌注损伤时间的延长而逐渐加重，Caspase3的表达(前体)和活化水平(切割片段)及PARP切割片段均相应增加。经免疫组化染色结果表明PARP阳性神经元密度及提示胶质细胞损伤的GFAP阳性细胞密度亦随损伤时间延长和细胞凋亡程度加重而增加。经直线相关分析证实，各组Caspase3切割片段、PARP切割片段、PARP阳性细胞密度与凋亡细胞密度彼此间均呈显著性正相关。GFAP阳性细胞密度亦与凋亡细胞密度间呈显著性正相关。
缺血前30min同侧侧脑室给予安慰剂DMSO对半暗带神经元凋亡程度无明显影响，上述检测指标亦无明显变化。而缺血前30min同侧侧脑室给予
C卿ase3抑制剂zAIEVD．ink后，半暗带区内神经元的凋亡明显减少，
Caspase3切割片段、PARP切割片段的含量降低；PAny阳性细胞密度、
GFAP阳性细胞密度减少。四者的变化趋势始终与凋亡变化趋势一致，故可以
说明caspase3活化及其介导的下游信号传导通路在启动缺血脑区半暗带神经
元损伤性凋亡过程中起了关键性作用。C卿ase3切割片段和PARP切割片段
多少能客观反映半暗带神经元的凋亡程度，且C呷ase3抑制剂可减少GFAP
阳性细胞密度，减轻胶质细胞损伤。可能由于本实验所用动物模型造成的脑
缺血损伤过强，未见到胶质细胞先于神经元损伤的病理改变。
同时实验结果显示，随损伤时间延长，RMIP蛋白含量逐渐增高，提示
细胞的修复能力逐渐增强。抑制剂组Caspase3前体和I？Alip蛋白增加尤其显
著。我们认为，一方面这是由于细胞凋亡和修复机能同时增强，使蛋白表达
增加所致；另一方面是由于z－DEVD．l？ill，’的应用抑制了Caspase3的切割激活，
进而使PABJ，切割失活减少所致。
本课题在动物实班实，0闷的％抑制剂可以有效删制O呸阳d活化，
减少PAllP切割失活，缓解半暗带神经元的凋亡程度；同时为细胞保存了能
量，增强了神经元的修复和存活。故Caspase3抑制剂有明显且持续的神经元
保护作用。本实验结果为应用C呷ase3抑制剂治疗缺血性脑血管病的可行性
及研制开发前景提供了客观的实验依据。
Ischemic cerebral vascular disease is still a serious disease which threats human health and life quality. Now it is generally agreed that apoptosis is the chief death pattern of neurons in penumbra after ischemia and reperfusion injury of brain. During apoptosis progress, firstly, caspaseS is activated by proteolysis as two fiagments. Then, poly(ADP-ribose)polymerase(PARP), a 116 KDa DNA repairing enzyme , served as CaspaseS's substrate, is cleaved in a 89KDa C -fiagment and a 24KDa N -fragment. This is the definit characterization of apoptosis. hi present study, apoptosis degree was observed by TUNEL as well as changes of expression and activity levels of Caspase3, and expression and cleavage of PARP in penumbra were detected the used rat model of middle cerebral artery filament occlusion and reperfusion (MCAO), Western Blotting and immunohistochemistry. Besides, we also investigated the protecting effect of z-DEVD.fink, a caspaseS inhibitor, on the neurons in penumbra. At the same time, we detected GFAP p
ositive cells density by immunohistochemistry which is a indicator of astrocytes ischemic injury. Especially, we analysed the correlation among above parameters one another. The results showed that , as a function of time intervals , the levels of expression and activity of CaspaseS, and quantities of expression and cleavage of PARP increased after MCAO, in concordance with the deterioration of apoptosis. After using z-DEVD.fink, the activity of CaspaseS and cleavage of PARP were decreased, but the expression were still increased The results were consistent with our previous expectation and suggested that caspaseS inhibitor could effectively protecte ischemic neurons and astrocytes and prevent them from apoptosis. We have not observed astrocytes injury before neurons pathological changes after cerebral ischemia.
引文
01 White BC, Sullivan JM, DeGracia DJ, et al. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J of Neurol Sci, 2000, 179(1-2) : 1-33．
02 Barber PA, Auer RN, Buchan AM, et al. Understanding and managing ischemia stroke. Can J Physiol Pharmacol, 2001, 79(3) : 283-296．
03 Yang GY, Pang L, Ge HL, et al. Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein. J Cereb Blood Flow Metab, 2001, 21(6) : 722-733．
04 Kato N, Morita H, Sugiyama T, et al. Evaluation of the poly(ADP-ribose)polymerase gene in human stroke. Atherosclerosis, 1999, 148(2) : 345-352．
05 Szabo C, Dawson VL. Role of poly(ADP-Ribose)synthetase in inflammation and ischaemia-reperrusion. Trends pharmacol sci, 1998,19(7) : 287-298．
06 Davidovic L, Vodenicharov M, Affar EB, et al. Importance of poly(ADP-ribose )glycohydrolase in the control of poly(ADP-ribose)metabolism. Exp Cell Res, 2001, 268(1) : 7-13．
07 Burns TF, Bernhard EJ, El-Deiry WS. Tissue specific expression of p53 target genes suggests a key role for KILLER/DR5 in p53-dependent apoptosis in vivo. Oncogene, 2001,20(34) : 4601-4612．
08 Kim JW, Won J, Sohn S, et al. DNA-binding activity of the N-tenninal cleavage product of poly(ADP-ribose)polymerase is required for UV mediated apoptosis. J of cell sci, 2000, 113(pt6) : 955-961．
09 Ariumi Y, Ueda K, Masutani M, et al. In vivo phosphorylation of poly(ADP-ribose)polymerase is independent of its activation. FEBS Lett, 1998, 436(2) : 288-292．
10 Cottet F, Blanche H, Verasdonck P, et al. New polymorphisms in the human
10 Cottet F, Blanche H, Verasdonck P, et al. New polymorphisms in the human poly(ADP-ribose)polymerase-1 coding sequencerlack of association with longevity or with increased cellular poly(ADP-ribosyl)ation capacity. J Mol Med, 2000, 78(8) : 431-440．
11 Uchida M, Hanai S, Uematsun N, et al. Genetic and functional analysis of PARP, a DNA strand breaking-binding enzyme. Mutat-Res, 2001,477(1-2) : 89-96．
12 Rhun YL, Kirkland JB, Shan GM. Cellular responses to DNA damage in the absence of poIy(ADP-ribose)polymerase. Biochem and Biophysi research communication, 1998,245(1) : 1-10．
13 Smulson ME, Pang D, Jung M, et al. Irreversible binding of poly(ADP-ribose)polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis. Cancer research, 1998, 58(16) : 3495-3498．
14 Rosenthal CMS, Rosenthal DS, Iyer S, et al. Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose)polymerase in the early stages of apoptosis. J of biological chemistry, 1998, 273(22) : 13703-13712．
15 O'Brien MA, Moravec RA, Kiss TL. Poly(ADP-ribose)polymerase cleavage monitored in situ in apoptotic cells. Biotechniques, 2001,30(4) : 886-891．
16 Boulares AH, Yalovlev AG, Ivanova V, et al. Role of Poly(ADP-ribose)polymerase cleavage in apoptosis. J of Biolog Chem, 1999, 274(33) : 22932-22940．
17 Green DR. Apoptotic pathways: paper wraps stone blunts scissors, Cell, 2000, 102(1) : 1-4．
18 Zheng TS, Flavell RA. Death by numbers. Nature biotechnology, 2000, 18(7) : 717-718．
19 Conde C, Mark M, Oliver FJ, et al. Loss of Poly(ADP-ribose)polymerase-1 causes increased rumor latency in p53-deficient mice. EMBO J, 2001, 20(13) : 3535-3543．
20 Plaschke K, Kopitz J, Weigand MA, et al. The neuroprotective effect of cerebral poly(ADP-ribose)polymerase inhibition in a rat model global ischemia. Neurosci Lett, 2000,284(1-2) : 109-112．
21 Guegan C, Sola B. Early and sequential recruitment of apoptotic effectors
after focal permanent ischemia in mice. Brain Research, 2000, (856) : 93-100．
22 Julio H, Yasuli Y,Chen H, et al. Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. American journal of pathology, 1993,142(2) : 623-635．
23 Chen J, Nagayama T, jin KL, et al. Induction of Caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. The journal of neuroscience, 1998, 18(13) : 4914-4928．
24 Cheng Y, Deshmuch M, Dcosta A, et al. Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypotic-ischemic brain injury. The American society for clinical investigation Inc, 1998, 101(9) : 1992-1999．
25 Ma JY, Endres M, Michael A, et al. Synergistic effects of caspase inhibitors and MK-801 in brain injury after transient focal cerebral ischaemia in mice. British journal of pharmacology, 1998,124(1) : 756-762．
26 Gillardon F, Kiprianovai, Sandkuhler J, et al. Inhibition of caspases prevents cell death of hippocampal CA1 neurons, but not impairment of hippocampal long-term potentiation following global ischemia. Neuroscience, 1999, 93(4) : 1219-1222．
27 Rabuffetti M, Sciorati C, Tarozzo G, et al. Inhibition of Caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-Chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. The journal of neuroscience , 2000, 20(12) : 4398-4404．
28 Silverstein FS. Can inhibition of apoptosis rescue ischemic brain? Clinical invest. 1998,101(9) : 1809-1810．
29 Namura S, Zhu JM, Fink K, et al. Activation and cleavage of Caspase-3 in apoptosis induced by experimental cerebral ischemia. The journal of neuroscience . 1998, 18(10) : 3659-3668．
30 Zea Longa E, Weinstein PR, Carlson S, Cummins R: Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989,20:84-91．
31 王维治，罗祖明。《神经病学》第四版，人民卫生出版社，122-142。
32 Goyal L. Cell death inhibition : keeping caspases in check. Cell . 2001,104(4) :805-808．
33． Michael O.H. Apoptosis : corralling the corpses. Cell, 2001,voll04: 325-328．
34． Paul T.A, Philip K.L, Chung Y.H. Immediate early gene expression in response to cerebral ischemia: friend or foe? Stroke, 1996,27(9) : 1682-1687．
35 Mary O.D, Michael C, Robert A.k, et al .The heterogeneous temporal evolution of focal ischemic neuronal damage in the rat. Acta neuropathol .1993,85:327-333．
36 汪家政，范明。《蛋白质技术手册》。科学出版社，2000，47-50。
02 Barber PA, Auer RN, Buchan AM, et al. Understanding and managing ischemia stroke. Can J Physiol Pharmacol, 2001, 79(3) : 283-296．
03 Yang GY, Pang L, Ge HL, et al. Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein. J Cereb Blood Flow Metab, 2001, 21(6) : 722-733．
04 Kato N, Morita H, Sugiyama T, et al. Evaluation of the poly(ADP-ribose)polymerase gene in human stroke. Atherosclerosis, 1999, 148(2) : 345-352．
05 Szabo C, Dawson VL. Role of poly(ADP-Ribose)synthetase in inflammation and ischaemia-reperrusion. Trends pharmacol sci, 1998,19(7) : 287-298．
06 Davidovic L, Vodenicharov M, Affar EB, et al. Importance of poly(ADP-ribose )glycohydrolase in the control of poly(ADP-ribose)metabolism. Exp Cell Res, 2001, 268(1) : 7-13．
07 Burns TF, Bernhard EJ, El-Deiry WS. Tissue specific expression of p53 target genes suggests a key role for KILLER/DR5 in p53-dependent apoptosis in vivo. Oncogene, 2001,20(34) : 4601-4612．
08 Kim JW, Won J, Sohn S, et al. DNA-binding activity of the N-tenninal cleavage product of poly(ADP-ribose)polymerase is required for UV mediated apoptosis. J of cell sci, 2000, 113(pt6) : 955-961．
09 Ariumi Y, Ueda K, Masutani M, et al. In vivo phosphorylation of poly(ADP-ribose)polymerase is independent of its activation. FEBS Lett, 1998, 436(2) : 288-292．
10 Cottet F, Blanche H, Verasdonck P, et al. New polymorphisms in the human
10 Cottet F, Blanche H, Verasdonck P, et al. New polymorphisms in the human poly(ADP-ribose)polymerase-1 coding sequencerlack of association with longevity or with increased cellular poly(ADP-ribosyl)ation capacity. J Mol Med, 2000, 78(8) : 431-440．
11 Uchida M, Hanai S, Uematsun N, et al. Genetic and functional analysis of PARP, a DNA strand breaking-binding enzyme. Mutat-Res, 2001,477(1-2) : 89-96．
12 Rhun YL, Kirkland JB, Shan GM. Cellular responses to DNA damage in the absence of poIy(ADP-ribose)polymerase. Biochem and Biophysi research communication, 1998,245(1) : 1-10．
13 Smulson ME, Pang D, Jung M, et al. Irreversible binding of poly(ADP-ribose)polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis. Cancer research, 1998, 58(16) : 3495-3498．
14 Rosenthal CMS, Rosenthal DS, Iyer S, et al. Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose)polymerase in the early stages of apoptosis. J of biological chemistry, 1998, 273(22) : 13703-13712．
15 O'Brien MA, Moravec RA, Kiss TL. Poly(ADP-ribose)polymerase cleavage monitored in situ in apoptotic cells. Biotechniques, 2001,30(4) : 886-891．
16 Boulares AH, Yalovlev AG, Ivanova V, et al. Role of Poly(ADP-ribose)polymerase cleavage in apoptosis. J of Biolog Chem, 1999, 274(33) : 22932-22940．
17 Green DR. Apoptotic pathways: paper wraps stone blunts scissors, Cell, 2000, 102(1) : 1-4．
18 Zheng TS, Flavell RA. Death by numbers. Nature biotechnology, 2000, 18(7) : 717-718．
19 Conde C, Mark M, Oliver FJ, et al. Loss of Poly(ADP-ribose)polymerase-1 causes increased rumor latency in p53-deficient mice. EMBO J, 2001, 20(13) : 3535-3543．
20 Plaschke K, Kopitz J, Weigand MA, et al. The neuroprotective effect of cerebral poly(ADP-ribose)polymerase inhibition in a rat model global ischemia. Neurosci Lett, 2000,284(1-2) : 109-112．
21 Guegan C, Sola B. Early and sequential recruitment of apoptotic effectors
after focal permanent ischemia in mice. Brain Research, 2000, (856) : 93-100．
22 Julio H, Yasuli Y,Chen H, et al. Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. American journal of pathology, 1993,142(2) : 623-635．
23 Chen J, Nagayama T, jin KL, et al. Induction of Caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. The journal of neuroscience, 1998, 18(13) : 4914-4928．
24 Cheng Y, Deshmuch M, Dcosta A, et al. Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypotic-ischemic brain injury. The American society for clinical investigation Inc, 1998, 101(9) : 1992-1999．
25 Ma JY, Endres M, Michael A, et al. Synergistic effects of caspase inhibitors and MK-801 in brain injury after transient focal cerebral ischaemia in mice. British journal of pharmacology, 1998,124(1) : 756-762．
26 Gillardon F, Kiprianovai, Sandkuhler J, et al. Inhibition of caspases prevents cell death of hippocampal CA1 neurons, but not impairment of hippocampal long-term potentiation following global ischemia. Neuroscience, 1999, 93(4) : 1219-1222．
27 Rabuffetti M, Sciorati C, Tarozzo G, et al. Inhibition of Caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-Chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. The journal of neuroscience , 2000, 20(12) : 4398-4404．
28 Silverstein FS. Can inhibition of apoptosis rescue ischemic brain? Clinical invest. 1998,101(9) : 1809-1810．
29 Namura S, Zhu JM, Fink K, et al. Activation and cleavage of Caspase-3 in apoptosis induced by experimental cerebral ischemia. The journal of neuroscience . 1998, 18(10) : 3659-3668．
30 Zea Longa E, Weinstein PR, Carlson S, Cummins R: Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989,20:84-91．
31 王维治，罗祖明。《神经病学》第四版，人民卫生出版社，122-142。
32 Goyal L. Cell death inhibition : keeping caspases in check. Cell . 2001,104(4) :805-808．
33． Michael O.H. Apoptosis : corralling the corpses. Cell, 2001,voll04: 325-328．
34． Paul T.A, Philip K.L, Chung Y.H. Immediate early gene expression in response to cerebral ischemia: friend or foe? Stroke, 1996,27(9) : 1682-1687．
35 Mary O.D, Michael C, Robert A.k, et al .The heterogeneous temporal evolution of focal ischemic neuronal damage in the rat. Acta neuropathol .1993,85:327-333．
36 汪家政，范明。《蛋白质技术手册》。科学出版社，2000，47-50。