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多肽药物MMI-0100在阿尔茨海默病小鼠模型中的作用机制研究

发布时间:2019-01-10 07:29  文章来源:笔耕文化传播
【摘要】:p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinases,p38MAPK)是MAPK激酶家族之一。已有大量研究报道p38MAPK/MK2信号通路参与调控细胞应激、炎症、凋亡等。针对该靶点,p38MAPK抑制剂(SB203580等)被开发研究,但因临床阶段出现的诸多安全隐患(肝毒性、心脏毒性、中枢神经系统毒性等),限制了其应用和发展。由此,下游底物MK2作为调控炎症反应的新型药物靶标成为目前研究的热点;近来设计合成的多肽药物MMI-0100是对MK2有抑制作用的外源治疗性多肽。且在外周炎症病变研究中表明有良好功效,基于此,MMI-0100靶向抑制p38MAPK/MK2信号通路可能对神经炎症病变也具有较好的调控作用。阿尔茨海默病(Alzheimer's disease,AD)是主要影响海马和皮质脑区的神经退行性疾病,病因复杂不明。但长期慢性的神经炎症反应引起神经胶质细胞持续激活和炎症介质的连续释放是导致神经元死亡并形成脑疾病的重要过程。本课题主要观察MMI-0100能否通过抑制神经炎症减轻小鼠AD模型中的认知损伤。结果:(1)采用小鼠新物体(NOR)和新位置(OLR)记忆识别模型,由Aβ42及LPS诱导神经炎症形成小鼠记忆损伤,发现给药MMI-0100 24h后,小鼠记忆损伤被减缓,并表现出与生理盐水组无显著性差异的记忆水平;(2)通过免疫组化实验,分析CD11b和GFAP蛋白表达量,确定小鼠海马中小胶质细胞及星形胶质细胞参与的炎症免疫应答情况,发现MMI-0100可以显著降低LPS引起的CD11b和GFAP的高表达,即MMI-0100能降低CNS中免疫细胞的持续激活,维持其处于静息状态;(3)在组织水平,实时定量PCR分析发现MMI-0100可以显著降低由Aβ42和LPS诱导的炎症因子(IL-6、IL-1β、TNF-α、i NOS)高表达,且在Western blot检测中发现MMI-0100可通过抑制ERK磷酸化来调控炎症反应,而p38MAPK可能在海马组织蛋白提取过程中被降解,因而未能确定其磷酸化水平;(4)在BV-2细胞系中检测发现MMI-0100为10-7M时能够显著降低LPS诱导的小鼠神经炎症中炎症因子(IL-6、IL-1β、i NOS及COX-2)表达,且能够浓度依赖性的降低ERK和p38MAPK磷酸化水平,表明MMI-0100可能是通过抑制ERK和p38MAPK通路来降低LPS引起的炎症反应;(5)MMI-0100是具有细胞穿膜活性的治疗性多肽,有趣的是,鼻黏膜给药MMI-0100后,由Aβ42和LPS引起的小鼠NOR和OLR记忆损伤模型检测发现,MMI-0100能够恢复小鼠记忆并与对照组无显著差异,为了说明此现象,合成荧光标记的Cy7.5-MMI-0100,进行小动物荧光成像,确定MMI-0100能利用其细胞穿膜活性通过鼻粘膜在脑区有大量分布,从而发挥抗炎作用。这将对多肽药物稳定性差、通过BBB率低等相关研究具有重要参考价值。
[Abstract]:P38 mitogen-activated protein kinase (p38 mitogen-activated protein kinases,p38MAPK) is one of the MAPK kinases. It has been reported that p38MAPK/MK2 signaling pathway is involved in regulation of cell stress, inflammation, apoptosis and so on. P38MAPK inhibitors (SB203580 et al.) have been developed for this target, but their application and development are limited by the safety risks (hepatotoxicity, cardiotoxicity, central nervous system toxicity, etc.) in the clinical stage. Therefore, the downstream substrate MK2 as a new drug target to regulate inflammatory response has become a hot topic. Recently, the peptide drug MMI-0100 has been designed and synthesized as an exogenous therapeutic peptide with inhibitory effect on MK2. In the study of peripheral inflammatory lesions, it has been shown that it has a good effect. Based on this, MMI-0100 targeting inhibition of p38MAPK/MK2 signaling pathway may also have a good regulatory effect on neuroinflammatory lesions. Alzheimer's disease (Alzheimer's disease,AD) is a neurodegenerative disease that mainly affects the hippocampus and cortical brain. But the sustained activation of glial cells and the continuous release of inflammatory mediators caused by chronic neuritis are the important processes of neuronal death and the formation of brain diseases. The aim of this study was to investigate whether MMI-0100 can attenuate cognitive impairment in AD model by inhibiting neuroinflammation. Results: (1) A 尾 42 and LPS induced neuritis induced memory impairment in mice by (NOR) and (OLR) recognition model. It was found that after 24 hours of administration of MMI-0100, the memory impairment was slowed down in mice. There was no significant difference in memory level between normal saline group and normal saline group. (2) the expression of CD11b and GFAP protein was analyzed by immunohistochemistry, and the inflammatory immune response of mouse hippocampal microglia and astrocytes was determined. It was found that MMI-0100 could significantly reduce the high expression of CD11b and GFAP induced by LPS. That is, MMI-0100 can reduce the sustained activation of immune cells in CNS and maintain them in a resting state. (3) at the tissue level, real-time quantitative PCR analysis showed that MMI-0100 could significantly reduce the overexpression of IL-6,IL-1 尾 (TNF- 伪, i NOS) induced by A 尾 42 and LPS. It was found in Western blot assay that MMI-0100 could regulate inflammatory response by inhibiting ERK phosphorylation, while p38MAPK might be degraded in the process of protein extraction from hippocampal tissue, so the phosphorylation level of p38MAPK could not be determined. (4) when MMI-0100 was 10-7 M, the expression of IL-6,IL-1 尾, i NOS and COX-2 in LPS induced neuroinflammation was significantly decreased in BV-2 cell line. Moreover, it can reduce the phosphorylation level of ERK and p38MAPK in a dose-dependent manner, suggesting that MMI-0100 may reduce the inflammatory response induced by LPS by inhibiting the ERK and p38MAPK pathways. (5) MMI-0100 is a therapeutic polypeptide with cellular membrane penetration activity. Interestingly, after nasal administration of MMI-0100, NOR and OLR memory damage models induced by A 尾 42 and LPS in mice were detected. MMI-0100 was able to restore the memory of mice without significant difference from the control group. In order to explain this phenomenon, a fluorescent labeled Cy7.5-MMI-0100, was synthesized for fluorescence imaging in small animals. It is confirmed that MMI-0100 can use its cell membrane transmembrane activity to distribute in the brain region through nasal mucosa, so as to play an anti-inflammatory effect. This will have important reference value for the study of low BBB rate and poor stability of polypeptide drugs.
【学位授予单位】:兰州大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R749.16;R-332

【参考文献】

相关期刊论文 前1条

1 李慧;;治疗阿尔茨海默病药物的临床研究进展[J];中国新药杂志;2017年06期



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