亚血红素多肽对脑出血后及脑缺血的保护作用
摘要
亚血红素多肽(DhHP-6)是以抗坏血酸过氧化物和微酶的结构为依据合成的一种含亚血红素多肽的六肽衍生物,作为一种过氧化物酶,其具有很强的抗氧化能力,可抑制和阻断氧化损伤的发生。本研究以手术方法制备脑出血及脑缺血模型来探讨亚血红素多肽对于脑出血后及脑缺血的保护作用。
实验一,利用大鼠脑立体定位仪,向大鼠脑内注入自体血制备大鼠实验性脑出血模型。观察DhHP-6对大鼠脑出血后3天的脑保护作用。结果表明:DhHP-6可明显抑制大鼠脑出血后脑组织中一氧化氮(NO)的升高,并能明显提高脑组织超氧化物歧化酶(SOD)活性,降低丙二醛(MDA)含量,同时能减轻大鼠的神经功能障碍和脑组织的病理损害。
实验二,用丝线结扎大鼠双侧颈总动脉制备大鼠急性不完全脑缺血模型。结果表明:DhHP-6可明显抑制大鼠不完全脑缺血术后脑组织中一氧化氮(NO)的升高,并可明显提高脑组织超氧化物歧化酶(SOD)活性,降低丙二醛(MDA)含量,同时能减轻脑组织的病理损害。
实验结果表明,亚血红素多肽对实验性脑出血后及不完全脑缺血具有脑保护作用,其作用机制可能为:DhHP-6增加SOD活性,发挥抗自由基作用,减少MDA生成,降低NO毒性,保护神经细胞膜的完整性,改善脑神经功能。
Cerebrovascular disease(CVD)is a devastating clinical condition,which severely do harm to human healthy.Both case-fatality rate and Mutilation rate are high.
The significance of the peroxidases is in the prevention and therapy of the diseases caused by free radicals, In spite of the therapeutical potential of natural peroxidases,it is severely limited by its source,difficult refinement,instability,molecular mass and other factors;thus enzyme mimetics of peroxidases are desirable. DhHP-6 is enzyme mimetics of peroxidases made by the method of mimic enzym.
In our study,to investigate the protective effects of DhHP-6 on intracerebral hemorrhage and ischemic in rats. The firt model of experimental intracerebral hemorrhage was induced in rats through infusion of 50 ul of fresh autologous blood drawn from caudal artery into the caudate uncleus.The changes behavior were observed.Histopathological change of rat brain subjected,Plasma SOD,MDA and NO,as well as brain tissue SOD,MDA and NO levels,were determined.
We made intracerebral hemorrhage model in rats to study the effects of DhHP-6 on neurocyte morphology changes and neurological scale. DhHP-6(1.0mg/kg,0.3mg/kg,0.1mg/kg)can reduced neurological scores(P<0.001、P<0.001、P<0.05), NO and MDA generation,decrease SOD activity After intracerebral hemorrhage. DhHP-6 significantly increased SOD content in plasma(P<0.05)and brain tissu(P<0.01,P<0.05)decreased MDA content in plasma(P<0.01, P<0.05)and brain tissue(P<0.01)as well as in plasma(P<0.05)and brain tissue(P<0.05,P<0.05)NO levels.
The second model of experimental, Through setting up incomplete cerebral ischemia. the effects of DhHP-6 on the level of SOD,MDA,NO,CK,LDH both in the brain tissue and serum of rats. At the same time,samples were taken to store in the fixation liquid for HE.
In our study,DhHP-6(1.0mg/kg)gnificantly increased SOD content in plasma(P<0.01)and brain tissu(P<0.01)decreased MDA content in plasm(P<0.01 ,P<0.01,P<0.05)and brain tissue(P<0.01,P<0.05)as well as in plasma(P<0.05)and brain tissue(P<0.05)CK and LDH levels.
DhHP-6 could obviously decrease the content of MDA and could increase the activity of SOD both in brain tissue and serum; .This indicated that DhHP-6 could increase the anti-oxidation activity of the enzyme, enhance the ability of removing free radicals and reduce the free radical from ischemia-reperfusion,so that it improve function of brain. The results of the experiment also demonstrated that DhHP-6 could strickingly reduce the content of NO in brain tissue,which showed that NaoxinKang Injection could ease the harm of NO.
The results of the experiment showed that DhHP-6 could significantly reduce the content of CK and LDH, wich indicates that DhHP-6 could relieve the damageand protect the integrity of cellular membrane.
实验一,利用大鼠脑立体定位仪,向大鼠脑内注入自体血制备大鼠实验性脑出血模型。观察DhHP-6对大鼠脑出血后3天的脑保护作用。结果表明:DhHP-6可明显抑制大鼠脑出血后脑组织中一氧化氮(NO)的升高,并能明显提高脑组织超氧化物歧化酶(SOD)活性,降低丙二醛(MDA)含量,同时能减轻大鼠的神经功能障碍和脑组织的病理损害。
实验二,用丝线结扎大鼠双侧颈总动脉制备大鼠急性不完全脑缺血模型。结果表明:DhHP-6可明显抑制大鼠不完全脑缺血术后脑组织中一氧化氮(NO)的升高,并可明显提高脑组织超氧化物歧化酶(SOD)活性,降低丙二醛(MDA)含量,同时能减轻脑组织的病理损害。
实验结果表明,亚血红素多肽对实验性脑出血后及不完全脑缺血具有脑保护作用,其作用机制可能为:DhHP-6增加SOD活性,发挥抗自由基作用,减少MDA生成,降低NO毒性,保护神经细胞膜的完整性,改善脑神经功能。
Cerebrovascular disease(CVD)is a devastating clinical condition,which severely do harm to human healthy.Both case-fatality rate and Mutilation rate are high.
The significance of the peroxidases is in the prevention and therapy of the diseases caused by free radicals, In spite of the therapeutical potential of natural peroxidases,it is severely limited by its source,difficult refinement,instability,molecular mass and other factors;thus enzyme mimetics of peroxidases are desirable. DhHP-6 is enzyme mimetics of peroxidases made by the method of mimic enzym.
In our study,to investigate the protective effects of DhHP-6 on intracerebral hemorrhage and ischemic in rats. The firt model of experimental intracerebral hemorrhage was induced in rats through infusion of 50 ul of fresh autologous blood drawn from caudal artery into the caudate uncleus.The changes behavior were observed.Histopathological change of rat brain subjected,Plasma SOD,MDA and NO,as well as brain tissue SOD,MDA and NO levels,were determined.
We made intracerebral hemorrhage model in rats to study the effects of DhHP-6 on neurocyte morphology changes and neurological scale. DhHP-6(1.0mg/kg,0.3mg/kg,0.1mg/kg)can reduced neurological scores(P<0.001、P<0.001、P<0.05), NO and MDA generation,decrease SOD activity After intracerebral hemorrhage. DhHP-6 significantly increased SOD content in plasma(P<0.05)and brain tissu(P<0.01,P<0.05)decreased MDA content in plasma(P<0.01, P<0.05)and brain tissue(P<0.01)as well as in plasma(P<0.05)and brain tissue(P<0.05,P<0.05)NO levels.
The second model of experimental, Through setting up incomplete cerebral ischemia. the effects of DhHP-6 on the level of SOD,MDA,NO,CK,LDH both in the brain tissue and serum of rats. At the same time,samples were taken to store in the fixation liquid for HE.
In our study,DhHP-6(1.0mg/kg)gnificantly increased SOD content in plasma(P<0.01)and brain tissu(P<0.01)decreased MDA content in plasm(P<0.01 ,P<0.01,P<0.05)and brain tissue(P<0.01,P<0.05)as well as in plasma(P<0.05)and brain tissue(P<0.05)CK and LDH levels.
DhHP-6 could obviously decrease the content of MDA and could increase the activity of SOD both in brain tissue and serum; .This indicated that DhHP-6 could increase the anti-oxidation activity of the enzyme, enhance the ability of removing free radicals and reduce the free radical from ischemia-reperfusion,so that it improve function of brain. The results of the experiment also demonstrated that DhHP-6 could strickingly reduce the content of NO in brain tissue,which showed that NaoxinKang Injection could ease the harm of NO.
The results of the experiment showed that DhHP-6 could significantly reduce the content of CK and LDH, wich indicates that DhHP-6 could relieve the damageand protect the integrity of cellular membrane.
引文
[1]惠汝太.脑卒中发病的分子机制[J].北京大学学报,2001,3(4):316-318.
[2]杨世杰.药理学[M].全国高等学校八年制临床医学专业规划教材,人民卫生出版社,2005,第一版:183-185.
[3]李惠.缺血性脑血管疾病[M].北京:北京科学技术出版社,2002:47-57
[4]Liu X F.Current status of neuron-protection in cerebrovas cular disease[J].Journal of Medical Postgraduates,2003,16(6):452-455.
[5]李凌波,李红,杨世杰.蒺藜皂苷对大鼠实验性脑出血的脑保护作用[J].哈尔滨医科大学学报,2006,40(2):99-102.
[6]Satvtore.Antioxidant therapy:a new pharmacological approach in shock,inflammation and ischemia reperfusion injury[J].Pharmacol Res,2001,53:135-159.
[7]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.
[8]Martindale JL,Holbrook NJ.Cellular response to oxidative stress:signaling for suicide and survival[J].J Cell Physiol,2002,192(1):1~15
[9]Yoon SO,Yun CH,Chunf AS.Dose effect of oxidative stress on signal transduction in aging[J].Mech Aging Dev,2002,123(12):1597~1604
[10]郑宏,刘冬立,朱陵群,等.偏瘫复康颗粒对脑缺血再灌注损伤的保护作用[J].中国实验方剂学杂志,2002,8(2):25-28.
[11]陈建华,刘晓娟,王启春.等.脑血管病患者血清内源性氧化剂和抗氧化剂水平变化的研究[J].中国危重病急救医学,2003,15(4):232-234.
[12]Sato ST,Tominaga T,Ohnishi T,et al.EPR spin-trapping study of NO formation dcring bilateral carotid occlusion in the rats[J].Biochim Biophys Aeta,1993,1181(2):195-199.
[13]Schmidly J W.Free radicals in central nervous system is chemia[J].Stroke,1990,25(3):7-9.
[14]Schmidley JW. Free raicats in central nerous system ischemia,Stroke,1990,21:1086.
[15]Yang GY, Schielke GP, Gong C, et al. Expression of TNF-a and ICAM-1 after focal cerebral ischemia in IL-βconverting enzyme deficient mice[J]. J Cereb Blood Flow Metab,1999,19:1109.
[16]Longa FZ,Weinstein PK, Carisons S, et a.l Reversible middle cerbral artery occlusion without craniotomy in rats[ J ]. Stroke, 1999,20: 34-42.
[17]Longa EZ,Weinstein PR,Carlson S.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[18]Joachim,Ricarda A.MD,et al.Neurokinin-1 Receptor Mediates Stress-Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness in Mice.Psychosomatic Medicine 2004;66(4):564—571.
[19]Ricciardolo,F.L.Mechanisms of citric acid-induced bronchoconstriction.Am J Med,2001;111 Suppl 8A,18S—24S.
[20]Joos,G.F.De Swert,K.O.&Pauwels,R.A.Airway inflammation and tachykinins:prospects for the development of tachykinin receptor antagonists.Eur J Pharmacol,2001;429(1-3):239—50.
[21]Hirotsugu Kohrogi,et al.The role of substance P release iin the lung with esophageal acid.Am J Med 2001;111:8A 25S—30S.
[22]徐娜.亚血红素多肽对心肌缺血再灌注损伤保护作用[D].吉林.吉林大学白求恩医学院,2008.
[23]李爽.亚血红素多肽对心肌细胞氧化损伤的保护作用及作用机制[D].吉林.吉林大学白求恩医学院,2008.
[24]赵玉,亚血红素多肽对大鼠脑缺血再灌注损伤的保护作用[D].吉林.延边大学,2008.
[25]林晓薇,王福森.自由基与脑血管病关系及对策.黑龙江医学,2004,28(1):18-19.
[26]郑荣梁.自由基生命科学进展.原子能出版社,1993,1:5.
[27]SuzukiH,WildhirtSM,DudekR R,e tal.Induction of Apoptosis in myocardial infarction and its possible relationship to nitric oxide synthase in macrophages.Tissue Cell.1996;28(l):89-97
[28]Szabolcs M,Michler RE,Yang X,et al.Apoptosis of card iacmyocytes during cardiac allograft rejection.Relation to induction of nitric oxide synthase.Circulation.1996;9 4(7):1665-1673
[29]Cai B,Roy DK,Sciacca R,et al.Efects of immunosuppressive therapy on expression of inducible nitric oxide synthase(iNOS) during cardiac allograft rejection.Int J Cardiol.1995;50(3):243-251.
[30]McLeod LL,Sevanian A.Lipid peroxidation and modification of lipid composition in an endothelial cell model of ischemia and reperfusion[J].Free Radic Biol Med,1997;23(4):680-694
[31]Prasad K.Increased oxygen free radical activity in patient on Cardio-pulmonary bypass surgery[J].Am Heart,1992,123:37
[32]Dianzani,MU.Free radicals in physiology and pathology[J].Boll Soc Ital Biol Sper.1992,68(8):491-511.
[33]Bjorksten J,J Gerontol.The crosslinkage theory of aging[J] Am Geriatr Soc,1956,11(3):298-300.
[34]Kevin L G,Novalija E,Stowe DF.Reactive oxygen speciesasmediators of cardiac injury and protection:the relevance toanesthesia practice[J].Anesth Analg,2005,101(5):1275-1287.
[35]Demerle-Pallardy C,Gillard-Roubert V,Marin JG,et al.In vitro antioxidant neurop rotective activity of BN80933,a dual inhibitor of neuronal nitric oxide synthase and lip id peroxidation[J].J Neuro-chem,2000,74(5):2079-2086.
[36]Tan S,Zhou F,Nielsen VG,et al.Sustained hypoxia-ischemia results in nitrogen and oxygen species production ang injury in the premature fetal rabbit brain[J].J Neuropathol Exp Neurol,1998,57(6):544-553.
[37]Kim JS.Cytokines and adhesion molecules in stroke and related diseases[J].Neurol Sci,1996,137(20):69-78.
[38]Lee WY,Han SH,Cho TS,etal.Effect of ursodeoxycholic acid on ischemia reperfusion injury in isolated rat heart[J].Arch Pharm Res,1999,22:479-484
[39]Moncada S , Palmer RM , H iggs EA.N itric oxide: physiology ,pathophysiology,and pharmacology[J]. Pharmacol Rev,1991,43(2):109-112.
[40]Sayama T,Suzuki S,FukuiM.Expression of inducible nitric oxide synthase in rats follow ing subarachnoid hemorrhage[J].N eurol Res,1998,20(1):79-83.
[41]N ishizawa S,Yamamoto S,Yokoyma T,et al.Dysfunc-tion of nitric oxide induces protein kinase C activation resulting in vasospasm after subarachnoid hemorrhage[J].N eurol Res,1997,19(5):558-562.
[42]Vaagenes P,Fodstad DT,Safar P.The use of cytosolic enzyme increase in cerebrosp inal fluid of patients resuscitated after cardiac arrest[J].Am J Em erg M ed,1994,12(6):621-5.
[2]杨世杰.药理学[M].全国高等学校八年制临床医学专业规划教材,人民卫生出版社,2005,第一版:183-185.
[3]李惠.缺血性脑血管疾病[M].北京:北京科学技术出版社,2002:47-57
[4]Liu X F.Current status of neuron-protection in cerebrovas cular disease[J].Journal of Medical Postgraduates,2003,16(6):452-455.
[5]李凌波,李红,杨世杰.蒺藜皂苷对大鼠实验性脑出血的脑保护作用[J].哈尔滨医科大学学报,2006,40(2):99-102.
[6]Satvtore.Antioxidant therapy:a new pharmacological approach in shock,inflammation and ischemia reperfusion injury[J].Pharmacol Res,2001,53:135-159.
[7]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.
[8]Martindale JL,Holbrook NJ.Cellular response to oxidative stress:signaling for suicide and survival[J].J Cell Physiol,2002,192(1):1~15
[9]Yoon SO,Yun CH,Chunf AS.Dose effect of oxidative stress on signal transduction in aging[J].Mech Aging Dev,2002,123(12):1597~1604
[10]郑宏,刘冬立,朱陵群,等.偏瘫复康颗粒对脑缺血再灌注损伤的保护作用[J].中国实验方剂学杂志,2002,8(2):25-28.
[11]陈建华,刘晓娟,王启春.等.脑血管病患者血清内源性氧化剂和抗氧化剂水平变化的研究[J].中国危重病急救医学,2003,15(4):232-234.
[12]Sato ST,Tominaga T,Ohnishi T,et al.EPR spin-trapping study of NO formation dcring bilateral carotid occlusion in the rats[J].Biochim Biophys Aeta,1993,1181(2):195-199.
[13]Schmidly J W.Free radicals in central nervous system is chemia[J].Stroke,1990,25(3):7-9.
[14]Schmidley JW. Free raicats in central nerous system ischemia,Stroke,1990,21:1086.
[15]Yang GY, Schielke GP, Gong C, et al. Expression of TNF-a and ICAM-1 after focal cerebral ischemia in IL-βconverting enzyme deficient mice[J]. J Cereb Blood Flow Metab,1999,19:1109.
[16]Longa FZ,Weinstein PK, Carisons S, et a.l Reversible middle cerbral artery occlusion without craniotomy in rats[ J ]. Stroke, 1999,20: 34-42.
[17]Longa EZ,Weinstein PR,Carlson S.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[18]Joachim,Ricarda A.MD,et al.Neurokinin-1 Receptor Mediates Stress-Exacerbated Allergic Airway Inflammation and Airway Hyperresponsiveness in Mice.Psychosomatic Medicine 2004;66(4):564—571.
[19]Ricciardolo,F.L.Mechanisms of citric acid-induced bronchoconstriction.Am J Med,2001;111 Suppl 8A,18S—24S.
[20]Joos,G.F.De Swert,K.O.&Pauwels,R.A.Airway inflammation and tachykinins:prospects for the development of tachykinin receptor antagonists.Eur J Pharmacol,2001;429(1-3):239—50.
[21]Hirotsugu Kohrogi,et al.The role of substance P release iin the lung with esophageal acid.Am J Med 2001;111:8A 25S—30S.
[22]徐娜.亚血红素多肽对心肌缺血再灌注损伤保护作用[D].吉林.吉林大学白求恩医学院,2008.
[23]李爽.亚血红素多肽对心肌细胞氧化损伤的保护作用及作用机制[D].吉林.吉林大学白求恩医学院,2008.
[24]赵玉,亚血红素多肽对大鼠脑缺血再灌注损伤的保护作用[D].吉林.延边大学,2008.
[25]林晓薇,王福森.自由基与脑血管病关系及对策.黑龙江医学,2004,28(1):18-19.
[26]郑荣梁.自由基生命科学进展.原子能出版社,1993,1:5.
[27]SuzukiH,WildhirtSM,DudekR R,e tal.Induction of Apoptosis in myocardial infarction and its possible relationship to nitric oxide synthase in macrophages.Tissue Cell.1996;28(l):89-97
[28]Szabolcs M,Michler RE,Yang X,et al.Apoptosis of card iacmyocytes during cardiac allograft rejection.Relation to induction of nitric oxide synthase.Circulation.1996;9 4(7):1665-1673
[29]Cai B,Roy DK,Sciacca R,et al.Efects of immunosuppressive therapy on expression of inducible nitric oxide synthase(iNOS) during cardiac allograft rejection.Int J Cardiol.1995;50(3):243-251.
[30]McLeod LL,Sevanian A.Lipid peroxidation and modification of lipid composition in an endothelial cell model of ischemia and reperfusion[J].Free Radic Biol Med,1997;23(4):680-694
[31]Prasad K.Increased oxygen free radical activity in patient on Cardio-pulmonary bypass surgery[J].Am Heart,1992,123:37
[32]Dianzani,MU.Free radicals in physiology and pathology[J].Boll Soc Ital Biol Sper.1992,68(8):491-511.
[33]Bjorksten J,J Gerontol.The crosslinkage theory of aging[J] Am Geriatr Soc,1956,11(3):298-300.
[34]Kevin L G,Novalija E,Stowe DF.Reactive oxygen speciesasmediators of cardiac injury and protection:the relevance toanesthesia practice[J].Anesth Analg,2005,101(5):1275-1287.
[35]Demerle-Pallardy C,Gillard-Roubert V,Marin JG,et al.In vitro antioxidant neurop rotective activity of BN80933,a dual inhibitor of neuronal nitric oxide synthase and lip id peroxidation[J].J Neuro-chem,2000,74(5):2079-2086.
[36]Tan S,Zhou F,Nielsen VG,et al.Sustained hypoxia-ischemia results in nitrogen and oxygen species production ang injury in the premature fetal rabbit brain[J].J Neuropathol Exp Neurol,1998,57(6):544-553.
[37]Kim JS.Cytokines and adhesion molecules in stroke and related diseases[J].Neurol Sci,1996,137(20):69-78.
[38]Lee WY,Han SH,Cho TS,etal.Effect of ursodeoxycholic acid on ischemia reperfusion injury in isolated rat heart[J].Arch Pharm Res,1999,22:479-484
[39]Moncada S , Palmer RM , H iggs EA.N itric oxide: physiology ,pathophysiology,and pharmacology[J]. Pharmacol Rev,1991,43(2):109-112.
[40]Sayama T,Suzuki S,FukuiM.Expression of inducible nitric oxide synthase in rats follow ing subarachnoid hemorrhage[J].N eurol Res,1998,20(1):79-83.
[41]N ishizawa S,Yamamoto S,Yokoyma T,et al.Dysfunc-tion of nitric oxide induces protein kinase C activation resulting in vasospasm after subarachnoid hemorrhage[J].N eurol Res,1997,19(5):558-562.
[42]Vaagenes P,Fodstad DT,Safar P.The use of cytosolic enzyme increase in cerebrosp inal fluid of patients resuscitated after cardiac arrest[J].Am J Em erg M ed,1994,12(6):621-5.