宫内炎性暴露致早产小鼠脑白质神经血管单元损伤机制及fgl2分子靶向干预

Intrauterine infection/inflammation-induced white matter injury (WMI) is an important risk factor for nervous system development in preterm infants, which may lead to severe neurological sequela such as epilepsies and cerebral palsy, and even cause direct mortality of infants. However, the molecular mechanism by which inflammation leads to WMI remains unclear, and until now there is no effective way to prevent or treat WMI. Our previous researches have found that the expression of fibrinogen-like protein 2 prothrombinase (fgl2) was increased in the LPS-stimulated rat brain, which was involved in the activated microglia-related inflammatory response. In the present study, after establishing a mouse model of intrauterine inflammation-induced WMI, we observe fgl2 expression in the preterm neonatal neurovascular unit and its associated injuries using immunofluorescence staining, DTI, MRS and two-photon confocal microscopy etc.. We then inhibit fgl2 expression by using fgl2 knockout mice to investigate potential protective effects on the components of the neurovascular unit in the white matter as well as the target cells and related signaling pathways. Further more, we try to understand the role of fgl2 in the LPS-induced oxidative stress of cerebrovascular endothelial cells and the inflammatory response of microglia in ex vivo white matter injury model. Studying the pathogenic mechnism and molecule-targeted intervention of WMI, may provide new strategies for a safe and effective therapeutic precept of WMI in preterm infants.

宫内感染/炎症所致脑白质损伤是影响早产儿神经系统发育的重要危险因素，可引起癫痫、脑瘫等严重后遗症，甚至直接威胁患儿生命。目前炎症导致脑白质损伤的具体机制尚不明确，对脑白质损伤仍无有效的防治手段。申请者前期研究发现纤维蛋白原样蛋白2凝血酶原酶(fgl2)在LPS刺激的大鼠脑组织中表达增加，并参与小胶质细胞活化相关的炎性反应。本项目拟在建立小鼠宫内炎性暴露致脑白质损伤模型的基础上，利用免疫荧光染色、磁共振弥散张量成像、磁共振波谱和双光子共聚焦等技术观察fgl2在早产鼠脑组织神经血管单元相关组份的表达以及各组份的损伤情况；采用fgl2-/-基因敲除小鼠抑制fgl2的表达，研究其可能的保护作用和相关信号转导通路；通过离体白质损伤实验进一步研究fgl2在LPS诱导的脑血管内皮细胞氧化应激和小胶质细胞炎性反应中的作用。探讨脑白质损伤的发病机制及分子靶向干预，为安全有效的防治早产儿脑白质损伤提供新策略。

宫内感染/炎症所致脑白质损伤是影响早产儿神经系统发育的重要危险因素，可引起癫痫、脑瘫等严重后遗症，甚至直接威胁患儿生命。目前炎症导致脑白质损伤的具体机制尚不明确，对脑白质损伤仍无有效的防治手段。本项目通过建立宫内炎性暴露致脑白质损伤动物模型，研究炎症对脑组织神经血管单元相关组份的影响，并探讨fgl2作为其中的纽带的作用机制。结果表明宫内炎性暴露后1日后胎鼠出现脑水肿，脑白质中TUNEL染色阳性细胞数显著增加神经细胞凋亡增加，TNF-α、IL-6、IL-1β等促炎性细胞因子表达增加，Iba-1表达增加且与fgl2表达增加相一致，NF-κB表达增加，提示宫内炎性暴露使胎鼠脑促炎性细胞因子增加，小胶质细胞活化，通过fgl2和NF-κB信号通路损伤神经血管单元。生后7日仔鼠EB检测血脑屏障渗透性增加，小胶质细胞活化增加，MBP免疫组化染色发现髓鞘化形成不良；生后14日仔鼠少突胶质细胞减少，少突胶质前体细胞增殖减少，Notch-1和Hes-1 表达明显降低、Wnt3α和β-catemin未见明显变化，提示宫内炎性暴露通过Notch信号通路使少突胶质细胞发育异常、髓鞘形成减少，引起脑白质损伤。采用fgl2-/-基因敲除小鼠抑制fgl2的表达，发现新生1日fgl2-/-基因敲除仔鼠脑组织促炎性细胞因子表达降低，小胶质细胞M1型活化减少，髓鞘化不良改善，NF-κB表达降低，提示抑制fgl2表达可以通过抑制NF-κB信号通路减少宫内炎性暴露对脑白质的损伤。此外，LPS干预原代内皮细胞和原代小胶质细胞，促炎性细胞因子和fgl2表达增加，小胶质细胞M1型活化增加M2型活化减少，提示fgl2-/-对脑白质损伤的保护作用涉及小胶质细胞、内皮细胞、少突胶质细胞等多个神经血管单元相关组份。本研究探究了神经血管单元作为整体在宫内炎症致脑白质损伤中的作用，抑制fgl2表达可能从多个靶点作用于神经血管单元，从而发挥宫内炎症致小鼠脑白质损伤的保护作用，为安全有效防治早产儿脑白质损伤提供新策略。