灯盏花素对脑复苏后Caspase-12及iNOS表达的影响
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摘要
目的：探讨灯盏花素(Breviscapine, Br)对Sprague-Dawley(SD)大鼠全脑缺血/再灌注(ischemia/reperfusion, I/R)损伤后大脑海马CA1区Caspase-12及iNOS表达的影响,进一步阐明其在脑复苏中的作用机制。方法：将90只SD大鼠随机分为假手术(SO)组30只；全脑缺血/再灌注(I/R)组30只；灯盏花素(Br)组30只；每组按其再灌注的时间又分为2、6、12、24、48小时(h)共5个观察时间点,采用简化的Pulsinelli等的四血管阻塞法建立大鼠脑I/R损伤的模型。假手术组与脑I/R组于再灌注后每6h腹腔注射0.9%的生理盐水1ml/kg,Br组则于再灌注后每6h腹腔注射Br 2.5mg/kg,然后在每个时间点断头取脑。用免疫组织化学SABC法检测海马CA1区锥体细胞中Caspase-12及iNOS的表达,用原位杂交法检测海马CA1区锥体细胞中Caspase-12mRNA的表达,测量其平均光密度值,用苏木精-伊红(HE)染色检测存活锥体细胞数,用TUNEL原位末端标记法检测凋亡锥体细胞数。结果：在SO组,Caspase-12蛋白及mRNA、iNOS表达的平均光密度值差异无统计学意义,存活神经元细胞数及凋亡细胞数均无明显变化,无统计学意义(p>0.05)。在I/R组,可见Caspase-12蛋白及mRNA平均光密度值在12小时升高,在24小时达高峰,后逐渐下降；iNOS蛋白在12小时平均光密度值开始升高,24小时达到高峰,到48小时开始下降；存活神经元细胞数目随再灌注时间的延长而减少(p均<0.01)；凋亡细胞数则随再灌注时间的延长而显著增加(p均<0.01)。在Br组,Caspase-12蛋白及Caspase-12mRNA平均光密度值6小时达到高峰,于12小时开始下降,在48小时达最低值；iNOS蛋白平均光密度值在6小时开始升高,在12小时达高峰,在24小时开始降低；存活神经元细胞数目随时间延长而逐渐减少,于12小时达最低值,此后细胞数目逐渐增多,并于48小时存活细胞数目达最多；凋亡细胞数目随时间延长而逐渐增加,在12小时达最高峰,此后凋亡细胞数目逐渐下降,于48小时达最低值。Br组各时间点与I/R组相同时间点相比,各值均有所降低,且存在统计学差异(p<0.05)。结论：大鼠全脑I/R后,灯盏花素可通过抑制Caspase-12及iNOS的表达,从而抗细胞凋亡,增加存活神经元的数目而起到脑保护作用。
Objective To investigate the expression of Caspase-12 and iNOS in hippocampus after the Breviscapine(Br) in intervention therapy the Sprague-Dawley (SD) rats with global cerebral ischemia/reperfusion (I/R) injury and testify the effect mechanism of Br during cerebral resuscitation. Methods 90 healthy SD rats were randomly divided into 3 groups: sham operation (SO) group (n=30), I/R group (n=30) and Br group (n=30). Then each group were assigned into 5 observing time-points:2,6,12,24,48 hours (h) according to reperfusion time. The SD rats model of global cerebral ischemia/reperfusion (including I/R and Br group) was produced by means of simple Pulsinelli-Brierley's four arteries occlusion method. SO and I/R groups were intraperitoneally injected with 0.9% Sodium chloride (dose:1ml/kg), and Br group with Breviscapine (dose:2.5mg/kg) one time per six hours after reperfusion. Then SD rats'cerebrums were taken out of their skull at each of observing time-point. The expression activation of Caspase-12 and iNOS protein in pyramidal cells in hippocampus comu ammonis (CA) 1 region were examined by immunohistochemical method (SABC) and Caspase-12mRNA by in situ hybridization, measured their average optical density(OD), and hematoxylin and eosin (HE) staining was also performed to detect the number of surviving pyramidal cells, and terminal-deoxynucleotidy transferase medatd dUTP nick end labeling (TUNEL) was used to detect apoptotic pyramidal cells (positive cells). Results In SO group, the average optical density of Caspase-12 protein and its mRNA, iNOS protein had no significance of difference. The number of surviving neurons and apoptic cells was the same condition(p>0.05). In I/R group, the average optical density of Caspase-12 protein and mRNA began to increase at 12h after reperfusion, arrived on peak at 24h, and then gradually decreased. The expression of iNOS protein was as same as Caspase-12 protein. With the reperfusion prolonged, the number of surviving neurons became more and more lower (all P<0.01), and more and more neurons emerged the phenomenon of cell-apoptosis, its apoptotic pyramidal cells then clearly rised, too(all P<0.01). In Br group,the average optical density of Caspase-12 protein and mRNA peaked at 6h, and then began to decrease at 12h, and reached the minimum at 48h. The average optical density of iNOS protein began to increase at 6h, reached the maximum at 12h, and then decreased gradually. With the reperfusion prolonged, the number of surviving neurons became more and more lower, and reached maximum at 12h, then gradually recoverd, and reached the maximum at 48h. The change of apoptic cells was just contrary. Comparing with I/R group, the average optical density in Br group was smaller at every corresponding time-point, and had statistically significant (p<0.05). Conclusions After global cerebral ischemia/reperfusion, Breviscapine could protect the brain from global cerebal ischemia/reperfusion injury by inhibiting neuronal apoptosis through inhibiting the generation of Casp ase-12 and iNOS, and incr ease the number of surviving neurons.
Objective To investigate the expression of Caspase-12 and iNOS in hippocampus after the Breviscapine(Br) in intervention therapy the Sprague-Dawley (SD) rats with global cerebral ischemia/reperfusion (I/R) injury and testify the effect mechanism of Br during cerebral resuscitation. Methods 90 healthy SD rats were randomly divided into 3 groups: sham operation (SO) group (n=30), I/R group (n=30) and Br group (n=30). Then each group were assigned into 5 observing time-points:2,6,12,24,48 hours (h) according to reperfusion time. The SD rats model of global cerebral ischemia/reperfusion (including I/R and Br group) was produced by means of simple Pulsinelli-Brierley's four arteries occlusion method. SO and I/R groups were intraperitoneally injected with 0.9% Sodium chloride (dose:1ml/kg), and Br group with Breviscapine (dose:2.5mg/kg) one time per six hours after reperfusion. Then SD rats'cerebrums were taken out of their skull at each of observing time-point. The expression activation of Caspase-12 and iNOS protein in pyramidal cells in hippocampus comu ammonis (CA) 1 region were examined by immunohistochemical method (SABC) and Caspase-12mRNA by in situ hybridization, measured their average optical density(OD), and hematoxylin and eosin (HE) staining was also performed to detect the number of surviving pyramidal cells, and terminal-deoxynucleotidy transferase medatd dUTP nick end labeling (TUNEL) was used to detect apoptotic pyramidal cells (positive cells). Results In SO group, the average optical density of Caspase-12 protein and its mRNA, iNOS protein had no significance of difference. The number of surviving neurons and apoptic cells was the same condition(p>0.05). In I/R group, the average optical density of Caspase-12 protein and mRNA began to increase at 12h after reperfusion, arrived on peak at 24h, and then gradually decreased. The expression of iNOS protein was as same as Caspase-12 protein. With the reperfusion prolonged, the number of surviving neurons became more and more lower (all P<0.01), and more and more neurons emerged the phenomenon of cell-apoptosis, its apoptotic pyramidal cells then clearly rised, too(all P<0.01). In Br group,the average optical density of Caspase-12 protein and mRNA peaked at 6h, and then began to decrease at 12h, and reached the minimum at 48h. The average optical density of iNOS protein began to increase at 6h, reached the maximum at 12h, and then decreased gradually. With the reperfusion prolonged, the number of surviving neurons became more and more lower, and reached maximum at 12h, then gradually recoverd, and reached the maximum at 48h. The change of apoptic cells was just contrary. Comparing with I/R group, the average optical density in Br group was smaller at every corresponding time-point, and had statistically significant (p<0.05). Conclusions After global cerebral ischemia/reperfusion, Breviscapine could protect the brain from global cerebal ischemia/reperfusion injury by inhibiting neuronal apoptosis through inhibiting the generation of Casp ase-12 and iNOS, and incr ease the number of surviving neurons.
引文
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[3]Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y. An endoplasmic reticulum stressspecific Caspase cascade in apoptosis. Cytochrome c-independent activation of Caspase-9 by Caspase-12.[J] Biol Chem.2002,277:34287-34294.
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[5]张兴毅,韩江全,宋国林,等.建立大鼠全脑缺血模型制作的方法改进.[J]西部医学,2010，22(1)：18-23.
[6]Ahmed BY, Chakravarthy S, Eggers R, et al. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors[J]. BMC Neurosci,2004,5(1):4-14.
[7]Vincenzo M, Michelangelo I, Carolina M, et al.The protective effect of M40401,a superoxide dismutase mimetic,on post-ischemic brain damage in Monglolian gerbils[J]. BMC Pharmacology,2003,6(16):1-9.
[8]Zhang M, Ma YF, Gan JX, et al.Basic fibroblast growth factor alleviates brain injury following global ischemia reperfusion in rabbits[J]. J Zhejiang Univ Sci B,2005,613 (7):637-643.
[9]Pulsinelli WA. A new model of bilateral hemispheric ischemia in the unanesthetised rat[J], Stroke,1979,10(3):267-272.
[10]Song CY, Li WZ, Yue ZY, et al. The protective effect of desflurane preconditioning on brain against ischemia-reperfusion injury in rats[J]. Chin J Anesthesiol,2006,26(3): 234-237.
[11]Sharma SS, Dhar A, Kaundal RK. Fe TPPS protects against global cerebral ischemic-reperfusion injury in gerbils [J]. Pharmacol Res,2007,55 (4):335-342.
[12]Lorrio S, Sobrado M, Arias E, et al. Galantamine postischemia provides neuroprotection and memory recovery against transient global cerebral ischemia in gerbils [J]. J Pharmacol Exp Ther,2007,322(2):591-599.
[13]卢晓梅,金玉楠,杜莉莉,等.灯盏花素对小鼠脑缺血再灌注损伤的影响[J].锦州医学院学报,2006,27(1)：44-46.
[14]张焰,陈群,颜学军,等.灯盏花素对沙土鼠脑缺血再灌注损伤的影响[J].临床麻醉学杂志,2001,17(11)：622-624.
[15]王雪松,阮旭中,刘买利.灯盏花素对缺血再灌注鼠脑损伤的脑保护作用研究[J].中成药,2002,24(12)：947-950.
[16]陈小夏,何冰,杜淇璋.灯盏花素对缺血再灌注大鼠脑组织ATPase活性的保护作用[J].中药新药与临床药理,1998,9(4)：214-216.
[17]韦佳,黄罗生,窦昌贵.灯盏花素脂质体注射液对大鼠脑缺血再灌注损伤的保护作用[J].中药药理与临床,2005：21(4)：20-22.
[18]唐省三.灯盏花素对大鼠脑缺血再灌注后凋亡相关蛋白的干预[J].中国临床康复,2005,9(17)：130-131.
[19]唐省三,马亚珍.灯盏花素注射液对大鼠脑缺血-再灌注脑损害的凋亡抑制作用[J].陕西医学杂志,2005,34(8)：918-922.
[20]赵泽燕.内质网应激与未成熟脑组织缺氧缺血性脑损伤.[J]中华医学写作杂志.2005，12：1236-1239.
[21]Nakagawa T, Zhu H, Morishima N,Li E, Xu J, Yankner BA, Yuan J.Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta.[J] Nature.2000,403:98-103.
[22]Mouw G, Zechel JL, Gamboa J, Lust WD, Selman WR, Ratcheson RA.Activation of Caspase-12, an endoplasmic reticulum resident Caspase, after permanent focal ischemia in rat.[J] Neuroreport.2003,14:183-186.
[23]张鸿,宋利春,贾春红,刘艳艳,吕永利,大鼠脑缺血／再灌注后神经无凋亡及Caspase-12mRNA和蛋白表达的改变.中国药理学通报.2008,24(8)：1069-1072.
[24]余昌咸,衰胜山,张骏.辛伐他汀对大鼠脑缺血再灌注后脑组织NOS表达的影响[J]贵州医药,2006,30(12)：1069-1071.
[1]Kao H, Takasawa R, Fukuda K, Shiokawa D, Sadanaga-Akiyoshi F, Ibayashi S,et al.DNA fragmentation in ischemia core and penumbra in focal cerebral ischemia in rats[J]. Mol Brain Res 2001;91:112-118.
[2]Saito A, Maier CM, Narasimhan P, et al. Oxidative stress and neuronal death/survival signaling in cerebral ischemia[J]. Mol Neurobiol,2005,31(1-3):105-116.
[3]Zhang F, Yin W, Chen J. Apoptosis in cerebral ischemia:executional and regulatory signaling mechanisms[J].Neurol Res,2004,26(8):835-845.
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[18]Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y. An endoplasmic reticulum stressspecific Caspase cascade in apoptosis. Cytochrome c-independent activation of Caspase-9 by Caspase-12. [J] Biol Chem.2002,277:34287-34294.
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[4]帅杰,董为伟.局灶性鼠脑缺血时一氧化氮及一氧化氮合酶抵制剂的作用机制[J].中华神经科杂志,1997，30(4)：227-231.
[5]张兴毅,韩江全,宋国林,等.建立大鼠全脑缺血模型制作的方法改进.[J]西部医学,2010，22(1)：18-23.
[6]Ahmed BY, Chakravarthy S, Eggers R, et al. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors[J]. BMC Neurosci,2004,5(1):4-14.
[7]Vincenzo M, Michelangelo I, Carolina M, et al.The protective effect of M40401,a superoxide dismutase mimetic,on post-ischemic brain damage in Monglolian gerbils[J]. BMC Pharmacology,2003,6(16):1-9.
[8]Zhang M, Ma YF, Gan JX, et al.Basic fibroblast growth factor alleviates brain injury following global ischemia reperfusion in rabbits[J]. J Zhejiang Univ Sci B,2005,613 (7):637-643.
[9]Pulsinelli WA. A new model of bilateral hemispheric ischemia in the unanesthetised rat[J], Stroke,1979,10(3):267-272.
[10]Song CY, Li WZ, Yue ZY, et al. The protective effect of desflurane preconditioning on brain against ischemia-reperfusion injury in rats[J]. Chin J Anesthesiol,2006,26(3): 234-237.
[11]Sharma SS, Dhar A, Kaundal RK. Fe TPPS protects against global cerebral ischemic-reperfusion injury in gerbils [J]. Pharmacol Res,2007,55 (4):335-342.
[12]Lorrio S, Sobrado M, Arias E, et al. Galantamine postischemia provides neuroprotection and memory recovery against transient global cerebral ischemia in gerbils [J]. J Pharmacol Exp Ther,2007,322(2):591-599.
[13]卢晓梅,金玉楠,杜莉莉,等.灯盏花素对小鼠脑缺血再灌注损伤的影响[J].锦州医学院学报,2006,27(1)：44-46.
[14]张焰,陈群,颜学军,等.灯盏花素对沙土鼠脑缺血再灌注损伤的影响[J].临床麻醉学杂志,2001,17(11)：622-624.
[15]王雪松,阮旭中,刘买利.灯盏花素对缺血再灌注鼠脑损伤的脑保护作用研究[J].中成药,2002,24(12)：947-950.
[16]陈小夏,何冰,杜淇璋.灯盏花素对缺血再灌注大鼠脑组织ATPase活性的保护作用[J].中药新药与临床药理,1998,9(4)：214-216.
[17]韦佳,黄罗生,窦昌贵.灯盏花素脂质体注射液对大鼠脑缺血再灌注损伤的保护作用[J].中药药理与临床,2005：21(4)：20-22.
[18]唐省三.灯盏花素对大鼠脑缺血再灌注后凋亡相关蛋白的干预[J].中国临床康复,2005,9(17)：130-131.
[19]唐省三,马亚珍.灯盏花素注射液对大鼠脑缺血-再灌注脑损害的凋亡抑制作用[J].陕西医学杂志,2005,34(8)：918-922.
[20]赵泽燕.内质网应激与未成熟脑组织缺氧缺血性脑损伤.[J]中华医学写作杂志.2005，12：1236-1239.
[21]Nakagawa T, Zhu H, Morishima N,Li E, Xu J, Yankner BA, Yuan J.Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta.[J] Nature.2000,403:98-103.
[22]Mouw G, Zechel JL, Gamboa J, Lust WD, Selman WR, Ratcheson RA.Activation of Caspase-12, an endoplasmic reticulum resident Caspase, after permanent focal ischemia in rat.[J] Neuroreport.2003,14:183-186.
[23]张鸿,宋利春,贾春红,刘艳艳,吕永利,大鼠脑缺血／再灌注后神经无凋亡及Caspase-12mRNA和蛋白表达的改变.中国药理学通报.2008,24(8)：1069-1072.
[24]余昌咸,衰胜山,张骏.辛伐他汀对大鼠脑缺血再灌注后脑组织NOS表达的影响[J]贵州医药,2006,30(12)：1069-1071.
[1]Kao H, Takasawa R, Fukuda K, Shiokawa D, Sadanaga-Akiyoshi F, Ibayashi S,et al.DNA fragmentation in ischemia core and penumbra in focal cerebral ischemia in rats[J]. Mol Brain Res 2001;91:112-118.
[2]Saito A, Maier CM, Narasimhan P, et al. Oxidative stress and neuronal death/survival signaling in cerebral ischemia[J]. Mol Neurobiol,2005,31(1-3):105-116.
[3]Zhang F, Yin W, Chen J. Apoptosis in cerebral ischemia:executional and regulatory signaling mechanisms[J].Neurol Res,2004,26(8):835-845.
[4]Desagher S,Martinou JC. Mitochondria as the central controlpoint of apoptosis[J]. TrendsCellBiol,2000,10:369-377.
[5]Celine C, Isabelle C, Eric D, et al. Apoptosis-inducing factor (AIF):a novel Caspase-independent death effector released from mitochondria[J]. Biochimie,2002,84(2-3):215-222.
[6]Bates B, Hirt, Thomas SS, et al. Neurotrophin-3 promotes cell death induced in cerebral ischmnia, oxygen-glucose deprivation, and oxidative stress:possible involvement of oxygen free radicals[J]. Neurobiol Dis,2002,9(1):24-37.
[7]de la Monte SM, Neely TR, Cannon J. el al. Oxidative stress and hypoxia-like iniury cause Alzheimer-type molecular abnor-inalities in central nervous system neurons[J].Cell Mol Life Sci.2000,57(10): 1471-1481.
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