脑立轻胶囊对拟血管性痴呆大鼠空间学习记忆影响的研究

【作者】 时文远；

【导师】 王正君；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2008， 硕士

【摘要】 血管性痴呆(vascular dementia,VD)系指在各种脑血管疾病基础上引起的脑组织损害,并产生以高级神经认知功能障碍为主的一组临床综合征,是老年期痴呆的主要类型之一,临床主要表现为认知功能、社会生活能力的减退及情感性格的改变等症状,学习记忆能力受损突出,致残率高,严重影响患者的生活质量,造成了沉重的经济和社会负担。现代医学对该病尚无特效治疗手段,长期治疗费用昂贵。随着近年来中医药基础和临床研究的深入,中医药在防治VD上日益显示出优势。脑立轻胶囊根据VD多以“肾精亏虚、瘀阻脑脉”为其主要病机特点,以“补益肾精、化瘀通络”为其治则,用肉苁蓉、山茱萸、三七、银杏叶、葛根组方而成,在临床中取得了较好疗效。本课题通过动物实验方法研究脑立轻胶囊对拟VD大鼠空间学习记忆的影响,探讨其作用机制,为临床用本药治疗VD提供理论依据。实验研究分为以下3部分:1选用清洁级雄性SD大鼠80只,双侧颈总动脉永久性结扎法(2-VO)建立VD大鼠模型,造模后动物随机分为脑立轻胶囊高、中、低剂量组和模型组、假手术组、对照组,每组10只。对照组选用尼麦角林作为对照药物,动物灌胃给药,治疗期30天。2治疗期结束后行Morris水迷宫实验,通过定位航行实验、空间探索实验、可见平台实验,检测治疗后模型大鼠的空间学习记忆能力,比较各组之间成绩的差异。从行为学上观察脑立轻胶囊对拟VD大鼠记忆的影响,探讨其治疗作用。3采用4%多聚甲醛经心脏灌注,待鼠尾变硬后取脑,石蜡包埋、切片,HE染色观察各组大鼠海马CA1区锥体细胞形态变化,计数锥体细胞,比较脑立轻胶囊对大鼠海马CA1区锥体细胞的影响;免疫组化法检测各组大鼠海马CA1区与记忆关系密切的酶-胆碱乙酰转移酶(ChAT)和与突触相关蛋白-突触素(Syn)的表达,比较组间差异,探讨脑立轻胶囊对ChAT和Syn表达的影响。大鼠经2-VO法造模70只,术后3天死亡19只;假手术组造模10只,无死亡。实验期间又陆续死亡6只。实验结果显示造模后各组大鼠的水迷宫成绩较假手术组明显下降(p＜0.01),海马CA1区锥体细胞的计数及胆碱乙酰转移酶(ChAT)和突触素(Syn)的表达与假手术组比较亦具有显著性差异(p＜0.01),说明经2-VOS法造模后大鼠的行为学成绩、海马CA1区锥体细胞及相关蛋白明显下降,证实造模成功。高剂量组和对照组的各项结果相比较无显著性差异(p＞0.05),高剂量组与模型组及低剂量组结果相比较具有显著性差异(p＜0.01),而低剂量组与模型组各项结果比较改善不明显(p＞0.05),中剂量组的各项结果介于高低剂量之间。实验结果说明双侧颈总动脉永久性结扎(2-VO)造成了大鼠学习记忆障碍和脑组织病理形态以及相关蛋白的改变,证明模型成功。中药脑立轻胶囊可以改善拟VD大鼠的空间学习记忆能力,其机制可能是:对低灌注环境下的大鼠海马CA1区神经元具有保护作用,减轻缺血对其造成的损伤;抑制缺血后模型大鼠海马CA1区ChAT和Syn的降低,提高模型大鼠海马CA1区ChAT和Syn表达,改善胆碱能神经元的功能,促进突触重塑;实验结果与剂量有关,高剂量明显优于中剂量和低剂量。同时也从动物实验证实脑立轻胶囊治疗VD确有疗效,但该药保护锥体细胞和对ChAT及Syn表达影响的具体机制有待进一步研究。更多还原

【Abstract】 Vascular dementia(VD)is an impairment of cognitive function caused by cerebrovascular disease,which is one of the main styles of the aged dementias. Cognitive impairment,decreased the ability of memory and change of the Psychological signs are its main clinical manifestation.Although there is not VD’s name in traditional Chinese medicine(YCM),VD belongs to "idiotia","foolish disease","stupid disease","mind disease","dementia","depressed syndrome" in TCM age-old record.Owing to so high incidence of VD,and it has the high incidence of Mutilation disease.the quality of life is affected seriously,which further results in great social and family burden.The modem medicine has maken great development in VD,many researches have been made on VD,but there is not effective methods and medicine for VD.To control the development of pathogenetic condition and to improve brain function and quality of life are main goals of treatment on clinically,and it will spend much expense for long term therapy.Recently,as more and more basic and clinic researches on Traditional Chinese Medicine(TCM)treating VD have been made,more and more privileges have appeared,k is significant for adopting traditional Chinese drug to treat VD.TCM has accumulated a lot of experience.Naoliqing capsule is according to that "deficiency of kidney-essence,obstruction of collaterals by blood stasis" is one of the main pathogenesis of VD and the therapeutical principle of VD should be "tonified kidney-essence,disperse blood stasis and dredge collateral" on the basis of clinical experience.Naoliqing capsule is made from Desertliving Cistanche,Asiatic Cornelian Cherry Frui,Sanchi,ginkgo leaf,Kudzuvine Root.Through animal experiment to expound the pharmacology of Naoliqing,we will reveal the mechanism how Naoliqing treat VD.Therefore,we established VD animal model to estimate the effects of cognitive dysfunction with Naoliqing treatment fromethology and histomorphology.The experiment is departed into three parts:1 This topic Chooses healthy male SD rats to established VD animal model.The rat models of vascular dementia were made by permanent bilateral occlusion of both commen carotid arteries(2-VO),the models were divided into six groups at random: sham-operated group,model group,nicergoline group and Naoliqing large, medium、small dose group.the medicine was given to the models by intragastric administration.2 Testing learning and memory by Morris water-maze when models were treated for 30 days.3 Fixed up via perfusion of 4%paraformaldehyde solution,brain samples with HE staining and immunohistorychemistry were applied to analyze the state of pyramidal cell and expression of ChAT and Syn.Take count of the cells and Observe the appearance of acethylcholine ChAT and Syn in CA1 district in hippocampus and compare it.We take the Escape latency of MWM as the evaluation target.Compared with model group,the large dose group and nicergoline group’s escape incubation period is shorter(P＜0.01);compared with small group,the large group and medium group’s escape incubation period were shorter(P＜0.01).The quantity of pyramidal cells in hippocampal CA1 region in model group was significantly less than other group’s. The large group’s and The medium group’s the positive expression of ChAT and Syn in hippocampal CA1 region is significantly higer than model group and small dose group(P＜0.01).there was no significantly difference between small dose group and model group(P＞0.05).The Results of experiment shows that:2-VO could cause the impairment of learning and memory ability of model rats and brain tissue degeneration of VD model rats,so it succeeded in establishing VD animal models.Treating VD rats with Naoliqing capsule can obviously improve the function of spatial learning and memory of VD animal models,alleviating the impairment of neuron ischemia in the hippocampal CA1 region and enhance expression of ChAT and Syn in hippocampal CA1 region maybe the histopathological base for improving the cognitive dysfunction of VD rats.更多还原