亚血红素多肽对大鼠脑缺血再灌注损伤的保护作用
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摘要
亚血红素多肽(DhHP-6)是以抗坏血酸过氧化物酶和微酶的结构为依据合成的一种含亚血红素的六肽衍生物,其作为过氧化物酶模拟物具有过氧化物酶活性及防止超氧离子和自由基的损害的作用。药理学研究表明,亚血红素多肽具有保护心肌缺血、舒张血管、干预动脉硬化的发生和发展、抑制白内障形成的作用。此外,在抗衰老、抗癌、增强机体免疫力等方面具有广泛的药理价值。但亚血红素多肽对脑缺血再灌注模型动物的影响尚未见报道。
本实验采用线栓法建立大鼠大脑中动脉脑缺血再灌注损伤模型,观察亚血红素多肽对缺血再灌注大鼠的血清中超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)含量的影响及对脑组织匀浆中超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)含量的影响,同时观察亚血红素多肽对大鼠的神经功能评分、脑组织病理形态学及脑梗死面积的影响,以探讨其对脑缺血再灌注损伤的影响,从而为亚血红素多肽临床应用于脑缺血的治疗提供理论依据。
实验结果显示,脑缺血再灌注后大鼠血清中MDA、LDH的含量明显升高,SOD的活性降低,亚血红素多肽高、中、低剂量组能明显降低大鼠脑缺血再灌注损伤后血清中LDH、MDA的含量(P＜0.01,P＜0.05),明显提高SOD的活性(P＜0.01,P＜0.05);能明显抑制大鼠脑缺血再灌注损伤后脑组织匀浆中NO、MDA的升高(P＜0.01,P＜0.05),显著提高SOD的活性(P＜0.01,P＜0.05)。大脑中动脉栓塞术后,缺血再灌注模型组大鼠表现出明显的运动功能障碍,神经功能评分增加,亚血红素多肽高、中、低剂量组能减低大鼠行为障碍的评分,不同程度地减轻大鼠的行为障碍(P＜0.001,P＜0.01,P＜0.05)。缺血再灌注后,神经细胞明显水肿,胞质着色较浅,胞核浓缩、深染。亚血红素多肽高、中剂量组能明显减轻上述改变,减轻细胞的水肿程度,维持细胞的正常形态。亚血红素多肽高、中剂量组能明显缩小脑梗死面积(P＜0.01,P＜0.05)。
上述结果表明,亚血红素多肽对脑缺血再灌注损伤的保护作用是通过以下机制实现的:亚血红素多肽能增强机体清除自由基能力,减轻脂质过氧化损伤,保护神经细胞膜的完整性和稳定性,并抑制NO的产生,减轻NO对脑组织及神经细胞的损伤,从而对脑缺血再灌注损伤具有保护作用。
Deuterohemin-His-Peptide 6(DhHP-6) is a six-peptide-derivation included deutohaemoglobin,which based on antiscorbic acid peroxydase and minienzyme composition.It was a simulacrum of peroxydase has the activity of peroxydase and to prevent damaging of hyperoxygen and free radical.Pharmacological research demonstrated that DhHP-6 cordis muscle with meager blood,inhibitting atherosclerotic occurrence and development.It also had the potent effects on restraining the formation of caligo lentis,antiaging,enhancing organism immunity.At present,little materials has been published about the effects of DhHP-6 on cerebral ischemia reperfusion injury.
The experiment rats cerebral ischemia reperfusion injury was induced by middle cerebral artery occlusion to observe the influence of SOD,MDA and LDH in serum;and the content of SOD,MDA and NO in cerebral tissue.We made ischemia reperfusion model in rats to study the effects of DhHP-6 on neurological scale,neurocyte morphology changes and infarct size to explore the mechanism,accordingly provided the theory of clinical application in cerebral ischemia.
Experimental result showed that,MDA,LDH released and decreased SOD activity after cerebral ischemia reperfusion in rats.DhHP-6 (1mg/kg,0.3mg/kg,0.1mg/kg) groups significantly inhibite MDA,LDH release(P＜0.01,P＜0.05) and improve SOD activity(P＜0.01,P＜0.05) in serum;and obviously restrain NO and MDA enhance(P＜0.01,P＜0.05), striking elevate SOD activity(P＜0.01,P＜0.05) in cerebral ischemia. After reperfusion,modle rats have distinct action deficits and increase neurological scores.DhHP-6(1mg/kg,0.3mg/kg,0.1mg/kg)groups can reduced neurological scores to differently extent lighten behavior deficits (P＜0.001,P＜0.01,P＜0.05 ),modle rats have distinct action deficits.As a result of the changes of neurocytes morphology,neurons were serious edema,cytoplasma was stained slightly,nucler were contracted and stained deeply after reperfusion.DhHP-6(1mg/kg,0.3mg/kg) groups educed cellular edema,maintained the neurocyte morphology. DhHP-6(1mg/kg,0.3mg/kg) groups could significantly deflate the infarct size(P＜0.01,P＜0.05)
Based on the above experiments,it has been demonstrated that DhHP-6 increased the ability of removing free radicals,decreased lipid peroxidation,protect both integrity and stability of cellular membrane, inhibited production of NO,decreased the damages of brain tissue and neurocyte then protected the neurons from cerebral ischemia reperfusion injury.
本实验采用线栓法建立大鼠大脑中动脉脑缺血再灌注损伤模型,观察亚血红素多肽对缺血再灌注大鼠的血清中超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)含量的影响及对脑组织匀浆中超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)含量的影响,同时观察亚血红素多肽对大鼠的神经功能评分、脑组织病理形态学及脑梗死面积的影响,以探讨其对脑缺血再灌注损伤的影响,从而为亚血红素多肽临床应用于脑缺血的治疗提供理论依据。
实验结果显示,脑缺血再灌注后大鼠血清中MDA、LDH的含量明显升高,SOD的活性降低,亚血红素多肽高、中、低剂量组能明显降低大鼠脑缺血再灌注损伤后血清中LDH、MDA的含量(P＜0.01,P＜0.05),明显提高SOD的活性(P＜0.01,P＜0.05);能明显抑制大鼠脑缺血再灌注损伤后脑组织匀浆中NO、MDA的升高(P＜0.01,P＜0.05),显著提高SOD的活性(P＜0.01,P＜0.05)。大脑中动脉栓塞术后,缺血再灌注模型组大鼠表现出明显的运动功能障碍,神经功能评分增加,亚血红素多肽高、中、低剂量组能减低大鼠行为障碍的评分,不同程度地减轻大鼠的行为障碍(P＜0.001,P＜0.01,P＜0.05)。缺血再灌注后,神经细胞明显水肿,胞质着色较浅,胞核浓缩、深染。亚血红素多肽高、中剂量组能明显减轻上述改变,减轻细胞的水肿程度,维持细胞的正常形态。亚血红素多肽高、中剂量组能明显缩小脑梗死面积(P＜0.01,P＜0.05)。
上述结果表明,亚血红素多肽对脑缺血再灌注损伤的保护作用是通过以下机制实现的:亚血红素多肽能增强机体清除自由基能力,减轻脂质过氧化损伤,保护神经细胞膜的完整性和稳定性,并抑制NO的产生,减轻NO对脑组织及神经细胞的损伤,从而对脑缺血再灌注损伤具有保护作用。
Deuterohemin-His-Peptide 6(DhHP-6) is a six-peptide-derivation included deutohaemoglobin,which based on antiscorbic acid peroxydase and minienzyme composition.It was a simulacrum of peroxydase has the activity of peroxydase and to prevent damaging of hyperoxygen and free radical.Pharmacological research demonstrated that DhHP-6 cordis muscle with meager blood,inhibitting atherosclerotic occurrence and development.It also had the potent effects on restraining the formation of caligo lentis,antiaging,enhancing organism immunity.At present,little materials has been published about the effects of DhHP-6 on cerebral ischemia reperfusion injury.
The experiment rats cerebral ischemia reperfusion injury was induced by middle cerebral artery occlusion to observe the influence of SOD,MDA and LDH in serum;and the content of SOD,MDA and NO in cerebral tissue.We made ischemia reperfusion model in rats to study the effects of DhHP-6 on neurological scale,neurocyte morphology changes and infarct size to explore the mechanism,accordingly provided the theory of clinical application in cerebral ischemia.
Experimental result showed that,MDA,LDH released and decreased SOD activity after cerebral ischemia reperfusion in rats.DhHP-6 (1mg/kg,0.3mg/kg,0.1mg/kg) groups significantly inhibite MDA,LDH release(P＜0.01,P＜0.05) and improve SOD activity(P＜0.01,P＜0.05) in serum;and obviously restrain NO and MDA enhance(P＜0.01,P＜0.05), striking elevate SOD activity(P＜0.01,P＜0.05) in cerebral ischemia. After reperfusion,modle rats have distinct action deficits and increase neurological scores.DhHP-6(1mg/kg,0.3mg/kg,0.1mg/kg)groups can reduced neurological scores to differently extent lighten behavior deficits (P＜0.001,P＜0.01,P＜0.05 ),modle rats have distinct action deficits.As a result of the changes of neurocytes morphology,neurons were serious edema,cytoplasma was stained slightly,nucler were contracted and stained deeply after reperfusion.DhHP-6(1mg/kg,0.3mg/kg) groups educed cellular edema,maintained the neurocyte morphology. DhHP-6(1mg/kg,0.3mg/kg) groups could significantly deflate the infarct size(P＜0.01,P＜0.05)
Based on the above experiments,it has been demonstrated that DhHP-6 increased the ability of removing free radicals,decreased lipid peroxidation,protect both integrity and stability of cellular membrane, inhibited production of NO,decreased the damages of brain tissue and neurocyte then protected the neurons from cerebral ischemia reperfusion injury.
引文
[1]刘煜敏,徐仁,黄怀钧等.兴奋性氨基酸、氧自由基与缺氧再灌注损伤关系的实验观察.卒中与神经疾病,2000,7(2):98-100
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[3]Longa EZ,Weinstein PR,Carlson S.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91
[4]George P.Smith.Filamentous Fusion Phage:Novel Expression Vectors That Display Cloned Antigens on the Virion Surface.Science,1985,228:1315-1317
[5]Geysen HM,Rodda SJ,Mason TJ.A priori delineation of a peptide which mimics a discontinuous antigenic determinant Mol Immunol.1986,23(7):709-15
[6]马丽炎,王春兰,张琪.三七皂甙对脑组织血液供应和能量代谢的影响.中国药理学通报,1998,14(1)27
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[8]Tsuehidated R.Regional cerebral blood flow during and after 2 hour middle cerebral artery occlusion in the rat[J].J Cere Blood Flow Metab,1997.17(2):1066-73
[9]王洁,张均田.脑缺血氧自由基清除剂与脑保护[J].中国药理学通讯,1998,14(1):6-10
[10]Chandan K S,Lester P.Antioxidant and redoxregulation of genetranscription[J].FASEBJ,1996.10:709
[11]郑彩酶.脑缺氧与缺血性脑血管病神经元损伤的研究进展.国外医学脑血管疾病分册,1993,1(1):6-9
[12]李宝民,张纪,殷国升.脑缺血再灌注自由基脑损害的影响和自由基清除作用.中华神经外科杂志,1990,69(3):2-6
[13]Pisani A,Calabresi P,Bernardi G.Hypoxia in striatal and cortical neurons:membrane potential and CA measurements.Neuroreport,1997,24(8):1143
[14]常学辉,张良芝.黄角汤对大鼠脑缺血损伤模型自由基影响的实验研究,中医药学.2003,31(1)48-49
[15]禹志领,张广钦,赵红旗.葛根总黄酮对脑缺血的保护作用[J]中国药科大学学报,1998,28(9):310-312
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[17]Samdani A F,Dawson T M,Dawson V L.Nitric oxide synthase in models of focal ischemia[J].Stroke,1997,28:1283-1288
[18]Dawson VL,Dawson TM,London ED,et al.Nitric oxide mediates glutamate neuroxicity in primary cortical culture[J].Proc Natl Acad Science U.S.A.1991,88(14):6368-6377
[19]Kuusisto J,Mykkanen L,Pyorala K,et al.Non-insulin-dependent diabetes and its metabolic contrao are important predictors of stroke in elderly subjects[J].Stroke,1994,25:1157
[20]张建新,张会欣,李兰芳,等.大鼠局灶性脑缺血后一氧化氮合酶基因表达的变化Chin J Appl Physicl 2005;21(3)277
[21]赵明,王洪新.脑缺血再灌注损伤机理及药物保护的研究进展.锦州医学院学报.2002,23(5)72-73
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[4]George P.Smith.Filamentous Fusion Phage:Novel Expression Vectors That Display Cloned Antigens on the Virion Surface.Science,1985,228:1315-1317
[5]Geysen HM,Rodda SJ,Mason TJ.A priori delineation of a peptide which mimics a discontinuous antigenic determinant Mol Immunol.1986,23(7):709-15
[6]马丽炎,王春兰,张琪.三七皂甙对脑组织血液供应和能量代谢的影响.中国药理学通报,1998,14(1)27
[7]Hertog MQ,Feskens EJ,Hollman PC,et al.Dietary antioxidant flavonoids and risk of coronary heart disease:the Zutphen Elderly Study Lancet,1993,342(8878):1007-1011
[8]Tsuehidated R.Regional cerebral blood flow during and after 2 hour middle cerebral artery occlusion in the rat[J].J Cere Blood Flow Metab,1997.17(2):1066-73
[9]王洁,张均田.脑缺血氧自由基清除剂与脑保护[J].中国药理学通讯,1998,14(1):6-10
[10]Chandan K S,Lester P.Antioxidant and redoxregulation of genetranscription[J].FASEBJ,1996.10:709
[11]郑彩酶.脑缺氧与缺血性脑血管病神经元损伤的研究进展.国外医学脑血管疾病分册,1993,1(1):6-9
[12]李宝民,张纪,殷国升.脑缺血再灌注自由基脑损害的影响和自由基清除作用.中华神经外科杂志,1990,69(3):2-6
[13]Pisani A,Calabresi P,Bernardi G.Hypoxia in striatal and cortical neurons:membrane potential and CA measurements.Neuroreport,1997,24(8):1143
[14]常学辉,张良芝.黄角汤对大鼠脑缺血损伤模型自由基影响的实验研究,中医药学.2003,31(1)48-49
[15]禹志领,张广钦,赵红旗.葛根总黄酮对脑缺血的保护作用[J]中国药科大学学报,1998,28(9):310-312
[16]Dawson D A.Nitric oxide and focal cerebral ischemia:multiplicity of actions and diverse outcome[J].Cerebrovasc Brain Metab Rev,1994,6(4):299-324
[17]Samdani A F,Dawson T M,Dawson V L.Nitric oxide synthase in models of focal ischemia[J].Stroke,1997,28:1283-1288
[18]Dawson VL,Dawson TM,London ED,et al.Nitric oxide mediates glutamate neuroxicity in primary cortical culture[J].Proc Natl Acad Science U.S.A.1991,88(14):6368-6377
[19]Kuusisto J,Mykkanen L,Pyorala K,et al.Non-insulin-dependent diabetes and its metabolic contrao are important predictors of stroke in elderly subjects[J].Stroke,1994,25:1157
[20]张建新,张会欣,李兰芳,等.大鼠局灶性脑缺血后一氧化氮合酶基因表达的变化Chin J Appl Physicl 2005;21(3)277
[21]赵明,王洪新.脑缺血再灌注损伤机理及药物保护的研究进展.锦州医学院学报.2002,23(5)72-73
[22]李盈盈等,家兔急性不完全性脑缺血模型和特点的实验研究,中华神经精神科杂志,1987,20(2):101
[23]Cechetti F,Rhod A,Simao F,et al.Effect of treadmill exercise on cell damage in rat hippocampal slices submitted to oxygen and glucose deprivation[J].Brain Res,2007,1157:121-125
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