MyD88在癫痫活动中的作用及机制研究

发布时间：2018-05-02 04:19

本文选题：MyD88 + ST2825　；参考：《重庆医科大学》2015年博士论文

【摘要】：第一部分 MyD88在难治性癫痫患者及癫痫动物中的表达目的：越来越多的证据表明脑内的炎症反应是癫痫发生发展的重要病理生理机制。髓样分化因子88(Myeloid differentiation factor 88, MyD88)是细胞内的接头分子,在机体免疫和炎症反应中起关键作用。本实验首先在难治性颞叶癫痫(temporal lobe epilepsy, TLE)患者及急、慢性癫痫动物模型中测定MyD88的表达规律,以探讨癫痫活动是否影响MyD88的表达。方法：1.从本课题组建立的癫痫脑库中随机抽取25例难治性TLE患者和15例对照组患者的颞叶新皮质；使用免疫荧光、免疫组化、和免疫印迹法测定MyD88的表达规律。2.使用氯化锂和皮罗卡品(25mg/kg)在SD大鼠中诱导急性癫痫发作模型,在癫痫发作后6h、24h、和72h,采用免疫荧光和免疫印迹法测定大鼠海马及周围皮质中MyD88的表达规律；建立急性戊四氮(pentylenetetrazol, PTZ)模型(65mg/kg),在PTZ注射后3h、6h、和24h,采用免疫印迹法测定大鼠海马中MyD88的表达规律。3.使用氯化锂和皮罗卡品在SD大鼠中建立慢性TLE模型,在皮罗卡品注射2个月后,采用免疫组化和免疫印迹法在有慢性自发性癫痫发作(spontaneous recurrent seizures, SRS)大鼠的海马及周围皮质中检测MyD88的表达规律；建立慢性PTZ点燃模型,采用免疫印迹法在点燃大鼠脑组织中测定MyD88的表达规律。结果：1.MyD88在难治性TLE患者脑组织中表达升高,主要表达于神经元的胞浆中。2.在急性皮罗卡品模型中,MyD88在癫痫发作后24h和72h表达升高,主要与神经元共表达,没有与胶质细胞共表达；在急性PTZ模型中,MyD88在癫痫发作后6h和24h表达升高。3.在慢性匹罗卡品模型中,在有SRS的大鼠海马和皮质中MyD88表达升高；在慢性PTZ点燃模型中,MyD88在点燃大鼠海马中表达也升高。结论：MyD88在难治性TLE患者和急、慢性癫痫大鼠脑组织中表达均升高,说明癫痫活动可影响MyD88的表达。第二部分MyD88在癫痫大鼠发作中的作用目的：为了测定MyD88对癫痫发作的影响,本部分在急性匹罗卡品和急性PTZ模型中测定MyD88的小分子抑制剂ST2825及刺激分子IL-1p对癫痫发作的影响。方法：1.在急性皮罗卡品模型中,在皮罗卡品注射前20min侧脑室注射不同剂量的MyD88抑制剂ST2825 (2.5μg、5μg、10μg),在皮罗卡品注射后90min,观察ST2825对癫痫发作的行为学影响,评价指标为SE发生率、SE潜伏期、癫痫发作严重程度、和累积发作评分。2.在急性皮罗卡品模型中,在皮罗卡品注射前20min给予10ggST2825,观察其对脑电图(electroencephalogram, EEG)上癫痫发作的影响,评价指标为EEG上第一次癫痫发作潜伏期、总的癫痫发作次数、和总的癫痫发作时间；检测10μg ST2825对小胶质细胞活化的影响。3.在急性PTZ模型中,在PTZ(65mg/kg)注射前20min给予10μgST2825,观察其对癫痫发作的影响,评价指标为第一次阵挛发作的潜伏期、全身强直-阵挛发作的潜伏期、和全身强直-阵挛发作的持续时间。4.在急性皮罗卡品模型中,在皮罗卡品注射前20min给予ST2825,前10min给予重组大鼠IL-1β,实验分为溶剂组、IL-1β组,ST+IL-1β组,观察IL-1β是否有促惊厥作用,及ST2825对IL-1β促惊厥作用的影响。5.在急性PTZ模型中,在PTZ注射前20min给予ST2825,前10min给予重组大鼠IL-1β,实验分为溶剂组、IL-1β组,ST+IL-1β组,观察IL-1β是否有促惊厥作用,及ST2825对IL-1β促惊厥作用的影响。结果：1.在急性皮罗卡品模型中,侧脑室注射5μg及10μg ST2825显著延长SE潜伏期,减弱癫痫发作的严重程度,降低累积发作评分。2.在急性皮罗卡品模型中,侧脑室注射10μg ST2825显著延长EEG上第一次癫痫发作的潜伏期,减少癫痫发作的总次数,缩短癫痫发作总的持续时间；此外,10μg ST2825显著减轻由癫痫发作诱导的小胶质细胞活化程度。3.在急性PTZ模型中,侧脑室注射10μg ST2825显著延长第一次阵挛发作的潜伏期、延长全身强直-阵挛发作的潜伏期、并缩短全身强直-阵挛发作的持续时间。4.在急性皮罗卡品模型中,侧脑室注射lng IL-1β显著缩短SE潜伏期,加重癫痫发作严重程度,增加累积发作评分；在IL-1β前10min给予ST2825, ST2825阻止IL-1β诱导的SE潜伏期缩短,减轻IL-1β诱导的癫痫发作程度加剧,减弱IL-1β诱导的累积发作评分增加。此外侧脑室注射lng IL-1β显著缩短EEG上第一次癫痫发作的潜伏期,增加总的癫痫发作时间；ST2825阻止IL-1β诱导的EEG上第一次发作潜伏期的缩短,减轻总的癫痫发作次数,减少IL-1β诱导的癫痫发作时间的增加。5.在急性PTZ大鼠模型中,i.c.v. lng IL-1β显著缩短全身强直-阵挛发作的潜伏期并延长全身强直-阵挛发作的持续时间。预先给予ST2825可阻断IL-1β诱导的潜伏期缩短及全身强直-阵挛发作持续时间的增加。结论：1.MyD88的小分子抑制剂ST2825在急性匹罗卡品模型中可减少行为学上及EEG上的癫痫发作,减轻小胶质细胞的活化；ST2825在急性PTZ模型中也能减少癫痫发作。2.IL-1β在急性皮罗卡品和急性PTZ模型中均具有促惊厥作用,ST2825能抑制IL-1β的促惊厥作用。第三部分MyD88参与癫痫发作的机制探讨目的：NR2B酪氨酸位点的磷酸化在NMDA受体功能和神经元兴奋性中发挥重要作用。上部分的研究说明MyD88在癫痫活动中起重要作用,本部分对NR2B酪氨酸的磷酸化水平改变是否参与这个过程进行探讨。方法：1.采用免疫印迹法检钡NMD A亚基NR2B酪氨酸磷酸化位点1472(NR2B Tyr1472)在不同干预条件下的磷酸化水平改变,分组：vehicle组、Pilo组、IL-1β组、IL-1β+Pilo组、ST+IL-1β+Pilo组、和ST+Pilo组。2.在急性皮罗卡品模型中,在匹罗卡品注射前20min给予NR2BTyr1472的抑制剂艾芬地尔,在皮罗卡品10min前给予重组大鼠IL-1β,实验分为vehicle组、IL-1β组、Ifen组、和Ifen+IL-1β组,观察抑制NR2B Tyr1472后对IL-1β促惊厥作用的影响。3.在急性PTZ模型中,在PTZ注射前20min给予艾芬地尔,在PTZ 10min前给予重组大鼠IL-1β,实验分为vehicle组、IL-1β组、Ifen组、和Ifen+IL-1β组,观察抑制NR2B Tyr1472后对IL-1β促惊厥作用的影响。结果：1.单独给予IL-1β或癫痫发作本身(仅皮罗卡品)增加NR2B Tyr1472位点的磷酸化,分别增加154%和134%(P0.05)。同时给予IL-1β和皮罗卡品时,与vehicle组相比,NR2B Tyr1472的磷酸化水平增加213%,说明IL-1β和癫痫发作对其磷酸化水平增加具有叠加效应。10μgST2825显著抑制由癫痫发作引起的NR2B Tyr1472磷酸化水平增高(P0.05)。此外,10μg ST2825有效阻止IL-1β+皮罗卡品诱导的NR2BTyr1472磷酸化水平增高(P0.01)。2.在急性皮罗卡品模型中,侧脑室注射lng IL-1β显著缩短SE潜伏期,加重癫痫发作严重程度,增加累积发作评分；在IL-1β前10min给予NR2B Tyr1472的抑制剂艾芬地尔,艾芬地尔阻止IL-1β诱导的SE潜伏期缩短,缓和IL-1β诱导的癫痫发作程度加剧,减弱IL-1p诱导的累积发作评分增加。3.在急性PTZ模型中,与vehicle组相比,IL-1β组显著缩短全身强直-阵挛发作的潜伏期并延长全身强直-阵挛的持续时间。相反,与vehicle组相比,艾芬地尔显著增加第一次阵挛发作和强直-阵挛发作的潜伏期,减少全身强直-阵挛的持续时间,具有抗惊厥作用。此外,艾芬地尔可阻断IL-1β诱导的潜伏期缩短和全身强直-阵挛发作持续时间的延长。结论：1.在癫痫发作中,NR2B Tyr1472的磷酸化水平增高,IL-1β可使NR2B Tyr1472的磷酸化水平进一步增高,ST2825可降低由癫痫发作诱导的NR2B Tyr1472磷酸化水平的增加。2.NR2B亚基的选择性抑制剂艾芬地尔可抑制癫痫发作活动,并可抑制IL-1β的促惊厥作用。第四部分MyD88对癫痫形成的影响目的：越来越多的证据表明脑内的炎症反应在癫痫形成中起重要作用,抗炎治疗有可能成为抗癫痫形成治疗的新策略。前部分的研究证实MyD88在癫痫发作中起重要作用,本部分进一步研究MyD88对癫痫形成的影响。方法：1.在SD大鼠中每天给予亚惊厥剂量的PTZ (33mg/kg)建立慢性PTZ点燃模型,IL-1β在PTZ前10min给予,ST2825在PTZ前20min给予,分Con组、vehicle组、IL-1β组、和ST2825组,记录每次PTZ注射后癫痫发作的最高级别和达到完全点燃的潜伏期,完全点燃是指大鼠在PTZ注射后连续3天达到4级或4级以上的癫痫发作。2.在慢性皮罗卡品模型中,在SE结束后24h,每天侧脑室注射IL-1β和ST2825,每天一次,连续给予7天。实验分为vehicle组、IL-1β组、和ST2825组。从SE后的第21-34天,使用高清视频系统监测SRS活动。分析指标为SRS发生率、SRS发作次数、和SRS发作严重程度。结果：1.在慢性PTZ点燃模型中,IL-1β组完全点燃的潜伏期与vehicle组相比明显缩短,IL-1β组的癫痫发作级别在第5、7-9、10、12天与vehicle组相比明显加重。相反,ST2825组完全点燃的潜伏期与vehicle组相比明显延长,ST2825组的癫痫发作级别在第6-8、13-18天明显减轻。此外,Con组与vehicle组相比,达到完全点燃的潜伏期及癫痫发作严重程度没有明显差别。2.在慢性皮罗卡品模型中,SRS发生率在vehicle组、IL-1β组、和ST2825组没有明显差异。与vehicle组相比,IL-1β组SRS每天发作次数明显增加,差异有统计学意义(p0.05),ST2825组SRS每天发作次数差别不显著(p0.05)。与vehicle组相比,SRS的严重程度在IL-1β组明显增加(p0.05),而在ST2825组差别不显著(p0.05)。结论：1.在慢性PTZ点燃模型中,IL-1β可缩短PTZ完全点燃的潜伏期,并缩短点燃过程。相反,ST2825可显著延长完全点燃的潜伏期并延缓点燃过程,具有一定的抗癫痫形成作用。2.在慢性皮罗卡品模型中,IL-1β可增加SRS发作频率并加重SRS发作严重程度,而ST2825对SRS发作频率及SRS发作严重程度没有影响,说明post-SE诱导的癫痫形成过程更难被修饰,多阶段或多药联合干预可能是未来的研究方向。
[Abstract]:Part one MyD88 expression in intractable epilepsy and epileptic animals: more and more evidence shows that the inflammatory reaction in the brain is an important pathophysiological mechanism in the development of epilepsy. Myeloid differentiation factor 88 (Myeloid differentiation factor 88, MyD88) is a cell joint molecule, in the immune and inflammatory response of the body. This experiment first measured the expression of MyD88 in intractable temporal lobe epilepsy (temporal lobe epilepsy, TLE) and acute and chronic epileptic animal models to investigate whether epileptic activity affects the expression of MyD88. Methods: 1. from the epileptic brain bank established in this group, 25 cases of refractory TLE patients and 15 cases of MyD88 were randomly selected. Group of patients with temporal Ye Xin cortex; using immunofluorescence, immunohistochemistry, and immunoblotting to determine the expression of MyD88.2. using lithium chloride and 25mg/kg to induce acute epileptic seizures in SD rats, 6h, 24h, and 72h after epileptic seizures, using immunofluorescence and Western blotting to determine My in the hippocampus and the surrounding cortex. The expression of D88, the establishment of an acute amyl four nitrogen (pentylenetetrazol, PTZ) model (65mg/kg), 3h, 6h, and 24h after PTZ injection, the expression of MyD88 in the hippocampus of rats was determined by immunoblotting.3. using lithium chloride and pilocarpine to establish a chronic TLE model in SD rats. After 2 months of injection, immunization and immunity were used. The expression of MyD88 in hippocampus and surrounding cortex of rats with chronic spontaneous seizures (spontaneous recurrent seizures, SRS) was detected by immunoblotting. A chronic PTZ kindling model was established. The expression of MyD88 in the brain tissue of kindled rats was determined by immunoblotting. Results: 1.MyD88 was in the brain tissue of refractory TLE patients. The expression of.2. in the cytoplasm of neurons was mainly expressed in the acute pilocarpine model, and the expression of 24h and 72h increased after epileptic seizures, and the expression of 24h and 72h was mainly co expressed with neurons. In the acute PTZ model, MyD88 increased.3. in the chronic pilocarpine model after epileptic seizures, and in the chronic pilocarpine model, there were SRS in the acute PTZ model. The expression of MyD88 in the hippocampus and cortex of rats increased. In the chronic PTZ kindling model, the expression of MyD88 in the hippocampus of the kindled rats also increased. Conclusion: the expression of MyD88 increased in the brain tissues of the refractory TLE patients and the acute and chronic epileptic rats, indicating that the epileptic activity could affect the expression of MyD88. Second part of the role of MyD88 in the seizure of epileptic rats Objective: to determine the effect of MyD88 on epileptic seizures, this part examines the effects of MyD88's small molecular inhibitor ST2825 and stimulator IL-1p on epileptic seizures in acute pilocarpine and acute PTZ models. Methods: 1. in the acute pilocarpine model, different doses of MyD88 inhibition were injected into the 20min lateral ventricle before the injection of pilocarpine. The effects of ST2825 on epileptic seizures were observed after injection of ST2825 (2.5, 5, 5, G, 10 g). The behavioral effects of ST2825 on epileptic seizures were observed. The evaluation indexes were SE incidence, SE latency, seizure severity, and cumulative episodes of.2. in acute pilocarpine models. Before injection, 20min was given 10ggST2825, and the electroencephalogram (electr) was observed. The effects of oencephalogram, EEG) on epileptic seizures, the evaluation index was the first seizure latency in EEG, the total number of epileptic seizures, and the total seizure time; the effects of 10 mu g ST2825 on the activation of microglia were detected in the acute PTZ model, and 10 micron gST2825 was 20min before PTZ (65mg/ kg) injection to observe its seizures. The evaluation index was the incubation period of the first clonic seizure, the latent period of the generalized tonic clonic seizure, and the duration of the generalized tonic clonic seizure.4. in the acute pilocarpine model, 20min was given to ST2825 before the injection of the pilocarpine, and the former 10min was given to the recombinant rat IL-1 beta, and the experiment was divided into the solvent group, the IL-1 beta group, ST+IL-1 beta group, and the view of the ST+IL-1 beta group. The effect of IL-1 beta on convulsion, and the effect of ST2825 on IL-1 beta convulsion,.5. in the acute PTZ model, 20min was given ST2825 before PTZ injection, and the anterior 10min was given to the recombinant rat IL-1 beta. The experiment was divided into solvent group, IL-1 beta group and ST+IL-1 beta group. 1. in the acute pilocarpine model, the lateral ventricle injection of 5 g and 10 mu g ST2825 significantly prolongs the SE latency, reduces the severity of the seizures, and reduces the cumulative attack score in the acute pilocarpine model. The lateral ventricle injection of 10 mu ST2825 significantly prolongs the incubation period of the first seizure on EEG, reducing the total number of epileptic seizures, and reducing the total number of seizures. In addition, 10 g ST2825 significantly alleviated the degree of activation of microglia induced by epileptic seizures in the acute PTZ model. The lateral ventricle injection of the lateral ventricle significantly prolonged the incubation period of the first clonic seizure, prolonged the latent period of the generalized tonic clonic seizure, and shortened the generalized tonic clonic seizure. Duration of.4. in the acute pilocarpine model, the lateral ventricle injection of LNG IL-1 beta significantly shortened the SE latency, aggravated the severity of epileptic seizures and increased the cumulative attack score; ST2825 prevented the IL-1 beta induced SE latency, reduced the severity of the IL-1 beta induced epileptic seizures, and reduced the involvement of IL-1 beta induced involvement in IL-1 beta 10min. LNG IL-1 beta injection in this lateral ventricle significantly shortened the incubation period of the first seizure on EEG and increased the total seizure time; ST2825 prevented the shortening of the first seizure latency on EEG induced by IL-1 beta, reduced the total number of epileptic seizures, and reduced the time of epileptic seizures induced by IL-1 beta.5. in acute PTZ In the rat model, i.c.v. LNG IL-1 beta significantly shortens the latent period of generalized tonic clonic seizures and prolongs the duration of generalized tonic clonic seizures. Pre administration of ST2825 can block the shortened latency induced by IL-1 beta and the duration of generalized tonic clonic seizures. Conclusion: the small molecular inhibitor of 1.MyD88, ST2825, is in the acute pion. In the card model, epileptic seizures on behavior and EEG can be reduced and the activation of microglia can be reduced; ST2825 can also reduce seizure.2.IL-1 beta in acute PTZ and acute PTZ models with convulsion, and ST2825 can inhibit the effect of IL-1 beta convulsion in acute PTZ model. Part MyD88 involved in epileptic seizures. Objective: the phosphorylation of NR2B tyrosine sites plays an important role in the function of NMDA receptor and neuronal excitability. The previous study shows that MyD88 plays an important role in epileptic activity. This part studies whether the phosphorylation level of NR2B tyrosine is involved in this process. Method: 1. by immunoblotting The phosphorylation level of barium NMD A subunit NR2B tyrosine phosphorylation site 1472 (NR2B Tyr1472) was changed under different intervention conditions. Groups: Vehicle Group, Pilo group, IL-1 beta group, IL-1 beta +Pilo group, ST+IL-1 beta +Pilo group, and in acute pilocarpine model, before pilocarpine injection, the inhibitor alfentil, The recombinant rat IL-1 beta was given before 10min, and the experiment was divided into vehicle group, IL-1 beta group, Ifen group, and Ifen+IL-1 beta group. The effect of NR2B Tyr1472 on IL-1 beta convulsion was observed..3. in the acute PTZ model was given before the PTZ injection. The effect of inhibition of NR2B Tyr1472 on IL-1 beta convulsion was observed in group beta, Ifen, and Ifen+IL-1 beta. Results: 1. the phosphorylation of IL-1 beta or epileptic seizures alone (only pilocarpine) increased the phosphorylation of NR2B Tyr1472 sites, increased by 154% and 134% (P0.05), respectively, while giving IL-1 beta and pilocarpine, compared with vehicle group, NR2B The level of phosphorylation increased by 213%, indicating that IL-1 beta and epileptic seizures had a superposition effect on its phosphorylation level, which significantly inhibited the increase of NR2B Tyr1472 phosphorylation level (P0.05) caused by epileptic seizures (P0.05). In addition, 10 mu g ST2825 effectively prevented IL-1 beta + pilocarpine induced NR2BTyr1472 phosphorylation level increased (P0.01).2. was in a hurry. In the sex pilocarpine model, the injection of LNG IL-1 beta in the lateral ventricle significantly shortened the SE latency, aggravated the severity of epileptic seizures, and increased the cumulative attack score. In the front of IL-1 beta, apifentel, a NR2B Tyr1472 inhibitor, prevented the SE latency induced by IL-1 beta, reducing the severity of epileptic seizures induced by IL-1 beta, weakening IL-1 and weakening IL-1. The P induced cumulative attack score increased.3. in the acute PTZ model. Compared with the vehicle group, the IL-1 beta group significantly shortened the latent period of the generalized tonic clonic seizure and prolonged the duration of the generalized tonic clonus. On the contrary, the latent period of the first clonic seizure and tonic clonic seizures increased significantly compared with the vehicle group, and decreased the total duration of the first clonic seizure and tonic clonic seizures. The duration of strong direct clonus has an anticonvulsant effect. In addition, Ifen can block the shortened latency induced by IL-1 beta and prolongation of the duration of generalized tonic clonic seizures. Conclusion: 1. in epileptic seizures, the level of phosphorylation of NR2B Tyr1472 increases, IL-1 beta can increase the level of phosphorylation of NR2B Tyr1472, ST2825 can be reduced. The low level of phosphorylation of NR2B Tyr1472 induced by epileptic seizures increased the activity of epileptic seizures by the selective inhibitor of.2.NR2B subunit and inhibited the convulsion of IL-1 beta. Fourth the effects of MyD88 on the formation of epilepsy: more and more evidence suggests that the inflammatory reaction in the brain plays an important role in the formation of epilepsy. Anti-inflammatory therapy may be a new strategy for antiepileptic therapy. Previous studies have confirmed that MyD88 plays an important role in epileptic seizures. This part further studies the effect of MyD88 on the formation of epilepsy. Methods: 1. a chronic PTZ kindling model was established in SD rats with PTZ (33mg /kg) every day in a dose of subconvulsive dose (33mg /kg), and IL-1 beta was in the PTZ pre 10m. In was given, ST2825 was given in pre PTZ 20min, Con group, vehicle group, IL-1 beta group, and ST2825 group, record the highest level of epileptic seizures and the latent period of complete kindling after each injection of PTZ. The complete kindling was the.2. in the chronic pilocarpine model in rats after 3 days of PTZ injection to 4 or more. After 24h, IL-1 beta and ST2825 were injected every day in the lateral ventricle for 7 days. The experiment was divided into vehicle, IL-1, and ST2825. SRS activity was monitored by HD video system on day 21-34 after SE. The analysis index was SRS incidence, SRS attack times, and SRS attack severity. Results 1. in chronic PTZ kindling model, 1. The latent period of complete kindling in the group was significantly shorter than that in the vehicle group. The level of epileptic seizures in the IL-1 beta group was significantly higher than that in the vehicle group on day 5,7-9,10,12. On the contrary, the latent period of the complete kindling in the ST2825 group was significantly longer than that in the vehicle group. The epileptic seizure level in the ST2825 group was significantly reduced on the day 6-8,13-18. In addition, the Con group and vehicle were observed. There was no significant difference in the incubation period and the severity of epileptic seizures in the group.2.. In the chronic pilocarpine model, the incidence of SRS was not significantly different in the vehicle group, in the IL-1 beta group and in the ST2825 group. Compared with the vehicle group, the number of SRS episodes in the IL-1 beta group increased significantly (P0.05), and the SRS of the ST2825 group was SRS every day. There was no significant difference in the number of episodes (P0.05). Compared with the vehicle group, the severity of SRS increased significantly in the IL-1 beta group (P0.05), but in the ST2825 group, the difference was not significant (P0.05). Conclusion: 1. in the chronic PTZ kindling model, IL-1 beta can shorten the incubation period of PTZ, and shorten the ignition process. On the contrary, ST2825 can significantly prolong the incubation period of complete kindling. And postpone the kindling process, with a certain antiepileptic effect of.2. in the chronic pilocarpine model, IL-1 beta can increase the frequency of SRS seizures and aggravate the severity of SRS seizures, and ST2825 has no effect on the frequency of SRS and the severity of SRS seizures, indicating that post-SE induced seizures are more difficult to be modified, multistage or multidrug combination Intervention may be the future research direction.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R742.1

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