沉默lncRNA-5657对脓毒症脑损伤的保护作用及机制研究

Inhibition of over-activation of microglia in brain is the main way to protect from Sepsis Encephalopathy (SE). The current studies showed that lncRNA was not only involved in the pathogen-related immune reaction, but also modulated the monocyte-phagocyte inflammatory response through the interaction with protein factors. However, there is no report on the roles of lncRNA in the microglia and inflammatory response in the brain of rats. Our previous studies showed the lncRNA-5657 expression in the microglia of LPS challenged was increased, which measured by HTseq and RT-qPCR. The LPS induced TNF-α release was decreased when silencing the lncRNA-5677. Thus we hypothesized the lncRNA-5657 might enhance the inflammatory reaction in the microglia through a special protein interaction. This study will explore the protective effects and mechanisms of lncRNA-5657 on SE using Lentivirus mediated lncRNA-5657 shRNA in the rat model. Additionally, we will use skin-derived induced pluripotent stem cells (iPSCs) from SE patients, non-SE patients and healthy volunteers, generate the patient-specific iPSCs-derived neurons model for study the regulation of inflammatory response by lncRNA-5657 silencing. and we will detect the special protein interacting with lncRNA-5657 by in vitro RNA-pulldown and MS analysis. This study will provide a new way in Sepsis Encephalopathy treatment through the lncRNA-5657 silencing protective effects and mechanisms.

抑制脑内小胶质细胞（RM）过度激活，减少炎症介质失控性释放是防治脓毒症脑损伤（SE）的根本措施，研究表明lncRNA与蛋白相互作用参与了单核-巨噬细胞炎症反应的调控，但lncRNA在RM炎症反应中的作用未见报道。我们前期采用高通量测序及RT-qPCR验证发现，lncRNA-5657在LPS诱导的RM炎症反应中表达上调，沉默lncRNA-5657能减少TNF-α等炎症因子释放。因此，我们假设lncRNA-5657通过与RM内某一特异蛋白相互作用，进而促进炎症反应。本项目拟在前期研究基础上，将慢病毒介导的lncRNA-5657 shRNA 转染大鼠SE模型及脓毒症患者来源的多能干细胞-神经元炎症模型中，探讨其对SE的保护作用；并在RM内通过RNA-pulldown实验联合质谱分析，鉴定与lncRNA-5657相互作用的蛋白，探讨lncRNA-5657的促炎机制，为治疗脓毒症脑损伤提供新途径。

抑制脑内小胶质细胞（RM）过度激活，减少炎症介质失控性释放是防治脓毒症脑损伤（SE）的根本措施，研究表明lncRNA与蛋白相互作用参与了单核-巨噬细胞炎症反应的调控，但lncRNA在RM炎症反应中的作用未见报道。本研究成果证实，lncRNA-5657在脓毒症细胞模型及动物模型炎症反应中表达上调，沉默lncRNA-5657能减少TNF-α、IL-1β、IL-6等炎症因子释放。明确了沉默 lncRNA-5657可以降低小胶质细胞炎症反应，对脓毒症大鼠脑损伤起到了保护作用。通过 RNA-pulldown实验联合质谱分析，明确了lncRNA-5657调控小胶质细胞炎症反应的作用机制，lncRNA-5657是通过与沉默信息调节因子1（SIRT1）相互作用，通过MEK-ERK信号通路，促进小胶质细胞的炎症反应。考虑到目前所有的细胞和动物模型都未能够忠实地模拟人体内的脓毒症脑损伤的发生过程及由之引起的神经损害机制，于是我们在脓毒症脑损伤患者来源的iPSCs-神经细胞炎症模型中继续研究lncRNA-5657的调控作用，我们发现，SE患者来源的神经元在相同应激下更易表现出神经元退行性变特点，提示获取的iPSCs-神经元具有SE患者的个体差异。再次验证了调控lncRNA-5657对脓毒症脑损伤患者神经元的保护作用，为脓毒症脑损伤的治疗提供新思路。