【摘要】：p38絲裂原活化蛋白激酶(p38 mitogen-activated protein kinases,p38MAPK)是MAPK激酶家族之一。已有大量研究報道p38MAPK/MK2信號通路參與調控細胞應激、炎癥、凋亡等。針對該靶點,p38MAPK抑制劑(SB203580等)被開發(fā)研究,但因臨床階段出現(xiàn)的諸多安全隱患(肝毒性、心臟毒性、中樞神經(jīng)系統(tǒng)毒性等),限制了其應用和發(fā)展。由此,下游底物MK2作為調控炎癥反應的新型藥物靶標成為目前研究的熱點;近來設計合成的多肽藥物MMI-0100是對MK2有抑制作用的外源治療性多肽。且在外周炎癥病變研究中表明有良好功效,基于此,MMI-0100靶向抑制p38MAPK/MK2信號通路可能對神經(jīng)炎癥病變也具有較好的調控作用。阿爾茨海默病(Alzheimer's disease,AD)是主要影響海馬和皮質腦區(qū)的神經(jīng)退行性疾病,病因復雜不明。但長期慢性的神經(jīng)炎癥反應引起神經(jīng)膠質細胞持續(xù)激活和炎癥介質的連續(xù)釋放是導致神經(jīng)元死亡并形成腦疾病的重要過程。本課題主要觀察MMI-0100能否通過抑制神經(jīng)炎癥減輕小鼠AD模型中的認知損傷。結果:(1)采用小鼠新物體(NOR)和新位置(OLR)記憶識別模型,由Aβ42及LPS誘導神經(jīng)炎癥形成小鼠記憶損傷,發(fā)現(xiàn)給藥MMI-0100 24h后,小鼠記憶損傷被減緩,并表現(xiàn)出與生理鹽水組無顯著性差異的記憶水平;(2)通過免疫組化實驗,分析CD11b和GFAP蛋白表達量,確定小鼠海馬中小膠質細胞及星形膠質細胞參與的炎癥免疫應答情況,發(fā)現(xiàn)MMI-0100可以顯著降低LPS引起的CD11b和GFAP的高表達,即MMI-0100能降低CNS中免疫細胞的持續(xù)激活,維持其處于靜息狀態(tài);(3)在組織水平,實時定量PCR分析發(fā)現(xiàn)MMI-0100可以顯著降低由Aβ42和LPS誘導的炎癥因子(IL-6、IL-1β、TNF-α、i NOS)高表達,且在Western blot檢測中發(fā)現(xiàn)MMI-0100可通過抑制ERK磷酸化來調控炎癥反應,而p38MAPK可能在海馬組織蛋白提取過程中被降解,因而未能確定其磷酸化水平;(4)在BV-2細胞系中檢測發(fā)現(xiàn)MMI-0100為10-7M時能夠顯著降低LPS誘導的小鼠神經(jīng)炎癥中炎癥因子(IL-6、IL-1β、i NOS及COX-2)表達,且能夠濃度依賴性的降低ERK和p38MAPK磷酸化水平,表明MMI-0100可能是通過抑制ERK和p38MAPK通路來降低LPS引起的炎癥反應;(5)MMI-0100是具有細胞穿膜活性的治療性多肽,有趣的是,鼻黏膜給藥MMI-0100后,由Aβ42和LPS引起的小鼠NOR和OLR記憶損傷模型檢測發(fā)現(xiàn),MMI-0100能夠恢復小鼠記憶并與對照組無顯著差異,為了說明此現(xiàn)象,合成熒光標記的Cy7.5-MMI-0100,進行小動物熒光成像,確定MMI-0100能利用其細胞穿膜活性通過鼻粘膜在腦區(qū)有大量分布,從而發(fā)揮抗炎作用。這將對多肽藥物穩(wěn)定性差、通過BBB率低等相關研究具有重要參考價值。
[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- 偽, 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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.16;R-332

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相關期刊論文 前1條

1 李慧;;治療阿爾茨海默病藥物的臨床研究進展[J];中國新藥雜志;2017年06期

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