健脑1方治疗血管性痴呆疗效观察及对NMDAR-CaMKⅡ通路的影响

发布时间：2018-01-02 18:28

本文关键词：健脑1方治疗血管性痴呆疗效观察及对NMDAR-CaMKⅡ通路的影响　出处：《广州中医药大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：血管性痴呆(Vascular Dementia, VD)是指缺血性、出血性脑血管疾病引起的脑损害导致的痴呆,它在各种老年期痴呆的病因中仅次于阿尔茨海默病(Alzheimer's disease, AD)和路易体痴呆(Dementia with Lewy body, DLB),约占老年期痴呆的20%左右。VD的患病率随年龄增长呈指数上升,严重影响到患者的生存质量,并给其家庭甚至社会带来了沉重负担。鉴于VD的可防治性,应及早干预治疗,预后良好。由于其病因及发病机制目前尚不清楚,且西药不良反应较多,运用中医药治疗VD越来越受到人们的关注,近年来,国内学者对中医药治疗本病进行了深入研究,发现中医药治疗VD的优势与特色在于可显著改善临床症状、减少不良反应。国医大师邓铁涛教授根据自己多年的临床经验,提出以“益气除痰活血法”治疗血管性痴呆的学术思想,邓老认为本病主要病机是气虚为本,痰瘀为标,由痰致瘀,以痰为主。治疗上牢抓中医辨证论治的“整体观”,认为本病虽病位在脑,但发病之根本在于脾,故强调本病治疗关键在于益气健脾,并主张治疗痰瘀同病时重在化痰,辅以祛瘀,自拟健脑1方在临床实践中疗效显著,值得深入研究及临床推广。为了更好地总结邓铁涛教授临床经验,探究其临证规律及学术特色,挖掘其科学内涵,冀以弘扬邓老学术思想,传承仁术。本课题是在中医理论的指导下,结合邓铁涛教授多年的临床经验,客观评价健脑1方治疗VD的临床疗效及其安全性,为本方良好的临床疗效提供客观数据支持,从而更好地继承名老中医学术经验,丰富中医学理论,继承和发扬中医药事业。并通过对大量国内外文献研究,提出“健脑1方通过干预NMDAR-CaMKⅡ信号传导通路调控一系列下游蛋白是其抗VD的可能作用机制”的假设。通过对VD大鼠海马NR2B和CaMKⅡ的基因表达深入研究有望阐明健脑1方影响VD大鼠的学习记忆能力主要的分子生物学作用机制,并为中医药治疗VD的研究提供新的靶点和思路。方法：临床研究：本课题采用平行、随机、对照实验方案,选择2014年1月至2014年12月就诊于广州中医药大学第一附属医院心内科及广东省第二中医院脑病科门诊及住院符合血管性痴呆(气虚痰瘀证)诊断标准患者60例,采用随机数字表法按照1：1的比例将60例患者随机均分成治疗组、对照组各30例,同时给予两组患者降压、降脂等一般治疗措施,其中治疗组给予健脑1方治疗,对照组予尼莫地平片治疗,治疗期间停用其它与治疗本病有关的内服药物,疗程共6周。治疗前与治疗6周后记录每例入组患者的MMSE评分、ADL评分、NPI评分、中医证候积分的变化,并记录治疗前后血常规、肝肾功能等安全性指标与治疗相关的不良反应事件,以客观准确评价健脑1方治疗血管性痴呆的临床疗效及安全性。实验研究：本研究采用双侧颈总动脉永久性结扎建立VD动物模型,将60只SD雄性大鼠随机分为假手术组、模型组、高剂量组、低剂量组及尼莫地平组,低、高剂量组、尼莫地平组大鼠分别灌服低剂量健脑1方、高剂量健脑1方和尼莫地平混悬液；模型组、假手术组均给予生理盐水灌胃,给药30天,用Morris水迷宫检测各组大鼠的学习记忆能力,HE染色观察大鼠海马组织学改变,用RT-PCR法检测大鼠海马NR2BmRNA及CaMKⅡmRNA表达水平,从而探讨邓铁涛教授健脑1方对血管性痴呆大鼠的学习记忆能力的影响,并从分子生物学角度探讨其可能的作用机制。成果：临床研究：试验结果显示：两组患者在性别、年龄、病程、病情及文化程度方面等因素构成比的差异性无统计学意义(P0.05),提示各组患者在性别、年龄、病程、病情及文化程度的构成方面基本均衡,具有可比性。两组患者治疗前MMSE、ADL、NPI及中医证候积分均未见有统计学差异(P0.05),具有可比性；最终研究结果可见：经过6周治疗,两组患者治疗后MMSE评分均较治疗前提高,具有显著性差异(P0.01)；治疗后两组患者MMSE评分比较无显著性差异(P0.05),两组治疗前后差值比较差异有统计学意义(P0.01)；治疗组总有效率80.0%,对照组总有效率53.3%,提示两组均能改善患者认知能力,且治疗组优于对照组(P0.05)。两组患者治疗后ADL评分均较治疗前降低,具有显著性差异(P0.01)；治疗后两组患者ADL评分及两组治疗前后差值比较均无显著性差异(P0.05)；治疗组总有效率23.3%,对照组总有效率30.0%,提示两组均能提高患者日常生活活动能力,且临床疗效相近(P0.05)。两组患者治疗后NPI评分与治疗前比较均无显著性差异(P0.05)；治疗后两组患者NPI评分及两组治疗前后差值比较均无显著性差异(P0.05)；治疗组总有效率6.67%,对照组总有效率6.67%,提示两组均未能显著改善患者精神行为症状(P0.05),且两组均不能有效减少VD患者照料者的苦恼程度(P0.05)。治疗组患者治疗后中医证候积分较治疗前降低,具有显著性差异(P0.01),对照组患者治疗后中医证候积分与治疗前比较无显著性差异(P0.05),治疗后两组患者中医证候积分及两组治疗前后差值比较均有显著性差异(P0.01)；治疗组总有效率为56.67%,对照组总有效率为10.0%,提示治疗组可显著改善患者中医证候,对照组患者中医证候改善甚微(P0.01)。参加本次研究的所有患者均未出现血常规及肝肾功能检查异常,两组患者出现的不良反应症状轻微,安全性好。实验研究：Morris水迷宫测试结果：随着训练天数的增加,各组大鼠逃避潜伏期逐渐缩短,说明各组大鼠在学习训练中逐渐学会寻找平台,但学习记忆能力存在显著差异,其中训练四天内每一天模型组大鼠逃避潜伏期均较假手术组明显延长(P0.05或P0.01),说明模型组大鼠空间学习记忆能力出现障碍,第三天,高剂量组大鼠逃避潜伏期较模型组明显下降(P0.05)；第四天,高剂量组及尼莫地平组大鼠逃避潜伏期均较模型组显著下降(P0.01和P0.05)。空间探索实验结果显示：120s内,高剂量组及假手术组大鼠穿越平台的次数明显高于模型组,差异具有统计学意义(P0.01)；各组大鼠停留在平台所在象限的游泳时间与模型组大鼠比较差异尚未发现统计学意义(P0.05),由此可见,实验结果说明高剂量组VD大鼠学习记忆能力显著提高。VD大鼠海马组织学改变：光镜下观察发现模型组大鼠海马神经细胞丢失；神经细胞核碎裂、核膜不清、核仁消失,神经细胞收缩,胞浆嗜酸性增强,胶质细胞反应性增生；病变集中在CA1、CA2及CA3区；尼莫地平组大鼠海马病变集中在CA1区,有神经元细胞损伤；高、低剂量组大鼠海马病变集中在CA1、CA3区,可见神经元细胞损伤,病变程度较模型组减轻,提示健脑1方及尼莫地平可减少VD大鼠海马组织神经元损伤范围及程度。大鼠海马NR2B mRNA及CaMKⅡmRNA表达：实验结果表明模型组大鼠海马NR2B mRNA的表达水平与假手术组相比明显降低(P0.01)。而与模型组相比较,尼莫地平组及高剂量组大鼠海马NR2B mRNA的表达水平显著增高,差异具有统计学意义(P0.05),低剂量组有增高NR2B mRNA表达的趋势。模型组大鼠海马CaMKⅡmRNA的表达与假手术组相比稍降低(P0.05),高剂量及尼莫地平组都能略微提高mRNA的表达水平,但差异无均统计学意义(P0.05)结论：临床研究结果提示健脑1方治疗VD在改善患者认知功能、提高患者日常生活活动能力方面具有良好疗效,还能明显改善患者的中医临床症状,但对VD患者精神行为症状未见明显改善,且不良反应少,安全性好。本课题研究印证了基于邓老提出的“益气除痰活血法”治疗血管性痴呆理念的正确性和科学性,以及基于邓老临床经验而创制的健脑1方的有效性和安全性。实验研究结果提示健脑1方可显著提高VD大鼠的学习记忆能力,并推测其作用机制可能与减少VD大鼠海马组织神经元损伤范围及程度,并可上调VD大鼠海马NR2B mRNA的表达水平有关。本实验发现VD大鼠海马CaMKⅡmRNA并没有得到同样的改善,提示CaMKⅡ可能不是健脑1方的重要作用靶点。
[Abstract]:Objective: vascular dementia (Vascular Dementia, VD) refers to ischemic brain damage, dementia, hemorrhagic cerebrovascular disease caused by it, after Alzheimer's disease in the etiology of senile dementia in (Alzheimer's disease AD) and Louis (Dementia with Lewy body body dementia, DLB), accounting for about old age 20%.VD the prevalence of dementia increased exponentially with the increase of age, seriously affect the patient's quality of life, and to their families and society a heavy burden. In view of prevention and treatment of VD, should be early intervention, the prognosis is good. Its etiology and pathogenesis is unclear, and many adverse reactions of Western medicine the use of Chinese medicine treatment of VD, more and more attention in recent years, domestic scholars on Chinese medicine treatment of this disease is studied, found the advantages and characteristics of traditional Chinese medicine in the treatment of VD is significantly improved by The clinical symptoms, reduce adverse reaction. The Chinese medicine master professor Deng Tietao according to their clinical experience for many years, put forward the idea of "Invigorating Qi and removing phlegm and activating blood" in the treatment of vascular dementia academic thought, Denglao thinks that its pathogenesis is the deficiency of Qi, phlegm stasis, the phlegm and blood stasis, phlegm in the treatment. Firmly grasp the dialectic of "wholeness", believe that although this disease disease in the brain, but the pathogenesis lies in the spleen, it emphasizes the treatment of this disease lies in Qi and spleen, and advocate the treatment of phlegm and blood stasis phlegm and removing blood stasis, when focusing on, with brain 1 party in clinical practice has obvious curative effect, worthy of further study and clinical application. In order to summarize the clinical experience of Professor Deng Tietao, to explore the clinical characteristics and academic characteristics, to explore its scientific connotation, in order to promote the academic thoughts of Deng, Renzhu inheritance. This paper is under the guidance of Chinese medicine theory, combined with Deng Tietao taught "Clinical experience for many years, the objective evaluation of 1 Jiannao Decoction on VD clinical curative effect and safety, to provide objective data support for the good clinical curative effect, so as to better inherit the old Chinese academic experience, enrich the theory of traditional Chinese medicine, inheritance and development of traditional Chinese medicine. Based on a large number of domestic and foreign literature research, put forward" 1 party intervention regulation by brain NMDAR-CaMK II signal transduction pathway downstream protein is its anti VD mechanism "hypothesis. Further research is expected to elucidate the molecular mechanism of brain 1 affect the ability of learning and memory in VD rats by mainly on VD rat hippocampal NR2B and CaMK II gene expression, and provide a new target and idea for the research of Chinese medicine for the treatment of VD. Methods: clinical study: this subject adopts a parallel, randomized, controlled experiment scheme, from January 2014 to December 2014 in Guangzhou Encephalopathy outpatient department of Cardiology the First Affiliated Hospital and the second hospital of Guangdong Province, University of traditional Chinese medicine and hospital with vascular dementia (Qi deficiency and phlegm and blood stasis syndrome) 60 cases of patients, randomly according to the ratio of 1:1 60 patients were randomly divided into treatment group and control group with 30 cases in each group, and given two patients with hypertension, cholesterol and other general treatment measures, the treatment group was treated with 1 brain decoction, control group received Nimodipine Tablets treatment, during treatment discontinuation and other treatment of the disease related to oral drugs, the treatment lasted for 6 weeks. Before treatment and after 6 weeks of treatment were recorded in groups of patients with MMSE score, ADL score, NPI score and the changes of TCM syndrome integral, and record the blood before and after treatment, liver and renal function indicators of adverse events associated with treatment, to objectively evaluate the clinical curative effect of 1 treatment of brain vascular dementia and ANN Full. Experimental research: animal model of VD was established by permanent bilateral common carotid artery ligation by this study, 60 male SD rats were randomly divided into sham operation group, model group, high dose group, low dose group and nimodipine group, low and high dose group, nimodipine group rats were fed low dose brain 1 Party, the 1 party and the high dose of Jiannao nimodipine suspension; model group, sham operation group were given normal saline, administered for 30 days, with the ability of learning and memory in Morris water maze test in rats, observe changes in hippocampus of rats with HE staining, RT-PCR assay in hippocampal NR2BmRNA and CaMK II mRNA expression level, so as to explore the influence of the ability of learning and memory of Professor Deng Tietao, the 1 Party of brain vascular dementia rats, and to explore its possible mechanism from the perspective of molecular biology. Results: the clinical study: the experimental results showed that the patients in the two groups of No, age, duration of illness, and the degree of cultural factors such as the ratio difference was not statistically significant (P0.05), suggesting that patients in gender, age, course of disease, the basic balance of composition condition and culture degree, comparable. Before MMSE, ADL two groups, NPI and TCM syndrome there were no statistically significant differences between the integral (P0.05), comparable; the final research results showed: after 6 weeks of treatment, improve the MMSE score of the two groups were lower than before treatment, with significant difference (P0.01); after treatment, MMSE scores between two groups had no significant difference (P0.05), there was statistical the significance of the two groups before and after treatment (P0.01); the difference between the treatment group total effectiveness 80%, control group total effective rate was 53.3%, suggesting that the two groups can improve the cognitive ability of the patients, and the treatment group than the control group (P0.05). The two groups of patients ADL score after treatment than before treatment Decreased, with significant difference (P0.01); after treatment, two groups of patients before and after treatment in the two groups of ADL score and the difference had no significant difference (P0.05); the total effective rate of treatment group 23.3%, control group total effective rate was 30%, suggesting that the two groups could improve the activities of daily living of patients, and the clinical curative effect is similar to (P0.05). NPI score of the two groups compared with before treatment showed no significant difference (P0.05); after treatment, two groups of patients before and after treatment in the two groups of NPI score and the difference had no significant difference (P0.05); the total effective rate of treatment group 6.67%, control group total effective rate was 6.67%, suggesting that the two groups no significant improvement in patients with behavioral and psychological symptoms (P0.05), and the two groups can not effectively reduce the distress of patients with VD caregivers (P0.05). The patients in the treatment group after treatment of TCM syndrome score lower than before treatment, with significant difference (P0.01), patients in the control group after treatment TCM syndrome score before treatment showed no significant difference (P0.05), two groups of patients before and after the TCM syndromes and treatment in the two groups the difference had significant difference after treatment (P0.01); the total efficiency of treatment group was 56.67%, control group total effective rate was 10%, indicating that the treatment group of patients with TCM syndrome significantly, the control group of TCM syndromes in patients with poor effect (P0.01). All patients in this study were not found in blood and liver function abnormalities and adverse reactions of two groups of patients with mild symptoms and good safety. Experimental research: Morris water maze test results: with the increase in the number of training days, rats the escape latency shortened gradually, that the rats in learning and training gradually learn to find the platform, but there are significant differences between the ability of learning and memory, including training four days every day the rats in the model group compared to the sham operation latency time Group significantly increased (P0.05 or P0.01), the model group of spatial learning and memory abilities of rats appeared disorder, third days, the escape latency of rats in high dose group decreased significantly compared with model group (P0.05); the fourth day, the high dose group and nimodipine group rats escape latency were significantly decreased compared with the model group (P0.01 and P0.05). Space exploration results showed: in 120s, the number of high dose group and sham operation group rats through the platform was significantly higher than the model group, the difference was statistically significant (P0.01); the rats staying in the platform quadrant and the rats in the model group the difference has not been found statistically significant (P0.05), thus the experimental results show that the high dose group of VD rats learning and memory ability to significantly improve the pathological changes of hippocampus in.VD rats: Lost nerve cells in the hippocampus of model group rats were observed under light microscope; nerve nuclei fragmentation, nuclear membrane Not clear, nucleolus disappeared, nerve cell shrinkage, cytoplasmic eosinophilic enhancement, astrocytes proliferation; lesion concentration in CA1, CA2 and CA3 region of hippocampus lesions; nimodipine group rats were concentrated in the CA1 area, a neuronal cell injury; high, low dose group rat hippocampal lesion concentration in CA1, CA3 District visible, neuronal cell injury, lesions alleviated as compared with model group, suggesting that the brain of 1 party and nimodipine can reduce the range and extent of neuronal damage in the hippocampus of VD rats. The expression of NR2B in hippocampus mRNA and CaMK II mRNA: the experimental results showed that the expression level of NR2B in hippocampus of mRNA model rats were significantly decreased compared with the sham operation group (P0.01). Compared with the model group, the expression level of nimodipine group and high dose group rat hippocampal NR2B mRNA increased significantly, the difference was statistically significant (P0.05), low dose group increased NR2B mRNA expression in the model group. The expression of CaMK in hippocampus of mRNA was slightly decreased compared with the sham operation group (P0.05), high dose group and nimodipine can slightly improve the expression level of mRNA, there were statistically significant but the difference (P0.05) conclusion: the clinical results suggest that brain 1 treatment of VD patients in improving the cognitive function, has good curative effect to improve the ability of daily life of patients, can significantly improve patients' clinical symptoms, but significantly improved for patients with behavioral and psychological symptoms were not VD, and less adverse reaction, good safety. This research confirms based on Deng's "Invigorating Qi and removing phlegm and activating blood" in the treatment of vascular dementia of the correct and scientific concept based on clinical experience, and Deng created brain 1 party is effective and safety. The experimental results suggest that the brain 1 party can significantly improve the learning and memory ability of VD rats, and its mechanism can be speculated It can be related to reducing the extent and extent of neuronal damage and increasing the expression level of NR2B mRNA in hippocampus of VD rats. It is found that the CaMK II mRNA in hippocampus of VD rats has not been improved similarly, suggesting that CaMK II may not be an important target for the 1 Party of brain strengthening.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R277.7

【参考文献】