【作者】 文继月；

【导师】 陈志武； 赵维中；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 硕士

【摘要】 缺血预处理（Ischemic preconditioning,IP）是指预先短暂性缺血或轻度缺氧后激发或动员机体内在的防护能力,使机体对随后严重的缺血、缺氧产生防御和保护现象。而药物预处理（pharmacological preconditioning,PP）是通过药物模拟缺血预处理激发机体内源性物质,如腺苷、NMDA受体阻断剂等而发挥保护作用。PP的研究开辟了脑保护研究的一个新领域。黄蜀葵花总黄酮（total flavones ofAbelmoschl Manihot,TFA）是锦葵属植物黄蜀葵的花中提取的有效部位。本室曾发现TFA药理性预处理（pharmacological preconditioning of TFA,PPA）对大鼠心肌缺血再灌注损伤有保护作用,但未见PPA对脑缺血再灌注损伤影响的研究报道,因此,本研究就PPA对脑缺血再灌注损伤的影响及其机制进行了初步研究。主要研究结果如下:1.PPA早期相对大鼠脑缺血再灌注损伤的影响PPA(20,40,80,160 mg·kg^-1)30min对大鼠MCAO 90min致缺血后脑梗死体积百分比增高有明显的抑制作用,可不同程度的降低血清中乳酸脱氢酶（lactatedehydrogenase,LDH）的活性、丙二醛（methylenedioxyamphetamine,MDA）、前列腺素E₂（Prostaglandin E₂,PGE₂）的含量,同时升高一氧化氮合酶（nitric oxide synthasc,NOS）的活力及血清中一氧化氮（nitric oxide,NO）的含量。2.PPA中间时相预处理对大鼠脑缺血再灌注损伤的影响PPA(80,160 mg·kg^-1)7h对大鼠MCAO 90min致脑梗死体积百分比增高无明显的影响。3.PPA延迟相预处理对大鼠脑缺血再灌注损伤的影响PPA(20,40,80,160 mg·kg^-1)36h对大鼠MCAO 90min致缺血后脑梗死体积百分比增高有明显的抑制作用,不同程度的降低血清中LDH的活性,MDA的含量,同时升高血清中一氧化氮的含量,其中160 mg·kg^-1PPA能显著提高脑组织中iNOSmRNA的表达。4.PPA对小鼠脑缺血再灌注损伤的影响小鼠PPA(25,50,100,200 mg·kg^-1)30min时对MCAO 90min致脑梗死体积百分比增高有明显的抑制作用,不同程度的降低血清中LDH、小鼠神经特异性烯醇化酶（nervous specific enolase,NSE）的活性及MDA的含量,同时升高血清中NO的含量。5.内皮依赖性超级化因子（Endothelium-derived hyperpolarizing factor,EDHF）在海马神经元缺氧复氧损伤的作用海马神经元培养至第8天,加入乙酰胆碱（Acetylcholine,Ach）和一段3mm脑基底环动脉后后行缺氧4h复氧24h.结果表明Ach（1μmol·L-1）+血管明显地抑制缺氧复氧后海马神经元活力的降低,同时能显著降低培养上清液中的LDH活性,但Ach及血管段单独均没有作用。加入前列腺环素（PGI₂）合成酶抑制剂吲哚美辛（indomethacin,Indo）及NO合酶抑制剂左旋硝基精氨酸甲酯（Nω-nitro-L-arginine-methyl-ester,L-NAME）预处理对Ach合用血管的作用无明显的影响,提示Ach介导的血管内皮EDHF释放对神经细胞缺氧再给氧损伤有保护作用。6.TFA对海马神经元缺氧复氧损伤的影响及可能的EDHF机制与单独应用TFA比较,联合使用TFA（3,10,30,100mg·L-1）和血管对缺氧复氧后海马神经元的活力有明显的升高作用,同时降低培养上清液中LDH的活性和海马神经元细胞内的游离Ca²⁺的增高。加入Indo及L-NAME预处理对TFA的保护作用无明显的影响,提示TFA对缺氧复氧致神经细胞损伤有一定的保护作用,其机制可能与EDHF有关。结论:TFA药理性预处理对脑缺血再灌注损伤有明显的保护作用,其机制可能与减轻细胞膜脂质过氧化、增加NO的生成、降低细胞内钙离子超载及释放EDHF等有关。更多还原

【Abstract】 The term "brain tolerance" describes as a phenomenon of transient resistance to a lethal insult evoked by preconditioning with a mild insult of short duration. Preconditioning stimulus,while inadequate to damage neurons,somehow increases their ability,and sometimes the ability of the whole organism as well,to survive subsequent lethal challenge.IP has been described to be a biphasic event:the acute phase is limited to 1-3 h after a brief ischemic stimulus,and the delayed phase emerges 24 h later and may last up to 72h.A series of recent studies have described another relevant phenomenon termed "chemical preconditioning".Abelmoschl Manihot is one of the plants that rich in flavones,such as quercetin,hyperin and rutin,Recent studies have shown that total flavones of Abelmoschl Manihot（TFA） have protective effects against cerebral ischemic injury in rabbit and rat.Also,pharmacological preconditioning of TFA（PPA） has been shown to possess a protective effect against myocardial ischemia-reperfusion injury in rabbit.However,there is no information reported about the effect of PPA on cerebral ischemic injury.By using rat,mice model of cerebral ischemia and the hypoxia-reoxygen model of foetal hippocamal neurons,we set out to investigate the effect of PPA on cerebral and its mechanism.1.Effects of PPA in the acute phase on the model of cerebral ischemic injury in ratIschemia and reperfusion（I/R） injury was induced by a middle cerebral artery occlusion（MCAO） for 90 minutes occlusion and 60 minutes of reperfusion in rat.PPA rats received 2 cycles of 5 minutes intravenous injection of TFA 20,40,80,160 mg·kg^-1 30 min before I/R.20,40,80,160 mg·kg^-1 PPA significantly reduced the infarction volume,markedly inhibited the elevated levels of serum LDH,MDA,PGE2 and decrement of serum NO and NOS.2.Effect of PPA of the middle phase on the model of cerebral ischemic injury in ratThe rat I/R injury was induced by MCAO for 90 minutes occlusion and 60 minutes of reperfusion.TFA(20,40,80,160 mg·kg^-1 )pretreatment 7 h has no significant effect on the infarction volume.3.Effect of PPA in the delayed phase on the model of cerebral ischemic injury in ratThe rat I/R injury was induced by MCAO for 90 minutes occlusion and 60 minutes of reperfusion.TFA or Nim pretreatment 36 h significantly decreased the infarction volume.PPA(20,40,80,160 mg·kg^-1) or Nim significantly inhibited the elevated levels of serum LDH and MDA,indicating a delayed protective effect of TFA preconditioning on cerebral ischemic reperfusion injury.In addition,the serum NO level and the cerebral iNOS mRNA were up-regulated.4.Effect of PPA in the acute phase on the model of cerebral ischemic injury in miceThe mouse I/R injury was induced by MCAO for 90 minutes occlusion and 60 minutes of reperfusion.PPA animals received 2 cycles of 5 minutes intravenous injection of TFA 25,50,100,200 mg·kg^-1 30 min before I/R.TFA or Nim pretreatment significantly decreased the infarction volume.Increases of serum LDH,NSE activity and MDA level were observed after I/R,but these changes were inhibited in mice pretreated with either TFA(25,50,100,200 mg·kg^-1) or Nim,In addition,the serum NO level were up-regulated.5.Effect of EDHF on hippocampal neurons subjected to oxygen deprivation/reperfusion in primary cultureTreatment with 1μmol·L^-1 Ach and a segment of cerebral artery significant increase the neuron viability（P＜0.01,compared with model）,and reduce the activity of LDH in culture medium.Plus with the Indo and the L-NAME didn’t influence the effect of Ach plus cerebral artery.6.Effect of TFA on hippocampal neurons subjected to oxygen deprivation/reperfusion in primary culture.Pretreatment with TFA（100 mg·kg（-1）） and cerebral artery significantly increase the neuron viability（P＜0.01,compared with model）,reduce the activity of LDH in culture medium.TFA(3,10,30,100 mg·kg^-1) plus with cerebral artery lowered the calcium concentration in neuron（compared with model）.Conclusion:PPA has marked protective effect on cerebral ischemia and reperfusion injury,the mechanisms of PPA might be related to attenuating oxygen free radicals, increasing of NO production,inhibiting the calcium concentration in neuron.更多还原