甘草甜素对幼鼠颞叶癫痫海马神经元的保护作用及其机制研究

发布时间：2018-05-23 20:05

本文选题：甘草甜素 + 海人酸　；参考：《郑州大学》2017年硕士论文

【摘要】：背景和目的癫痫是儿童时期常见的慢性神经系统疾病,70-80%的癫痫患者通过抗癫痫药物的治疗能控制癫痫的发作,但仍有20-30%左右的癫痫患者在接受规范合理的抗癫痫药物治疗后依然有发作而成为难治性癫痫。颞叶癫痫在难治癫痫中最常见,一直以来都是癫痫治疗的难点。大量研究表明,颞叶癫痫的反复发作可致海马区神经元的丢失及海马硬化,这不仅对病人的生活、工作产生巨大的影响,也给患者的家庭和社会造成了很大的负担。目前的抗癫痫药物主要作用于离子通道,抑制神经元的兴奋性,进行对症治疗。深入研究癫痫的病因和发病机制,针对发作机制中的关键环节发现新药物、寻求新的治疗途径,是癫痫治疗未来发展的方向。近年来越来越多的研究表明:癫痫与炎症密切相关,高迁移率族蛋白1(High mobility group protein 1,HMGB1)是一种重要的细胞炎症因子,正常情况下主要存在于细胞核中,参与维持核小体的结构和基因转录的调控,当发生组织损伤时迅速从损伤的神经元、小胶质细胞和星形胶质释放到细胞外、脑脊液及血清中,它的释放是触发免疫反应的危险信号,在脓毒症、关节炎等多疾病中具有重要的作用,最新研究发现其与癫痫的发生也有着紧密的联系。Toll样受体4(Toll-like receptor 4,TLR4)是一种识别病原相关分子模式(Pathogen-associated molecular patterns,PAMPs)的受体,能识别损伤或应激所致的“内源性危险信号”如:HMGB1,激活核转录因子kappa B(Nuclear factor-k-gene binding,NF-κB)发生磷酸化,磷酸化的NF-κB(Phosphorylated Nuclear factor-k-gene binding,p-NF-κB)进入细胞核,诱导促炎因子基因表达,脑内炎症导致癫痫反复发作。而甘草类药物有较好的抗炎、抗过敏和细胞膜保护作用,并且有较高的安全性和耐受性,临床上已广泛用来治疗肝炎、皮肤病等,最近国内外有研究表明甘草甜素(Glycyrrhizin,GL)作为HMGB1的小分子抑制剂,在炎症抑制方面有一定作用,并且有较好的安全性和耐受性,且部分药物可穿过血脑屏障,有望成为抗癫痫药物的候选者,但关于甘草类药物的神经元保护及抗癫痫作用的机制,目前还缺乏临床及实验数据的支持。本研究在用海人酸(Kainic acid,KA)诱导建立幼鼠颞叶癫痫模型的基础上,采用甘草甜素(Glycyrrhizin,GL)预处理,通过观察行为学表现,探讨甘草甜素对海人酸诱导幼鼠癫痫发作的敏感性及严重性的影响,利用Western blot与Q-RT-PCR分别从蛋白质与mRNA水平检测海马组织HMGB1/TLR4/p-NF-κB的表达,运用HE染色及免疫组化从细胞凋亡水平观察海马区神经元的丢失情况,探讨甘草甜素可能的抗癫痫及神经保护机制,及其与脑内免疫炎性反应的关系,有望老药新用,找到癫痫治疗的新方向并为其提供理论依据。材料与方法出生21天的SD大鼠,随机分为对照组、模型Ⅰ组、模型Ⅱ组。模型Ⅰ组用KA诱导癫痫发作,模型Ⅱ组在应用KA前30min腹腔注射GL,模型Ⅰ组根据观察时间点不同分为3h、12h、24h、7d四个亚组,模型Ⅱ组根据GL不同剂量分为10mg/kg、50mg/kg、100mg/kg三个亚组,每个亚组3只动物,行为学表现按照Racine评分量表进行评分,RT-PCR检测急性期(3h、12h、24h)海马区HMGB1/TLR-4 mRNA的表达,Western blot检测HMGB1/TLR-4/p-NF-κB蛋白的表达,Elisa检测血清中HMGB1蛋白的表达变化,免疫组化检测慢性期(7d)海马神经元抗核抗体(Neu-N)的表达。结果1.行为学结果:模型Ⅰ组SE发生率为75%,死亡4只,存活率为:67.3%,SOT:24.08±1.98min;模型Ⅱ组SE发生率为64.3%,总共死亡6只,存活率85.7%,SOT:33.39±2.66min;生理盐水对照组大鼠均未死亡,存活率为100%;GL预处理模型Ⅱ组与模型Ⅰ组比较,SE发生率降低,存活率增高,SOT时间延长。2.癫痫急性期海马区HMGB1/TLR-4基因表达结果:模型Ⅰ组与对照组比较,随着观察时间(3h、12h、24h)的延长,HMGB1、TLR-4基因表达升高,其中12h时达到峰值(差异具有统计学意义,P0.05),12h时间点,其中模型Ⅱ组HMGB1、TLR-4基因表达量较模型Ⅰ组显著降低(差异具有统计学意义,P0.05),不同剂量的干预的模型Ⅱ组之间无显著差异(P0.05)。3.癫痫急性期海马区HMGB1/TLR-4/p-NF-κB蛋白表达结果:模型Ⅰ组与对照组比较,随着观察时间(3h、12h、24h)的延长,TLR-4、p-NF-kB蛋白表达升高,其中12h时达到峰值(差异具有统计学意义,P0.05),HMGB1的蛋白表达没有明显变化(P0.05)。在12小时的时间点,模型Ⅱ组TLR-4、p-NF-k B蛋白表达量较模型Ⅰ组显著降低(差异具有统计学意义,P0.05),不同剂量的干预的模型Ⅱ组之间差异不大(P0.05)。4.癫痫急性期血清中HMGB1表达结果:模型Ⅰ组与对照组比较,血清中HMGB1浓度增高,其中12h时明显(差异具有统计学意义,P0.05),在12h的时间点,模型Ⅱ组与模型Ⅰ组比较,血清中HMGB1的浓度显著降低(差异具有统计学意义,P0.05),不同剂量的干预的模型Ⅱ组之间差异不大(P0.05)。5.慢性期海马区Neu-N表达结果:模型Ⅰ组与对照组比较,海马区神经元显著丢失(差异具有统计学意义,P0.05),模型Ⅱ组与模型Ⅰ组比较,海马区神经元的丢失显著减少(差异具有统计学意义,P0.05),不同剂量的干预的模型Ⅱ组之间差异不大(P0.05)。结论1.GL预处理可以延长幼鼠癫痫发作的潜伏期,降低癫痫发作的易感性,减轻幼鼠癫痫的发作。2.幼鼠癫痫急性期海马HMGB1/TLR-4基因表达增加,TLR-4的蛋白表达增高,诱导NF-κB的磷酸化。3.GL预处理可以降低HMGB1的基因合成及释放,降低胞外HMGB1浓度,降低TLR-4的基因表达及蛋白合成,抑制NF-κB的磷酸化。4.GL预处理能减轻癫痫所致的海马区病理性损害,预防神经元的丢失,发挥一定的神经保护作用。
[Abstract]:Background and objective epilepsy is a common chronic nervous system disease in childhood. Epilepsy patients in 70-80% can control epileptic seizures by antiepileptic drugs, but there are still about 20-30% of epileptic patients who still have seizures after receiving standardized and reasonable antiepileptic drugs. Temporal lobe epilepsy is difficult to treat epilepsy. A large number of studies have shown that recurrent seizures of temporal lobe epilepsy can cause loss of neurons in the hippocampus and hippocampus sclerosis, which not only affects the life and work of the patients, but also causes a great burden on the family and society. The current antiepileptic drugs are mainly responsible for the effect of antiepileptic drugs. The ion channel, which inhibits the excitatory of neurons, carries out symptomatic treatment, studies the etiology and pathogenesis of epilepsy, finds new drugs and seeks new ways of treatment in the key link of the seizure mechanism. It is the direction of the future development of epilepsy treatment. In recent years, more and more studies have shown that epilepsy is closely related to inflammation and high mobility group. Protein 1 (High mobility group protein 1, HMGB1) is an important cell inflammatory factor, which is normally present in the nucleus and is involved in the maintenance of the structure of the nucleosome and the regulation of gene transcription. When tissue damage occurs, it is quickly released from the damaged neurons, microglia and astrocytes to the extracellular, cerebrospinal fluid and serum. Its release is a dangerous signal to trigger the immune response and plays an important role in many diseases such as sepsis and arthritis. The latest research has found that it is closely related to the occurrence of epilepsy..Toll like receptor 4 (Toll-like receptor 4, TLR4) is a type of Pathogen-associated molecular patterns, PAM (PAM). Ps) receptors can identify "endogenous risk signals" caused by injury or stress, such as HMGB1, the activation of the nuclear transcription factor kappa B (Nuclear factor-k-gene binding, NF- kappa B), and the phosphorylation of NF- kappa B into the nucleus, inducing the gene expression of proinflammatory factors, and inflammation in the brain. It causes recurrent seizures, and Glycyrrhiza drugs have good anti-inflammatory, anti allergy and cell membrane protection, and have high safety and tolerance. It has been widely used in the treatment of hepatitis, dermatosis and so on. Recently, studies have shown that Glycyrrhizin (GL) is a small molecule inhibitor of HMGB1, in the area of inflammation inhibition. There is a certain effect, good safety and tolerance, and some drugs can pass through the blood brain barrier, which is expected to be a candidate for antiepileptic drugs. However, the mechanism of the protective and antiepileptic effects of Glycyrrhiza drugs is still lacking the support of clinical and experimental data. This study was induced by Kainic acid (KA). On the basis of the model of temporal lobe epilepsy in young rats, the effects of glycyrrhizin on the susceptibility and severity of epileptic seizures in young rats induced by glycyrrhizin were investigated by Glycyrrhizin (GL) preconditioning, and HMGB1/TLR4/p-NF- kappa B in hippocampus was detected from protein and mRNA levels by Western blot and Q-RT-PCR. Using HE staining and immunohistochemistry to observe the loss of neurons in the hippocampus from the level of apoptosis, explore the possible antiepileptic and neuroprotective mechanisms of glycyrrhizin, and its relationship with the immunological reaction in the brain. It is hopeful that the old drug can be used to find new directions for the treatment of epilepsy and provide a theoretical basis for it. Materials and methods are born. 21 days of SD rats were randomly divided into control group, model I group, model II group. Model I group used KA to induce epileptic seizures. Group II group was injected GL before KA 30min, model I group was divided into 3h, 12h, 24h, 7d subgroups according to the observation time points, and the model II group was divided into 10mg/kg, 50mg/kg, and three subgroups according to GL different doses, each group. In the subgroup, 3 animals were scored according to the Racine scale. RT-PCR was used to detect the expression of HMGB1/TLR-4 mRNA in the hippocampus of the acute phase (3H, 12h, 24h), the expression of HMGB1/TLR-4/p-NF- kappa B protein in Western blot, the expression of the protein in the Elisa test serum, and the immunohistochemical detection of the anti nuclear antibody of the hippocampal neurons in the chronic period. Results 1. the results of 1. behavior: the incidence of SE in model I group was 75%, and 4 died, the survival rate was 67.3%, SOT:24.08 + 1.98min; the incidence of SE in the model II group was 64.3%, the total mortality was 6, the survival rate was 85.7%, SOT:33.39 + 2.66min; the normal saline control group was not dead and the survival rate was 100%; GL Preconditioning Model II group and model I group were compared. In comparison, the incidence of SE decreased, the survival rate increased, and the SOT time prolonged the expression of HMGB1/TLR-4 gene in the hippocampus of.2.: model I was compared with the control group. With the prolongation of the observation time (3H, 12h, 24h), the expression of HMGB1 and TLR-4 increased, in which 12h reached the peak value (the difference was statistically significant, P0.05), and the 12h time point, model II Group HMGB1, TLR-4 gene expression was significantly lower than that in model I group (the difference was statistically significant, P0.05). There was no significant difference between the model II groups with different doses of intervention (P0.05) the expression of HMGB1/TLR-4/p-NF- kappa B protein in the hippocampus of.3. epileptic period of.3.: the model I group was compared with the control group, with the prolongation of the observation time (3H, 12h, 24h), TLR-4, The expression of p-NF-kB protein increased, of which 12h reached its peak value (the difference was statistically significant, P0.05), and there was no significant change in the protein expression of HMGB1 (P0.05). In the 12 hour time point, the expression of p-NF-k B protein in model II group was significantly lower than that in the model I group (the difference was statistically significant, P0.05), and the model II Group intervened in different doses. The results of HMGB1 expression in serum of P0.05.4. epileptic acute phase: in model I group, the concentration of HMGB1 in serum was higher than that in control group, of which 12h was significant (the difference was statistically significant, P0.05). In the time point of 12h, the concentration of HMGB1 in the model II Group was significantly reduced (the difference was statistically significant, P0., P0., P0.). 05), there was no significant difference between the model II groups in different doses of intervention (P0.05).5. chronic hippocampal Neu-N expression results: compared with the control group, the hippocampal neurons were significantly lost in model I group (the difference was statistically significant, P0.05). The loss of neurons in the hippocampus was significantly reduced in model II group and model I group (the difference was statistically significant. P0.05), there was little difference between the model II groups with different doses of intervention (P0.05). Conclusion 1.GL pretreatment could prolong the incubation period of epileptic seizures in young rats, reduce the susceptibility of epileptic seizures, reduce the expression of HMGB1/TLR-4 gene expression in hippocampus of young rats with epileptic seizures, increase the expression of TLR-4 protein and induce the phosphorus of NF- kappa B. Acidified.3.GL pretreatment can reduce the gene synthesis and release of HMGB1, reduce the concentration of extracellular HMGB1, reduce the gene expression and protein synthesis of TLR-4, and inhibit the phosphorylated.4.GL pretreatment of NF- kappa B to reduce the pathological damage in the hippocampus, prevent the loss of neurons and play a certain neuroprotective effect.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.1

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