组蛋白介导腺泡细胞RAGE/TLR4-NLRP3炎性通路加剧急性胰腺炎胰腺坏死的机制探索

Multiple organ dysfunction syndrome (MODS) and pancreatic necrosis are predominant factors for disease severity and prognosis of acute pancreatitis (AP). We have previously demonstrated that circulating histones, a category of damage-associated molecular pattern molecules, were key inflammatory mediators in AP-MODS that could also acerbate pancreatic necrosis. This conclusion is further strengthened by the clinical observations that MODS normally occurred prior to the appearance of marked necrosis in patients with AP-MODS, and when it persisted the necrosis developed. In parallel, we have recently revealed that plasma levels of soluble receptor for advanced glycation end products (sRAGE) were significantly decreased in patients with AP-MODS on admission, significantly and negatively correlated with circulating histones. These findings led us to ask whether histones can bind to RAGE, a cell surface pattern recognition receptor, to activate inflammatory pathway in pancreatic acinar cells. We found that histone H4 bond to RAGE by using immunoprecipitation and further confirmed this result by surface plasmon resonance: the KD between these two proteins was 19.3 nM. In freshly isolated mouse pancreatic acinar cells, sRAGE and anti-RAGE antibody significantly reduced histone-induced cell death; histones dose-dependently caused elevation of interlukin-1β, implying the activation of NLRP3 inflammasome. As there is a well-established role of Toll-like receptor 4 (TLR4) in AP, in combination of our results, we propose that histone-mediated pancreatic acinar cell RAGE/TLR4-NLRP3 inflammatory pathway is the key mechanism in acerbating pancreatic necrosis in AP-MODS. This project seeks to verify the binding of RAGE and histone subtypes (H1, H2A, H2B and H3); to further elucidate the mechanism of RAGE/TLR4-NLRP3 in histone-mediated pancreatic acinar cell death; and to explore the therapeutic values of either using sRAGE in neutralizing histones or concomitantly blocking RAGE and TLR4 in AP-MODS. This project will improve our understanding of the role and mechanism of circulating histones in AP-MODS and pancreatic necrosis. It will also provide novel strategies for treating acute inflammatory diseases based on histone-mediated inflammatory pathway.

我们在发现循环组蛋白是急性胰腺炎多器官功能不全（AP-MODS）的关键介质并可加剧胰腺坏死的基础上，结合MODS普遍发生于胰腺坏死出现之前，并在其持续过程中坏死加剧的临床现象，通过预实验证明AP-MODS病人入院时血浆可溶性晚期糖基化终产物受体（sRAGE）显著降低并与组蛋白水平呈负相关；组蛋白H4能与RAGE结合，sRAGE和抗RAGE抗体减少组蛋白引起的腺泡细胞死亡；组蛋白呈浓度依赖性引起IL-1β升高；再结合组蛋白通过TLR4激活炎性体（NLRP3）的已知发现，提出“组蛋白介导腺泡细胞RAGE/TLR4-NLRP3炎性通路是加剧AP-MODS胰腺坏死的关键机制”科学假说。拟通过研究各亚型组蛋白与RAGE结合；RAGE/TLR4-NLRP3介导组蛋白引起腺泡细胞死亡的机制；sRAGE和阻断RAGE/TLR4对AP-MODS的疗效验证该假说，从而为探索治疗急性炎性疾病提供创新思路和策略。

晚期糖基化终产物受体（RAGEs）与急性胰腺炎发生多器官功能不全综合征（AP-MODS）的有关，但其机制尚不清楚。我们的数据证实，重组可溶性晚期糖基化终产物受体（sRAGE）可以和不同组蛋白亚型结合。sRAGE可以保护组蛋白对小鼠胰腺腺泡的细胞毒性。组蛋白刺激时，野生型和TLR4-/-小鼠腺泡细胞RAGE表达升高，抑制RAGE能保护组蛋白细胞毒性。NLRP3抑制剂无明显保护作用，铁死亡抑制剂有一定的保护作用。组蛋白引起腺泡细胞丙二醛（MDA）浓度依赖性升高。在组蛋白引起的多器官功能不全动物模型（HIS-MODS）中，各器官组织Nrf2和GPX4 mRNA下调。.因此，我们认为细胞外组蛋白结合RAGE受体，激活Nrf2-GPX4通路，通过脂质过氧化及铁死亡加重AP-MODS，sRAGE可通过竞争性结合组蛋白抑制其细胞毒性，从而减轻AP-MODS/HIS-MODS。