大黄素甲醚对大鼠脑缺血再灌注损伤的保护作用
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摘要
脑血管病是临床常见病，其发病率高，预后较差，在全球已成为第一致残和前三位致死原因。缺血性脑血管病在其中占有重大比例，寻找适当的治疗措施是临床与实验研究的重要课题之一。现已证实，溶栓治疗是一项积极有效的治疗措施，但脑缺血再灌注往往引起较缺血本身更为严重的损伤，大量研究表明，脑缺血再灌注后的炎性反应和脑损伤后的细胞凋亡起着十分重要的作用。本研究在用线拴法堵塞大鼠大脑中动脉，建立鼠脑缺血再灌注模型的基础上，利用放射免疫及免疫组织化学技术定量分析炎性细胞因子白细胞介素-1β（IL-1β）、细胞间粘附分子-1（ICAM-1）和Caspase-3在脑缺血再灌注后的表达变化，以探讨其对脑缺血再灌注损伤的作用，以及大黄素甲醚对它们表达的影响，为临床上大黄素甲醚的应用提供实验及理论依据。
1 大黄素甲醚对大鼠脑缺血再灌注IL-1β表达的影响
目的：探讨大黄素甲醚对大鼠脑缺血再灌注IL-1β表达的影响
方法：成年健康雄性SD大鼠105只，随机分为正常组(Norm)，假手术组(Sham)和缺血再灌注组（Model）：分为再灌注6小时、12小时、24小时、48小时各时段组，药物对照组(NS)、小剂量（大黄素甲醚20mg/kg）药物治疗组(LD)、大剂量（大黄素甲醚40mg/kg）药物治疗组(HD)且每组又分为脑缺血再灌注12小时、24小时两组，每组均为
7只。本研究采用线栓法制备大鼠右侧大脑中动脉脑缺血再灌注模型，尼龙丝线经右侧颈内动脉轻柔推进阻塞大脑中动脉,假手术组尼龙丝线进入颈内动脉但不阻塞大脑中动脉。药物治疗组于缺血前48小时、24小时、术前将大黄素甲醚溶于2ml生理盐水鼻饲，药物对照组则仅鼻饲等量生理盐水。实验结束后断头处死取右侧大脑半球研成匀浆，采用放射免疫分析法测定脑组织中IL-1β的含量。
结果：本实验局灶性脑缺血再灌注各个时间段脑组织IL-1β含量明显升高，经单因素方差分析有显著性差异（F=293.049，P<0.01），在时间和数值上保持一致性，而且与相应时间点的假手术组和正常组比较有显著性差异（P<0.01）。再灌注6小时达高峰（1.295±0.0352ng/ml，与假手术组比较P<0.01），随之开始下降，在脑缺血再灌注48小时（0.9030±0.0212 ng/ml, P<0.01）仍处于较高的水平,假手术组和正常组比较没有显著性差异（P>.05）。大黄素甲醚治疗组与相应的药物对照组比较病变侧脑组织IL-1β含量降低，大剂量治疗各组及小剂量治疗组脑缺血再灌注12小时有显著性差异（P<0.01）。
结论：IL-1β在脑缺血再灌注损伤中起重要作用，大黄素甲醚通过抑制脑缺血再灌注IL-1β的表达，减轻脑缺血再灌注损伤。
2 大黄素甲醚对大鼠脑缺血再灌注ICAM-1和Caspase-3表达的影响
目的：探讨大黄素甲醚对大鼠脑缺血再灌注ICAM-1和Caspase-3表达的影响
方法：成年健康雄性SD大鼠105只，随机分为正常组，
假手术组：分为再灌注6小时、12小时、24小时、48小时各时段组，缺血再灌注组：分为再灌注6小时、12小时、24小时、48小时各时段组，药物对照组、小剂量药物治疗组（大黄素甲醚20mg/kg）、大剂量药物治疗组（大黄素甲醚40mg/kg）且每组又分为脑缺血再灌注12小时、24小时两组，每组均为7只。本研究采用线栓法制备大鼠右侧大脑中动脉脑缺血再灌注模型，尼龙丝线经右侧颈内动脉轻柔推进阻塞大脑中动脉，假手术组尼龙丝线进入颈内动脉但不阻塞大脑中动脉。药物治疗组于缺血前48小时、24小时、术前将大黄素甲醚溶于2ml生理盐水鼻饲。药物对照组则仅鼻饲等量生理盐水。实验结束取材，石蜡包埋，常规切片，HE染色和免疫组织化学染色检测ICAM-1和Caspase-3蛋白表达。电镜标本用2.5%戊二醛固定，锇酸后固定，树脂包埋，超薄切片，电镜观察并拍片。
结果：（1）光镜（HE染色）：脑缺血再灌注各组均可见皮质和纹状体区出现细胞肿胀，胞浆嗜酸性变，细胞间隙增大，胞核碎裂、溶解、固缩，淋巴细胞和中性粒细胞浸润，以再灌注24小时上述变化明显。假手术组和正常组未见异常变化。大黄素甲醚治疗组上述变化减轻。假手术组和正常组未见异常变化。
（2）电镜：模型组神经元细胞质高度水肿，细胞器稀疏散在，电子密度减低，线粒体高度水肿，大部分嵴排列紊乱或消失，粗面内质网扩张脱颗粒，部分神经元细胞出现核染色质边集；正常组神经元核膜圆形，规整，核仁大而明显，胞浆内可见各种细胞器，电子密度均一，细胞形态正常。
（3）免疫组化结果：①ICAM-1：脑缺血再灌注各时间
点病变侧蛋白表达明显增高，积分光密度值（F=15.707）和面密度值(F=18.343)经单因素方差分析有显著性差异（P<0.01）,在时间上和数值上保持一致性，而且与相应时间点的假手术各组和正常组比较有显著性差异（P<0.01）。脑缺血再灌注24小时达高峰（积分光密度值31.89±4.38，面密度值 0.0185±0.0031），脑缺血再灌注48小时仍持续高水平表达。大黄素甲醚治疗组各组蛋白表达的积分光密度和面密度较同时段药物对照组降低，且有显著性差异（P<0.05或P<0.01）。②Caspase-3：脑缺血再灌注各时间点病变侧蛋白表达明显增高，积分光密度值（F=8.272）和面密度值(F=329.894)经单因素方差分析有显著性差异（P<0.01）,在时间上和数值上保持一
Cerebrovascular disease is a kind of common disease in clinic attacked by higher morbidity and unfavorable prognosis . It is seriously harm for the human health due to its first disability and lethality rate among three major causes of death ,the rate of cerebral ischemia is higher . Therefore , it is the one of the most important project of the clinical and experimental research to study pathophysiologic mechanisms and find the suitable treatment method for cerebral ischemia. It has been proved that thrombolytic therapy is first selected and effective measure for the patients with cerebral thromboembolism. However cerebral injury caused by ischemia-reperfusion(IR) would be more serious than ischemic injury itself . Ishchemic cerebral injury is a result of joint action of multiple phathophysiologic mechanisms with complicated procedure, in wihich the inflammatory response and apoptosis are very important processes. The rat model of focal cerebral ischemia was created by middle cerebral artery occlusion (MCAO) in the experiment. The level of on IL-1β by radioimmunoassay and the expression of ICAM-1 and Caspase-3 by immunohistochemical technology in the brain at the different reperfusion time (6h, 12h, 24h, 48h) together with
effects of Physcion were observed. The research aimed to detect the effects of Physcion on inhibiting inflammatory cytokines and apoptosis in different reperfusion time after cerebral ischemia at early stage . The experimental results may be regared as the reliable basis of experiment and theory of clinic applying Physcion .
1 The effect of Physcion on the level of IL-1β from cerebral ischemia-reperfusion in rats
objective: To discuss the effect of physcion on the level of IL-1β from cerebral ischemia-reperfusion in rats
Meathods: The 105 healthy male SD adult rats were selected, and were randomly into normal group(Norm), sham-operated group(Sham) and cerebral ischemia-reperfusion group (Model) with 6 hours、12 hours、24 hours and 48 hours after ischemia reperfuson(IR), drug-control (NS) group 、low-dosage (20mg/kg) Physcion treating group (LD) and high-dosage (40mg/kg) Physcion treating group (HD) at 12 hours or 24 hours after ischemia-reperfusion. There were seven rats in every group. In the focal cerebral ischemia-reperfusion model , the right MCA was blocked up by a expanded-ended nylon filament for two hours ,and then the filament was pulled out . In the Sham group the nylon filament did not block up the MCA. The rats in drug treating group were administrated Physcion by stomach tube at 48h、24h before operation and just before operation. The rats in drug-control groups was administrated the same dosage’s phydiological saline at the
corresponding time point. At the end of the experiment the right part of the rats’ brain was grinded into homogenate for detecting IL-1β by radioimmunoassay.
Results : The level of IL-1β at the different time point of Model group increased and not only had significant difference by one-way ANVON (F=293.049 P<0.01) but also were consistent in the time and quantity, and had significant difference compared with the corresponding time point of Sham group and Norm group(P<0.01). The level of IL-1β was at peak at 6h (1.295±0.0352ng/ml , compared with the Sham group P<0.01)after IR，then decreased gradually but reminded a higher level at 48h (0.9030±0.0212ng/ml, P<0.01) after IR. Compared with the corresponding time point of the NS group the level of IL-1β in Physcion treating group reduced and had significant difference (P<0.01).
Conclusion: IL-1β plays an important role in the cerebral ischemia-reperfusion injury. Physcion can inhibit the level of IL-1β from IR to protect the brain from ischemia-reperfusion injury
2 The effect of Pyscion on the expression of ICAM-1 and Caspase-3 from cerebral ischemia-reperfusion in rats
Objective: To explore the effect of Pyscion on the expression of ICAM-1 and Caspase-3 from cerebral ischemia-reperfusion in rats
Methods: The 105 healthy male SD adult rats were select
1 大黄素甲醚对大鼠脑缺血再灌注IL-1β表达的影响
目的：探讨大黄素甲醚对大鼠脑缺血再灌注IL-1β表达的影响
方法：成年健康雄性SD大鼠105只，随机分为正常组(Norm)，假手术组(Sham)和缺血再灌注组（Model）：分为再灌注6小时、12小时、24小时、48小时各时段组，药物对照组(NS)、小剂量（大黄素甲醚20mg/kg）药物治疗组(LD)、大剂量（大黄素甲醚40mg/kg）药物治疗组(HD)且每组又分为脑缺血再灌注12小时、24小时两组，每组均为
7只。本研究采用线栓法制备大鼠右侧大脑中动脉脑缺血再灌注模型，尼龙丝线经右侧颈内动脉轻柔推进阻塞大脑中动脉,假手术组尼龙丝线进入颈内动脉但不阻塞大脑中动脉。药物治疗组于缺血前48小时、24小时、术前将大黄素甲醚溶于2ml生理盐水鼻饲，药物对照组则仅鼻饲等量生理盐水。实验结束后断头处死取右侧大脑半球研成匀浆，采用放射免疫分析法测定脑组织中IL-1β的含量。
结果：本实验局灶性脑缺血再灌注各个时间段脑组织IL-1β含量明显升高，经单因素方差分析有显著性差异（F=293.049，P<0.01），在时间和数值上保持一致性，而且与相应时间点的假手术组和正常组比较有显著性差异（P<0.01）。再灌注6小时达高峰（1.295±0.0352ng/ml，与假手术组比较P<0.01），随之开始下降，在脑缺血再灌注48小时（0.9030±0.0212 ng/ml, P<0.01）仍处于较高的水平,假手术组和正常组比较没有显著性差异（P>.05）。大黄素甲醚治疗组与相应的药物对照组比较病变侧脑组织IL-1β含量降低，大剂量治疗各组及小剂量治疗组脑缺血再灌注12小时有显著性差异（P<0.01）。
结论：IL-1β在脑缺血再灌注损伤中起重要作用，大黄素甲醚通过抑制脑缺血再灌注IL-1β的表达，减轻脑缺血再灌注损伤。
2 大黄素甲醚对大鼠脑缺血再灌注ICAM-1和Caspase-3表达的影响
目的：探讨大黄素甲醚对大鼠脑缺血再灌注ICAM-1和Caspase-3表达的影响
方法：成年健康雄性SD大鼠105只，随机分为正常组，
假手术组：分为再灌注6小时、12小时、24小时、48小时各时段组，缺血再灌注组：分为再灌注6小时、12小时、24小时、48小时各时段组，药物对照组、小剂量药物治疗组（大黄素甲醚20mg/kg）、大剂量药物治疗组（大黄素甲醚40mg/kg）且每组又分为脑缺血再灌注12小时、24小时两组，每组均为7只。本研究采用线栓法制备大鼠右侧大脑中动脉脑缺血再灌注模型，尼龙丝线经右侧颈内动脉轻柔推进阻塞大脑中动脉，假手术组尼龙丝线进入颈内动脉但不阻塞大脑中动脉。药物治疗组于缺血前48小时、24小时、术前将大黄素甲醚溶于2ml生理盐水鼻饲。药物对照组则仅鼻饲等量生理盐水。实验结束取材，石蜡包埋，常规切片，HE染色和免疫组织化学染色检测ICAM-1和Caspase-3蛋白表达。电镜标本用2.5%戊二醛固定，锇酸后固定，树脂包埋，超薄切片，电镜观察并拍片。
结果：（1）光镜（HE染色）：脑缺血再灌注各组均可见皮质和纹状体区出现细胞肿胀，胞浆嗜酸性变，细胞间隙增大，胞核碎裂、溶解、固缩，淋巴细胞和中性粒细胞浸润，以再灌注24小时上述变化明显。假手术组和正常组未见异常变化。大黄素甲醚治疗组上述变化减轻。假手术组和正常组未见异常变化。
（2）电镜：模型组神经元细胞质高度水肿，细胞器稀疏散在，电子密度减低，线粒体高度水肿，大部分嵴排列紊乱或消失，粗面内质网扩张脱颗粒，部分神经元细胞出现核染色质边集；正常组神经元核膜圆形，规整，核仁大而明显，胞浆内可见各种细胞器，电子密度均一，细胞形态正常。
（3）免疫组化结果：①ICAM-1：脑缺血再灌注各时间
点病变侧蛋白表达明显增高，积分光密度值（F=15.707）和面密度值(F=18.343)经单因素方差分析有显著性差异（P<0.01）,在时间上和数值上保持一致性，而且与相应时间点的假手术各组和正常组比较有显著性差异（P<0.01）。脑缺血再灌注24小时达高峰（积分光密度值31.89±4.38，面密度值 0.0185±0.0031），脑缺血再灌注48小时仍持续高水平表达。大黄素甲醚治疗组各组蛋白表达的积分光密度和面密度较同时段药物对照组降低，且有显著性差异（P<0.05或P<0.01）。②Caspase-3：脑缺血再灌注各时间点病变侧蛋白表达明显增高，积分光密度值（F=8.272）和面密度值(F=329.894)经单因素方差分析有显著性差异（P<0.01）,在时间上和数值上保持一
Cerebrovascular disease is a kind of common disease in clinic attacked by higher morbidity and unfavorable prognosis . It is seriously harm for the human health due to its first disability and lethality rate among three major causes of death ,the rate of cerebral ischemia is higher . Therefore , it is the one of the most important project of the clinical and experimental research to study pathophysiologic mechanisms and find the suitable treatment method for cerebral ischemia. It has been proved that thrombolytic therapy is first selected and effective measure for the patients with cerebral thromboembolism. However cerebral injury caused by ischemia-reperfusion(IR) would be more serious than ischemic injury itself . Ishchemic cerebral injury is a result of joint action of multiple phathophysiologic mechanisms with complicated procedure, in wihich the inflammatory response and apoptosis are very important processes. The rat model of focal cerebral ischemia was created by middle cerebral artery occlusion (MCAO) in the experiment. The level of on IL-1β by radioimmunoassay and the expression of ICAM-1 and Caspase-3 by immunohistochemical technology in the brain at the different reperfusion time (6h, 12h, 24h, 48h) together with
effects of Physcion were observed. The research aimed to detect the effects of Physcion on inhibiting inflammatory cytokines and apoptosis in different reperfusion time after cerebral ischemia at early stage . The experimental results may be regared as the reliable basis of experiment and theory of clinic applying Physcion .
1 The effect of Physcion on the level of IL-1β from cerebral ischemia-reperfusion in rats
objective: To discuss the effect of physcion on the level of IL-1β from cerebral ischemia-reperfusion in rats
Meathods: The 105 healthy male SD adult rats were selected, and were randomly into normal group(Norm), sham-operated group(Sham) and cerebral ischemia-reperfusion group (Model) with 6 hours、12 hours、24 hours and 48 hours after ischemia reperfuson(IR), drug-control (NS) group 、low-dosage (20mg/kg) Physcion treating group (LD) and high-dosage (40mg/kg) Physcion treating group (HD) at 12 hours or 24 hours after ischemia-reperfusion. There were seven rats in every group. In the focal cerebral ischemia-reperfusion model , the right MCA was blocked up by a expanded-ended nylon filament for two hours ,and then the filament was pulled out . In the Sham group the nylon filament did not block up the MCA. The rats in drug treating group were administrated Physcion by stomach tube at 48h、24h before operation and just before operation. The rats in drug-control groups was administrated the same dosage’s phydiological saline at the
corresponding time point. At the end of the experiment the right part of the rats’ brain was grinded into homogenate for detecting IL-1β by radioimmunoassay.
Results : The level of IL-1β at the different time point of Model group increased and not only had significant difference by one-way ANVON (F=293.049 P<0.01) but also were consistent in the time and quantity, and had significant difference compared with the corresponding time point of Sham group and Norm group(P<0.01). The level of IL-1β was at peak at 6h (1.295±0.0352ng/ml , compared with the Sham group P<0.01)after IR，then decreased gradually but reminded a higher level at 48h (0.9030±0.0212ng/ml, P<0.01) after IR. Compared with the corresponding time point of the NS group the level of IL-1β in Physcion treating group reduced and had significant difference (P<0.01).
Conclusion: IL-1β plays an important role in the cerebral ischemia-reperfusion injury. Physcion can inhibit the level of IL-1β from IR to protect the brain from ischemia-reperfusion injury
2 The effect of Pyscion on the expression of ICAM-1 and Caspase-3 from cerebral ischemia-reperfusion in rats
Objective: To explore the effect of Pyscion on the expression of ICAM-1 and Caspase-3 from cerebral ischemia-reperfusion in rats
Methods: The 105 healthy male SD adult rats were select
引文
Rothwell NJ. Functions and mechanisms of IL-1 in the brain. Trends Phamacol Sci, 1991, 12:430~436
Satio K, Suyama K, Nishida K,et al. Early increases in TNF-α, IL-6 and IL-1 beta levels following transient cerebral ischemia in gerbil brain. Neurosci Lett, 1996, 206:149~152
Long EZ, Weistein PR, Carlson S,et al, Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989, 20(1):84~91
Rothwell NJ, Luheshi G. Pharmacology of interleukin-1α actions in the brain , Adv Pharmacol, 1994, 25: 1~20
Wang X, Yue TL, Barone FC. Concomitant cortical expression of TNF-alpha and IL-1 beta mRNAs follows early response gene expression in transient focal ischemia, Mol Chem Neuropathol, 1994, 23(2-3): 103~114
Yamaski Y, Matsuura N,Shozuhara H,et al Interleukin-1 as a pathogenetic mediator on ischemic brain damage in rats. Stroke, 1995, 26(4): 676~681
Del Zoppo GJ, Schmid–Schonbein GW, Mori E,et al. Polymorphonuclear leukocytes occulude capillaries following middle cerebral artery occlusion and reperfusion in baboons, Stroke, 1991, 22(10):1276~1283
Betz AL,Schielke GP,Yang GY. Interleukin-1 in cerebral ischemia . keio J Med, 1996, 45(3): 230~238
苏立凯，王淑仙，代瑞廷，等. 大黄素甲醚对沙土鼠脑缺血损伤时肿瘤坏死因子、白介素-1表达的影响. 中国中西医结合急救杂志，2002，7（1）：46~48
Satio K, Suyama K, Nishida K,et al. Early increases in TNF-α, IL-6 and IL-1 beta levels following transient cerebral ischemia in gerbil brain. Neurosci Lett, 1996, 206:149~152
Long EZ, Weistein PR, Carlson S,et al, Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989, 20(1):84~91
Rothwell NJ, Luheshi G. Pharmacology of interleukin-1α actions in the brain , Adv Pharmacol, 1994, 25: 1~20
Wang X, Yue TL, Barone FC. Concomitant cortical expression of TNF-alpha and IL-1 beta mRNAs follows early response gene expression in transient focal ischemia, Mol Chem Neuropathol, 1994, 23(2-3): 103~114
Yamaski Y, Matsuura N,Shozuhara H,et al Interleukin-1 as a pathogenetic mediator on ischemic brain damage in rats. Stroke, 1995, 26(4): 676~681
Del Zoppo GJ, Schmid–Schonbein GW, Mori E,et al. Polymorphonuclear leukocytes occulude capillaries following middle cerebral artery occlusion and reperfusion in baboons, Stroke, 1991, 22(10):1276~1283
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