LPS诱导巨噬细胞释放包含HMGB1外泌体引起肝细胞焦亡在肝衰竭中的机制研究

Exosomes have emerged as important vectors of intercellular cross talk. Lipopolysaccharide (LPS) plays a pivotal role in the pathogenesis of acute liver failure caused by sepsis. Through the expression of Toll-like receptor 4 (TLR4), macrophage can efficiently take up LPS, leading to inflammation and hepatocyte injury. To date, the underlying mechanism of how LPS activated macrophage derived exosomes mediate hepatocyte injury remains unclear. Preliminary studies showed LPS induced secretion of significant higher concentrated HMGB1-bearing exosomes from macrophage, contributing to inflammasome activation and pyroptosis of hepatocyte. Moreover, we found angiotensin (1-7) alleviated this inflammation activity, which needed further investigation. Therefore, we assumed that LPS could induce the release of HMGB1 contained exosomes from macrophages which triggered the pyroptosis of hepatocytes through NLRP3 inflammasome pathway and leading to liver failure. Angiotensin (1-7) alleviated these effect induced by LPS. In this study, we plan to investigate the mechanism of how LPS induces macrophage to assemble HMGB1 into exosomes via RAGE-dependent pathway in vitro and in vivo as well as how these HMGB1-bearing exosomes trigger hepatocyte pyroptosis with macrophage phenotype regulation in return. Furthermore, the effect of angiotensin (1-7) on LPS will be explored. This project provides novel insight into pathogenesis of liver failure during sepsis.

基于外泌体的细胞间通讯是目前的研究热点。LPS是脓毒症促进肝衰竭的重要介质，巨噬细胞受LPS刺激后可促进肝细胞损伤。外泌体如何介导上述细胞间通讯鲜见报道。我们的前期研究发现，LPS刺激的巨噬细胞分泌的外泌体中HMGB1含量显著增高，该外泌体可引起肝细胞NLRP3炎症小体活化及焦亡；血管紧张素1-7（Ang（1-7））可抑制LPS诱发的肝损伤，然而具体机制不明。因此，我们设想：LPS诱导巨噬细胞释放外泌体中的HMGB1引起肝细胞NLRP3炎症小体活化及细胞焦亡，进而促进肝衰竭；Ang（1-7）对LPS有抑制作用。本研究拟从体内、外层面探讨LPS诱导巨噬细胞HMGB1以RAGE依赖途径装配至外泌体的机理；HMGB1外泌体经NLRP3-ASC-Caspase1轴诱导肝细胞焦亡，进而调节巨噬细胞表型的分子机制；观察Ang（1-7）对LPS的抑制作用。本研究为拓展外泌体在肝衰竭的调控机制提供新思路。

LPS是脓毒症促进肝衰竭的重要介质，巨噬细胞受LPS刺激后可促进肝细胞损伤。外泌体如何介导上述细胞间通讯鲜见报道。本项目从体内、外探讨外泌体HMGB1水平与脓毒症肝衰竭相关性，LPS刺激巨噬细胞释放的外泌体HMGB1的调控机制以及LPS-外泌体-HMGB1对肝细胞功能的影响及Ang（1-7）的保护作用。体内研究发现：肝细胞是体内摄取外泌体的主要细胞。LPS-巨噬细胞-外泌体可引起小鼠肝损伤、组织学检测进一步发现，LPS-巨噬细胞-外泌体的小鼠肝脏NLRP3炎性小体的表达增加。此外，我们还发现LPS-巨噬细胞外泌体中包含的HMGB1 可诱导肝细胞焦亡并激活NLRP3炎症小体。脓毒症患者的血清EVs表达高HMGB1并通过激活NLRP3炎性小体导致小鼠肝脏损伤。体外研究发现：与对照组相比，LPS增加了巨噬细胞外泌体中341种（6.74%）蛋白质的表达（其中CXCL2、CCL22、TNF、CCL3、CXCL10、CD40、IL1rn、Gbp2明显的上调）。我们还发现巨噬细胞EVs中含有HMGB1。LPS-巨噬细胞-外泌体导致肝细胞损伤和NLRP3炎症体的激活。临床数据方面，我们发现HMGB1+血清EVs的水平与患者的肝脏损伤程度和炎症指数（C反应蛋白（CRP）和降钙素（PCT））呈正相关。机制研究方面，我们发现包装到EVs中的HMGB1依赖于细胞质的RAGE。EVs通过转铁蛋白介导的内吞作用将HMGB1运送到肝细胞。 LPS-EV中的HMGB1可通过激活NLRP3炎性小体促进肝细胞的焦亡。本项目拟研究 LPS 调节巨噬细胞外泌体释放HMGB1并促进肝衰竭的分子机理及 Ang（1-7）的治疗作用，为拓展脓毒症合并肝衰竭机制提供新的思路，为临床干预预脓毒症合并肝衰竭提供新的靶点和策略。