【作者】 郭岩；

【导师】 陈志武； 赵维中；

【作者基本信息】 安徽医科大学 ， 药理学， 2008， 博士

【摘要】 脑缺血性疾病是严重危害人类生命健康的常见病和多发病,具有很高的死亡率和致残率,但脑缺血的药物治疗效果却不尽如人意。因此,开发预防和治疗脑缺血损伤有效药物是国内外医药界特别关注的热点。映山红总黄酮（total flavonesof rhododendra,TFR）是从映山红中提取的主要有效部位,其主要成分包括金丝桃甙、槲皮素和映山红素等,有较强的抗心肌缺血损伤作用。但迄今为止,尚无对映山红总黄酮抗脑缺血作用的系统研究,因此,为加快开发利用映山红总黄酮,本研究将从整体水平、细胞水平评价TFR对局灶脑缺血再灌注损伤、全脑缺血再灌注损伤和海马细胞缺氧复氧损伤的保护作用,分析TFR与神经元缺血缺氧损伤后NOS mRNA表达、ERK和JNK通路、Ca²⁺和EDHF的关系。目的:观察TFR对小鼠和大鼠局灶性脑缺血再灌注损伤是否具有保护作用,以及保护作用是否与脂质过氧化和NO有关。观察TFR对大鼠全脑缺血再灌注损伤是否具有保护作用,以及保护作用是否与ERK和JNK信号转导通路有关。在细胞水平,探讨TFR对大鼠海马神经元体外缺氧复氧的保护作用,以及TFR作用与Ca²⁺和EDHF的关系。方法:1.采用线栓法制作小鼠和大鼠大脑中动脉阻塞再灌注模型（middle cerebralartery occlusion,MCAO）,脑缺血2 h再灌注24 h后进行小鼠神经功能缺失评分;干湿法测定脑含水量;TTC染色显示大鼠再灌注后的梗塞灶,计算梗塞灶体积;测定血清和脑组织中LDH活力、MDA和NO含量及脑组织中SOD和GSH-PX活力;用RT-PCR法测定脑缺血再灌注后梗塞半球的iNOS、nNOS、eNOS mRNA表达的变化。2.采用Pulsinelli等介绍的四血管结扎法（4-vessel occlusion,4-VO）建立大鼠全脑缺血再灌注损伤模型。记录脑缺血前、缺血10 min和再灌注后5min、10 min、15 min、30 min、45 min、60 min的脑电图改变;采用HE染色法观察脑组织病理结构改变,进行脑组织病理评分;检测血清中LDH活性和NSE含量的变化;采用蛋白免疫印迹方法;测定大鼠脑皮质和海马组织中ERK1/2、p-ERK1/2、JNK1/2和p-JNK1/2在脑缺血再灌注后表达的变化,以及TFR对此变化的影响。3.采用大鼠原代培养的海马神经元缺氧4 h再复氧24 h模型。用MTT染色来评价神经元存活率;收集细胞上清液,进行LDH活性、NO和MDA含量测定;以Fluo-3/AM染色观察海马神经元内Ca²⁺的变化。4.在海马细胞缺氧前分别用脑血管段和脑微血管内皮细胞预处理,然后行海马神经元MTT染色率和上清液中LDH活性、NO含量的测定。结果:1.在小鼠MCAO模型上,TFR（30、60、120 mg/kg）能明显改善神经功能障碍（P＜0.05,P＜0.01）,明显降低缺血侧脑组织的含水量,使缺血侧脑水肿明显减轻（P＜0.01）,显著降低血清中LDH活性,MDA含量和NO含量（P＜0.05,P＜0.01）。2.在大鼠MCAO模型上,TFR（15、30、60 mg/kg）缺血再灌注半球梗塞体积百分比减小（P＜0.05）,使脑组织中LDH活性和MDA含量及NO含量下降,SOD和GSH-PX活性均升高（P＜0.05,P＜0.01）。3.在大鼠4-VO模型上,脑缺血时大鼠的脑电图幅度迅速下降,再灌注后脑电幅度逐渐恢复。TFR（15、30、60 mg/kg）在再灌注5 min时脑电图幅度与NS组比较无差异:在再灌注10 min、15 min和30 min时TFR（15、30、60 mg/kg）脑电图幅度尽管比NS组幅度高,但无统计学意义;再灌注45 min和60 min时,TFR（15、30、60 mg/kg）脑电图幅度与NS组比较有显著差异（P＜0.05,P＜0.01）。结果提示TFR可促进再灌注时大鼠脑电的恢复。同时TFR（15、30、60 mg/kg）能不同程度地改善脑缺血再灌注大鼠脑组织病理结构的改变,能减少血清中LDH的活力和NSE的含量（P＜0.05,P＜0.01）。4.在大鼠局部脑缺血2 h再灌注24 h模型上,30和60 mg/kg TFR可以显著增加脑组织eNOS mRNA的表达,降低脑组织iNOS mRNA和nNOS mRNA表达（P＜0.05,P＜0.01）,减少NO的生成。6.30和60mg/kg TFR对大鼠全脑缺血再灌注损伤脑皮质和海马组织ERK1/2和JNK1/2总体蛋白水平没影响,但对其激活产生了影响,促进ERK1/2信号通路的激活,抑制JNK1/2信号通路的激活。6.在大鼠原代海马神经元缺氧4 h复氧24 h模型上,TFR（10、20、40、80、160 mg/L）显著降低神经元的MTT染色和减少海马细胞LDH的漏出（P＜0.05,P＜0.01）,TFR（40、80、160 mg/L）显著减少上清液NO和MDA的含量（P＜0.05,P＜0.01）。7.在大鼠原代海马神经元缺氧4h复氧24h模型上,与模型组相比,TFR（10、20、40、80、160 mg/L）显著抑制海马细胞内钙离子的增加（P＜0.05,P＜0.01）,并呈一定的浓度依赖性。8.在大鼠原代海马神经元缺氧4 h复氧24 h模型上,TFR（10、20、40、80、160 mg/L）进一步升高加入脑血管段和脑微血管内皮细胞的海马神经元的活力,减少海马细胞LDH的漏出（P＜0.05,P＜0.01）,对NO的产生无影响;再加入Indo和L-NAME,神经元的活力和海马细胞LDH的漏出无进一步改变,NO产生减少。结论:1.TFR对小鼠和大鼠局部缺血再灌注损伤的脑组织具有保护作用。2.TFR对大鼠全脑缺血再灌注损伤具有保护作用。3.TFR可以提高海马神经元对于缺氧复氧的耐受性。4.TFR增加大鼠脑组织eNOS mRNA的表达,降低iNOS mRNA和nNOS mRNA表达,从而影响血清和脑组织中NO的含量;增加大鼠脑组织ERK1/2的激活,抑制JNK1/2在缺血再灌注后的激活;抑制海马细胞缺氧复氧时细胞内钙离子的增加;脑血管内皮,尤其是非NO和非PGI₂,即假定的EDHF可能参与了TFR的作用。5.TFR作为抗脑缺血再灌注损伤的新药开发有潜在的临床应用价值。更多还原

【Abstract】 Cerebrovascular ischemic disease is a source of mortality and profound morbidity that remains pervasive in the modern world.Neuroprotective agents reduce the injuries in ischemic penumbra and promote the recovery of brain function.Finding more effective neuroprotective agents with fewer risks has been a challenge.Some traditional Chinese medicines have been proven to be effective in alleviating symptoms that are similar to those induced by cerebral ischemia.It was found that some traditional Chinese medicines,especially those contained flavones,have protective effects against cerebral ischemic injury.Rhododendra is one of the plants that is rich in flavones,such as quercetin,hyperin and rutin,and has been used for treating patients with bronchitis for hundreds of years in China.Recent studies have shown that most flavones have protective effects against cerebral ischemic injury.Also,total flavones of rhododendra （TFR） have protective effects against myocardial ischemic injury by scavenging oxygen fiee radicals and inhibiting nitric oxide.However,there is very limited information about the protective effect of TFR on animal models of cerebral ischemic reperfusion injury.To determine the protective effect and mechanism of TFR on cerebral ischemic reperfusion injury,the models of local cerebral ischemia reperfusion in mice and rats, global ischemia reperfusion in rats and anoxia and reoxygenation in rat hippocampal neurons were used in this study.Influence of TFR on expression of NOS mRNA,ERK and JNK signal pathway,Ca²⁺ and EDHF were analyzed to clarify the possible mechanism. Purpose:In the experiments in vivo,it was determined whether TFR has protective effects on injury induced by local cerebral ischemia reperfusion and by global cerebral ischemia reperfusion.In the experiments in vitro,it was determined whether TFR protects neuron from injury induced by anoxia and reoxygenation in rat primary hippocampal neurons. To clarify the possible mechanism,the effects of TFR on lipid peroxidation,NOS mRNA expression intracellular calcium and EDHF were observed.Methods:1.Middle cerebral artery occlusion（MCAO） was performed to induce local brain ischemia reperfusion model by an intraluminal filament in mice and rats.MCA was occluded for 2h and the brain was reperfused for 24h at that moment neurologic deficiency scores were assessed,and infarct volume was measured by TTC staining. Brain water content,plasma and brain levels of malondialdehyde,nitric oxide contents, lactate dehydrogenase activity,brain levels of superoxide dismutase and glutathione peroxidase activities were evaluated.Expression levels of inducible nitric oxide synthase mRNA,neuronal nitric oxide synthase mRNA and endothelial nitric oxide synthase mRNA were detected by RT-PCR at reperfusion 24h after cerebral ischemia.2.Cerebral ischemia reperfusion was induced by 4-vessel occlusion（4-VO） in rat. Changes in electroencephalograph was recorded before and after ischemia 10min and after reperfusion 5min,10min,15min,30min,45min and 60min.HE staining was used to observe brain pathologic changes.Measuring the releases of lactate dehydrogenase and NSE content.The activation extracellular-signal regulated kinase1/2 and c-Jun N-terminal protein kinase1/2 were investigated by western blot.3.The injury of hippocampai neurons after anoxia and reoxygenation and the protective effects of TFR were observed on cultured neonatal rat primary hippocampal neurons. Cell viability of hippocampal neurons was quantified by measuring MTT staining,the releases of LDH and the content of NO and MDA were measured.The intracellular calcium indicated by the fluorescence in neurons was detected.The effect of brain vessle or cerebral micorvessel endothelial cell on anoxia injury of hippocampal neurons was observed by using MTT staining method and measurement of LDH and NO.Results:1.On MCAO model in mice,TFR（30,60,120 mg/kg） significantly ameliorated the neurological deficit（P＜0.05 or P＜0.01） and reduced the brain water content （P＜0.05）.The activity of LDH and the contents of MDA and NO in plasma were decreased（P＜0.05 or P＜0.01）.2.On MCAO model in rats,TTC staining showed infarct volume of rats was significantly smaller in the TFR（15,30,60 mg/kg） group after 24 h reperfusion compared with NS control group（P＜0.05）.The activities of LDH,SOD and GSH-PX in brain were enhanced,while the contents of MDA and NO in brain were decreased （P＜0.05 or P＜0.01）.3.On 4-VO model in rats,compared with sham-operate group,EEG amplitude decrease significantly after ischemia and recovery after reperfusion in NS control and TFR（15, 30,60 mg/kg） groups.At 5 min after reperfusion,there was no difference between the EEG amplitude in treated groups of 15,30,60 mg/kg TFR and that in untreated group （NS control）（P＞0.05）.EEG amplitud of TFR（15,30,60 mg/kg） groups was higher than that of NS control at 10 min,15 min and 30 min after reperfusion,however it did not reach the level of statistical significance.At 45 min and 60 min,EEG amplitud of TFR（15,30,60 mg/kg） groups was significantly higher than that of NS control（P＜0.05, P＜0.01）.TFR（15,30,60 mg/kg） could significantly improve brain pathologic changes, and inhibite the increases of LDH relasing and NSE content. 4.On primarily cultured hippocampal neurons hypoxia and reoxygenation model,TFR （10,20,40,80,160 mg/L） significantly reduced MTT staining of rat primary hippocampal neurons compared with NS control group（P＜0.01）.TFR mardedly inhibited A/R-induced increases of LDH and NO release（P＜0.05）5.TFR（30,60 mg/kg） significantly suppressed the expression of iNOS and nNOS mRNA.Meanwhile,the expression of eNOS mRNA in brain was up-regulated in the TFR（30,60 mg/kg） group（P＜0.05 or P＜0.01）.6.On the global cerebral ischemia reperfusion in rats,TFR（30,60 mg/kg） could not change total protein level of ERK 1/2 and JNK1/2,but TFR could increase the activation of ERK and inhibit the ctivation of JNK in ischmeia reperfusion.7.After hippocampal neurons anoxia and reoxygenation,TFR（10,20,40,80,160 mg/L） markedly decreased the intracellular calcium from 499.02±53.87（control group） to 397.28±94.82,335.89±75.09,238.51±38.40,147.56±17.76 and 88.50±7.03, respectively.8.Before anoxia and reoxygenation hippocampal neurons pretreatment with cerebral vessel or cerebral micorvessel endothelial cell respectively,TFR（20,40,80,160 mg/L） significantly increase the neuron viability and reduce the activity of LDH furtherly,but NO content don’t change in culture medium.When adding with Indo and L-NAME, neuron viability and LDH don’t change furtherly,but NO content was reduced in culture medium.Conclusion:1.TFA has remarkable protective effects on local cerebral ischemic reperfusion injury in mice and rats.2.TFA has remarkable protective effects on global cerebral ischemic reperfusion injury in rats. 3.TFR protects neuron from anoxia and reoxygenation injury in vitro.4.The mechanisms that TFR protect cerebral ischemia and reperfusion injury include anti-lipid peroxidation,up-regulating eNOS mRNA expression and down-regulating iNOS mRNA and nNOS mRNA expression,inceasing the activation of ERK1/2 and inhibiting the activation of JNK1/2,inhibiting the neurons calcium overload.Vascular endothelium,especially non-NO-non-PGI₂（supposed EDHF）,might involve the protection of TFR partially.5.TFR may be of value as a new drug against cerebral ischemia reperfusion for clinical application.更多还原