清热活血法对大鼠急性脑缺血损伤神经保护作用的研究
|
摘要
目的：探讨以清热活血法为主要组方原则的清热活血方对急性脑缺血大鼠海马及大脑皮质IL-1β、ICAM-1、PMNL、EAA、脑组织含水量的影响及对梗死区病理形态学脑损害的预保护性作用。
方法：采用随机分组对照实验，以在老龄及高脂血症基础上结扎双侧颈总动脉造成大鼠持续性脑缺血模型，各组大鼠于造模后3h、6h、12h、24h四个时间点分别用ELISA法及免疫组化法等方法检测海马区及大脑皮质的IL-1β、ICAM-1、PMNL、EAA的含量及脑组织含水量，光镜下观察海马不同分区组织病理形态学改变，并对海马CA1区神经元丢失做定量计数。
结果：①大鼠脑缺血3h后，海马区IL-1β开始上升，24h达高峰；而其顶叶皮质ICAM-1表达及白细胞浸润6h后开始上升，24h后继续增加，较IL-1β明显滞后。②清热活血方可显著减轻脑组织IL-1β、ICAM-1、PMNL的含量，与模型组及川芎嗪对照组比较均有显著性差异（P<0.05），该方并可显著降低缺血脑组织兴奋性氨基酸的含量。③药物干预组大鼠海马区以神经元变性为主，轻度神经元坏死、丢失及胶质增生；模型组大鼠可见海马区严重神经元坏死、丢失及胶质增生和脑水肿；模型组CA1区锥体细胞计数及额叶皮质含水量与药物干预组、假手术组比较差异有统计学意义（P<0.05），且大剂量中药组优于药物对照组（P<0.05）。
结论：①清热活血方下调IL-1β、ICAM-1的含量，抑制脑内PMNL聚集，降低兴奋性氨基酸的含量及脑组织含水量，从而减轻缺血大鼠脑损伤，可能是该方保护脑梗死后脑损伤的作用机理之一。②IL-1β介导的ICAM-1、PMNL参与脑梗死后脑损伤病理过程。
[Objective]: To explore the protective effect of QRHX prescription based on the main principle of Qing Re Huo Xue on cerebral ischemia (CI) injury.
[Methods]: The rat models with CI were prepared and randomly devided into normal group, sham—operated group, CI group, treatment group with different dosage of QRHX prescription, and tetramethylpyrazine control group. The changes in the following parameters, including the contents of interleukin—1β(IL—1β), Intercellular adhesive molecule(ICAM—1), Polymorphonuclear leukocyte (PMNL), excitatory amino acid(EAA) and water in brain tissue were observed, with methods of ELISA and immunohistochemistry and HPLC respectively at 3h,6h,12h,24h after operation. Meanwhile, the different pathologic changes were observed by light microscope and the neuron number in the hippocampus CA1 was counted at those points respectively.
[Results]: QRHX prescription was able to decrease the contents of IL-1β, ICAM-1, and EAA in brain tissue obviously (P<0.05).The difference was significant as compared with QRHX high dose group and tetramethylpyrazine control group(p<0.05). The neuronal degeneration and lightly neucrosis and glue hyperplasia occurred in hippocampus areas of the rats pretreated by drugs, but the damage was more severe in the model group. The number statistics difference of neuron in CA1 area between the
model group，Sham-operated group and drug-pretreated was significant (p<0.05).
[Conclusions]: The results suggest an important role for IL-1β、ICAM-1 mediated PMNL adhesion in the evolution of brain injury following CI. Downregulating the expression levels of IL-1β and ICAM-1 protein, and inhibiting the accumulation of PMNL may ascribe to the protective effects of QRHX prescription on inflammatory brain injury following CI.
方法：采用随机分组对照实验，以在老龄及高脂血症基础上结扎双侧颈总动脉造成大鼠持续性脑缺血模型，各组大鼠于造模后3h、6h、12h、24h四个时间点分别用ELISA法及免疫组化法等方法检测海马区及大脑皮质的IL-1β、ICAM-1、PMNL、EAA的含量及脑组织含水量，光镜下观察海马不同分区组织病理形态学改变，并对海马CA1区神经元丢失做定量计数。
结果：①大鼠脑缺血3h后，海马区IL-1β开始上升，24h达高峰；而其顶叶皮质ICAM-1表达及白细胞浸润6h后开始上升，24h后继续增加，较IL-1β明显滞后。②清热活血方可显著减轻脑组织IL-1β、ICAM-1、PMNL的含量，与模型组及川芎嗪对照组比较均有显著性差异（P<0.05），该方并可显著降低缺血脑组织兴奋性氨基酸的含量。③药物干预组大鼠海马区以神经元变性为主，轻度神经元坏死、丢失及胶质增生；模型组大鼠可见海马区严重神经元坏死、丢失及胶质增生和脑水肿；模型组CA1区锥体细胞计数及额叶皮质含水量与药物干预组、假手术组比较差异有统计学意义（P<0.05），且大剂量中药组优于药物对照组（P<0.05）。
结论：①清热活血方下调IL-1β、ICAM-1的含量，抑制脑内PMNL聚集，降低兴奋性氨基酸的含量及脑组织含水量，从而减轻缺血大鼠脑损伤，可能是该方保护脑梗死后脑损伤的作用机理之一。②IL-1β介导的ICAM-1、PMNL参与脑梗死后脑损伤病理过程。
[Objective]: To explore the protective effect of QRHX prescription based on the main principle of Qing Re Huo Xue on cerebral ischemia (CI) injury.
[Methods]: The rat models with CI were prepared and randomly devided into normal group, sham—operated group, CI group, treatment group with different dosage of QRHX prescription, and tetramethylpyrazine control group. The changes in the following parameters, including the contents of interleukin—1β(IL—1β), Intercellular adhesive molecule(ICAM—1), Polymorphonuclear leukocyte (PMNL), excitatory amino acid(EAA) and water in brain tissue were observed, with methods of ELISA and immunohistochemistry and HPLC respectively at 3h,6h,12h,24h after operation. Meanwhile, the different pathologic changes were observed by light microscope and the neuron number in the hippocampus CA1 was counted at those points respectively.
[Results]: QRHX prescription was able to decrease the contents of IL-1β, ICAM-1, and EAA in brain tissue obviously (P<0.05).The difference was significant as compared with QRHX high dose group and tetramethylpyrazine control group(p<0.05). The neuronal degeneration and lightly neucrosis and glue hyperplasia occurred in hippocampus areas of the rats pretreated by drugs, but the damage was more severe in the model group. The number statistics difference of neuron in CA1 area between the
model group，Sham-operated group and drug-pretreated was significant (p<0.05).
[Conclusions]: The results suggest an important role for IL-1β、ICAM-1 mediated PMNL adhesion in the evolution of brain injury following CI. Downregulating the expression levels of IL-1β and ICAM-1 protein, and inhibiting the accumulation of PMNL may ascribe to the protective effects of QRHX prescription on inflammatory brain injury following CI.
引文
[1] Brown RD, Whisnant JP, Sicks JD, et al. Stroke incidence, Prevalence and survival: secular trends in Rochester. Minnesota, through 1989. Stroke，1996，27:373
[2] Cheng XM, Ziegler DK, Lai YHC et al. Stroke 1986 through 1990. stroke,1995,26:1990
[3] Medenlow AD. Mechanisms of ischemic brain damage with intracerebral hemorrhage. Stroke, 1993,24(Suppl 1):I-115-I-177
[4] Dyker AG, Lee KR. Duration of neuroprotective treatment for ischemic stroke. Stroke, 1998,29:535
[5] 王永炎. 关于提高脑血管疾病疗效难点的思考. 中国中西医结合杂志, 1997,17:195
[6] 刘茂才. 关于中风病治疗的难点与突破口的思考. 中国中西医结合杂志, 1997,17:451
[7] yamasaki y, Matsuura NK, Shozahar HK, et al. Interleukin-1 as pathogenetic mediator of ischemic brain damage in rats. Stroke, 1995,26(4): 676
[8] Jean WC. The progression and topographic distribution of inerleukin-1 beta expression after permanent middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab, 1999,19:87
[9] 胡国恒，王行宽，黄保民，等. 康脑神颗粒治疗急性缺血性中风临床研究. 中国中医急症,2001,10(6):317
[10] 胡国恒，陈劲云，李艳，等. 康脑神及清开灵对大鼠脑缺血再灌注损伤防治作用的对比研究. 中华实用中西医杂志, 2002,15:19
[11] 贺石林，王键，王净净主编. 中医科研设计与统计学[M]. 长沙：湖南科学技术出版社,2001,48
[12] 徐叔云，卞如濂，陈修主编.药理实验方法学.人民卫生出版社,2002,1063
[13] Hsu SM, Raine L, and Fanger H. Use of avidin-biotin-peroxdase
complex (ABC) in immunoperoxidase techniques: A Comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem. 1981,29(4):577
[14] 欧阳一冰,杨同书. 迟发性神经元损伤的实验研究. 白求恩医科大学学报，1991;17(2):120
[15] Chen BM, Xia Lw, Zhao RQ. Determination of Nc-dimethy larginine in human plasma by high performance liquid chromatogphy. [J] Chromatogphy J. Biomed Sci Appl, 1997,69(2):467
[16] 王炜，刘朝，江卫国，等. HPLC测定脑组织与脑脊液四种氨基酸类神经递质含量的方法的改良研究[J]. 同济医科大学学报，1999,28(2):23
[17] Liu T , MC Donnel Pc, Young PR,et al .Interleukin—1βm RNA expression in ischemic rat cortex .Stroke,1997,24:1746
[18] 李岚，马学英，夏松青等.大鼠局灶性脑缺血再灌注模型中IL—Iβ表达及其与白细胞浸润关系。临床军医杂志，2000,28(3)：1
[19] 张岚，李向红，谷志远等。大鼠脑缺血一再灌注损伤诱导IL—Iβ表达。中国病理生理杂志，1999，15，（2）：107
[20] Jean WC. THE progression and topgrahie ditribution of interteurin—1 beta expression after permanent middle cerebral artery occlusion in the rat.J cereb Blood Flow metab,1999,19:87
[21] 杜柏岩，关秀茹，高光强，等. IL-1β对大鼠脑缺血再灌注损伤的意义. 哈尔滨医科大学学报，2000,34(2):124
[22] Hara H, fried lander RM ,Gagliardini V,et/al.Inhibitor of IL-1β convertimg enzyme family proteases reduces ischemic and excitotoxic neuronal damaege. Proc Natl A cad Sci USA ,1997.94:2007
[23] Loddick SA, Mackenzie A,Rothwell NJ An ICE inhibitor z-VAD-DCB attenuates ischemic brain damage in the rat.neuroreport, 1996,7:1465
[24] Barone FC,Hillegass LM,Price WJ, etal.Polymorphionuclear leukocyte infiltration into cerebral local is che mic tissue :myeloperoxidase activity assay and histological verification. J Neurosci Res, 1991,29:336
[25] Zhang RL, Chopp M, chen H, et al. Temporal profile of ischemic tissue damage, neutrophil response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat. J Neuuol Sci, 1994,125:3
[26] Del Zoppo GJ, Schmid-schonbein GW, Mori E, et al. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artey occlusion and reperfusion in baboons. Stroke, 1991,22:1276
[27] Mori E, Del Zoppo GJ, Chambers JD, et al. Inhibiton of polymorhponuclear leuocyte adherence suppresses no-re-flow after focal cerebral ischemia in baboons. Stroke, 1992,23:712
[28] Kochanek PM, Hallenbeck JM. Polymorphounclear leukocyte and monocytes/macrophages in the pathogenesi of cerebral ischemia and stroke. Stroke,1992,23:1367
[29] Lindsberg PJ, Hallenbeck JM, Feuerstein GZ. Platelet-activating-factor in troke and brain injury. Ann Neurol, 1991,30:117
[30] Adams D, Shaw S. Leucocyte-endothelial interactions and regulation of leucocyte migration. Lancet, 1994,343:831
[31] Jander S, Kraemer M, Schroeter M, et al. Lymphocytic infiltration and expression of ICAM-1 in photochemically induced ischemia of the rat cortex. J Cereb Blood Flow Metab, 1995,15:42
[32] 张金涛，吴卫平，刘洁晓. 丹参对鼠脑缺血后再灌注IL-1β转化酶表达的影响. 中华老年心脑血管杂志，2000,2:124
[33] 李岚，赵光东，张健. 川芎嗪对局部脑缺血再灌注模型纹状体内IL-1β的影响. 中国临床神经科学,2001,9(1):36
[34] 卢尚岭，李明忠，蔡剑前，等. 中风病机浅淡. 中医急症研究，1988,343
[35] yang Gy, Gong C, Qin Z, et al. Inhibition of TNF-α attenuate infarct volume and ICAM-1 expression in ischemia mouse brain. Neuro Report, 1998,9:2131
[36] yang Gy, Mao y, Zhou LF, et al. Attenuation of temporary focal
cerebral ischemic injury in the mouse following transfection with IL-1 recepor antagonist. Brain Res Mol Brain Res, 1999,72:129
[37] yang Gy, Schielke GP, Gong C, et al. Expression of TNF-α and ICAM-1 after focal cerebral ischemia in IL-1β converting enzyme deficient. J Cereb Blood Flow Metab, 1999,19:1109
[38] Schneider D, Berrouschot J, Brandt T, et al. Satety pharmacokinetics and biological activicty of enlimomab (anti-IL-1β antibody): an open-label, dose escalation study in patient hospitalized for acute stroke. Eur Neurol, 1998,40(2):78
[39] 徐叔云，卞如濂，陈修主编. 药理实验方法学.人民卫生出版社, 2002,1060
[40] 黄如训. 脑卒中实验研究与临床相关性. 中国神经精神疾病杂志, 2001,27(3):237
[41] 黄芳，万海桐. 中医药防治缺血性中风的治则治法研究. 浙江中医杂志, 2000,35(5):220
[2] Cheng XM, Ziegler DK, Lai YHC et al. Stroke 1986 through 1990. stroke,1995,26:1990
[3] Medenlow AD. Mechanisms of ischemic brain damage with intracerebral hemorrhage. Stroke, 1993,24(Suppl 1):I-115-I-177
[4] Dyker AG, Lee KR. Duration of neuroprotective treatment for ischemic stroke. Stroke, 1998,29:535
[5] 王永炎. 关于提高脑血管疾病疗效难点的思考. 中国中西医结合杂志, 1997,17:195
[6] 刘茂才. 关于中风病治疗的难点与突破口的思考. 中国中西医结合杂志, 1997,17:451
[7] yamasaki y, Matsuura NK, Shozahar HK, et al. Interleukin-1 as pathogenetic mediator of ischemic brain damage in rats. Stroke, 1995,26(4): 676
[8] Jean WC. The progression and topographic distribution of inerleukin-1 beta expression after permanent middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab, 1999,19:87
[9] 胡国恒，王行宽，黄保民，等. 康脑神颗粒治疗急性缺血性中风临床研究. 中国中医急症,2001,10(6):317
[10] 胡国恒，陈劲云，李艳，等. 康脑神及清开灵对大鼠脑缺血再灌注损伤防治作用的对比研究. 中华实用中西医杂志, 2002,15:19
[11] 贺石林，王键，王净净主编. 中医科研设计与统计学[M]. 长沙：湖南科学技术出版社,2001,48
[12] 徐叔云，卞如濂，陈修主编.药理实验方法学.人民卫生出版社,2002,1063
[13] Hsu SM, Raine L, and Fanger H. Use of avidin-biotin-peroxdase
complex (ABC) in immunoperoxidase techniques: A Comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem. 1981,29(4):577
[14] 欧阳一冰,杨同书. 迟发性神经元损伤的实验研究. 白求恩医科大学学报，1991;17(2):120
[15] Chen BM, Xia Lw, Zhao RQ. Determination of Nc-dimethy larginine in human plasma by high performance liquid chromatogphy. [J] Chromatogphy J. Biomed Sci Appl, 1997,69(2):467
[16] 王炜，刘朝，江卫国，等. HPLC测定脑组织与脑脊液四种氨基酸类神经递质含量的方法的改良研究[J]. 同济医科大学学报，1999,28(2):23
[17] Liu T , MC Donnel Pc, Young PR,et al .Interleukin—1βm RNA expression in ischemic rat cortex .Stroke,1997,24:1746
[18] 李岚，马学英，夏松青等.大鼠局灶性脑缺血再灌注模型中IL—Iβ表达及其与白细胞浸润关系。临床军医杂志，2000,28(3)：1
[19] 张岚，李向红，谷志远等。大鼠脑缺血一再灌注损伤诱导IL—Iβ表达。中国病理生理杂志，1999，15，（2）：107
[20] Jean WC. THE progression and topgrahie ditribution of interteurin—1 beta expression after permanent middle cerebral artery occlusion in the rat.J cereb Blood Flow metab,1999,19:87
[21] 杜柏岩，关秀茹，高光强，等. IL-1β对大鼠脑缺血再灌注损伤的意义. 哈尔滨医科大学学报，2000,34(2):124
[22] Hara H, fried lander RM ,Gagliardini V,et/al.Inhibitor of IL-1β convertimg enzyme family proteases reduces ischemic and excitotoxic neuronal damaege. Proc Natl A cad Sci USA ,1997.94:2007
[23] Loddick SA, Mackenzie A,Rothwell NJ An ICE inhibitor z-VAD-DCB attenuates ischemic brain damage in the rat.neuroreport, 1996,7:1465
[24] Barone FC,Hillegass LM,Price WJ, etal.Polymorphionuclear leukocyte infiltration into cerebral local is che mic tissue :myeloperoxidase activity assay and histological verification. J Neurosci Res, 1991,29:336
[25] Zhang RL, Chopp M, chen H, et al. Temporal profile of ischemic tissue damage, neutrophil response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat. J Neuuol Sci, 1994,125:3
[26] Del Zoppo GJ, Schmid-schonbein GW, Mori E, et al. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artey occlusion and reperfusion in baboons. Stroke, 1991,22:1276
[27] Mori E, Del Zoppo GJ, Chambers JD, et al. Inhibiton of polymorhponuclear leuocyte adherence suppresses no-re-flow after focal cerebral ischemia in baboons. Stroke, 1992,23:712
[28] Kochanek PM, Hallenbeck JM. Polymorphounclear leukocyte and monocytes/macrophages in the pathogenesi of cerebral ischemia and stroke. Stroke,1992,23:1367
[29] Lindsberg PJ, Hallenbeck JM, Feuerstein GZ. Platelet-activating-factor in troke and brain injury. Ann Neurol, 1991,30:117
[30] Adams D, Shaw S. Leucocyte-endothelial interactions and regulation of leucocyte migration. Lancet, 1994,343:831
[31] Jander S, Kraemer M, Schroeter M, et al. Lymphocytic infiltration and expression of ICAM-1 in photochemically induced ischemia of the rat cortex. J Cereb Blood Flow Metab, 1995,15:42
[32] 张金涛，吴卫平，刘洁晓. 丹参对鼠脑缺血后再灌注IL-1β转化酶表达的影响. 中华老年心脑血管杂志，2000,2:124
[33] 李岚，赵光东，张健. 川芎嗪对局部脑缺血再灌注模型纹状体内IL-1β的影响. 中国临床神经科学,2001,9(1):36
[34] 卢尚岭，李明忠，蔡剑前，等. 中风病机浅淡. 中医急症研究，1988,343
[35] yang Gy, Gong C, Qin Z, et al. Inhibition of TNF-α attenuate infarct volume and ICAM-1 expression in ischemia mouse brain. Neuro Report, 1998,9:2131
[36] yang Gy, Mao y, Zhou LF, et al. Attenuation of temporary focal
cerebral ischemic injury in the mouse following transfection with IL-1 recepor antagonist. Brain Res Mol Brain Res, 1999,72:129
[37] yang Gy, Schielke GP, Gong C, et al. Expression of TNF-α and ICAM-1 after focal cerebral ischemia in IL-1β converting enzyme deficient. J Cereb Blood Flow Metab, 1999,19:1109
[38] Schneider D, Berrouschot J, Brandt T, et al. Satety pharmacokinetics and biological activicty of enlimomab (anti-IL-1β antibody): an open-label, dose escalation study in patient hospitalized for acute stroke. Eur Neurol, 1998,40(2):78
[39] 徐叔云，卞如濂，陈修主编. 药理实验方法学.人民卫生出版社, 2002,1060
[40] 黄如训. 脑卒中实验研究与临床相关性. 中国神经精神疾病杂志, 2001,27(3):237
[41] 黄芳，万海桐. 中医药防治缺血性中风的治则治法研究. 浙江中医杂志, 2000,35(5):220