卡托普利与氯沙坦对血管性痴呆大鼠的作用及机制研究

发布时间：2018-06-02 09:24

本文选题：卡托普利 + 氯沙坦　；参考：《泸州医学院》2012年硕士论文

【摘要】：目的：分别观察肾素一血管紧张素系统抑制药卡托普利和氯沙坦对血管性痴呆(VD)模型大鼠的学习记忆的改善及治疗效果,并初步探讨其可能作用机制。方法：Morris水迷宫筛选学习记忆功能正常的雄性SD大鼠60只；随机分手术组(48只)和假手术组(12只)；手术组采用不同时点分别永久性结扎大鼠左、右侧颈总动脉法建立VD模型。术后4周,筛选制模成功大鼠36只,随机分为2.5mg/Kg卡托普利组、2.0mg/Kg氯沙坦组和模型组,每组12只。用药组每天1次灌胃给药,模型组与假手术组给予等容量生理盐水,连续4周。药后4周,Morris水迷宫测试各组大鼠学习记忆能力。部分大鼠4%多聚甲醛灌注固定,石蜡包埋脑组织,连续冠状切片,HE染色和免疫组织化学染色(SABC法),TUNEL法检测海马神经元凋亡,光镜观察海马神经元的损伤、Bcl-2与Bax蛋白表达及海马神经元凋亡情况。GD-10.0多媒体彩色病理图文分析系统分析Bcl-2及Bax阳性表达的平均灰度值,灰度值越高表达产物越多。部分大鼠断头取脑,分离皮质和海马,冰上匀浆,3000r/min转速离心10min制备组织匀浆上清液。放射免疫法测定大脑皮层及海马组织中TNF-α、IL-1β含量；采用羟胺法测定大脑皮层及海马组织中超氧化物歧化酶(SOD)活性；硫代巴比妥酸(TBA)法测定大脑皮层及海马组织中丙二醛(MDA)含量；硝酸还原酶法测定大脑皮层及海马组织中一氧化氮(NO)含量；化学比色法测定大脑皮层及海马组织中一氧化氮合酶(NOS)和海马组织中胆碱脂酶(AChE)活性。腹主动脉取血,全自动血流变快测仪测定检测血液流变学。结果：(1)手术后4周：①定位航行实验：随着训练次数的增加,各组大鼠平均逃避潜伏期均缩短。与假手术组比较,手术组大鼠逃避潜伏期均明显延长(P0.05)。②空间搜索实验：与假手术组比较,模型组在120s内穿越平台的次数明显减少(P0.05)。(2)药后4周：①定位航行实验：经过5天水迷宫训练,大鼠逃避潜伏期,卡托普利和氯沙坦治疗组与模型组比较明显缩短(P0.05)；②空间搜索实验：120s内穿越平台的次数卡托普利和氯沙坦治疗组与模型组比较均有明显增多(P0.05)。(3)病理组织学观察(HE染色)：假手术组海马组织神经元排列整齐,形态完整；细胞数量较多,细胞形态正常,体积较大,核居中,大而圆,染为淡蓝色,核仁核膜清晰,核仁染为紫色,胞质着色浅而均匀。模型组大鼠海马组织神经元排列紊乱,数目减少,胞核固缩浓染。各用药组均能改善上述神经元损伤。(4)TUNEL法检测：假手术组大鼠海马可见少数细胞胞核呈棕黄色,模型组大鼠海马许多细胞胞核着成棕黄色,不均匀,并呈环指样结构,与假手术组比较,其凋亡细胞阳性率显著增强(P0.05)。卡托普利和氯沙坦治疗组组海马胞核着成棕黄色的细胞数明显减少,凋亡细胞阳性率亦明显降低(P0.05)。(5)免疫组织化学检测：各组大鼠海马Bal-2、Bax阳性神经元染色均以胞浆有棕黄色物质沉积为主,也有部分胞膜及核膜着色,并可显示神经元轮廓。假手术组大鼠海马Bal-2、Bax均呈少量表达。模型组大鼠Bcl-2及Bax蛋白表达均明显增加,与假手术组比较有显著意义(P0.05)。卡托普利组和氯沙坦组大鼠Bcl-2及Bax蛋白表达有明显改善(P0.05)。(6)皮层及海马组织匀浆：卡托普利组和氯沙坦治疗组大脑皮质和海马SOD活性较模型组明显升高(P0.05),两治疗组大脑皮层及海马MDA含量较模型组明显降低(P0.05)。卡托普利组和氯沙坦治疗组大脑皮质及海马NOS活力及NO含量较模型组明显降低(P0.05)。卡托普利和氯沙坦治疗组对大脑皮质及海马TNF-α含量和IL-1p含量较模型组明显下降(P0.05)。与模型组比较,卡托普利和氯沙坦治疗组大鼠可降低大脑海马AChE活性(P0.05)。(7)血液流变学：与模型空白组比较,卡托普利与氯沙坦组大鼠全血黏度的低切、中切和血浆黏度均有显著性降低(P0.05),对血液流变学具有一定改善作用。结论：(1)不同时点分别永久性结扎大鼠左右侧颈总动脉成功制备了VD模型。(2)卡托普利及氯沙坦可改善VD模型大鼠学习记忆能力,减轻海马神经元损伤；其机制与以下因素有关：①可能通过影响Bcl-2及Bax蛋白表达,减少海马神经元凋亡；②抑制脑组织AChE活性,减少ACh的降解；③增加SOD活性增强氧自由基的清除,降低NOS活性,减少NO的含量,减轻氧自由基和NO对脑组织的损害；④减少脑组织TNF-α和IL-1β含量,减轻免疫炎症反应对神经元的损伤；⑤改善血液流变学,增加脑组织的供血供氧。
[Abstract]:Objective: To observe the improvement and therapeutic effect of renin and angiotensin system inhibitor Kato Pury and losartan on the learning and memory of vascular dementia (VD) model rats, and to explore its possible mechanism. Methods: 60 male rats with normal learning and memory function were screened by Morris water maze, and 48 rats were randomly divided into operation group (48 rats). And sham operation group (12); VD model was established in the operation group with permanent ligation of rats and right common carotid artery method. 4 weeks after operation, 36 rats were selected and divided into 2.5mg/Kg captopril group, 2.0mg/Kg losartan group and model group, 12 rats in each group. The drug group was administered 1 times a day, model group and sham operation group. The learning and memory ability of rats in each group was tested by Morris water maze for 4 weeks after 4 weeks. Some rats were perfused with 4% polyformaldehyde, paraffin embedded brain tissue, continuous coronal section, HE staining and immunohistochemical staining (SABC). TUNEL method was used to detect the apoptosis of hippocampal neurons, and the damage of hippocampal neurons was observed by light microscope. The expression of Bcl-2 and Bax protein and the apoptosis of hippocampal neurons were analyzed by.GD-10.0 multimedia color pathological graphic analysis system. The average gray value of Bcl-2 and Bax positive expression was analyzed. The higher the gray value, the more products were expressed. Some rats broke head to take brain, separated cortex and hippocampus, on ice homogenate, and 3000r/min rotational speed centrifugation 10min was used to prepare tissue homogenate supernatant. Radioimmunoassay was used to determine the content of TNF- alpha and IL-1 beta in the cerebral cortex and hippocampus; the activity of superoxide dismutase (SOD) in the cerebral cortex and hippocampus was measured by hydroxylamine method; the content of malondialdehyde (MDA) in the cerebral cortex and hippocampus was measured by thiobarbituric acid (TBA); the nitrate reductase method was used to determine the cerebral cortex and hippocampus. Nitric oxide (NO) content; chemical colorimetric assay for the activity of nitric oxide synthase (NOS) and hippocampal cholinesterase (AChE) in the cerebral cortex and hippocampus. Blood rheology was measured by abdominal aorta and automatic hemorrheology rapid measuring instrument. Results: (1) 4 weeks after operation: (1) positioning navigation experiment: with the increase of training times, each The average escape latency of the rats in the group was shortened. Compared with the sham operation group, the escape latency of the operation group was significantly prolonged (P0.05). (2) the space search experiment: compared with the sham group, the number of the model group passed the platform in 120s significantly decreased (P0.05). (2) 4 weeks after the medicine: (1) the positioning navigation experiment: after 5 days water maze training, rats escape The incubation period, Kato Pury and losartan treatment group was significantly shorter than the model group (P0.05); (2) the spatial search experiment: the number of Kato Pury and losartan treatment groups in 120s and losartan group were significantly increased (P0.05). (3) histopathological observation (HE staining): the hippocampal neurons in the sham operation group were arranged neatly and shape. Complete state, more cell number, normal cell morphology, large volume, large and round nucleus, large and round, dyed light blue, clear nucleolus membrane, nucleolus dye purple, light and homogeneous cytoplasm. The hippocampus neurons in the model group were arranged in disorder, the number was reduced, and the nuclei were condensed and concentrated. (4) TUNEL In the sham operation group, the hippocampus of the rats in the sham operation group was found to be brown and yellow, and the nucleus of many cells in the model group were brown and yellow, inhomogeneous and ring finger like structure. Compared with the sham operation group, the positive rate of apoptotic cells increased significantly (P0.05). The hippocampus nucleus of captopril and losartan group was brownish yellow cells. The number of apoptotic cells decreased significantly (P0.05). (5) immuno histochemical detection: Bal-2 and Bax positive neurons in hippocampus of rats in each group were stained mainly with brown yellow substance in cytoplasm, and some membrane and nuclear membrane were coloured, and the neurons of the neurons were displayed. The hippocampus Bal-2 in the sham operation group was a small amount of expression of Bax. The expression of Bcl-2 and Bax protein in the rat model group increased significantly (P0.05). The expression of Bcl-2 and Bax protein in the captopril group and losartan group was significantly improved (P0.05). (6) the cortex and hippocampus homogenate: the cerebral cortex and hippocampal SOD activity in the captopril group and the losartan treatment group were significantly higher than those in the model group. The content of MDA in cerebral cortex and hippocampus in the two treatment group was significantly lower than that in the model group (P0.05). The NOS activity and NO content in the cerebral cortex and hippocampus of the Kato Pury group and the Losartan group were significantly lower than that of the model group (P0.05). The content of TNF- A and the IL-1p content in the cortex and hippocampus of the cerebral cortex and the hippocampus in Kato Pury and losartan groups were significantly lower than those in the model group. (P0.05). Compared with the model group, the rats of Kato Pury and losartan treatment group could reduce the AChE activity in the hippocampus of the brain (P0.05). (7) Hemorheology: compared with the model blank group, the blood viscosity of the whole blood in the rats of Kato Pury and losartan group was lower than that of the rats in the Losartan group. The viscosity of both the middle and the plasma decreased significantly (P0.05), and the blood rheology was improved. Conclusion: (1) the VD model was successfully prepared by ligating the left and right common carotid artery in rats at different time points. (2) Kato Pury and losartan could improve the learning and memory ability of VD model rats and reduce the damage of hippocampal neurons. The mechanism is related to the following factors: (1) the expression of Bcl-2 and Bax protein may be affected and the hippocampal neurons can be reduced. Apoptosis; (2) inhibiting the activity of AChE in brain tissue and reducing the degradation of ACh; (3) increasing the activity of oxygen free radicals by increasing SOD activity, reducing the activity of NOS, reducing the content of NO, reducing the damage of oxygen free radicals and NO on brain tissue; reducing the content of TNF- A and IL-1 beta in the brain tissue and reducing the damage of the immune inflammatory reaction to the neurons; and 5. Increase the blood supply and oxygen supply in the brain tissue.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.13

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