TLR2靶向调控Tregs在早产小鼠炎症性脑损伤中的作用机制研究

Staphylococcus epidermidis (SE) infection is closely related to brain damage in premature infants, which seriously endanger infant's health and intelligence development, but its specific mechanism is not clear. For the first time, we have confirmed that SE can induce brain damage in premature mice by TLR2 mediated inflammatory reaction. Frontiers studies have shown that Tregs and TLR2/PI3k/AKT signaling pathways play an important role in inflammation which mediated brain damage. Based on this, we propose a scientific hypothesis that SE promotes the involvement of Tregs in brain damage through the TLR2 mediated PI3K/AKT signaling pathway. Uponding on flow cytometry, Western Blot, qRT-PCR, Luminex and gene knockout technology, this project will use the 5 day old newborn premature mice to establish the optimization model of infection combined with hypoxia ischemia brain injury model, from animal、tissue and cell levels, to study the mechanism of TLR2 targeting Tregs in inflammatory brain injury in premature mice and to get target point downstream effect. It provides a theoretical basis and drug target for the treatment of brain injury caused by SE infection in premature infants.

表皮葡萄球菌（Staphylococcus epidermidis ,SE）感染与早产儿脑损伤密切相关，严重危害婴儿的健康和智力发育，但是其具体作用机制尚不清楚。我们前期工作首次证实SE可通过TLR2介导炎症反应引起早产小鼠脑损伤。前沿报道表明Tregs与TLR2/PI3k/AKT信号通路在炎症介导的脑损伤中发挥重要作用。基于此，我们提出科学假设：SE通过TLR2介导的PI3K/AKT信号通路促进Tregs参与脑损伤。本项目拟采用5日龄新生早产小鼠，建立优化的感染联合缺氧缺血模型，通过流式细胞分选、Western Blot、qRT-PCR、Luminex及基因敲除，在细胞、组织、动物水平三个层面展开，进一步深入探讨TLR2靶向调控Tregs的作用方式和调控分子机制，解析下游效应途径及靶点，为治疗SE感染导致的早产儿脑损伤提供理论依据和药物靶点。

近年随着早产儿存活率明显上升，早产儿后遗症尤其是早产儿脑损伤越来越成为社会严重问题。感染与早产儿脑损伤关系密切，我们既往研究证实了TLR2介导的炎症反应可引起早产小鼠脑损伤，而前沿报道表明Tregs与TLR2/PI3K/AKT信号通路在炎症介导的脑损伤中发挥重要作用，但其具体作用机制尚不清楚。.由于早产儿不但容易合并感染，且常常伴有不同程度的缺氧，所以感染联合缺氧缺血（HI）能更好地模拟早产儿生后的临床状态。因此本项目采用5日龄新生早产小鼠，通过腹腔注射TLR2激动剂Pam3CSK4（pam）联合缺氧缺血，建立优化的小鼠炎症性脑损伤模型，通过免疫组化、qRT-PCR、Western Blot及Luminex细胞因子检测等方法，探讨TLR2靶向调控Tregs的作用方式和调控分子机制。.结果表明，HI联合pam组较HI联合生理盐水（saline）组脑白质损伤程度更重，而单纯pam或saline注射两组小鼠脑白质未见明显异常。建模后24小时，HI+Pam组中小鼠脑组织IL-6和CCL2水平较HI+saline组及假手术组均明显升高，而IL17有下降趋势，但无统计学意义（P＞0.05）。作为Treg的转录因子，Foxp3的mRNA水平在建模后24小时明显下调，促进Treg分化的细胞因子TGF-β也有明显下降，而Treg分泌的细胞因子IL-10基因的表达未发现显著变化（P＞0.05）。同时Western Blot结果表明，HI+pam组和HI+saline组相比，HI+pam组TLR2信号通路下游相关蛋白AKT、STAT3蛋白表达量上升了20%, 15%，而CD4、CD25及foxp3蛋白表达量分别下降了14%、16%和18%。.综上所述，通过小鼠炎症性联合缺氧缺血性脑损伤模型表明，TLR2可介导PI3K/AKT/STAT3信号通路靶向调控Tregs参与脑损伤，其可能是通过作用于转录因子Foxp3从而下调Tregs，导致体内细胞因子分泌失衡，而失衡的外周细胞因子通过循环浸润到脑组织从而参与脑损伤的发生发展过程。通过阐明 TLR2信号通路与Tregs的作用机制，为将来TLR2信号通路抑制剂或免疫调节剂应用于早产脑损伤提供了方向和前景。