TLR4-CCR2-CXCR4信号轴促进中性粒细胞骨髓动员加重肝缺血再灌注损伤

Liver ischemia/reperfusion (I/R) injury is a clinically important phenomenon encountered during elective liver surgical procedures, solid organ transplantation, trauma, and hypovolemic shock. Polymorphonuclear leukocytes (PMNs, neutrophils) are highly specialized cellular effectors in host defense and immune surveillance. CXCR4-CXCL12 axis regulates mobilization of mutilple cells in the bone marrow. Under the conditions of infection and rheumatoid arthritis, neutrophils up-regulate the expression of CCR2 to infiltrate into inflammed site and cause tissus damage. Our previous study showed that neutrophil mobilization decreased in the bone marrow and liver damage alleviated in TLR4 KO and CCR2 KO mice during hepatic IRI process; TLR4 regulates the activation of CCR2 on neutrophils. Based on these findings, we speculate that HMGB1 released from injured tissue binds to TLR4 on neutrophils to activate CCR2, which go through endocytosis and form heterodimers with CXCR4 to decrease the sensitivity to CXCL12, leading to the mobilization of neutrophils. We adopt neutrophil culture and in vivo study of WT、TLR4 KO and CCR2 KO mice to investigate the role of TLR4-CCR2-CXCR4 signaling on the neutrophil mobilization and explore the pro-inflammatory mechnisms of neutrophils and potential target on the treatment of IRI.

中性粒细胞(PMN)骨髓动员是导致肝脏IRI主要机制之一，其机理有待研究。骨髓腔内CXCL12活化CXCR4促进PMN驻留。在多种炎症应激时，PMN改变其表面受体表达及活性，促使其骨髓动员，浸润至靶器官引起损伤。我们研究提示，在TLR4或CCR2基因敲除小鼠，肝IRI过程中骨髓PMN动员显著减少，肝脏PMN浸润降低伴损伤减轻，外周血HMGB1浓度下降；TLR4缺陷还可抑制PMN表面CCR2活化。故推测，肝IRI过程中，应激受损细胞释放HMGB1与PMN表面TLR4结合将促使CCR2内吞并与CXCR4形成异二聚体，导致CXCR4对CXCL12敏感性降低，从而促进PMN从骨髓动员。本课题拟用WT、TLR4 KO和CCR2 KO小鼠，通过PMN原代培养、刺激和在体动物实验，研究TLR4-CCR2-CXCR4信号轴对PMN动员的影响，分析促进炎症反应和损伤肝细胞的机制，为临床IRI防治提供新靶点。

肝脏缺血再灌注损伤（ischemia/reperfusion injury, IRI）常见于肝脏部分切除术、失血性休克、创伤、肝脏移植等，是造成手术失败和患者死亡的重要原因。我们按照预期对本项目展开研究，充分证明了CCR2/TLR4/p38信号轴的作用及机制。由于这些信号的调控涉及应激状态下表观遗传学的机制，因此我们对相关的microRNA的作用进行了探索，发现miR191通过调控经典的HIF-1alpha/ZONAB影响肝脏IR的进程，而miR210则通过形成mrR210/SMAD4的环路实现对IR过程的精细调控，这些发现均发表于知名的期刊，希望能为肝脏IR损伤的防治提供可能的作用靶点。