右美托咪定预处理通过抑制MMP9及丙泊酚引起的发育期海马星形胶质细胞凋亡的机制研究

Multiple evidence of animal studies indicates that most general anesthetics may exert neurotoxic effects on developing neurocytes during the vulnerable period of brain growth spurt. Propofol, the most widely used anaesthetic in paediatric patients’ surgery and diagnostic procedure, causes widespread neurodegeneration (particularly apoptosis) in rodent pups. Recent evidence has demonstrated that astrocytes were critical participants in every major aspect of mammalian brain development. So far, relatively little is known about the association between exposure of developing astrocytes to brief propofol anaesthesia and risk for its impairment. By microarray analysis, it has shown that propofol caused a significant expression change of microRNAs (miRNAs) of immature cultured hippocampal astrocytes of 7-day-old Sprague Dawley (SD) rats. Of these miRNAs, miR-665 was significantly differentially expressed after both short-term exposure to high-concentration propofol (10 μg/ml for 1 h) and long-term exposure to low-concentration propofol (0.9 μg/ml for 48 h). Of particular interest, miR-665 has been demonstrated to negatively regulate BCL2L1 protein expression via post-transcriptional mechanism. It involved in the neurotoxicity induced by propofol via a cleaved caspase-3 (CC3) mediated mechanism by negatively regulating BCL2L1. Apoptosis of developing hippocampal astrocytes was thus significantly influenced by propofol or miR-665, or both. Herein, in cultured astrocytes, the project first aims at investigating whether the propofol-related neurotoxicity was followed BCL2L1-MMP9-CC3 signalling pathway under the regulation of miR-665. On the other hand, before injection intraperitoneally of propofol, some rats were injected lentivirus labeled by glial fibrillary acidic protein (GFAP) promoter intraventricularly, aiming at further elucidation the association between miR-665 and miRNA-related gene regulatory pathway (BCL2L1-MMP9-CC3 signalling pathway) in the developing astrocytes. These findings could have significant implications to reveal the anesthesia-related neurotoxicity in clinical practice. Then we will focus on the involvement of astrocytes in the synaptic plasiticity underlying learning and memory to further clarify the vital role of miR-665/BCL2L1-MMP9-CC3 pathway, which significantly influenced by propofol and attenuated by dexmedetomidine. Using a combination of non-coding RNAs, astrocytes, developing brains and behavior, we provide the first evidence for the mechanisms of miR-665/BCL2L1-MMP9-CC3 pathway in the apoptotic process of astrocytes, impairment of synaptic plasiticity, and neurological deterioration in propofol application. Dexmedetomidine might act as an effective therapeutic drug for treatment of neurological disorders in peadiatric anesthesia or sedation with propofol clinically. In conclusion, this study lead to develop new strategies for preventing neurotoxic effects caused by general anesthtics propofol to developing astrocytes and synaptic plasiticity.

丙泊酚对发育大脑有神经毒性作用。星形胶质细胞积极参与大脑发育过程，但尚缺乏丙泊酚对其作用机制的研究。本课题组前期验证丙泊酚显著增加海马区星形胶质细胞内miR-665的表达，干扰BCL2L1水平引起新生大鼠海马星形胶质细胞凋亡；腹腔注射丙泊酚引起新生大鼠星形胶质细胞凋亡和神经行为学异常。本项目在确认丙泊酚调控miR-665表达关键位点基础上，采用特定基因的高效选择性抑制剂，阐明miR-665/BCL2L1-MMP9-CC3通路在丙泊酚引起星形胶质细胞凋亡的作用；而后通过电生理及电镜实验确立发育期海马星形胶质细胞凋亡对海马突触可塑性的影响；最后通过体外基因和体内功能实验，明确右美托咪定预处理是否阻断miR-665/BCL2L1-MMP9-CC3通路的传导，发挥其神经保护的作用。本研究为前期系列研究的深入与拓展，为探讨发育大脑损伤与神经行为学异常的相关性提供新思路和新靶点。

背景：本课题组已证明通过激活rno-miR-665介导的凋亡通路，丙泊酚可促进新生大鼠脑发育高峰期海马区星形胶质细胞凋亡。由于星形胶质细胞的数量对突触发育及功能完善等方面发挥重要的作用，所以丙泊酚促星形胶质细胞凋亡作用可能会影响发育大脑突触的结构和功能。另外，星形胶质细胞上含有α2受体，而右美托咪定恰恰是高选择性的α2肾上腺素能受体激动药。目前，关于二者的相关研究还十分欠缺，故本课题拟探讨右美托咪定预处理对丙泊酚麻醉后发育期星形胶质细胞及突触的影响，为全麻期间脑保护策略提供可靠依据。.主要研究内容：本研究从凋亡信号通路、突触可塑性和学习记忆能力三方面着手：在离体细胞水平，采用病毒介导的RNAi/过表达、特异性基因干扰等策略明确丙泊酚和右美托咪定的分子调控机制。在整体动物水平，用real-time RT-PCR和western blot检测凋亡基因的变化；用电子显微镜对神经细胞和突触的形态进行观察；用电生理技术对在体脑片突触传递可塑性进行评价；用水迷宫实验观察大鼠远期学习和记忆能力的变化。.结果：丙泊酚激活新生大鼠海马区星形胶质细胞中rno-miR-665/Bcl2-like 1 (Bcl2l1)-cleaved caspase 9 (CC9)- cleaved caspase 3 (CC3)信号通路，增加海马区星形胶质细胞的凋亡，matrix metalloprotein 9 (MMP9)水平的下降进一步抑制Bcl2l1的表达，造成神经细胞的凋亡进一步增多；同时，丙泊酚还损伤海马区突触形态以及抑制一月龄大鼠突触传递可塑性。右美托咪定抑制丙泊酚激活的rno-miR-665/Bcl2l1-CC9-CC3凋亡通路，MMP9表达上升，进而缓解抗凋亡基因Bcl2l1的下降，星形胶质细胞凋亡减少，海马区突触形态和功能得到恢复，发挥了抗丙泊酚促凋亡的作用。另外，我们发现无论是否与右美托咪定合用，在新生期接受丙泊酚单次注射的大鼠，均未出现远期学习和记忆能力下降。.关键数据：丙泊酚引起发育期星形胶质细胞凋亡、突触结构异常、突触传递可塑性下降。右美托咪定发挥脑保护的作用机制是抑制丙泊酚引起的rno-miR-665/Bcl2l1-CC9-CC3通路激活。.科学意义：本课题为探寻右美托咪定对全麻后发育大脑的脑保护作用机制提供新的研究思路，为提高临床儿科全麻的安全性和可控性做出贡献。