【作者】 袁恒杰；

【导师】 娄建石； 康毅； 刘艳霞； 高卫真； 焦建杰；

【作者基本信息】 天津医科大学 ， 药理学， 2009， 博士

【摘要】 目的:研究无创性延迟肢体缺血预适应(NDLIP)对大鼠脑缺血再灌注(I/R)损伤的保护作用。方法:健康雄性Wistar大鼠随机分为4组。(1) Sham组:颈正中切口,暴露左侧颈总动脉、颈外动脉及颈内动脉,游离左侧颈总动脉,但不夹闭。(2) I/R组:建立大鼠MCAO模型,实施1h缺血/24h再灌注。(3) ECIP+I/R组:左侧颈总动脉先行3次5min缺血/5min再灌注,随后实施1h缺血/24h再灌注。(4) NDLIP+I/R组:左后肢实施3次5min缺血/5min再灌注,每天1次,连续3天,第4天建立MCAO模型,对脑实施1h缺血/24h再灌注。从神经功能行为评分、能量代谢、脑细胞坏死和凋亡、抗氧化能力、血管内皮功能等方面评价NDLIP的脑保护作用。大鼠实施1h缺血/24h再灌注后进行行为评分观察神经功能变化;HPLC法测定皮层中ATP、ADP和AMP含量,并计算总腺苷酸(TAN)和能荷(EC) ; TTC染色法测定脑梗死面积,HE染色观察脑细胞形态学改变,TUNEL法检测皮层和海马细胞凋亡,Real-time PCR法检测皮层和海马凋亡相关基因Fas、FasL mRNA表达水平,Western Blot法检测皮层Fas、FasL蛋白含量;比色法测定脑组织髓过氧化物酶(MPO)、总超氧化物歧化酶(T-SOD)、锰-超氧化物歧化酶(Mn-SOD)、谷胱甘肽过氧化物酶(GSH-PX)、黄嘌呤氧化酶(XOD)活力,丙二醛(MDA)含量以及血清一氧化氮(NO)浓度;ELISA法检测血清ET-1含量和t-PA及PAI-1活力。结果:1 NDLIP对大鼠脑I/R损伤后行为评分和能量代谢的影响与I/R组相比、ECIP+I/R组和NDLIP+I/R组行为评分降低(P<0.01) ;ECIP+I/R组和NDLIP+I/R组皮层ATP (P<0.01)、ADP (P<0.01)和AMP(P<0.05)含量显著升高,TAN (P<0.01)和EC (P<0.01)显著升高。2 NDLIP对大鼠脑I/R损伤后细胞死亡和凋亡的影响与I/R组比较,ECIP+I/R组和NDLIP+I/R组脑梗死面积缩小(P<0.05),MPO活性降低(P<0.01),大脑皮层细胞凋亡(TUNEL: P<0.01)和海马细胞凋亡(TUNEL: P<0.01)降低。皮层细胞和海马锥体细胞损伤明显改善。3 NDLIP对大鼠脑I/R损伤保护作用的凋亡机制与I/R组相比,ECIP+I/R组和NDLIP+I/R组大鼠皮层组织Fas(P<0.01)和FasL(P<0.01) mRNA水平明显降低,海马组织Fas(P<0.01)和FasL(P<0.01)mRNA水平明显降低;ECIP+I/R组和NDLIP+I/R组大鼠皮层组织Fas(P<0.05)和FasL (P<0.05)蛋白含量下降。4 NDLIP对大鼠脑I/R损伤后抗氧化能力的影响与I/R组相比,ECIP+I/R组和NDLIP+I/R组皮层T-SOD (P<0.01)、Mn-SOD (P<0.01)和GSH-PX (P<0.01)及海马T-SOD (P<0.01)和Mn-SOD(P<0.01)活力升高;皮层(P<0.01)和海马(P<0.01) Mn-SOD mRNA表达升高;XOD活性(P<0.01)及皮层(P<0.01)和海马(P<0.01) MDA含量降低。5 NDLIP对大鼠脑I/R损伤前后血管内皮功能的影响缺血前,与I/R组比较,ECIP+I/R组和NDLIP+I/R组血清NO浓度升高(P<0.05), ET-1/NO比值降低(P<0.05),各组间血清ET-1浓度未见差异。再灌注后,与I/R组比较,ECIP+I/R组和NDLIP+I/R组血清ET-1降低(P<0.01), NO浓度升高程度增加(P<0.01), ET-1/NO比值降低(P<0.05)。6 NDLIP对大鼠脑I/R损伤前后纤溶系统的影响缺血前,各组纤溶指标无显著性差异。再灌注后,与I/R组相比,ECIP+I/R组和NDLIP+I/R组t-PA活力升高(P<0.01), PAI-1活力降低(P<0.01)。与缺血前相比,I/R组,ECIP+I/R组和NDLIP+I/R组t-PA活力显著降低(P<0.01), PAI-1活力显著升高(P<0.01)结论:NDLIP可以降低大鼠脑I/R后的行为评分,改善大鼠神经功能缺损;升高大鼠脑I/R后皮层组织的ATP、ADP和AMP含量,提高腺苷酸池水平,增加EC水平,改善脑I/R后脑组织的能量代谢,从而保护I/R损伤的大脑组织。NDLIP可以缩小大鼠脑I/R损伤后的梗塞面积,改善脑细胞受损形态,显著降低脑I/R损伤后的皮层和海马的凋亡指数,其作用强度与ECIP相当。NDLIP能够显著下调大脑皮层和海马组织Fas、FasL mRNA水平,同时下调脑组织Fas、FasL蛋白表达水平,NDLIP减少脑I/R后脑组织凋亡的发生。NDLIP可以降低大鼠脑I/R后的脑组织MDA含量,降低脑组织中XOD活性,升高脑I/R后脑组织SOD、GSH-PX活性,增强脑I/R损伤后的抗氧化能力,减轻过氧化损伤,提示其脑保护作用与抗氧化、清除自由基作用有关。NDLIP可增加基础状态下血清NO含量,明显增加I/R损伤后NO含量,减少I/R损伤诱导的ET-1释放增加,对基础状态下的ET-1含量不影响,因此,使基础状态和I/R损伤后ET-1/NO比值下降。此作用与增强脑组织抗氧化能力的作用、抑制凋亡相辅相成,共同介导NDLIP脑保护效应。NDLIP能够升高大鼠I/R后血清t-PA活力,降低大鼠I/R后血清PAI-1活力,改善纤溶/抗纤溶系统功能,抑制血栓的形成,改善纤溶系统功能。更多还原

【Abstract】 Objective:To study the protection of noninvasive delayed limb ischemicpreconditioning (NDLIP)against cerebral ischemia reperfusion (I/R)injury inrats.Method:Healthy male Wistar rats were divided randomly into 4 groups.(1)Sham group:Left common carotid artery was dissociated,while was notoccluded.(2)I/R group:rats were subjected to 1h occlusion of middle cerebrealartery followed by 24h ofreperfusion.(3)ECIP+I/R group:rats were subjected to3 cycles of 5 min of ischemia and reperfusion on the common carotid artery beforelh of ischemia and 24h of reperfusion.(4)NDLIP+I/R group:rats weresubjected to 3 cycles of 5 min of ischemia and reperfusion on the left hind limbfor 3 days to induce NDLIP.On the forth day,l h of ischemia and 24h ofreperfusion was performed.Delayed protections of NDLIP were estimated inthe light of the changes of neural behavior mark,energy metabolism,cerebral celldeath and apoptosis,cerebral tissue antioxidative ability,blood vessel endothelialfunction and fibrinolysis system function.Neurological functions were studiedthrough observing neural behaviors.Determinations of ATP,ADP and AMP werecarried out by HPLC.TAN and EC were calculated.Cerebral infarct size wasdetermined based on 2,3,5-triphenyltetrazolium chloride staining.Cerebral cellapoptosis was detected using the TUNEL method.Expressions ofapoptosis-associated genes Fas and FasL were measured using Real-time PCR andexpressions of apoptosis-associated proteins Fas and FasL were measured usingWestern blot method.Changes of cerebral cortex and hippocampus morphologywere observed after HE staining.Activity of myeloperoxidase (MPO),total-superoxide dismutase (T-SOD),manganese-superoxide dismutase (Mn-SOD),glutathione peroxidase (GSH-PX)and xanthine oxidase (XOD)in cerebral tissue,content of malonaldehyde (MDA)and concentration of nitric oxide (NO)inserum were detected by spectrophotometer.Mn-SOD mRNA was measured byRT-PCR method.Content of endothelin-l(ET-1)and activities of tissueplasminogen activator(t-PA)and plasminogen activator inhibitor-1(PAI-1)were measured by ELISA.Results:1 Effects of NDLIP on neural behaviors and energy metabolism inducedby cerebral I/R injuryCompared with I/R group,the neural behaviors marks of ECIP+I/R groupand NDLIP+I/R group decreased (P＜0.01),the contents of ATP (P＜0.01),ADP (P＜0.01)and AMP (P＜0.05)in ECIP+I/R group and NDLIP+I/R groupincreased.The calculation value of TAN (P＜0.01)and EC (P＜0.01)inECIP+I/R group and NDLIP+I/R group increased,too.2 Effects of NDLIP on cerebral cell death and apoptosis induced by cerebral I/RinjuryCompared with I/R group,cerebral infarct size were diminished (P＜0.05)in ECIP+I/R group and NDLIP+I/R group.Activities of MPO were decreased(P＜0.01),apoptosis of cortex cell (TUNEL:P＜0.01)and hippocampus cell(TUNEL:P＜0.01)in ECIP+I/R group and NDLIP+I/R group decreased.Theanti-death protection of NDLIP was as effective as that of ECIP.3 Apoptosis mechanism of prevention of NDLIP against cerebral I/RinjuryCompared with I/R group,expressions of apoptosis gene Fas (P＜0.01)and FasL (P＜0.01)in cortex in ECIP+I/R group and NDLIP+I/R groupdecreased,expressions of Fas (P＜0.01)and FasL (P＜0.01)in hippocampus ofECIP+I/R group and NDLIP+I/R group decreased.Compared with I/R group,expressions of apoptosis protein Fas (P＜0.05)and FasL (P＜0.05)in cortex ofECIP+I/R group and NDLIP+I/R group decreased.4.Effects of NDLIP on cerebral antioxidative activity after cerebral I/R injuryCompared with I/R group,the activities of T-SOD (P＜0.01),Mn-SOD(P＜0.01)and GSH-PX (P＜0.01)in cortex and activities of T-SOD (P＜0.01)andMn-SOD mRNA(P＜0.01)in hippocampus of ECIP+I/R group and NDLIP+I/Rgroup increased,the expression of Mn-SOD in cortex (P＜0.01)and inhippocampus (P＜0.01)of ECIP+I/R group and NDLIP+I/R group increased.The activities of XOD (P＜0.01)and MDA content in cortex (P＜0.01)and in hippocampus (P＜0.01)of ECIP+I/R group and NDLIP+I/R group decreased.5.Effects of NDLIP on blood vessel endothelial function before and aftercerebral I/R injuryBefore ischemia,there was no difference on the concentration of NO inserum among I/R group,ECIP+I/R group and NDLIP+I/R group.But comparedwith I/R group,NO in serum increased (P＜0.05)and ratio of ET-1 and NOdecreased (P＜0.05)in ECIP+I/R group and NDLIP+I/R group.After 24h of reperfusion,compared with I/R group,the concentration ofET-1 in serum decreased (P＜0.01),the concentration of NO in serum increased(P＜0.01)and the ratio of ET-1 and NO decreased (P＜0.05)in ECIP+I/R groupand NDLIP+I/R group.The endothelial protection of NDLIP was as effective asthat of ECIP.6 Effects of NDLIP on fibrinolysis system before and after cerebral I/R injuryBefore ischemia,there was no any difference on fibrinolytic indicatorsamong the groups.After 24h of reperfusion,compared with I/R group,theactivities of t-PA (P＜0.01)increased and the activities of PAI-1 decreased(P＜0.01)in ECIP+I/R group and NDLIP+I/R group.Conclusion:1.NDLIP decreased neural behavior marks and improved neurologicaldeficits of rats after cerebral I/R.NDLIP increased contents of ATP,ADP andAMP and calculation value of TAN and EC,improve energy metabolism aftercerebral I/R.2.NDLIP decreased cerebral infract size and cell apoptosis,attenuatedcortex and hippocampus morphology injury.The anti-cell death effect ofNDLIP was as effective as that of ECIP.The mechanism was involved in thedecrease of MPO activity and the down regulation of expressions of apoptosisassociated protein Fas and FasL.3.The mechanism of the prevention of NDLIP on cerebral I/R was involvedin the down regulation of expressions of apoptosis associated protein Fas andFasL.4.NDLIP increased cerebral antioxidative ability after I/R injury, decreased peroxidative damage.The antioxidative protection of NDLIP was aseffective as that of ECIP.5.In normal condition,NDLIP increased the concentration of NO inserum,slanted the balance between ET-1 and NO toward NO.After I/R injury,NDLIP decreased the disturbance of endothelial function,increased NO inserum,decreased ET-1 concentration and maintained the balance betweenET-1 and NO to approach normal.The endothelial protection of NDLIP was aseffective as that of ECIP.6.NDLIP increased t-PA activities and decreased PAI-1 activities.Theeffect on fibrinolysis system of NDLIP was as effective as that of ECIP.更多还原