石甘散对戊四氮致痫大鼠海马神经元脑源性神经营养因子及离子通道的影响
发布时间：2017-12-30 20:15
本文关键词：石甘散对戊四氮致痫大鼠海马神经元脑源性神经营养因子及离子通道的影响 出处：《黑龙江中医药大学》2017年博士论文 论文类型：学位论文
【摘要】：目的通过观察戊四氮致痫大鼠经石甘散治疗前后,实验大鼠一般状态、行为学、海马组织病理形态、离子通道电流幅度及采用免疫组织化学方法检测BDNF在海马内表达的不同,研究石甘散的抗痫的机制,为石甘散在临床上用来预防及治疗癫痫,提供必要的理论依据。方法选取110只健康成年SD大鼠,随机选取15只作为空白组,其余大鼠用戊四氮(PTZ)建立大鼠癫痫动物模型,造模成功后记录癫痫大鼠行为学表现,造模成功大鼠随机分为模型组、西药组、石甘散低剂量组、石甘散中剂量组、石甘散高剂量组,西药组以丙戊酸钠15.75mg/Kg灌胃治疗,中药组分别予以石甘散0.4g/Kg、0.8g/Kg、1.6g/Kg灌胃,空白组给予蒸馏水3ml/kg灌胃,疗程为21天,观察治疗后大鼠行为学改变、发作级别变化、体重改变,通过尼氏染色观察戊四氮致痫大鼠治疗后海马神经元病理组织变化,采用免疫组织化学方法检测BDNF在海马内表达的变化,通过全细胞膜片钳技术记录治疗后各组大鼠海马神经元电压依赖性Na~+通道、K~+通道电流幅度的改变。结果1.各组大鼠治疗前后行为学改变:(1)体重变化:造模成功后各组大鼠体重与空白组比较均显著下降(P0.01),治疗后,模型组大鼠体质量与空白组相比显著降低(P0.01),其余各组大鼠与空白组比较无差异(P0.05);与治疗前相比,各治疗组体重增加,石甘散低剂量组具有显著统计学意义(P0.05),西药组、石甘散中、高剂量组统计学意义极显著(P0.01)。(2)痫性发作潜伏期(Epileptic seizure latency,ESL)变化:治疗前,各组之间痫性发作潜伏期时间比较无显著差异(P0.05)。治疗后,模型组痫性发作潜伏期时间缩短,其余各组发作潜伏期时间均增加,其中,西药组和石甘散低、中、高剂量组发作潜伏期时间显著增加,统计学有其显著意义(P0.01)。(3)强直性惊厥潜伏期(Tonic convulsion latency,TCL)变化:各组治疗后与治疗前相比,模型组大鼠强直性惊厥潜伏期显著降低(P0.05),而其余各组大鼠潜伏期均增加,石甘散低剂量组潜伏期延长,有显著统计学意义(P0.05),西药组、石甘散中、高剂量组有显著意义(P0.01)。(4)强直性惊厥发作率(Tonic seizure rate)变化:治疗前后组间比较,模型组强直性惊厥发作率显著升高(P0.01),西药组和石甘散中、高剂量组发作率比治疗前显著降低(P0.01),石甘散低剂量组与疗前比较变化不明显(P0.05)。2.尼氏染色方法观察海马神经元病理形态改变:(1)形态改变:正常组大鼠CAI区神经元锥体细胞排列紧密有序,尼氏体深染,形态规则,数量正常;模型组大鼠锥体细胞形态皱缩,数量明显减少且呈现紊乱排列,尼氏体浅染且有部分溶解;西药组神经元形态可,细胞形状较规则,部分神经元形态不完整且皱缩,数量有部分丢失,尼氏体深染;石甘散低剂量组神经元锥体细胞数量减少,尼氏体浅染溶解,锥体细胞排列松散且形态不规则;石甘散中剂量组锥体细胞数量略有减少,形态略皱缩,尼氏体染色略浅;石甘散高剂量组神经元锥体细胞呈有序排列,形态尚可,数量略微减少,尼氏体呈深染。(2)尼氏阳性细胞数比较:与空白组比较,模型组和石甘散低剂量组尼氏阳性细胞数明显降低,有显著统计学意义(P0.01);与模型组比较,空白组、西药组和石甘散中、高剂量组的尼氏阳性细胞数均显著增加(P0.01);各组组间尼氏阳性细胞数比较,模型组和石甘散低剂量组细胞数明显减少(P0.01),其余各组细胞数与空白组比较差异不大,西药组和石甘散高剂量组阳性细胞数相对较多,且两者无明显统计学差异(P0.05)。3.治疗后PTZ大鼠海马神经元脑源性神经营养因子BDNF表达(1)与空白组比较,模型组、西药组、石甘散低、中、高剂量组大鼠BDNF的平均灰密度(AGD)均增加,模型组最显著(P0.01),模型组、西药组、石甘散低、中、高剂量组大鼠BDNF的平均光密度(AOD)均降低(P0.05),模型组最显著(P0.01)。(2)与模型组比较,其余各治疗组的AGD均降低,西药组和石甘散中、高剂量组AGD显著降低(P0.05),石甘散低剂量组无统计学意义(P0.05);各组AOD数值均增加,西药组和石甘散中、高剂量组AOD增加(P0.05),石甘散低剂量增量无统计学意义(P0.05)。(3)组间比较结果:模型组和石甘散低剂量组的AGD与其余各组比较增加明显,其中模型组最显著(P0.01),空白组、西药组和石甘散中、高剂量组之间AGD无显著差异(P0.05);模型组和石甘散低剂量组的AOD与其余各组比较降低明显,其中模型组最显著(P0.01),空白组、西药组和石甘散中、高剂量组之间AOD无显著差异(P0.05)。4.离子通道电流幅度的影响实验后,与空白组相比,模型组大鼠海马神经元电压依赖性持续性Na~+通道电流幅度(INaP)显著升高(P0.05),与模型组比较,西药组和石甘散低、中、高剂量组均能降低INaP,差异有统计学意义(P0.05),但石甘散低、中、高剂量组和西药组对电压依赖性瞬时性Na~+通道(INat)无显著改变,统计学无意义(P0.05)。与空白组相比,模型组大鼠海马神经元电压瞬时外向K~+通道(IA)显著降低(P0.05),与模型组比较,西药组和石甘散低、中、高剂量组均能升高降低的IA,差异有统计学意义(P0.05),但石甘散低、中、高剂量组和西药组对延迟整流K~+通道(IK)无显著改变,统计学无显著差异(P0.05)。结论1.石甘散低、中、高剂量组和丙戊酸钠均可以有效增加戊四氮致痫大鼠的体重。2.石甘散低、中、高剂量组与丙戊酸钠均能明显延长戊四氮大鼠阵发性痉挛的潜伏期时间;石甘散中、高剂量组和丙戊酸钠能延长大鼠强直性惊厥的潜伏期时间,并降低大鼠强直性惊厥的发生率,提示石甘散具有与丙戊酸钠相似的抗癫痫效应,且石甘散中、高剂量作用最明显。3.石甘散中、高剂量组和丙戊酸钠均可以增加戊四氮模型大鼠尼氏阳性细胞数量,改善其病理形态,提示两者对神经元均具有保护作用。4.石甘散中、高剂量组和丙戊酸钠均可以降低戊四氮模型大鼠BDNF平均灰密度(Average gray density),增加其平均光密度(Average optical density),说明两者对大鼠海马神经元具有相似的保护作用。5.石甘散低、中、高剂量组和丙戊酸钠均能显著下调戊四氮致痫大鼠海马神经元电压依赖性持续性Na~+通道电流幅度(INaP),上调瞬时外向K~+通道(IA)电流幅度,对电压依赖性瞬时性Na~+通道(INat)和延迟整流K~+通道(IK)电流无显著影响,说明石甘散可能通过对离子通道的调控发挥抗痫作用。6.石甘散可能通过增加脑源性神经营养因子BDNF含量影响离子通道电流幅度,调节静息膜电位,抑制神经突触传递兴奋性,发挥其抗痫作用。
[Abstract]:Objective To observe the e four before and after nitrogen from epileptic rats Gan powder in the treatment of experimental rats, the general state, behavior, hippocampal tissue morphology, ion channel current amplitude and to detect the expression of BDNF in hippocampus by immunohistochemistry method, study of Shi Gan powder anti epilepsy mechanism for Shi Gan powder at clinic for the prevention and treatment of epilepsy, provide the necessary theoretical basis. Methods: 110 healthy adult SD rats, 15 rats were randomly selected as the blank group, the rest rats with e four nitrogen (PTZ) to establish a rat animal model of epilepsy, the successful model of postscript recorded behaviors of rats with epilepsy manifestations, modeling the rats were randomly divided into model group, western medicine group, Shi Gan San Shi Gan powder low dose group, middle dose group, Shi Gan San high dose group, western medicine group with sodium valproate 15.75mg/Kg gavage treatment, traditional Chinese medicine group were given Shi Gan powder 0.4g/Kg, 0.8g/Kg, 1.6g/Kg by gavage, the blank group to Distilled water 3ml/kg orally, 21 days for a course, change the behavior of rats were observed after treatment, onset level changes, weight change, observe the e four nitrogen treatment of epileptic rats after the pathological changes of hippocampal neurons by Nissl staining, changes by immunohistochemical method to detect the expression of BDNF in the sea horse, through the whole cell patch clamp recording after treatment of hippocampal neurons in rats of voltage dependent Na~+ channel, K~+ channel current amplitude changes. The change results before and after treatment of 1. rats in each group: (1) weight change: afterinjecting weight and blank group rats were significantly decreased (P0.01), after treatment, model body mass of rats decreased significantly compared with the control group (P0.01), no difference in other groups with the control group (P0.05); compared with before treatment, the treatment group increased weight, Shi Gan powder low dose group was statistically significant (P0.05), western medicine group, Shi Gan powder in high dose group, significant differences (P0.01). (2) seizure latency (Epileptic seizure, latency, ESL) change: before treatment, the seizure latency time between groups showed no significant difference (P0.05). After treatment, the epileptic model group seizure latency time is shortened, the other groups were increased seizure latency time, among them, the western medicine group and Shi Gan powder, low and high dose group seizure latency time significantly increased, with the statistical significance (P0.01). (3) tetanic convulsion latency (Tonic convulsion, latency, TCL) change: compared each group after treatment with before treatment, tetanic convulsion latency of rats in model group was significantly decreased (P0.05), and the rest of the rats increased latency, Shi Gan powder of low dose group latency was statistically significant (P0.05), western medicine group, Shi Gan powder, are significant in high dose group (P0.0 1). (4) ankylosing convulsion rate (Tonic seizure rate) changes: compared before and after treatment group, model group of ankylosing convulsion rate increased significantly (P0.01), western medicine group and Shi Gan powder in high dose group was significantly lower than before treatment onset (P0.01), low dose of Shi Gan powder group before treatment did not change significantly (P0.05) to observe the pathological changes of hippocampal neurons.2. and Nissl staining method: (1) the morphological changes of neurons in rats: normal group CAI pyramidal cells arranged closely, deep staining, Nissl body shape, the number of normal pyramidal cell shrinkage; morphology of the rats in the model group, the number of decreased and showed a disorganized, Nissl body shallow dyeing and partial dissolution; western medicine group of neurons, the cell shape is regular, some incomplete neurons and the number of shrinkage, partial loss, deep dyeing and Nissl body; Shi Gan powder quantity low dose group of neurons decreased, Nissl body shallow dye dissolved, pyramidal cells arranged in loose and irregular shape; Shi Gan powder quantity in middle dose group, pyramidal cell morphology decreased slightly, slightly wrinkled, Nissl body was slightly shallow; Shi Gan San high dose group of neurons arranged orderly, can form, the number of slightly less, Nissl bodies were anachromasis. (2) comparison of Nissl positive cells: compared with the blank group, model group and Shi Gan powder of low dose group Nissl positive cells decreased significantly, there was statistical significance (P0.01); compared with the model group, blank group, western medicine group and Shi Gan powder, the number of Nissl positive cells the high dose group were significantly increased (P0.01); comparison between each group of Nissl positive cells, model group and Shi Gan powder of low dose group decreased (P0.01), the number of cells in other groups compared with the control group had little difference, the western medicine group and Shi Gan powder relatively high dose group the number of positive cells and, two 鑰呮棤鏄庢樉缁熻瀛﹀樊寮，
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