人参皂苷Rb1对癫痫大鼠海马神经元凋亡和β-catenin蛋白表达的影响

发布时间：2018-06-28 16:06

  本文选题：人参皂苷Rb1 + 癫痫　； 参考：《泸州医学院》2014年硕士论文

【摘要】：目的：探讨人参皂苷Rb1对青霉素诱导癫痫大鼠海马神经元凋亡和β-catenin蛋白表达的影响。方法：雄性Sprague-Dawley(SD)大鼠30只（体重250±50g），随机分为4组：空白对照(BC)组（n=6）、人参皂苷Rb1对照(GC)组（n=8）、青霉素致痫(PE)组（n=8）和人参皂苷Rb1干预致痫(GPE)组（n=8）。人参皂苷Rb1对照组和人参皂苷Rb1干预致痫组大鼠在模型制备前1h腹腔注射1%人参皂苷Rb1注射液3ml/Kg (30mg/Kg)，空白对照组和青霉素致痫组大鼠则腹腔注射等剂量生理盐水。大鼠癫痫模型的制备采用腹腔注射青霉素760万U/Kg的方法，即青霉素致痫组和人参皂苷Rb1干预致痫组大鼠腹腔注射80万U/ml青霉素注射液9.5ml/Kg，空白对照组和人参皂苷Rb1对照组大鼠则腹腔注射等剂量的生理盐水。模型制备后观察大鼠行为学改变，并记录癫痫发作等级。模型制备后3h时，各组大鼠腹腔注射3％戊巴比妥钠1ml/Kg麻醉，开胸暴露心脏，剪开右心房，将穿刺针经左心室刺入升主动脉，用生理盐水快速灌注冲洗，然后用4%多聚甲醛500ml灌注固定，开颅，取出脑组织浸泡于4%多聚甲醛8～12h后固定。常规酒精脱水，石蜡包埋，连续切片（厚度4μm）。苏木精-伊红染色法（HE）染色，光镜下观察海马神经元组织学结构变化；脱氧核糖核苷酸末端转移酶介导的缺口末端标记（TUNEL）法测海马神经元凋亡；免疫组化法测海马神经元β-catenin蛋白的阳性表达。结果：⑴大鼠癫痫发作的行为学改变：空白对照组和人参皂苷Rb1对照组大鼠无痫样发作表现；青霉素致痫组大鼠癫痫发作明显，发作等级达Ⅲ级及以上，其中Ⅲ级3只(3/8), Ⅳ级2只(2/8)，Ⅴ级3只(3/8)；人参皂苷Rb1干预致痫组大鼠癫痫发作程度较青霉素致痫组明显减轻，发作等级仅为Ⅰ-Ⅱ级，，其中Ⅰ级3只(3/8)，Ⅱ级5只(5/8)。⑵海马神经元组织结构改变：空白对照组和人参皂苷Rb1对照组大鼠海马神经元形态结构正常；青霉素致痫组和人参皂苷Rb1干预致痫组大鼠海马神经元出现变性和坏死损伤，但后者损伤程度较轻。(3)海马神经元细胞凋亡变化：各组大鼠海马CA1、CA2、CA3和齿状回区均有神经元凋亡。空白对照组、人参皂苷Rb1对照组、青霉素致痫组和人参皂苷Rb1干预致痫组大鼠海马CA1区凋亡神经元计数（个）分别是2.5±1.05、1.75±0.71、3.5±0.93和1.63±0.74；CA2区凋亡神经元计数分别是3.33±1.03、1.50±0.76、6.13±1.23、2.75±0.71； CA3区凋亡神经元计数分别是3.83±1.17、3.75±0.71、8.38±1.3、5.38±1.19；齿状回凋亡神经元计数分别是3.5±1.38、3.38±0.92、21.75±3.85、10.88±3.36。与空白对照组相比，青霉素致痫组和人参皂苷Rb1干预致痫组大鼠海马区凋亡阳性细胞数明显增高，差异具有统计学意义（P0.05）。与青霉素致痫组比较，人参皂苷Rb1干预致痫组海马区凋亡阳性细胞数明显减少（P0.05)。与人参皂苷Rb1对照组比较，人参皂苷Rb1干预致痫组海马区凋亡阳性细胞数明显增高（P0.05)。(4)海马神经元β-catenin蛋白表达变化：空白对照组、人参皂苷Rb1对照组、青霉素致痫组和人参皂苷Rb1干预致痫组大鼠海马β-catenin蛋白表达OD值的平均值（AO）分别是(16.94±0.17)×10-2、(20.39±2.64)×10-2、(18.79±0.67)×10-2和(17.07±0.5)×10-2。与空白对照组相比，人参皂苷Rb1对照组和青霉素致痫组β-catenin蛋白阳性表达高于空白对照组（P0.05）；与青霉素致痫组相比，人参皂苷Rb1干预致痫组β-catenin蛋白阳性表达明显减低（P0.05）；与人参皂苷Rb1对照组比较，人参皂苷Rb1干预致痫组β-catenin蛋白阳性表达明显减低（P0.05），差异均具有统计学意义。结论：青霉素诱导癫痫大鼠海马神经元凋亡增加和β-catenin蛋白表达升高；人参皂苷Rb1能够减轻青霉素致痫大鼠的癫痫发作、减少海马神经元细胞凋亡和抑制β-catenin蛋白表达的上调。
[Abstract]:Objective: To investigate the effect of ginsenoside Rb1 on the apoptosis and the expression of beta -catenin protein in the hippocampus of penicillin induced epileptic rats. Methods: 30 male Sprague-Dawley (SD) rats (weight 250 + 50g) were randomly divided into 4 groups: blank control (BC) group (n=6), ginsenoside Rb1 control (n=8), penicillin induced epilepsy (PE) group (n=8) and ginsenoside 1 intervention induced epilepsy (GPE) group (n=8). Ginsenoside Rb1 control group and ginsenoside Rb1 intervention induced epileptic group rats before the model preparation of 1H intraperitoneal injection of 1% ginsenoside Rb1 injection 3ml/Kg (30mg/Kg), the blank control group and penicillin induced epilepsy rats were intraperitoneally injected with equal dose of physiological salt water. The preparation of the rat model of epilepsy was injected penicillin 7 in the abdominal cavity. 600 thousand U/Kg method, i. e. penicillin induced epilepsy group and ginsenoside Rb1 intervention, rats were intraperitoneally injected with 800 thousand U/ml penicillin injection 9.5ml/Kg, the blank control group and the ginsenoside Rb1 control group were intraperitoneally injected with equal dose of saline. After the model preparation, the rat behavior changes were observed and the seizure grade was recorded. Model preparation was made. After 3h, the rats were injected with 3% pentobarbital sodium 1ml/Kg anaesthesia, open the heart to expose the heart, cut open the right atrium, puncture the needle through the left ventricle into the ascending aorta and rinse with the saline rapid perfusion, then use 4% polyoxymethylene 500ml perfusion fixation, craniotomy, and remove the brain tissue after 4% polyformaldehyde 8 ~ 12h fixation. Conventional alcohol dehydration, Paraffin embedded, continuous slice (thickness of 4 m), hematoxylin eosin staining (HE) staining, observation of histological structure of hippocampal neurons under light microscope, apoptosis of hippocampal neurons by deoxyribonucleotide terminal transferase mediated nick end labeling (TUNEL), and immunohistochemical method to detect the positive expression of beta -catenin protein in hippocampal neurons. The behavioral changes of epileptic seizures in rats: there was no epileptic seizure in the blank control group and the ginsenoside Rb1 control group; the epileptic seizures in the group of penicillin induced epileptic rats were obvious, the level of the seizures reached grade III and above, of which 3 levels (3/8), grade IV (2/8), grade V 3 (3/8), and ginsenoside Rb1 intervention in epileptic group induced epileptic seizures in rats Compared with penicillin induced epilepsy group, the level of seizures was only grade I - II, including grade I 3 (3/8) and grade II 5 (5/8). (2) hippocampal neuron structure change: blank control group and ginsenoside Rb1 control group were normal in hippocampal neurons; penicillin induced epilepsy group and ginsenoside Rb1 intervention in epileptic group rats hippocampus neurons Degeneration and necrosis were observed, but the damage degree of the latter was mild. (3) apoptosis of hippocampal neurons: neuron apoptosis in hippocampus CA1, CA2, CA3 and dentate gyrus of rats in each group. The blank control group, the ginsenoside Rb1 control group, the penicillin induced epilepsy group and the Rb1 dry preeclampsia group of ginsenoside Rb1 rats' hippocampal CA1 region apoptotic neuron count () The number of apoptotic neurons in the CA2 region was 3.33 + 1.03,1.50 + 0.76,6.13 + 1.23,2.75 + 0.71, respectively, 2.5 + 1.05,1.75 + 0.71,3.5 + 0.93 and 1.63 + 0.74 respectively. The apoptotic neuron count in CA3 region was 3.83 + 1.17,3.75 + 0.71,8.38 + 1.3,5.38 + 1.19, and the number of apoptotic neurons in the dentate gyrus was 3.5 +. Compared with the blank control group, the number of apoptotic cells in hippocampus of epilepsy group induced by penicillin and ginsenoside Rb1 was significantly higher than that in epilepsy group (P0.05). Compared with penicillin induced epilepsy group, the number of apoptotic cells in hippocampus of epilepsy group induced by ginsenoside Rb1 was significantly decreased (P0.05). The control group of ginsenoside Rb1 was compared with the group of ginsenoside Rb1 (P0.05). The number of apoptotic cells in hippocampus of epileptic group was significantly increased by ginsenoside Rb1 (P0.05). (4) the changes in the expression of beta -catenin protein in hippocampal neurons: blank control group, ginsenoside Rb1 control group, penicillin induced epilepsy group and ginsenoside Rb1 intervention in epileptic group rats hippocampus beta -catenin protein expression o value (AO) was (16.94) + 0.17) * 10-2, (20.39 + 2.64) x 10-2, (18.79 + 0.67) x 10-2 and (17.07 + 0.5) x 10-2. compared with the blank control group, the positive expression of beta -catenin protein in the ginsenoside Rb1 control group and penicillin induced epilepsy group was higher than that in the blank control group (P0.05). Compared with the penicillin induced epilepsy group, the positive expression of beta -catenin protein in the epileptic group induced by ginsenoside Rb1 was significantly reduced. Lower (P0.05); compared with the ginsenoside Rb1 control group, the positive expression of beta -catenin protein in the epileptic group induced by ginsenoside Rb1 was significantly decreased (P0.05), and the difference was statistically significant. Conclusion: the apoptosis of hippocampus neurons in the epileptic rats induced by penicillin was increased and the beta -catenin protein table was increased, and ginsenoside Rb1 could reduce the large amount of penicillin induced epilepsy. Seizures in rats reduce the apoptosis of hippocampal neurons and inhibit the up regulation of the expression of beta -catenin protein.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

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