S100B/神经酰胺介导脓毒症脑病线粒体损伤的机制研究

Sepsis associated encephalopathy (SAE) is a common and severe complication of sepsis. Understanding the mechanism of SAE is the precondition and the key for its prevention and treatment. In previous studies, we found that S100B gene played an important role in SAE, and exogenous high concentration of S100B led to mitochondrial injury and ceramide expression. These results strongly suggest that S100B and ceramide induce mitochondrial injury, which further increase the secretion of damage associated molecular patterns (DAMPs). The S100B/ceramide signaling pathway is a potential important target for the treatment of SAE. This study will use mouse cerebral microglia cells and SAE mouse models, stimulate them with high concentration of exogenous S100B or inhibit the expression of S100B, to study the function of S100B in SAE. Then, we will inhibit the expression of RAGE, which is the receptor of S100B in brain, use signal chips and high content screening system cell imaging technology to reveal the effect of S100B/ceramide signaling pathway on SAE related molecule and the expression of ceramide. Further, we will use a sphingomyelinase inhibitor to decrease the expression of ceramide and inhibit the S100B/ceramide signaling pathway, to explore a new therapy for SAE. Finally, we will study the protective effect of mitochondrial antioxidant peptides SS-31 on mitochondrial injury in SAE. This study will deepen our understanding on the mechanisms of SAE and mitochondrial injury, and provide theory evidence for the prevention and treatment of SAE and the development of new drugs.

脓毒症脑病（SAE）是脓毒症的严重并发症，阐明其发病机制是有效防治的关键。申请者前期工作发现S100B是SAE相关的重要分子，已初步证实S100B蛋白可导致线粒体损伤，并与神经酰胺密切相关，提示S100B及神经酰胺可能通过调控线粒体损伤参与SAE发生发展。为此，推测S100B-神经酰胺轴是SAE治疗潜在的重要干预靶点。本课题拟首先通过S100B蛋白刺激及抑制S100B表达，明确S100B介导SAE的作用；然后通过干扰S100B受体RAGE表达，采用信号芯片、高内涵筛选细胞成像等方法，探索S100B/RAGE信号通路下游信号分子；进而采用鞘磷脂酶抑制剂减少神经酰胺产生拮抗S100B/RAGE信号通路的效应，探索SAE新治疗方法。最后，探索应用线粒体抗氧化肽SS-31对SAE线粒体损伤的保护作用。研究结果将加深对SAE脑线粒体损伤分子机制的理解，为SAE防治及新药研制开发提供理论依据。

脓毒症脑病（Sepsis associated encephalopathy, SAE）是脓毒症的常见并发症之一，可导致病情恶化、增加死亡风险。目前对于SAE的发病机制尚未完全阐明，线粒体功能障碍与脓毒症脑细胞能量供应受损相关，被认为是SAE发生发展的关键环节。S100B是定位于神经系统的相对特异性蛋白，申请者在前期的前瞻性临床研究中发现血清 S100B 蛋白水平与 SAE 严重程度及预后相关，也进一步在脓毒症脑病大鼠模型中得到证实。前期细胞实验发现LPS 可引起小鼠小胶质细胞培养上清 S100B 蛋白水平明显增高，细胞线粒体结构明显受损；采用高浓度 S100B 重组蛋白刺激鼠小胶质细胞系 BV-2，也可以观察到 BV-2 细胞线粒体损伤。鉴于线粒体损伤在 SAE 中的重要作用和前期工作观察到的结果， 申请者推测 S100B 可能通过介导线粒体损伤参与了 SAE 的发生发展。在本项目的实施过程中，我们证明了阻断S100B/RAGE/ NF-kB信号通路不仅可以改善脓毒症脑病小鼠海马结构的神经炎症反应，还可以通过降低一氧化氮合酶(iNOS)和环氧合酶-2 (COX-2)的蛋白水平来减轻氧化应激；揭示了线粒体靶向肽SS-31通过稳定线粒体形态来减轻脂多糖(LPS)诱导的小鼠小胶质细胞(BV-2)的炎症和氧化应激反应，线粒体分裂抑制剂Mdivi-1通过抑制 Drp1下调LPS诱导的GTPase，从而使线粒体损伤、脑组织凋亡减少；通过前瞻性的临床研究证明了SAE患者血清GFAP和UCH-L1水平相比于非SAE组显著升高，GFAP水平较高SAE患者的长期日常活动较差，而UCH-L1水平较高SAE患者具有更明显的长期疼痛；通过回顾性研究揭示了脓毒症患者发生脓毒症脑病（SAE）的概率约为42.3%，SAE组的28天和180天死亡率显著高于非SAE组。总的来说，本项目不仅在细胞实验和动物实验水平证明了S100B在SAE发生发展中的重要作用，阻断S100B可以减轻线粒体损伤和脑组织损伤，而且在临床实验中验证了S100B的重要意义。