祛风通窍方对血管性痴呆模型大鼠作用机制的研究

发布时间：2018-05-30 00:15

本文选题：血管性痴呆 + 祛风通窍方　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:通过观察祛风通窍方对血管性痴呆(VD)大鼠定位航行、空间探索能力,海马CA1区神经元细胞形态,ChAT、Tau、Aβ、VEGF蛋白表达,IL-1β、IL-6、TNF-α浓度的影响,探讨祛风通窍方对血管性痴呆模型大鼠学习记忆能力的保护作用机制。方法:1.分组:水迷宫筛选符合实验要求大鼠,随机分为假手术组、尼莫地平组、模型组、祛风通窍方高、中、低剂量组,每组大鼠各10只。2.给药方法:高、中、低剂量组分别给予祛风通窍方26.8g/kg/d、13.4g/kg/d、6.7g/kg/d灌胃(按照等体积3ml的标准计算高、中、低剂量组大鼠灌胃药物的浓度)。模型组、假手术组予以3ml生理盐水灌胃。尼莫地平组予以尼莫地平混悬液6.25mg/kg/d灌胃。每天上午灌胃一次,连续30天。3.取材方法:配置戊巴比妥钠浓度1%,对实验大鼠进行腹腔麻醉。大鼠仰卧位固定于消毒实验台,开胸取血,离心,取上清液,-20℃冰箱保存,用于ELISA检测IL-1β、IL-6、TNF-α的浓度。采集血液后,一部分大鼠迅速断头取出新鲜脑组织,将脑组织置于EP管中保存,用于半定量PCR及Western Blot检测VEGF蛋白。另一部分大鼠进行灌注取脑,用于HE染色和免疫组化检测ChAT、Tau、Aβ蛋白。4.观察指标:Morris水迷宫检测大鼠定位航行、空间探索能力;HE染色光镜观察大鼠海马CA1区神经元细胞形态;免疫组化检测ChAT、Tau、Aβ蛋白表达;ELISA法检测IL-6、IL-1β、TNF-α浓度;半定量PCR法测定VEGFmRNA表达;Western Blot法检测VEGF蛋白表达。结果:1.行为学:与假手术组比较,高、中、低剂量组、模型组、尼莫地平组大鼠EL延长,第i象限游泳的时间缩短,经过原平台范围的次数减少,差异具有统计学意义(p0.05),高剂量组大鼠el随着训练时间延长而缩短,从第四天开始接近于假手术组。与模型组比较,高、中、低剂量组、尼莫地平组大鼠的el随着训练时间延长而缩短,第i象限游泳的时间延长,经过原平台范围的次数增加,差异具有统计学意义(p0.05)。与尼莫地平组比较,低剂量组el随着训练时间延长而缩短,第i象限游泳的时间延长,经过原平台范围的次数增加,但差异无统计学意义(p0.05);高、中剂量组大鼠el随着训练时间延长而缩短,第i象限游泳的时间延长,经过原平台范围的次数增加,差异具有统计学意义(p0.05)。其中,以高剂量组大鼠行为学改善更为明显,与中剂量组比较,差异具有统计学意义(p0.05)。2.he染色:同倍率光镜下观察,假手术组海马ca1区神经元细胞排列整齐,胞质均质红染,胞核大而圆,核仁清晰明显。模型组海马ca1区神经细胞排列紊乱,数量减少,细胞轮廓、胞核模糊,部分甚至看不到细胞。高、中剂量组神经细胞排列比较规则,细胞轮廓、核膜与核仁比较清晰,且高剂量组神经细胞形态优于中剂量组,更为接近正常形态。低剂量组和尼莫地平组的神经元细胞形态介于模型组与中剂量组之间。3.chat、tau、aβ蛋白检测:chat、tau、aβ阳性细胞均表现为胞浆内呈现棕黄色的沉淀物。(1)chat蛋白:与假手术组比较,模型组、各给药组chat蛋白mod值有所降低(p0.05)。与模型组比较,各给药组chat蛋白mod值有所升高(p0.05)。与尼莫地平组比较,低剂量组chat蛋白mod值升高,但差异无统计学意义(p0.05);高、中剂量组chat蛋白mod值有所升高,差异有统计学意义(p0.05)。其中,以高剂量组升高最为明显,与中剂量比较,差异有统计学意义(p0.05)(2)tau蛋白:与假手术组比较,模型组、各给药组tau蛋白mod值有所升高(p0.05)。与模型组比较,各给药组tau蛋白mod值降低(p0.05)。与尼莫地平组比较,低剂量组tau蛋白mod值降低,但差异无统计学意义(p0.05);高、中剂量组tau蛋白mod值有所降低,差异有统计学意义(p0.05)。其中,以高剂量组降低最为明显,与中剂量组比较,差异有统计学意义(p0.05)。(3)aβ蛋白:与假手术组比较,模型组、各给药组aβ蛋白mod值有所升高(p0.05)。与模型组比较,各给药组aβ蛋白mod值有所降低(p0.05)。与尼莫地平组比较,低剂量组aβ蛋白mod值降低,但差异无统计学意义(p0.05);高、中剂量组aβ蛋白mod值有所降低,差异有统计学意义(p0.05)。其中,以高剂量组降低最为明显,与中剂量组比较,差异有统计学意义(p0.05)。4.il-6、il-1β、tnf-α检测:与假手术组比较,模型组、各给药组il-6、il-1β、tnf-α浓度有所升高(p0.05)。与模型组比较,各给药组il-6、il-1β、tnf-α浓度降低(p0.05)。与尼莫地平组比较,高、中、低剂量组il-6、il-1β、tnf-α浓度有所降低(p0.05)。其中,以高剂量组降低最为明显,与中剂量组比较,差异具有统计学意义。5.vegfmrna表达检测:与假手术组相比,模型组、各给药组vegfmrna表达有所增加(p0.05)。与模型组比较,各给药组vegfmrna表达有所增加(p0.05)。与尼莫地平组比较,低剂量组vegfmrna表达增加,但差异无统计学意义(p0.05);高、中剂量组vegfmrna表达有所增加,差异有统计学意义(p0.05)。其中,以高剂量组增加最为明显,与中剂量组比较,差异有统计学意义(p0.05)。6.vegf蛋白表达检测:与假手术组相比,模型组、各给药组vegf蛋白表达有所增加(p0.05)。与模型组比较,各给药组vegf蛋白表达有所增加(P0.05)。与尼莫地平组比较,低剂量组VEGF蛋白表达增加,但差异无统计学意义(P0.05);高、中剂量组VEGF蛋白表达有所增加,差异有统计学意义(P0.05)。其中,以高剂量组增加最为明显,与中剂量组比较,差异有统计学意义(P0.05)。结论:1.血管性痴呆大鼠存在学习记忆能力的下降;海马CA1区神经元细胞形态紊乱,ChAT蛋白表达降低,Tau、Aβ蛋白表达升高,IL-6、IL-1β、TNF-α的浓度升高,VEGF表达增加。2.祛风通窍方能有效改善VD大鼠的行为学,保护VD大鼠海马CA1区神经元细胞形态。3.祛风通窍方对血管性痴呆模型大鼠的作用机制可能与以下有关:(1)上调VD大鼠ChAT的表达,改善胆碱能系统的功能;(2)抑制VD大鼠脑内Tau蛋白过度磷酸化,降低Aβ蛋白表达,减少神经元的损害;(3)降低VD大鼠血清中炎性细胞因子IL-6、IL-1β、TNF-α的浓度,从而抑制VD大鼠缺血区的炎性反应;(4)能刺激VEGF的表达,从而促进缺血时血管的新生,修复缺血损伤的神经。4.祛风通窍方改善血管性痴呆大鼠学习记忆能力存在一定量效关系。
[Abstract]:Objective: To observe the effect of dispel Tong Qiao Decoction on the location of vascular dementia (VD) rats, space exploration ability, neuron cell morphology in hippocampus CA1 region, ChAT, Tau, A beta, VEGF protein expression, IL-1 beta, IL-6, and TNF- alpha concentration, and explore the mechanism of protective effect of dispelling wind and Tong Fang on learning and memory ability of vascular idiot model rats. Methods: 1. groups: The water maze screening rats were randomly divided into sham operation group, nimodipine group, model group, high, low dose group, and 10.2. methods in each group: high, middle, and low dose groups were given 26.8g/kg/d, 13.4g/kg/d, and 6.7g/kg/d respectively (high, medium and low dosage according to the standard of equal volume 3ml. The model group, the model group, the sham operation group was given 3ml physiological saline for gastric perfusion. Nimodipine group was treated with nimodipine suspension 6.25mg/kg/d. One time every morning, 30 days of continuous.3. extraction method: the concentration of pentobarbital sodium was 1%, the experimental rats were anesthetized with abdominal cavity. The supine position of rats was fixed at the disinfectant test table. Blood, centrifuge, centrifuge, take the supernatant, save the supernatant, -20 C refrigerator and used for ELISA to detect the concentration of IL-1 beta, IL-6, TNF- alpha. After collecting the blood, some rats quickly cut out the fresh brain tissue and put the brain tissue in the EP tube and used for the semi quantitative PCR and Western Blot detection VEGF protein. Another part of the rats was perfused to take the brain for HE dyeing and in the HE staining. Immunohistochemistry test ChAT, Tau, A beta protein.4. observation index: Morris water maze test rats navigation, space exploration ability; HE staining light microscope observation of the hippocampus CA1 region neuron cell morphology; immunohistochemistry detection ChAT, Tau, A beta protein expression; ELISA method for IL-6, beta, alpha concentration; semi quantitative determination method The OT method was used to detect the expression of VEGF protein. Results: 1. behavior: compared with the sham group, the EL in the high, middle, low dose group, the model group, the nimodipine group was prolonged, the time of the swimming in the I quadrant shortened, the difference was statistically significant (P0.05) after the frequency of the original platform range (P0.05), and the El in the high dose group shortened with the training time, from fourth. Compared with the model group, the El in the high, middle and low dose group, the rats in Nimodipine group shortened with the time of training, the time of swimming in the I quadrant was prolonged, and the difference was statistically significant (P0.05). Compared with the nimodipine group, the low dose group El was prolonged with the training time. The duration of swimming in the I quadrant was lengthened, and the number of swimming in the original platform increased, but the difference was not statistically significant (P0.05). The El in the middle dose group shortened with the training time, the time of the I quadrant was prolonged, and the difference was statistically significant (P0.05) through the number of the original platform range (P0.05). The behavior improvement of rats was more obvious. Compared with the middle dose group, the difference was statistically significant (P0.05).2.he staining: under the same rate light microscope, the neuron cells in the hippocampal CA1 area of the sham operation group were arranged neatly, the cytoplasm homogeneous red dye, the nucleus large and round, the nucleolus clear and obvious. The neurons in the hippocampus CA1 area were arranged in disorder, and the number of cells decreased. The outline, the nucleus of the cell was blurred, and the cells were not even visible. The nerve cells in the middle dose group were arranged more regularly, the cell outline, the nuclear membrane and the nucleolus were clearer, and the morphology of the nerve cells in the high dose group was better than the middle dose group, and it was closer to the normal form. The neurons in the low dose group and the nimodipine group were between the model group and the middle dose group. .3.chat, tau, a beta protein detection: chat, tau, a beta positive cells showed brown yellow sediment in the cytoplasm. (1) chat protein: compared with the sham group, the model group, the mod value of chat protein in each group decreased (P0.05). Compared with the model group, chat protein Mod value increased. The mod value of chat protein in the group increased, but the difference was not statistically significant (P0.05). The mod value of chat protein in the middle dose group was higher, the difference was statistically significant (P0.05). Among them, the high dose group was the most obvious, and the difference was statistically significant (P0.05) (2) tau protein: the comparison with the sham operation group, the model group, and the tau protein m in each administration group. The OD value increased (P0.05). Compared with the model group, the tau protein mod value of each group was reduced (P0.05). Compared with the nimodipine group, the tau protein mod value of the low dose group decreased, but the difference was not statistically significant (P0.05); the value of tau protein mod value in the middle dose group decreased, and the difference was statistically significant (P0.05). Compared with the middle dose group, the difference was statistically significant (P0.05). (3) a beta protein: compared with the sham group, the mod value of a beta protein in the model group was higher (P0.05). Compared with the model group, the A beta protein mod value decreased (P0.05). Compared with the nimodipine group, the A beta protein mod value decreased in the low dose group, but the difference was not statistically significant (p0.). 05); high, middle dose group A beta protein mod value decreased, the difference was statistically significant (P0.05), among which, the most obvious reduction in high dose group, compared with the middle dose group, the difference was statistically significant (P0.05).4.il-6, IL-1 beta, tnf- alpha detection: compared with the sham operation group, the model group, the concentration of IL-6, IL-1 beta, tnf- alpha in each administration group increased (P0.05). And model Compared with the nimodipine group, the concentration of IL-6, IL-1 beta, and tnf- a decreased (P0.05) in the high, middle and low dose groups compared with the nimodipine group (P0.05). Compared with the high dose group, the concentration of IL-6, IL-1 beta and tnf- alpha was lower (P0.05). Compared with the middle dose group, the difference was statistically significant.5.vegfmrna expression detection: compared with the sham operation group, the model group was compared with the sham group, and the model group was compared with the sham group. The expression of VEGFmRNA in the drug group increased (P0.05). Compared with the model group, the expression of VEGFmRNA in each group increased (P0.05). Compared with the nimodipine group, the expression of VEGFmRNA in the low dose group increased, but the difference was not statistically significant (P0.05); the expression of VEGFmRNA in the middle dose group increased, and the difference was statistically significant (P0.05). Among them, the high dose group was in the high dose group. The increase was most obvious, compared with the middle dose group, the difference was statistically significant (P0.05).6.vegf protein expression detection: compared with the sham group, the expression of VEGF protein in the model group was increased (P0.05). Compared with the model group, the expression of VEGF protein in each group was increased (P0.05). The expression of VEGF protein in the low dose group was compared with the nimodipine group and the expression of VEGF protein in the low dose group. The difference was not statistically significant (P0.05), and the expression of VEGF protein in the middle dose group increased, and the difference was statistically significant (P0.05). Among them, the increase in the high dose group was the most obvious, and the difference was statistically significant (P0.05) compared with the middle dose group (P0.05). Conclusion: 1. the decrease of learning and memory ability in the rats with vascular dementia and the neurons in the hippocampus CA1 area neurons. The cell morphologic disorder, the expression of ChAT protein, the increase of Tau, A beta protein expression, the increase of IL-6, IL-1 beta, TNF- alpha, the increase of VEGF expression to improve the behavior of VD rats, and protect the mechanism of the neuronal cell morphology of the CA1 region of the hippocampus of VD rats to the mechanism of vascular dementia model rats may be the following. (1) up regulating the expression of ChAT in VD rats and improving the function of the cholinergic system; (2) inhibiting the excessive phosphorylation of Tau protein in the brain of VD rats, reducing the expression of A beta protein and reducing the damage of neurons; (3) reducing the concentration of inflammatory cytokines IL-6, IL-1 beta and TNF- alpha in serum of VD rats, thus inhibiting the inflammatory response in the ischemic region of VD rats; (4) stimulates VEGF. The expression of.4. can promote the regeneration of blood vessels during ischemia, and repair the ischemic injury of the nerve to improve the learning and memory ability of vascular dementia rats.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332

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