【作者】 王雪；

【导师】 孟红梅；

【作者基本信息】 吉林大学 ， 神经病学， 2019， 博士

【摘要】 本研究分为三部分进行实验:实验一:谷氨酸诱导HT22细胞发生Parthanatos死亡机制的研究目的:多聚(ADP-核糖)聚合酶1(PARP-1)依赖的细胞死亡,也称为Parthanatos死亡,是一种新的独特的细胞死亡方式,在癫痫研究领域中却鲜有报道。本部分研究拟在癫痫体外模型中探讨Parthanatos死亡是否存在于癫痫诱导的神经元损伤机制中,为癫痫的神经保护治疗提供新的靶点。方法:体外培养小鼠海马神经元细胞系HT22细胞,使用CCK8法检测GLU的不同浓度以及不同作用时间对HT22细胞损伤情况,以选取最理想的细胞损伤模型。利用PARP-1抑制剂PJ34和si RNA沉默PARP-1基因技术对GLU诱导的HT22细胞损伤进行干预,使用LDH释放试验、Western blot、免疫荧光、流式细胞术等技术观察相关指标,探讨其作用机制。结果:(1)GLU诱导HT22细胞发生损伤呈浓度和时间依赖性,随着GLU作用时间的延长,细胞死亡率呈递增趋势(p<0.01),细胞线粒体的膜电位进行性下降(p<0.01),细胞内PARP-1表达升高,PAR的合成增多,线粒体AIF发生核转位增加;(2)与GLU作用HT22细胞12h和24h的死亡率相比,相应的PJ34预处理均可明显降低细胞的死亡率(12h,p<0.05;24h,p<0.01)。与此同时,PJ34的应用可明显降低GLU诱导的HT22细胞内PARP-1的表达和PAR的合成,并减少线粒体AIF向核转位。PJ34还可以减轻GLU诱导的HT22细胞内线粒体膜电位的下降(p<0.01);(3)si RNA沉默PARP-1基因后,可明显减少GLU诱导的HT22细胞死亡(p<0.01),并降低细胞内PARP-1的表达和PAR的合成,减少线粒体AIF向核发生转位,还会减轻GLU诱导的HT22细胞内线粒体膜电位的下降(p<0.01)。结论:(1)GLU诱导HT22细胞发生死亡呈浓度和时间依赖性;(2)GLU诱导的HT22细胞死亡存在Parthanatos死亡机制;(3)PARP-1可以作为神经保护治疗的潜在干预靶点。实验二:氧化应激在谷氨酸诱导HT22细胞发生Parthanatos死亡中的作用目的:探讨谷氨酸诱导HT22产生的ROS与Parthanatos死亡的关系,明确ROS在Parthanatos死亡机制中的始动作用。方法:体外培养小鼠海马神经元细胞系HT22细胞,使用GLU诱导HT22细胞来模拟癫痫后细胞损伤体外模型。利用LDH释放试验、Western blot、流式细胞术、酶标法以及ELISA法进行细胞死亡率、ROS、8-OHd G、还原型谷胱甘肽(Glutathione,GSH)以及相关蛋白的检测,明确氧化应激在谷氨酸诱导HT22细胞发生Parthanatos死亡中的作用,并证实NAC预处理对细胞的保护作用。结果:(1)GLU诱导的HT22细胞生成的ROS和DNA损伤标志物8-OHd G的水平呈时间依赖性,NAC预处理可有效降低ROS的生成(p<0.01)和8-OHd G的表达(p<0.01),同时可以提高细胞内GSH的水平(p<0.01);(2)NAC预处理可以明显减少GLU诱导的HT22细胞死亡(p<0.01),同时降低GLU诱导HT22细胞内PARP-1的表达和PAR的合成,并减少线粒体AIF向核转位。结论:(1)ROS可能是GLU诱导HT22细胞发生Parthanatos死亡的始动因素;(2)NAC可能是通过提高细胞内GSH水平发挥抗氧化损伤的作用;(3)NAC可有效抑制GLU诱导HT22细胞发生Parthanatos死亡。实验三:NAC及PJ34对海人酸诱导的大鼠癫痫模型的神经保护作用及机制研究目的:探讨抗氧化剂NAC及PARP-1抑制剂PJ34在癫痫体内研究中的作用及机制。方法:对Wistar大鼠右侧杏仁核注射KA来制备大鼠癫痫模型,观察大鼠行为学特征及病理学变化(HE染色)。应用NAC及PJ34进行预处理,使用FJB染色、Western blot、酶标法以及ELISA等技术进行相关指标测定,来探索其在体内癫痫模型中的作用机制。结果:(1)杏仁核注射KA诱导大鼠癫痫模型的行为学特征与人类癫痫发作相似,这种模型造模成功率高且死亡率低;(2)在KA点燃大鼠癫痫模型中,海马组织CA1和CA3区可见神经元的损害,其中以CA3区损害为主;(3)PJ34预处理可明显减轻大鼠癫痫发作后海马神经元的损害,具有神经保护作用(p<0.01),并抑制海马组织PARP-1的表达和PAR的合成,同时减少线粒体AIF向核转位;(4)NAC预处理明显减轻大鼠癫痫后海马神经元的损害(p<0.01)以及DNA的损伤(p<0.01),并增加海马组织内GSH的含量(p<0.01),同时,NAC预处理可抑制海马组织PARP-1的表达和PAR的合成,并减少线粒体AIF向核转位。结论:(1)在KA点燃大鼠癫痫模型中,海马组织病理改变以CA1和CA3区神经元损害为主(CA3区更为严重);(2)PJ34对大鼠癫痫后海马神经元的损害具有保护作用,其可抑制海马神经元发生Parthanatos死亡;(3)NAC对大鼠癫痫后海马神经元的损害具有保护作用,其可能是通过提高组织GSH的量而减轻癫痫发作造成海马组织的DNA损伤,另外,NAC亦可抑制海马神经元发生Parthanatos死亡。更多还原

【Abstract】 This study is divided into 3 parts:PART 1: Study on the mechanism of Parthanatos induced by glutamate in HT22 cellsObjective: Polymer(ADP-ribose)polymerase 1(PARP-1)dependent cell death,also known as Parthanatos,is a new and unique way of cell death,but rarely reported in the field of epilepsy.This study aims to investigate whether Parthanatos was involved in the mechanism of epilepsy-induced neuronal damage in vitro study,and to provide new targets for neuroprotective treatment of epilepsy.Methods: HT22 cells,cultured in vitro,were stimulated by GLU at different concentrations and at different times.CCK8 kit was used to detect mortality of HT22 cells induced by GLU to choose the best cell injury model.In addition,PARP-1 inhibitor PJ34 and si RNA silencing PARP-1 gene technology were used to intervene in GLU-induced HT22 cell injury.Then,LDH release test,Western blot,immunofluorescence,flow cytometry and other techniques were used to observe the relevant indicators to explore the function and the mechanism of PJ34 and si RNA silencing PARP-1 gene on GLU-induced HT22 cell injury.Results:(1)GLU induced the damage of HT22 cells in a concentration and time dependent manner.With the prolonged treatment time of GLU on HT22 cells,the mortality of the cell was progressively increased(p < 0.01),mitochondrial membrane potential was progressively decreased(p < 0.01),the expression of intracellular PARP-1 and the synthesis of PAR were significantly increased,and the translocation of AIF from mitochondrial to the nucleus was also increased.(2)Compared with the mortality of HT22 cells treated with GLU for 12 h and 24 h,the corresponding pretreatment of PJ34 significantly decreased the mortality of HT22 cells(12 h,P < 0.05;24 h,P < 0.01).Meanwhile,application of PJ34 significantly reduced the expression of intracellular PARP-1 and the synthesis of PAR in GLU-induced HT22 cells,and inhibited the translocation of AIF from mitochondrial to thenucleus.Also,PJ34 inhibited the decrease of mitochondrial membrane potential in GLU-induced HT22 cells(p < 0.01).(3)Silencing PARP-1 gene by si RNA technology significantly reduced the mortality of GLU-induced HT22 cells(p < 0.01)and the expression of intracellular PARP-1 and the synthesis of PAR.It also inhibited the translocation of AIF from mitochondrial to the nucleus and the decline of mitochondrial membrane potential in GLU-induced HT22 cells(p < 0.01).Conclusion:(1)GLU induces the death of HT22 cells in a concentration and time dependent manner.(2)Parthanatos is involved in the pathway of GLU-induced HT22 cell death.(3)PARP-1 can be a potential intervention target for neuroprotective therapy.PART 2: Role of oxidative stress in Parthanatos induced by glutamate in HT22 cellsObjective: This study aims to investigate the relationship between ROS produced by glutamate on HT22 and Parthanatos,and to clarify the initial role of ROS in the mechanism of Parthanatos.Methods: HT22 cells,cultured in vitro,were stimulated by GLU to simulate the cell damage model in vitro after epilepsy.LDH release test,Western blot,flow cytometry,enzyme labeling and ELISA were used to detect cell mortality,ROS,8-OHd G,glutathione(GSH)and related proteins to clarify the role of oxidative stress in Parthanatos induced by GLU in HT22 cells,and to confirm the protective effect of NAC on HT22 cells.Results:(1)With the prolonged treatment time of GLU on HT22 cells,the levels of ROS and 8-OHd G(DNA damage marker)were progressively increased(p < 0.01).Application of NAC significantly reduced the production of ROS(p < 0.01)and the expression of 8-OHd G generated from GLU-induced HT22 cells(p < 0.01).Meanwhile,NAC also increased the level of GSH(p < 0.01).(2)Pretreatment of NAC significantly reduced the mortality of HT22 cells induced by GLU(p < 0.01),decreased the expression of intracellular PARP-1 and the synthesis of PAR,and inhibited the translocation of AIF from mitochondrial to the nucleus.Conclusion:(1)ROS may be the initial factor of Parthanatos in HT22 cells induced by GLU.(2)NAC plays an antioxidant role by increasing the level of GSH in GLU-inducedHT22 cells.(3)NAC can effectively inhibit the occurrence of Parthanatos in GLU-induced HT22 cells.PART 3: Neuroprotective effects and mechanisms of NAC and PJ34 on epilepsy rats induced by kainic acidObjective: This study aims to investigate the role and mechanism of antioxidant NAC and PARP-1 inhibitor PJ34 on epilepsy model in vivo.Methods: kainic acid(KA)was injected into the right amygdala of Wistar rats to establish epilepsy model,and the behavioral characteristics and pathological changes(HE staining)were observed.NAC and PJ34 were used for pretreatment,FJB staining,Western blot,enzyme labeling and ELISA were used to determine the related indicators to explore their function and mechanism in epilepsy model induced by KA.Results:(1)The behavioral characteristics of the epilepsy rats induced by KA were similar to those of patients with epilepsy.This model had a high success rate and low mortality rate.(2)The epilepsy rats induced by KA showed neurons damage located in CA1 and CA3 areas of hippocampus,especially in CA3 area.(3)Application of PJ34 significantly reduced the damage of hippocampal neurons after epileptic seizures in rats(p < 0.01).It also reduced the expression of PARP-1 and the synthesis of PAR in hippocampus,and inhibited the translocation of AIF from mitochondrial to the nucleus.(4)Application of NAC significantly reduced the death of hippocampal neurons(p < 0.01)and DNA damage(p <0.01)after epileptic seizures in rats(p < 0.01),accompanied with the increased level of GSH in hippocampus(p < 0.01).Meanwhile,NAC also reduced the expression of PARP-1 and the synthesis of PAR in hippocampus of epilepsy rats,and inhibited the translocation of AIF from mitochondrial to the nucleus.Conclusion:(1)In KA-induced epilepsy model,pathological changes in hippocampus were the damage of neurons located in CA1 and CA3 regions(more severe in CA3 regions).(2)PJ34 has protective effect on the damage of hippocampal neurons after epilepsy in rats,and inhibits the occurrence of Parthanatos in hippocampal neurons.(3)NAC has protective effect on the damage of hippocampal neurons after epilepsy in rats,and alleviates DNAdamage of hippocampal tissues caused by epilepsy by increasing the level of GSH.In addition,NAC also inhibit the occurrence of Parthanatos in hippocampal neurons.更多还原