氟西汀对IL-1β诱导的小鼠皮层神经元损伤的保护作用及机制

发布时间：2018-05-05 09:20

本文选题：氟西汀 + 凋亡　；参考：《南京医科大学》2016年硕士论文

【摘要】：神经炎症是多种神经系统疾病的发病机制之一,参与缺血性脑卒中、神经退行性病变、抑郁症等神经精神疾病的发生、发展。研究表明,白介素-1β(interleukin-1p, IL-1p)、肿瘤坏死因子-a(tumor necrosis factor-a, TNF-a)以及白介素-6(interleukelin-6, IL-6)等炎症因子以不同方式诱导神经损伤。抗炎治疗己经探索性地应用于脑卒中、癫痫以及阿尔兹海默病(Alzheimer's disease,AD)中。因此在神经系统疾病治疗策略中,寻找有效的抗神经炎症靶点以及有力的神经保护剂显得尤为重要。氟西汀(Fluoxetine)是一种经典的选择性5-羟色胺(Serotonin,5-HT)再摄取抑制剂(Selective serotonin reuptake inhibitor, SSRI)。除了抗抑郁症作用外,氟西汀尚有显著的抗炎、抗肿瘤及神经保护作用。越来越多的证据显示,氟西汀对缺血性脑卒中患者以及脑卒中模型动物均具有神经保护作用。研究表明氟西汀减少缺血性脑卒中模型大鼠脑梗死体积并降低其神经功能评分。此外,氟西汀减少缺血性脑卒中模型大鼠脑内TNF-a和IL-1p的释放、抑制核转录因子-kB (Nuclear factor-kB, NF-kB)活性、抵抗生物胺的丢失。氟西汀对缺血性脑损伤的保护作用可能与其抗炎、抗凋亡作用相关,但其确切机制尚未阐明。p53是一种肿瘤抑制基因。野生型p53蛋白由373个氨基酸组成,包括四个功能域：①N端功能域,又分为转录活化区和脯氨酸富集区；②DNA结合域,负责与DNA序列的结合；③寡聚化结构域；④C端调控域,大部分的转录后修饰均发生于此。生理条件下,由于鼠双微基因2(Mouse double minute 2 homolog, MDM2)的泛素化作用及蛋白酶体的降解,p53低水平表达。一旦发生DNA损伤,双键断裂促进FOXO3a、E2F1、YB1等转录因子的表达,促使ATM或ATR分别活化CHK2和CHK1,进而活化p53,调控下游靶基因的表达(尤其是凋亡蛋白Bax的表达)。p53功能广泛,参与细胞周期阻滞、凋亡、老化、DNA损伤修复等,在肿瘤的发生、发展中发挥着重要作用。近年来,研究发现p53也参与神经精神疾病的发生、发展,如缺血性脑卒中、神经退行性病变、抑郁症以及癫痫等。例如,在缺血性脑卒中模型中,p53通过死亡相关蛋白激酶1(Death-associated protein kinase 1, DAPK1)信号通路诱导神经元凋亡。炎症因子,尤其是IL-1β,通过诱导NF-κB核转位,进而活化p53上调凋亡调控因子(p53-upregulated modulator of apoptosis, PUMA),后者促进p53与BcI-XL解离。游离的p53活化Bax,最终导致凋亡的发生。上述研究提示p53可能参与调控氟西汀的抗凋亡作用,然而其在氟西汀对缺血性脑损伤的神经保护作用中的意义尚未见报道。本文通过在体与离体实验研究氟西汀的神经保护作用与p53的相关性及机制。研究发现,氟西汀抑制短暂性大脑中动脉阻塞(Transient middle cerebral artery occlusion, tMCAO)模型小鼠脑内神经元凋亡、IL-113释放及p53表达下降。离体培养原代皮层神经元以及N2a细胞株,进一步离体研究发现氟西汀通过p38-p53信号通路发挥神经保护作用。本文工作证实了氟西汀对缺血性脑卒中的神经保护作用,并揭示了p53在其中的重要意义和调控机制,深化了对氟西汀药理作用的认识,为氟西汀治疗神经系统疾病提供了实验依据和学术基础。目的：研究氟西汀对小鼠皮层神经元的保护作用及其与p53的相关性。方法：1.氟西汀对小鼠脑缺血再灌注急性期损伤的治疗作用。应用3月龄C57BL/6J雄性小鼠,建立tMCAO模型,缺血1 h,复灌1 h后腹腔注射(i.p.,bid.)氟西汀(40 mg/kg)。24 h后行神经功能评分,TTC染色测定脑梗死体积,免疫组织化学计算NeuN阳性细胞数,TUNEL法观察凋亡细胞,Western Blot检测Bax、Bcl-2、p53、IL-1p及proIL-1p蛋白表达。2.氟西汀对原代皮层神经元的抗凋亡作用。(1)离体培养小鼠原代皮层神经元,氟西汀(0.1、1、10μM)预处理1 h后,给予IL-1β(30 ng/ml)刺激24 h。应用Hoechst 33342荧光染色观察细胞凋亡,显微镜明场观察神经元突起,LDH法检测细胞活力,Western Blot检测Bax、Bcl-2、NF-kB、p53蛋白表达,Real-time PCR检测p53 mRNA表达,细胞免疫化学观察p53分布。(2)离体培养小鼠原代皮层神经元,cyanopindolol (10μM)或酮舍林(1 μM)预处理30 min后,给予上述相同药物刺激。应用Hoechst33342荧光染色观察细胞凋亡,Western Blot观察Bax、Bcl-2蛋白表达变化。3.氟西汀通过p38-p53信号通路发挥抗凋亡作用。(1)培养N2a细胞株,转染pcDNA3.1(-)-3HA-p53,氟西汀(1μM)预处理1 h后,给予IL-1β(30 ng/m1)刺激24 h。应用Hoechst 33342荧光染色观察细胞凋亡,LDH法检测细胞活力,流式细胞术观察细胞凋亡,Western Blot观察Bax、Bcl-2、NF-kB、p53蛋白表达。(2)培养N2a细胞株,氟西汀(1μM)预处理1 h后,给予茴香霉素(10 nM)或SB203580(10州)刺激11 h。WesternBlot观察MAPK、p53蛋白表达,Real-time PCR检测p53、FOXO3a mRNA表达。结果：1.氟西汀改善小鼠脑缺血再灌注急性期损伤。氟西汀(40mg/kg,i.p.,bid.)显著改善tMCAO导致的神经功能障碍,减小脑梗死体积,抑制神经元丢失,缓解细胞凋亡,抑制IL-1p释放。2. 氟西汀减少IL-1β导致的原代皮层神经元凋亡和炎症。离体培养小鼠原代皮层神经元,氟西汀(1μM)预处理1 h,显著抑制IL-1β(30 ng/ml)导致的神经元突起缩短、LDH释放、细胞核固缩、细胞凋亡、NF-κB信号通路活化。3.氟西汀通过抑制p38-p53信号通路的激活发挥抗凋亡及抗炎作用氟西汀下调原代皮层神经元和N2a细胞株p53表达。p53过表达取消氟西汀的抗炎及抗凋亡作用。氟西汀下调p53的同时伴随抑制p38磷酸化。p38激动剂茴香霉素可取消氟西汀对p53的下调作用。反之,p38抑制剂SB203580可模拟氟西汀对p53的下调作用。结论：1.氟西汀抗炎和对缺血性脑损伤的神经保护作用与调节p53表达相关。2.氟西汀通过抑制p38磷酸化调节p53。综上所述,本文工作的主要创新之处在于：1.证实氟西汀改善缺血再灌注急性期损伤本文研究发现氟西汀急性期用药显著减轻小鼠神经功能障碍、脑梗死体积、神经元丢失及细胞凋亡,提示氟西汀显著改善缺血再灌注急性期损伤,为氟西汀用于脑卒中急性期治疗提供实验基础和学术依据。2.阐明氟西汀通过p38-p53信号通路发挥神经保护作用氟西汀抑制IL-1β介导的原代皮层神经元和N2a细胞株细胞损伤,该保护作用在p53过表达后被取消,且氟西汀通过抑制p38信号通路发挥了对p53的下调作用。本研究首次提出氟西汀通过抑制p38-p53信号通路发挥神经保护作用,揭示了氟西汀神经保护的新机制,为氟西汀治疗脑卒中以及其他神经系统疾病提供新思路。
[Abstract]:Neuroinflammation is one of the mechanisms of a variety of nervous system diseases. It participates in the occurrence and development of neuropsychiatric disorders such as ischemic stroke, neurodegenerative disease, depression and other neuropsychosis. Studies have shown that interleukin -1 beta (interleukin-1p, IL-1p), tumor necrosis factor -a (tumor necrosis factor-A, TNF-a), and interleukin -6 (interleukelin-6, IL-6) Inflammatory factors induce nerve damage in different ways. Anti-inflammatory therapy has been applied to stroke, epilepsy and Alzheimer's disease (AD). Therefore, it is particularly important to find effective anti neuroinflammatory targets and powerful neuroprotectant in the treatment strategy of nervous system disease. Fluoxetine (Fluox Etine) is a classic selective 5- serotonin (Serotonin, 5-HT) reuptake inhibitor (Selective serotonin reuptake inhibitor, SSRI). Besides antidepressant effect, fluoxetine has significant anti-inflammatory, anti-tumor and neuroprotective effects. More and more evidence shows that fluoxetine has an ischemic stroke patient and a stroke model. The study shows that fluoxetine reduces the volume of cerebral infarction in ischemic stroke model rats and reduces the neurological function score. In addition, fluoxetine reduces the release of TNF-a and IL-1p in the brain of ischemic stroke model rats, inhibits the activity of nuclear transcription factor -kB (Nuclear factor-kB, NF-kB), and resists biogenic amines. The protective effect of fluoxetine on ischemic brain injury may be related to its anti-inflammatory and anti apoptotic effects, but its exact mechanism has not yet elucidated that.P53 is a tumor suppressor. The wild type p53 protein consists of 373 amino acids, including four functional domains: (1) the N terminal functional domain, and also a transcriptional activation area and a proline enrichment region; (2) DNA binding Domain, which is responsible for the combination of DNA sequences; (3) oligomeric domains; (4) C terminal regulation domain, most of the posttranscriptional modification occurs. Under physiological conditions, the ubiquitination of mouse double microgene 2 (Mouse double minute 2 homolog, MDM2) and the degradation of proteasome, p53 low level expression. Once DNA damage occurs, double bond fracture promotes FOXO. The expression of transcription factors, such as 3a, E2F1 and YB1, urges ATM or ATR to activate CHK2 and CHK1 respectively, and then activate p53 and regulate the expression of the downstream target gene (especially the expression of apoptotic protein Bax).P53 functions widely, and participate in cell cycle arrest, apoptosis, aging, DNA damage repair and so on. It plays an important role in the development of tumor. In recent years, research has been studied. It is found that p53 also participates in the occurrence and development of neuropsychiatric disorders, such as ischemic stroke, neurodegenerative disease, depression, and epilepsy. For example, in ischemic stroke model, p53 induces neuronal apoptosis through the signal pathway of death related protein kinase 1 (Death-associated protein kinase 1, DAPK1). Inflammatory factors, especially IL-1 beta, are induced. By inducing the transposition of NF- kappa B, then activating p53 to regulate the apoptosis regulating factor (p53-upregulated modulator of apoptosis, PUMA), and the latter promotes the dissociation of p53 and BcI-XL. The free p53 activation Bax, which eventually leads to the apoptosis, may be involved in the regulation of the anti apoptosis effect of fluoxetine, however, it is in fluoxetine for ischemic brain. The significance of the neuroprotective effect of injury has not yet been reported. In this paper, the correlation and mechanism of the neuroprotective effect of fluoxetine to p53 was studied in vivo and in vitro. It was found that fluoxetine inhibited neuronal apoptosis in the brain of Transient middle cerebral artery occlusion (tMCAO) model mice, IL-1 13 release and p53 expression decline. In vitro culture of primary cortical neurons and N2a cells. Further in vitro studies have found that fluoxetine plays a neuroprotective role through the p38-p53 signaling pathway. This work confirms the neuroprotective effect of fluoxetine on ischemic stroke, and reveals the significance and regulatory mechanism of p53 in it. The pharmacological effects of fluoxetine were known to provide experimental basis and academic basis for the treatment of fluoxetine in the treatment of nervous system diseases. Objective: To study the protective effect of fluoxetine on mouse cortical neurons and the correlation with p53. Methods: the therapeutic effect of 1. fluoxetine on acute cerebral ischemia reperfusion injury in mice. 3 month old C57BL/6J was used. In male mice, tMCAO model was established, ischemia was 1 h, and after reperfusion for 1 h, after intraperitoneal injection of (i.p., bid.) fluoxetine (40 mg/kg).24 h, the nerve function was scored, the volume of cerebral infarction was measured by TTC staining, the number of NeuN positive cells was calculated by immunohistochemistry, and apoptotic cells were observed by TUNEL method. The antiapoptotic effect on primary cortical neurons. (1) the primary cortical neurons of mice were cultured in vitro. After 1 h preconditioning with fluoxetine (0.1,1,10 mu M), IL-1 beta (30 ng/ml) was given to 24 h. To observe cell apoptosis by Hoechst 33342 fluorescence staining, microscopic field observation of neuron protuberance, LDH method to detect cell viability, Western Blot detection Bax, Bcl-2, Bcl-2. -kB, p53 protein expression, Real-time PCR detection of p53 mRNA expression, cell immuno chemical observation of p53 distribution. (2) in vitro culture of primary cortical neurons in mice, cyanopindolol (10 mu) or ketone shlin (1 mu M) pretreated 30 min, give the same drug stimulation. Protein expression change.3. fluoxetine exerts anti apoptosis effect through p38-p53 signaling pathway. (1) culture N2a cell line, transfection pcDNA3.1 (-) -3HA-p53, fluoxetine (1 M) pretreatment 1 h, IL-1 beta (30 ng/m1) stimulation 24 h. application Hoechst 33342 fluorescence staining to observe cell apoptosis, LDH method to detect cell viability, flow cytometry observation cell apoptosis, Estern Blot observed Bax, Bcl-2, NF-kB, and p53 protein expression. (2) culture N2a cell lines, and fluoxetine (1 micron M) pretreated 1 h, and gave anisomycin (10 nM) or SB203580 (10 state) to stimulate 11 h.WesternBlot to observe and express. Results: 1. fluoxetine improved the acute phase of cerebral ischemia reperfusion injury in mice. 40mg/kg (i.p., bid.) significantly improved the neurological dysfunction caused by tMCAO, reduced the volume of cerebral infarction, inhibited neuron loss, alleviated apoptosis, and inhibited the release of.2. fluoxetine to reduce the apoptosis and inflammation of primary cortical neurons induced by IL-1 beta, in vitro culture of the primary cortical neurons in mice, and the preconditioning of fluoxetine (1 mu M) was 1 h in vitro. Inhibition of IL-1 beta (30 ng/ml) induced neurite protuberance, LDH release, nuclear condensation, cell apoptosis, NF- kappa B signaling pathway activating.3. fluoxetine to inhibit apoptosis and anti-inflammatory effects of fluoxetine down regulation of fluoxetine down regulation of primary cortical neurons and N2a cells p53 expression by.P53 overexpression and canceling the anti inflammation of fluoxetine And antiapoptotic effect. Fluoxetine downregulates p53 while inhibiting p38 phosphorylation.P38 agonist anisomycin cancels fluoxetine's down regulation of p53. Conversely, p38 inhibitor SB203580 can mimic the down-regulation of fluoxetine to p53. Conclusion: 1. fluoxetine and the neuroprotective effect on ischemic brain injury are associated with the regulation of p53 expression. The main innovations of this work are: 1. the main innovations of this work are: 1. the findings of fluoxetine to improve the acute phase of ischemia-reperfusion injury in fluoxetine showed that the acute phase of fluoxetine significantly alleviated the dysfunction of the nerve in mice, the volume of cerebral infarction, the loss of neurons and the apoptosis of the cells, suggesting that fluoxetine was significant. Improving the acute phase of ischemia-reperfusion injury, providing experimental basis and academic basis for the treatment of acute phase of cerebral apoplexy by fluoxetine,.2. clarifies that fluoxetine exerts neuroprotective effect on the p38-p53 signaling pathway to inhibit IL-1 beta mediated primary cortical neurons and N2a cell cell damage, and the protective effect is taken after p53 overexpression. In addition, fluoxetine plays a downregulation effect on p53 by inhibiting the p38 signaling pathway. This study first proposed that fluoxetine played a neuroprotective role by inhibiting the p38-p53 signaling pathway and revealed a new mechanism for the protection of fluoxetine, providing new ideas for fluoxetine in the treatment of stroke and other nervous system diseases.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R743

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