【作者】 季伟；

【导师】 梅和珊；

【作者基本信息】 河北医科大学 ， 药理学， 2010， 硕士

【摘要】 缺血再灌注脑组织自由基大量产生和血小板活化因子等活性物质的释放与神经元损伤密切相关,开发具有清除自由基和对抗血小板活化因子作用的神经保护剂对减轻缺血再灌注损伤具有重要意义。银杏内酯B(ginkgolide B, GinB)为银杏提取物的主要活性成分之一,是血小板活化因子特异性的拮抗剂,研究显示银杏内酯B通过抑制血小板活化因子功能而抑制缺血所致炎性反应、改善脑能量代谢和脑组织病理学改变,保护脑组织免受缺血再灌注损伤。本研究拟采用大鼠局灶性脑缺血再灌注模型,通过观察银杏内酯B对缺血后脑组织形态学、血清中丙二醛(MDA)含量、羟自由基(·OH)产生能力、超氧化物歧化酶(SOD)活力及脑组织中超氧化物歧化酶(SOD)和诱导型一氧化氮合酶(iNOS)的表达,评价银杏内酯B对缺血再灌注脑损伤的神经保护作用,并对其作用机制进行探讨。目的:评价银杏内酯B在大鼠局灶性脑缺血再灌注损伤中的神经保护作用。方法:SD大鼠随机分为溶剂对照组、假手术组、GinB 1mg·kg-1组,GinB 2mg·kg-1组,GinB 4mg·kg-1组和金纳多12 mg·kg-1组。采用线拴法闭塞大脑中动脉(MCAO)制备大鼠局灶性脑缺血再灌注模型,并于插入拴线后尾静脉立即分别注射溶剂生理盐水、金纳多和不同剂量的银杏内酯B(GinB),假手术组行手术通路,除不插线外,其余操作步骤同手术组。手术后6小时进行神经功能评分、TTC染色法测定脑梗塞体积、HE染色法观察脑组织形态学变化、黄嘌呤氧化酶法测定血清中超氧化物歧化酶(SOD)活性、硫代巴比妥酸法丙二醛(MDA)含量、Fenton反应测定羟自由基(·OH)产生能力、免疫组织化学法观察脑缺血再灌注损伤后大鼠脑组织中超氧化物歧化酶(SOD)和诱导型一氧化氮合酶(iNOS)的表达及银杏内酯B对超氧化物歧化酶和诱导型一氧化氮合酶的表达的影响。结果:1.大鼠脑缺血再灌注后,假手术组未见行为异常,神经功能评分为0分;溶剂对照组大鼠于缺血再灌注后出现明显的行为改变,神经功能评分最高,为9.50±0.53分,金纳多与银杏内酯B 1、2和4 mg·kg-1可不同程度的改善大鼠的神经症状,神经功能评分分别降低至6.38±0.92分、8.38±0.52分、7.50±0.53分和6.38±1.06分,与溶剂对照组比较均具有显著性差异(P<0.01,P<0.05)。2.手术后6小时,除假手术组外,其余各组大鼠大脑皮质和纹状体均出现明显的梗死灶,银杏内酯B 1、2和4 mg·kg-1可明显缩小脑缺血再灌注损伤后脑梗死体积,脑梗死体积分别为(20.89±3.19)%、(19.65±3.27)%和(17.38±2.62)%,与溶剂对照组比较(27.97±2.16)%梗死体积分别降低25.31%、29.75%和37.86%,差异具有显著性(P<0.01);金纳多可产生与银杏内酯B相似的作用,梗死体积降至(19.29±3.59)%,与溶剂对照组相比有显著性差异(P<0.01)。3.溶剂对照组大鼠脑缺血再灌注后脑含水量为(81.1±1.2)%,明显高于假手术组(78.3±1.2)%,两者比较有显著性差异(P<0.01)。4 mg·kg-1 GinB可明显降低大鼠脑缺血再灌注损伤后脑含水量,与溶剂对照组相比较有极显著性差异(P<0.01)。金纳多、1 mg·kg-1和2 mg·kg-1银杏内酯B虽有一定降低脑含水量的作用,但与溶剂对照组比较无显著性差异(P>0.05)。4.脑缺血再灌注损伤后,溶剂对照组血清超氧化物歧化酶(SOD)活力为148±33 U·ml-1,明显低于假手术组401±87 U·ml-1,降低幅度达63%;金纳多和银杏内酯B 4mg·kg-1可明显对抗脑缺血再灌注损伤所致SOD活力的降低,SOD活力分别为315±79 U·ml-1和250±78 U·ml-1,与溶剂对照组比较有显著性差异(P<0.01);银杏内酯B1 mg·kg-1和2 mg·kg-1虽可增加血清SOD活力(分别为166±28 U·ml-1和202±35 U·ml-1),但与溶剂对照组比较无显著性差异(P>0.05)。5.大鼠大脑中动脉闭塞后,溶剂对照组血清MDA含量为20.97±2.92 nmol·ml-1,明显高于假手术组14.77±1.80 nmol·ml-1(P<0.01),金纳多和不同剂量的银杏内酯B均可显著降低脑缺血再灌注损伤引起的脂质过氧化产物MDA含量的升高,分别为:金纳多组17.20±2.20 nmol·ml-1、银杏内酯B 1 mg·kg-1组18.24±3.12 mmol·ml-1、银杏内酯B 2 mg·kg-1组17.95±2.81 mmol·ml-1、银杏内酯B4 mg·kg-1组16.12±2.24 mmol·ml-1,降低幅度分别达17.98%、13.02%、14.40%和23.13%,与溶剂对照组比较具有显著性差异(P<0.05,P<0.01)。6.手术后,假手术组血清羟自由基生成能力为145±52 U·ml-1,明显低于溶剂对照组294±40 U·ml-1(P<0.01),金纳多和银杏内酯B具有清除羟自由基的作用,可明显降低脑缺血再灌注损伤后血清·OH水平的升高,降低幅度达35.71%、13.95%、29.25%和31.63%,金纳多和银杏内酯B2 mg·kg-1、4 mg·kg-1清除血清·OH作用强,为189±37 U·ml-1、208±21 U·ml-1和201±28 U·ml-1,与溶剂对照组比较有极显著性差异(P<0.01),银杏内酯B 1mg·kg-1虽可降低·OH的生成能力,但与溶剂对照组比较无显著性差异(253±31 U·ml-1,P>0.05)。7.组织形态学观察结果显示,溶剂对照组缺血侧大脑皮层和纹状体出现明显坏死病灶,神经元大量消失;金纳多和银杏内酯B组可见神经元变性,神经元细胞核增大、浓染,核仁、核膜不清等病理改变,但与溶剂对照组比较有明显的改善;银杏内酯B组随着剂量增加,坏死神经元数目明显减少;假手术组未见明显的组织病理学改变,大脑皮层细胞形态完整,神经细胞核仁清晰,核圆形,核膜完整,神经胶质细胞少,且散在分布。8.免疫组织化学检测显示,假手术组神经元胞浆可见Mn-SOD弱阳性表达;溶剂对照组神经元变性,可见不同程度Mn-SOD阳性表达;金纳多组神经元阳性表达明显增强;不同剂量银杏内酯B组变性神经元阳性表达较溶剂对照组组增强,但阳性表达明显较金纳多组弱。9.各组脑组织在脉络膜上皮细胞均有iNOS阳性表达,大脑皮层神经元和神经胶质细胞偶见表达,假手术组皮质内小血管内皮细胞弱阳性表达;溶剂对照组可见皮质内小血管内皮细胞表达增强,金纳多组内皮细胞阳性表达较溶剂对照表达减弱,但较假手术组表达增强。银杏内酯B组随着剂量的增大,内皮细胞NO表达逐渐减弱。结论:银杏内酯B对脑缺血再灌注损伤有明显的保护作用,其作用机制与拮抗血小板活化因子受体,抑制和减少体内脂质过氧化产物和自由基的产生,增强机体内源性抗氧化酶系统功能有关。更多还原

【Abstract】 The pathogenesis of brain ischemia-reperfusion injury results from the generation of many active substances, including free radicals and platelet-activating factor. It is of significance against ischemia reperfusion injury to develop effective neuroprotectors through scavenging free radicals and antagonizing platelet-activating factor.Ginkgolide B, one of the main active component of the Ginkgo biloba extract, is a specific antagonist of platelet-activating factor. Experimental studies demonstrated that ginkgolide B could inhibit inflammatory reaction, improve energy metabolism and the pathological changes after ischemia reperfusion injury through antagonism of platelet-activating factor. In present study the brain histomorphology, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), the generation capacity of hydroxyl radical, the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in brain were taken to evaluate the neuroprotective effects and the mechanisms of ginkgolide B action on the transient focal cerebral ischemia in rats.Objective: To evaluate the neuroprotective effects of ginkgolide B on focal cerebral ischemia-reperfusion injury.Methods: The rats were randomly divided into 6 groups, which were solvent group, sham group, GinB 1mg·kg-1 group, GinB 2mg·kg-1 group, GinB 4mg·kg-1 group and ginaton 12mg·kg-1 group. Focal cerebral ischemia-reperfusion was produced by introducing a nylon suture above the bifurcation of the internal carotid artery (ICA) to block the blood flow to the middle cerebral artery (MCA) for 2 hours and drawing the nylon suture and keeping the reperfusion for 4 hours. The rats in sham group underwent the same surgical procedure without inserting nylon suture into the ICA. The rats in other groups were injected intravenously solvent, ginaton or different dosages of ginkgolide B (GinB)immediately after inserting nylon suture. The infarct volume, the histological changes of brain tissue, the neurological score, the brain water content, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), the generation capacity of hydroxyl radical, the expression of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in cerebral cortex were determined respectively at 6 hour after the middle cerebral artery occlusion (MCAO) to evaluate the effects of ginkgolide B.Results:1. At 6 hour after MCAO, the neurological score of sham group was 0. The rats in solvent group showed apparente neurologic deficits with the highest neurological score. Ginaton, GinB 1mg·kg-1, 2mg·kg-1 and 4 mg·kg-1 could ameliorate the neurologic deficits and the neurological score were reduced to (6.38±0.92), (8.38±0.52), (7.50±0.53) and (6.38±1.06) respectively, as compared to (9.50±0.53) in solvent group (P<0.01, P<0.05).2. All the rats developed obvious infarction in the ischemic hemisphere after MCAO except those in sham operation group. GinB 1, 2 and 4 mg·kg-1 showed a 25.31%, 29.75% and 37.86% reduction in cerebral infarct volume, compared with solvent group (P<0.01). A similar effect of reducing cerebral infarct volume was also observed in ginaton-treated group.3. The brain water content was(81.1±1.2)% in solvent group after MCAO, significantly higher than that in sham group (P<0.01). GinB4 mg·kg-1 could remarkedly reduce the brain water content(P<0.01). A reduction of brain water content were observed in the other group, but there was no significant difference compared to solvent group (P>0.05).4. The activity of superoxide dismutase(SOD)in serum was (148±33)U·ml-1 in solvent group after MCAO, much lower than (401±87) U·ml-1 in sham group, (315±79) U·ml-1 in ginaton-treated group and (250±78) U·ml-1 in GinB 4mg·kg-1 -treated group (P<0.01). GinB 1 and 2mg·kg-1 could raise SOD activity too, but there was no significant difference compared with solvent group (P>0.05).5. MDA content in sham group was 14.77±1.80 nmol·ml-1. After MCAO, the MDA content in serum raised to 20.97±2.92 nmol·ml-1 in solvent group. Ginaton and different dosages of GinB could significantly decreased the MDA content (17.20±2.20nmol·ml-1 in ginaton group with 17.98% reduction, 18.24±3.12 mol·ml-1 in GinB 1mg·kg-1 group with 13.02% reduction, 17.95±2.81 mmol·ml-1 in GinB 2mg·kg-1 group with 14.40% reduction and 16.12±2.24 mmol·ml-1 in GinB 4mg·kg-1 group with 23.13% reduction) in serum, compared with solvent group(P<0.05,P<0.01).6. A generation capacity of hydroxyl radicals in serum was 145±52 U·ml-1 in sham group, lower than 294±40 U·ml-1 in solvent group(P<0.01). Ginaton and GinB1, 2 and 4 mg·kg-1 could inhibit the generation of hydroxyl radical after MCAO and showed 35.71%, 13.95%, 29.25% and 31.63% reduction of hydroxyl radical in serum, respectively. Ginaton, GinB 2mg·kg-1 and 4mg·kg-1 showed significant effects on the clearance of hydroxyl radical as compared with solvent group(P<0.01), but GinB 1mg·kg-1 did not.7. In HE staining, cerebral tissue structure in solvent group showed ischemic damage both in cortex and striatum characterized by the degeneration and necrosis of neurons. Ginaton and GinB could improve the pathological changes induced by ischemia-reperfusion although the neuronal degeneration was observed. The number of necrotic cell in ginaton and GinB-treated group was less than that in solvent group. No pathological changes were observed in sham group.8. The immunochemical positive expression for Mn-SOD appeared in cytoplasm. A weak positive expression for Mn-SOD was observed in sham group. Compared with solvent group, the positive staining remarkedly increased in ginaton and GinB group. The positive expression in GinB group was weaker than that in ginaton group.9. There was positive expression for iNOS in cytoplasm. The positive expression notably appeared in chorioidal epithelium, but was rare in neuron and glia in all experimental group. The positive staining was obvious within vascular endothelial cell in solvent group than those in sham group. The positive expression for iNOS became weak in ginaton-treated and GinB-treated group compared with solvent group, and still a little stronger than that in sham group. With the dosage increasing,the iNOS expression became weak in GinB-treated group.Conclusion: Ginkgolide B showed significant protective effects against cerebral ischemic-reperfusion injury. The mechanism maybe related to antagonizing platelet-activating factor receptor, inhibiting the production of lipid peroxidants and the generation of free radicals, enhancing endogenous antioxidant enzyme activity.更多还原