锂盐对LPS诱导大鼠脑炎和脑炎后癫痫发作的影响及GSK-3β在其中的作用

发布时间：2018-05-23 10:19

本文选题：脑炎 + GSK-3β　；参考：《川北医学院》2017年硕士论文

【摘要】：目的:通过侧脑室注射脂多糖(LPS)构建成年SD大鼠脑炎模型,并检测海马组织中的炎性因子及GSK-3β的表达变化。观察不同剂量锂盐对该模型大鼠脑组织病理生理变化、GSK-3β表达及痫性发作敏感性的影响;应用GSK-3β激动剂WT、其间接抑制剂VPA进一步干预模型大鼠,观察GSK-3β在脑炎及脑炎后癫痫发作中的作用。以期初步探讨锂盐对脑炎和脑炎后癫痫发作的影响及可能机制,并为其防治提供新思路。方法:(1)LPS颅内感染大鼠模型的制备:将20只成年雄性SD大鼠随机分为对照组和LPS组,每组10只,LPS组大鼠侧脑室注射LPS50μg/5ul,对照组注射同等体积生理盐水(NS)。给药24小时后,每组随机选取5只大鼠麻醉,断头取脑,分离双侧海马组织,提取蛋白,采用ELISA检测IL-1β及TNF-α;采用Western-blot方法检测P-GSK-3β及GSK-3β。各组剩余5只大鼠灌注取脑,制作石蜡切片用作免疫组化检测脑组织的病理变化。(2)不同剂量锂盐对LPS致炎和癫痫发作的影响:取48只成年雄性SD大鼠,随机分为6组:对照组、LPS组、LPS-10mg Li组、LPS-20mgLi组、LPS-40mgLi组、LPS-80mgLi组,每组各8只,采用上述方法脑炎模型组侧脑室注入LPS 50μg/5μl,对照组注射同等体积生理盐水。3小时后腹腔分别注射5ml/kg的生理盐水(NS)、NS、10、20、40、80mg/Kg的Licl,每24小时一次,连续3天。然后行腹腔注射匹鲁卡品(Pilo),观察各组大鼠癫痫行为学表现。(3)不同剂量锂盐对lps颅内感染大鼠海马病理生理与癫痫发作的影响及其可能机制:取126只成年雄性sd大鼠,随机分为lps-10mgli组、lps-20mgli组、lps-40mgli组、lps-80mgli组、lps-vpa组、lps-wt组和lps组,每组18只,采用上述方法脑炎模型组侧脑室注入lps50μg/5μl,3小时后腹腔分别注射10、20、40、80mg/kglicl、30mg/kgvpa、0.6μg/kgwt和5ml/kg生理盐水,每24小时一次,连续3天。每组随机选取8只大鼠腹腔注射pilo,行颅内电极脑电图观察基础脑电频谱、能谱、发作后高频振荡活动变化及癫痫发作潜伏期与严重程度观察;另外每组随机选取5只大鼠麻醉断头取双侧海马组织,提取蛋白,采用elisa检测il-1β、tnf-α、il-10的浓度,western-blot检测p-gsk-3β及gsk-3β的表达量;剩余的大鼠采用免疫组化检测海马神经元和小胶质细胞表达。结果:(1)与对照组相比:lps组大鼠体温升高,进食、活动减少,且海马组织中il-1β、tnf-α的浓度明显升高(p0.05);lps组大鼠对pilo的致痫性增强,se潜伏期明显缩短(p0.05),同时该组大鼠海马组织中gsk-3β的含量较对照组明显升高,而p-gsk-3β的含量较对照组明显降低(p0.05);海马ca1区有大量活化增生的小胶质细胞,神经元排列松散、紊乱、脱失、形态模糊,染色变浅,形态不完整,与对照组比较有显著差异(p0.05)。(2)与lps组比较:小剂量锂干预(lps-10mgli组、lps-20mgli组)海马局部场电位ripple震荡功率谱显著降低,se潜伏期明显延长,lps-40mgli组大鼠se潜伏期有所延长但无统计学意义(p0.05),而lps-80mgli组ripple震荡功率谱密度及振幅明显增高,se潜伏期明显缩短(p0.05)。同时与LPS组比较,LPS-10mgLi、LPS-20mgLi组、LPS-VPA组大鼠海马组织中p-GSK-3β/GSK-3β比值明显变大(p0.05),海马组织中致炎因子IL-1β、TNF-α的含量显著降低(p0.05),抗炎因子IL-10显著升高(p0.05),伴随较少激活的小胶质细胞(p0.05),海马CA1区神经元出现排列致密、整齐、脱失少、形状清楚,染色较深,神经元形状更完整;而LPS-80mg Li组、LPS-WT组出现相反结果。结论:LPS可诱导SD大鼠脑炎改变,并导致大鼠CA1区神经元丢失,小胶质细胞活化;颅内炎症促进癫痫发作,小剂量的锂具有抑制炎性活动,抑制小胶质细胞的增生作用,减轻脑炎后脑损伤。大剂量的锂则作用相反。小剂量锂盐可延长脑炎后癫痫放电潜伏期,降低ripple震荡振幅,降低海马神经元兴奋性,提高癫痫发作阈值,降低脑炎后癫痫发作的敏感性,对脑炎后癫痫可能具有一定的修饰作用。小剂量锂盐抑制大鼠海马组织中GSK-3β的表达,抑制炎症因子,减轻组织病理损伤,提示GSK-3β可能参与锂修饰脑炎后癫痫的机制。
[Abstract]:Objective: to construct an adult SD rat model of encephalitis by injection of lipopolysaccharide (LPS) in the lateral ventricle and detect the changes in the expression of inflammatory factors and GSK-3 beta in the hippocampus. The effects of different doses of lithium salt on the pathophysiological changes of the brain tissue, the expression of GSK-3 beta and the sensitivity of epileptic seizures in the model rats were observed. The indirect inhibition of the GSK-3 beta agonist WT was used. The effect of GSK-3 beta in the epileptic seizures after encephalitis and encephalitis was observed by VPA. The effect and possible mechanism of lithium salt on the seizures of encephalitis and encephalitis were preliminarily discussed. Methods: (1) the preparation of LPS rat model of intracranial infection: 20 adult male SD rats were randomly divided into control Group and group LPS, 10 rats in each group were injected with LPS50 mu g/5ul in the lateral ventricle of group LPS, and the control group was injected with the same volume of physiological saline (NS). After 24 hours of administration, 5 rats were randomly selected to extract the brain, separate the bilateral hippocampal tissues, extract the protein, detect the IL-1 beta and TNF- alpha by ELISA, and detect P-GSK-3 beta and GSK-3 beta by Western-blot method. The remaining 5 rats were perfused to take the brain, and the paraffin sections were used as immunohistochemistry to detect the pathological changes of brain tissue. (2) the effects of different doses of lithium salts on LPS induced inflammation and seizures: 48 adult male SD rats were randomly divided into 6 groups: control group, group LPS, group LPS-10mg Li, group LPS-20mgLi, LPS-40mgLi group, LPS-80mgLi group, and 8 rats in each group, with 8 rats in each group, using 8 rats in each group. The lateral ventricle of the model group of encephalitis was injected with LPS 50 mu g/5 Mu L, and the control group injected the same volume of physiological saline.3 hours after.3 hours, respectively, and injected 5ml/kg in normal saline (NS), Licl of NS, 10,20,40,80mg/Kg, once every 24 hours for 3 days. Then the intraperitoneal injection of pilocarpine (Pilo) was performed. (3) different agents were observed. The effect and possible mechanism of lithium salt on hippocampal pathophysiology and epileptic seizures in LPS rats: 126 adult male SD rats were randomly divided into group lps-10mgli, group lps-20mgli, group lps-40mgli, lps-80mgli group, lps-vpa group, lps-wt group and LPS group, 18 rats in each group, and the lateral ventricle of encephalitis model group was injected lps50 mu g/5 L, 3 The intraperitoneal injection of 10,20,40,80mg/kglicl, 30mg/kgvpa, 0.6 u g/kgwt and 5ml/kg saline, once every 24 hours, for 3 days for 3 days. Each group randomly selected 8 rats by intraperitoneal injection of pilo to observe the basic EEG spectrum of intracranial electroencephalogram, spectrum, changes of high frequency vibration after seizures and the latency and severity of epileptic seizures In addition, each group selected 5 rats randomly to pick up bilateral hippocampal tissues and extract the protein. The concentration of IL-1 beta, tnf- a, IL-10 was detected by ELISA, and the expression of p-gsk-3 beta and GSK-3 beta was detected by Western-blot. The remaining rats were immunohistochemistry to detect the expression of hippocampal neurons and microglia. Results: (1) compared with the control group, the group of LPS group was larger than the control group. The concentration of IL-1 beta and tnf- alpha in the hippocampus increased significantly (P0.05) in the hippocampus (P0.05), and in the LPS group, the se latency was significantly shortened (P0.05), and the content of GSK-3 beta in the hippocampus of the rats was significantly higher than that in the control group, but the content of p-gsk-3 beta was significantly lower than that in the control group (P0.05); C in the hippocampus; C in the hippocampus. There were a large number of activated microglia cells in the A1 area. The neurons were loosely arranged, disordered, lost and blurred, and the morphology was shallow, and the morphology was not complete. Compared with the control group, there were significant differences (P0.05). (2) the local field potential ripple shock power spectrum of the hippocampus decreased significantly in the small dose lithium intervention (group lps-10mgli, lps-20mgli group), and the se latent period was clear. In group lps-40mgli, the latency of Se was prolonged, but there was no statistical significance (P0.05), while the power spectrum density and amplitude of ripple concussion in the lps-80mgli group increased significantly and the latency of Se shortened obviously (P0.05). Meanwhile, the ratio of p-GSK-3 beta beta in the hippocampus of rats of LPS group was significantly higher than that in LPS group. The content of inflammatory factors IL-1 beta, TNF- a in hippocampus decreased significantly (P0.05), anti inflammatory factor IL-10 increased significantly (P0.05), with less activated microglia (P0.05), the neurons of the hippocampal CA1 region appeared dense, neatly, less lost, clear in shape, deep in color, and more complete in the shape of neurons, while LPS-80mg Li group, LPS-WT group appeared opposite. Results. Conclusion: LPS can induce the changes of encephalitis in SD rats, and lead to the loss of neurons in the CA1 area and the activation of microglia, the intracranial inflammation promotes the seizure. The small dose of lithium can inhibit the inflammatory activity, inhibit the proliferation of microglia and reduce the brain injury after encephalitis. The small dose lithium salt can prolong the brain. The latent period of epileptic discharge after inflammation reduces the amplitude of ripple oscillation, reduces the excitability of hippocampal neurons, increases the threshold of epileptic seizures, reduces the sensitivity of epileptic seizures after encephalitis, and may have some modifier effect on postencephalitis epilepsy. Small dose of lithium salt inhibits the expression of GSK-3 beta in the hippocampus of rats, inhibits inflammatory factors and reduces histopathology. Injury suggests that GSK-3 beta may be involved in the mechanism of lithium modifying epilepsy after encephalitis.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1

【参考文献】