肝脏巨噬细胞亚群及HIF1α/IKBKE信号在无菌炎症性肝损伤修复中的功能机制研究

Macrophages are defined as plastic and polyfunctional cells, which serve important functions in phagocytosis, metabolism, and tissue homeostasis. Our previous findings show that hepatic resident and infiltrating macrophages played important roles in drug induced and alcohol liver injury and repair, where they induced liver sinusoidal endothelial cells proliferation and migration, therefore contributed to vessel repair. Hypoxia-inducible factor (HIF) might be the key molecule. In preliminary experiments, using a drug induced acute hepatic injury murine model, we found that IKBKE deficient mice presented more severe liver injury and inflammation than those in wild type mice, as well as the expression levels of the necrosis related protein RIP1 and phosphorylated JNK. Moreover, HIF target genes, MCP-1 and PAI-1 levels were significantly increased. Therefore, it is speculated that hepatic macrophage subsets and IKBKE gene regulate liver injury and repair, where IKBKE might be involved in HIF signaling pathway. We have successfully constructed a transgenic mouse strain whose hepatic resident macrophages specifically express Cre recombinase, and will further explore the molecular mechanisms of sterile inflammatory liver injury and repair by knocking down IKBKE, specifically depleting macrophage subsets or their HIF1α gene in mice. In addition, we will also analyze the clinical correlation between liver damage and MCP-1 and PAI-1, which will provide novel diagnostic and therapeutic strategies for hepatic injury.

巨噬细胞是一种高度可变的多功能细胞，在吞噬、代谢和组织稳态中发挥关键作用。前期工作中我们发现，肝脏定居和浸润性巨噬细胞在药物和酒精性肝损伤中也具有重要功能，能够协同促进肝窦内皮细胞增殖和迁移，帮助血管修复，缺氧诱导因子（HIF）可能是关键分子。预实验结果显示，药物肝损伤模型中IKBKE基因缺陷小鼠的肝损伤和炎症反应程度比野生型小鼠严重，坏死相关蛋白RIP1及磷酸化JNK表达水平增加，而且HIF靶基因MCP-1和PAI-1也显著升高。据此，我们推测肝脏巨噬细胞亚群及IKBKE分子能够调控肝损伤修复，而IKBKE可能涉及HIF信号通路。我们首次构建了肝脏定居巨噬细胞特异性表达Cre重组酶的转基因小鼠，本项目拟通过IKBKE基因敲除、特异性去除巨噬细胞亚群及其HIF1α基因的小鼠，研究无菌炎症性肝损伤修复分子机制，同时检测分析MCP-1和PAI-1与临床肝损伤的相关性，为肝损伤的诊治提供新策略。

IKBKE是一种丝氨酸/苏氨酸激酶，作为非典型的IκB激酶家族成员，其激活后能够磷酸化干扰素反应因子（IRF）-3和7，以及信号转导和激活因子（STAT）-1，也能够直接磷酸化AKT Thr308/Ser473部位，因而在炎症反应中发挥重要作用。本项目利用对乙酰氨基酚（APAP）或植物刀豆蛋白A（ConA）诱导的小鼠药物性或免疫性肝损伤模型，以及TNF-α诱导的无菌性炎症和肝损伤模型，研究IKBKE基因在肝损伤和炎症反应中的功能。内容包括：①研究IKBKE基因在APAP诱导的急性肝损伤中的作用；②研究IKBKE基因在ConA诱导的免疫性肝损伤中的作用；③研究IKBKE基因在TNF-α诱导的无菌性炎症和肝损伤中的作用。.1.在APAP诱导的小鼠急性肝损伤模型中，野生型（WT）小鼠肝脏中的IKBKE基因表达显著增高，集中在中央静脉区；与WT小鼠相比，IKBKE基因缺陷小鼠肝损伤显著加重，但APAP的代谢酶CYP2E1和代谢产物的表达无差异，而肝脏的线粒体完整性更差，消耗谷胱甘肽更多。IKBKE基因缺失可能直接上调RIPK1的表达，导致持续的JNK激活以及RIPK1/JNK的线粒体定位。此外，IKBKE基因缺失增强了促炎因子的表达和肝脏炎症细胞（中性粒细胞和单核细胞）的浸润。利用RIPK1的抑制剂Necrostatin-1可以显著减轻APAP诱导的肝损伤；.2.在ConA诱导的小鼠免疫性肝损伤模型中，IKBKE基因敲除导致肝损伤加重，肝脏IL-6、TNF-α、IL-4和MCP-1基因的表达升高；同时，IKBKE-/-小鼠的肝脏中性粒细胞、Ly6C高表达单核细胞、Kupffer细胞和CD4+ T细胞的比例显著高于WT小鼠，T细胞和NK细胞的激活增强；另外，IKBKE-/-小鼠肝脏中剪切的caspase-3和8、p-RIPK1、p-IKKα/β和p-STAT3表达也增加；RIPK1抑制剂显著减轻ConA诱导的小鼠肝损伤.3.在TNF-α模型中，与WT小鼠相比，IKBKE-/-小鼠肝损伤增强，肝脏IL-6、TNF-α、IL-4和MCP-1的表达升高，肝脏剪切的caspase-3和p-JNK水平显著增高；RIPK1抑制剂能显著降低小鼠的肝损伤。.本研究揭示了IKBKE基因在急性肝损伤及炎症反应中发挥重要的调节作用，为炎性肝损伤的治疗提供了新的手段和靶点。