【作者】 袁利邦；

【导师】 熊利泽； 董海龙；

【作者基本信息】 第四军医大学 ， 麻醉学， 2010， 硕士

【摘要】 研究背景缺血性脑血管病是导致人类死亡和致残的最严重疾病之一,尽管大量的科研人员在此领域进行了不懈的研究,但除了溶栓治疗之外,还没有任何一种治疗措施及药物被临床验证疗效确实可靠。由于溶栓治疗的时间窗很短,所以目前寻找一种确切并行之有效的神经保护剂成为较迫切的任务。本实验室长期致力于寻找和筛选具有脑保护作用的药物和措施。由于脑缺血再灌注损伤是由一连串的有害级联反应导致的,涉及多个病理环节,既往研究已证实针对某一单一环节的神经保护剂其临床疗效均不确切,因此神经保护剂联合应用或多作用靶点药物的研究成为当前脑保护研究的一种新途径。自由基损伤一直是脑缺血损害中的重要病理环节。在新药靶点研究中,针对自由基损伤的药物很可能成为脑保护新的候选药物。自由基分为氧自由基(OFR)和氮自由基(NFR)。氧自由基主要包括超氧阴离子(·O2-)、过氧化氢(H2O2)和羟自由基(·HO)。氮自由基主要指内源性一氧化氮(NO)、二氧化氮(NO2)及由NO与·O2-反应所生成的过氧亚硝基阴离子(peroxynitrite,ONOOˉ)。内源性NO在脑缺血再灌注损伤中的作用主要取决于合成NO的一氧化氮合酶(NOS)的类型,尽管由内皮细胞型一氧化氮合酶(eNOS)在缺血早期合成的少量NO可起到一定的神经保护作用,但由神经元型一氧化氮合酶(nNOS)、诱导型一氧化氮合酶(iNOS)在再灌注过程中大量产生的NO的神经毒性作用使得内源性NO在脑缺血损害中主要发挥负面效应。最新有研究提示外源性NO可抑制由内源性NO介导的c - jun N末端激酶3 (JNK3)磷酸化所导致的神经元凋亡产生保护作用,此外,外源性NO还可促进血管形成及神经细胞分化来发挥神经保护作用。同样有大量研究证实脑缺血再灌注过程中氧自由基爆发性释放在脑缺血再灌注损伤中扮演了多重有害作用,而氧自由基清除剂可通过清除氧自由基发挥一定的神经保护作用。以往针对自由基的新药研发均是将氧自由基与氮自由基两者分割开来分别进行。我们根据趋利避害,扶正抑邪的理念,前期已利用氧自由基清除剂阿魏酸(FA,图1A)和外源性NO供体单硝酸异山梨醇酯(ISMN,图1B)这两种药物设计并合成了可同时清除氧自由基和释放外源性NO的双靶点药物乙酰阿魏酸单硝酸异山梨醇酯(acetyl ferulaic isosorbide,AFI,图2),本研究旨在研究其脑保护作用并探讨相关机制。中药单体人参皂甙Rd(Gs-Rd)是用于治疗缺血性脑血管病的国家一类新药,目前正在进行临床三期研究。活血类中药单体川芎嗪、葛根素都已被大量研究证实具有脑保护效应,人参皂甙Rd与川芎嗪、葛根素联合应用后是否能发挥更加强大的脑保护作用值得进行进一步研究。本研究分两部分,实验一:研究可在体内同时清除OFR和外源性释放NO的双靶点药物——乙酰阿魏酸单硝酸异山梨醇酯(AFI)在脑缺血再灌注损伤中的保护效应,并探讨其抑制神经元凋亡的相关机制。实验二:探讨中药单体人参皂甙Rd与葛根素和(或)川芎嗪联合应用的脑保护效应。实验一AFI的脑保护效应及机制研究目的AFI对脑缺血再灌注损伤的保护作用及机制进行研究方法1 AFI对大鼠局灶性脑缺血再灌注损伤的保护作用60只SD大鼠,随机分为6组:对照(control)组,乙酰阿魏单硝酸异山梨醇酯(AFI)组,单硝酸异山梨醇酯(ISMN)组,阿魏酸钠(SF)组,阿魏酸钠+乙酰单硝酸异山梨醇酯(SF+ISMN)组,依达拉奉(edavarone)组。各组动物制备右侧MCAO模型,各给药组均于再灌注前10min腹腔注射相应药物,对照组给予相应体积的溶剂(1ml, 20%丙酮)。再灌注后72h神经功能学评分,并处死动物,取脑行2,3,5-氯化三苯四哇(TIC)染色,以测量脑梗死容积。2 AFI脑保护作用的剂量效应关系研究40只SD大鼠,随机分为四组:对照组,AFI 1mg/kg组,AFI 3mg/kg组,AFI 9mg/kg组。各组动物制备右侧MCAO模型,均于再灌注前10min给予相应剂量的AFI(1,3,9mg/kg)或溶剂。再灌注后72h神经功能学评分,并处死动物,取脑行2,3,5-氯化三苯四哇(TIC)染色,以测量脑梗死容积。3 AFI对脑缺血再灌注损伤后caspase-3活性的影响24只SD大鼠,随机分为假手术组,对照组和AFI组,各组动物制备右侧MCAO模型(假手术组不栓线,其余操作与其他两组相同),均于再灌注前10min给予AFI 3mg/kg或溶剂,再灌注后24h处死动物,分别取半暗带皮层和纹状体行caspase-3活性测定。4 AFI对神经元凋亡的影响36只SD大鼠,随机分为假手术组,对照组和AFI组,各组动物制备右侧MCAO模型,均于再灌注前10min给予AFI 3mg/kg或溶剂,分别于再灌注后4h和24h经左心室进行灌注固定,石蜡包埋切片,行TUNEL染色,每组6只动物,高倍镜下在相应部位取4个视野行TUNEL染色阳性神经元计数,计算均数及标准差。结果1神经功能学评分及脑梗死容积测定再灌注后72h,相比于对照组,AFI组,SF组,SF+ISMN组,edaravone组神经功能学均有改善(P<0.05),其中edaravone组改善最明显(P<0.001),AFI组次之(P<0.01),而ISMN组与对照组相比无明显统计学差异。再灌注后72h,相比于对照组,AFI组,SF组,SF+ISMN组,edaravone组梗死容纳百分比均有明显下降(P<0.05),其中edaravone组梗死容积最小(P<0.001),AFI组次之(P<0.01),而ISMN组与对照组相比无明显统计学差异。2剂量效应关系再灌注后72h, AFI 3mg/kg组及AFI 9mg/kg组均可以提高神经功能学评分(P<0.05),减少脑梗死容积(P<0.05),AFI 1mg/kg组神经功能学评分及脑梗死容积百分比与对照组相比并无明显统计学差异。3 AFI对脑缺血再灌注损伤后caspase-3活性的影响。再灌注后24h,想比于对照组,AFI可以明显降低半暗带皮层和纹状体的caspase-3活性(P<0.01)。4 AFI对神经元凋亡的影响相比于对照组,再灌注后4h,AFI可以明显减少半暗带皮层和纹状体TUNEL染色阳性神经元的数目(P<0.05),再灌后24h减少更明显(P<0.01)。结论AFI可以改善大鼠脑缺血再灌注损伤后的神经功能,降低脑梗死容积,进一步研究发现其可以通过降低caspase-3的活性,减少神经元的凋亡发挥神经保护作用。实验二Gs-Rd及其联合用药的脑保护效应研究目的探讨人参皂甙Rd及人参皂甙Rd与川芎嗪和(或)葛根素联合应用是否具有脑保护效应并比较单独使用人参皂甙Rd与联合用药之间的保护效应有无差异。方法50只雄性SD大鼠随机分为5组(每组10只)。各给药组按设定时间和剂量分别单独或联合腹腔注射相应药物,对照组给予相应体积的溶剂(20%丙二醇1ml)。各组行局灶性脑缺血,2h后恢复灌注至72h。在再灌注后72h进行神经功能评分(NBS),72h评分后取脑行TTC(2, 3, 5-氯化三苯四唑)染色计算脑梗死容积百分比。结果单独使用人参皂甙Rd及人参皂甙Rd与川芎嗪和(或)葛根素联合使用均能显著改善大鼠局灶性脑缺血再灌注损伤的神经功能评分(P<0.05),减少脑梗死容积(P<0.05),各给药组间相比均无统计学差异。结论单独使用人参皂甙Rd和人参皂甙Rd与川芎嗪和(或)葛根素联合使用均具有明显的脑保护效应,但联合用药组没有表现出强于单独给药组的协同保护效应。更多还原

【Abstract】 BackgroundIschemic cerebrovascular disease is one of the causes that result in human death and disability. Although a large number of researchers in this field have made unremitting studies, in addition to thrombolytic therapy, there are not any kind of treatment measures or medicines that their therapeutic effect have been proved reliable in clinic. As the time window of thrombolytic therapy is short, to find some effective neuroprotective agents become more urgently.We always focuse on finding and screening the drugs and measures which may have brain protection. Because cerebral ischemia-reperfusion injury is a cascade that include a series of insult factors involving several pathological aspects and previous studies have shown that almost all of the neuroprotective agents for a single insult factor are not effective in clinical research, the combination of neuroprotective agents or to develop multi-target drugs have become the new approach of cerebral protection. The role of nitric oxide (NO) is a hot issue in the study of cerebral ischemia-reperfusion injury. The majority view is that the role of endogenous NO depends on the nitricoxide synthase (NOS) type. the NO generated from the endothelium-nitricoxide synthase (eNOS) has the neuroprotective effect, that generated from induced-nitricoxide synthase (iNOS) and neuronal-nitricoxide synthase (nNOS) have neurotoxic effects. Several current studies suggests that exogenous NO can inhibit the harmful effects of endogenous NO, promote angiogenesis and neurogenesis to play a neuroprotective effect. The multiple harmful effects of the large number of OFR produced by cerebral ischemia-reperfusion injury is confirmed by a number of researches, so we designed and synthesized the double-target drug named acetyl ferulaic isosorbide using OFR scavenger FA and exogenous NO donor ISMN, and investigate it’s neuroprotective effect and the related mechanisms.Chinese traditional medicine play an important role in ischemic stroke treatment. Monomer ginsenoside Rd is a new drug for the treatment of ischemic cerebrovascular disease and it’s clinical phase III study is ongoing. The neuroprotective effects of tetramethylpyrazine and puerarin have been demonstrated, whether the combination of these three drugs have more stronger neuroprotective effects is worthy to further study.This study include two parts. Part I: To investigate the cerebral protective effect and related mechanism of the double-target drugs: acetyl ferulaic isosorbide (AFI); Part II: To study the whether the combination of these three drugs have more stronger neuroprotective effects than singles.Part I the neuroprotective effect of AFI: a novel ROS-eliminating and NO-releasing drug ObjectiveTo investigate the neuroprotective effect of AFI and related mechanism in rat.Method1 The neuroprotection of AFISixty male SD rats weighing 280 - 320 g were randomly assigned into 6 groups ( n = 10, each group): control group, AFI group, ISMN group, SF group, SF + ISMN group and Edavarone group. rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 72 h. 10 min before reperfusion, the drug or vehicle were respectively infused intraperitoneally. The neurological behavior scores (NBS) were assessed at 72 h after reperfusion. The percentage of infarct volume was determined at 72 h after reperfusion by using TTC stain.2 the dose-effect relationship of AFIForty male SD rats weighing 280 - 320 g were randomly assigned into 4 groups ( n = 10, each group): control group, AFI-1mg/kg group, AFI-3mg/kg group, AFI-9mg/kg group. Rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 72 h. 10 min before reperfusion, the AFI(1,3,9mg/kg) or vehicle were respectively infused intraperitoneally. The neurological behavior scores (NBS) were assessed at 72 h after reperfusion. The percentage of infarct volume was determined at 72 h after reperfusion by using TTC stain.3 The effect of AFI on caspase-3 activityTwenty four male SD rats weighing 280 - 320 g were randomly assigned into 3 groups ( n = 8, each group): sham group, control group, AFI-3mg/kg group. rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 24 h. 10 min before reperfusion, the AFI(3mg/kg) or vehicle were respectively infused intraperitoneally. 24 h after reperfusion, the animals were sacrificed and the penumbra cortex and striatum of right hemisphere were dissected for the measurement caspase-3 activity.4 The effect of AFI on neuron apoptosisFor immunohistochemistry and TUNEL analysis, 36 animals were randomly assigned into 3 groups ( n = 12, each group): sham group, control group, AFI-3mg/kg group. Rat were anesthetized with an overdose of 2% sodium pentobarbital and perfusion-fixed via the left ventricle using 4% paraformaldehyde at 4 h and 24 h after MCAO. The brain blocks were embedded in paraffin and cut into 5μm coronal sections and were used for and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. TUNEL-positive cell numbers were counted in a blind fashion in four fields in the penumbra cortex and striatum of right hemisphere at high-power microscopic magnification (×400) and expressed as the number of positive cells per high-power field. Data from six animals in each group at corresponding time point were analyzed.Result1 The AFI induced neuroprotection on neurologic scores and infarct volume.AFI, SF, Edaravone, the combined application of SF and ISMN all can siganifacantly elevate the neurological behavior scores (NBS) and reduce the infart volume of rats after reperfusion than those of control group ( P< 0.05). Edaravone has the best effect (P<0.001) and AFI has the better(P<0.01). There are no siginicant difference between ISMN group and control group on NBS and infart volume 2 The dose-effect relationship of AFICompare with the control group, AFI with the dose of 3 mg/kg and 9 mg/kg both elevate the neurological behavior scores (NBS) and reduced brain infarct volume after reperfusion(P<0.05), AFI with the dose of 1 mg/kg has not produce significant neuroprotection.3 The effect of AFI on caspase-3 activityAFI(3mg/kg) treatment can inhibit the caspase-3 activity in the the penumbra cortex and striatum of right hemisphere as compare with control group(P<0.01).4 The effect of AFI on neuron apoptosisIn the the penumbra cortex and striatum of right hemisphere, the number of apoptotic neurons in the AFI treatment (3 mg/kg) group was significantly lower than that in the control group at 4 h (P<0.05) and 24 h after reperfusion(P<0.01). ConclusionAFI has neuroprotective effect against cerebral ischemia-reperfusion injury. The neuroprotection of AFI may be mediated by it’s inhibition of caspase-3 and decrease the number of neuron apoptosis.Part II Neuroprotective effects study of ginsenoside Rd and it’s combined application with othersObjectiveTo investigate the neuroprotective effect of ginsenoside Rd and the combined application of ginsenoside Rd with puerarin and(or) tetramethylpyrazine in rats. Compare whether combination of ginsenoside Rd with puerarin and (or) tetramethylpy -razine have more effective protection than ginsenoside Rd.MethodFifty male SD rats weighing 280 - 320 g were randomly assigned into 5 groups ( n = 10, each group). Drugs were respectively infused intraperitoneally as single or combined method for medicine administration groups. The control group received infusions of vehicle. rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 72 h. The neurological behavior scores (NBS) were assessed at 72 h after reperfusion. The percentage of infarct volume was determined at 72 h after reperfusion. ResultEither ginsenoside Rd or the combined application of ginsenoside Rd with puerarin and(or) tetramethylpyrazine can siganifacantly elevate the NBS and reduce the infart volume after reperfusion than those of control group ( P< 0. 05). No significant difference was found in NBS and infarct volume among drug administration groups.ConclusionGinsenoside Rd and the combined application of ginsenoside Rd with puerarin and(or) tetramethylpyrazine have similar neuroprotective effects. The combined application of ginsenoside Rd with puerarin and (or) tetramethylpyrazine did not generate synergistic effect.更多还原