白细胞整合素CD11b对脓毒症时高迁移率族蛋白B1（HMGB1）调控机制的研究

As an important late proinflammatory mediator, high mobility group protein B1 (HMGB1) plays a pivotal role in the pathogenesis of sepsis and septic shock. It has been known that immune cells such as macrophages, mononuclear cells and neutrophils, can actively secret HMGB1 when stimulated by lipopolysaccharide (LPS)and tumour necrosis factor alpha (TNF-α), however the underlying mechanisms remains largely unknown. .In a sepsis model of rat reproduced by cecal ligation and perforation (CLP), we found that lactosyl derivative Gu-4, a specific inhibitor of CD11b that we have identified previously, greatly improved the survival of the animals. Interestingly, HMGB1 level in the serum of Gu-4 treated CLP animals was significantly decreased compared to untreated CLP animal, which led us to hypothesize that CD11b signaling pathway may be involved in the process of HMGB1 release during sepsis. To our knowledge, there are no data at present showing that CD11b is involved in HMGB1 release yet. .To test our hypothesis, we will use tools from molecular biology field including RNA interference, flow cytometry, quantitative real time polymerase chain reaction (QPCR)，laser confocal microscopy, ELISA, Western blotting, comparative proteomic analysis and protein chip analysis to understand whether CD11b signaling is responsible for HMGB1 release and how this function is achieved. Our work is divided into three parts: .First, we plan to generate CD11b knockdown (or knockout) cells followed by performing a series of experiments on these cells to observe the changes of HMGB1 in transcription, translation, posttranslation modification, intracellular distribution and extracellular release. These data will help us to determine whether CD11b is (mainly or partly) responsible for the production and the release of HMGB1. .Second, we plan to perform comparative proteomic analysis and inhibitory analysis to identify the signaling molecule (s) and signaling pathway (s) that play essential roles in CD11b associated HMGB1 release. This will allow us to find out how the CD11b signaling pathway controls the production and release of HMGB1 in sepsis. .Third, we plan to observe the survival of CD11b knockout mice which subjected to CLP or lethal endotoxemia, thereby determining the role of CD11b in sepsis. .By identifying novel participants that regulate HMGB1 in sepsis, our studies will help us to clarify the detail mechanism underlying this important pathological process.In addition, our work will also help us to identify targets for the treatment of sepsis, a serious problem that endangering human health.

HMGB1是脓毒症时重要的晚期炎症介质。LPS、TNF-α等可诱导炎症细胞主动释放HMGB1，但机制不清。用前期发现的CD11b抑制剂乳糖衍生物Gu-4处理脓毒症大鼠，我们发现动物存活率明显提高，且其血液中HMGB1水平明显降低，提示CD11b信号通路与脓毒症时HMGB1释放相关。目前尚无HMGB1调控与CD11b存在关联的报道。本研究内容包括：在CD11b被干扰（或敲除）的细胞考察LPS、TNF-α等刺激后HMGB1在转录、翻译、翻译后修饰、胞内分布、胞外释放的变化，明确CD11b是否参与HMGB1调控；分析CD11b信号通路调控HMGB1的机制，包括CD11b与TLR4和TNF-α信号通路之间的交互作用关系，阐明CD11b如何调控HMGB1；用CD11b 敲除小鼠复制脓毒症模型，在整体水平考察CD11b与HMGB1的关系。本研究将拓展对脓毒症时HMGB1调控分子机制的理论认识。

本研究通过盲肠结扎穿刺（CLP）脓毒症动物模型在体实验以及体外细胞模型实验，采用RNAi、核质分离、激光共聚焦、流式细胞技术、ELISA、免疫共沉淀、Western bolt等手段，首次发现：（1）用靶向抑制CD11b的乳糖衍生物Gu-4，可提高脓毒症模型动物的存活率，改善肺组织损伤，且证实与循环中脓毒症晚期炎症因子HMGB1的水平相关；（2）在体外细胞模型，应用不同CD11b拮抗剂以及CD11b RNAi或CD11b敲除细胞，证明抑制CD11b能够降低LPS诱导的HMGB1主动分泌，揭示CD11b参与LPS诱导HMGB1分泌的调控；（3）查明CD11b通过影响LPS诱导的HMGB1核质转位，从而调控HMGB1的主动释放；（4）CD11b通过抑制cPKC对HMGB1的磷酸化修饰影响HMGB1与CRM1的结合，从而抑制LPS引起的HMGB1出核；（5）证明CD11b调控LPS诱导的HMGB1的释放的分子机制与Src-Syk-PKC信号通路相关；（6）用CD11b敲除小鼠复制的CLP脓毒症模型表现出较高的存活率，并伴随血清中低HMGB1水平。上述结果证实CD11b参与脓毒症时HMGB1的释放调控，通过靶向抑制CD11b调控HMGB1释放可能是治疗脓毒症的新途径。