可溶性Tim-3调控单核巨噬细胞分化对慢加急性肝衰竭的作用及其机制研究

The early immune injury of acute-on-chronic liver failure (ACLF) patients is closely related to the activation of monocytes. The soluble Tim-3 (sTim-3) can negatively regulate the monocyte activation. Our previous studies showed that sTim-3 level in ACLF patients significantly increased, and the in vitro study indicated that sTim-3 can inhibit the HMGB1 translocation/release and increase the autophagic level of monocytes. Some studies reported that autophagy can regulate the release of HMGB1 and macrophage phenotypes, but the mechanism of role played by sTim3 on monocyte/macrophage differentiation in ACLF development has not been clear. So in the present study, on the basis of autophagic regulation for HMGB1, in vitro experiments will be performed to investigate the mechanisms of sTim-3 regulation for monocyte/macrophage differentiation phenotypes; in vivo experiments will be conducted for the effects of sTim-3 regulation for monocyte/macrophage differentiation on hepatic injury; and clinical investigation will be performed to demonstrate the relationship between sTim-3 and development and prognosis of ACLF. The aims of present study were to establish an immune therapy strategy of monocyte as target, to inhibit the early activation of monocytes, to remould monocyte/macrophage differentiation phenotypes, and to provide a new theoretical basis for the early diagnosis and treatment of ACLF.

慢加急性肝衰竭患者的早期免疫损伤与单核细胞过度活化密切相关，可溶性Tim-3可负向调控单核细胞的活化状态。我们前期研究发现慢加急性肝衰竭患者血清sTim-3水平显著升高，体外实验发现sTim-3抑制内毒素引起损伤相关模式分子HMGB1转位释放，提高单核细胞自噬水平。文献报道自噬又可调控HMGB1释放和巨噬细胞表型，但sTim-3对慢加急性肝衰竭发生中单核巨噬细胞分化的作用及其机制尚不明确。为此，本课题拟从自噬调控HMGB1入手，开展体外实验来研究sTim-3调控单核巨噬细胞分化表型的机理；从动物实验来研究sTim-3调控单核巨噬细胞分化对肝脏损伤的影响；通过临床研究来验证血清sTim-3与慢加急性肝衰竭肝脏炎症发生和预后的关系，建立以单核细胞为靶标的免疫治疗策略，抑制其早期过度活化，重塑肝脏单核巨噬细胞分化表型，为慢加急性肝衰竭的早期诊治提供新的理论依据。

慢加急性肝衰竭患者的过度炎症状态对机体损伤起着重要作用，其中单核细胞/巨噬细胞调节在肝衰竭机制中起着重要作用，但仍未完全阐明。可溶性T细胞免疫球蛋白域粘蛋白域蛋白-3 (soluble T-cell immunoglobulin domain and mucin domain-containing molecule-3, sTim-3)可负向调控单核细胞/巨噬细胞的活化状态，在炎症应答中起重要作用。通过检测临床上不同程度肝脏炎症患者包括慢加急性肝衰竭患者、失代偿期肝硬化患者、代偿期肝硬化患者和慢性乙型肝炎患者血浆的sTim-3表达水平，发现慢加急性肝衰竭组sTim-3血浆水平显著比其他组更高，与疾病进展相关。此外，体外细胞研究发现，内毒素（脂多糖，LPS）活化从健康人群外周血分离的高纯度CD14+单核细胞，入胞重组sTim-3蛋白通过自噬诱导机制来抑制单核细胞释放多种炎症介质包括细胞因子（IL-1β、IL-8、IL-12和G-CSF）和HMGB1转位释放。体内动物研究发现，重组sTim-3蛋白减轻D-氨基半乳糖胺(D-galactosamine, D-GalN))/脂多糖(lipopolysaccharide, LPS) 引起的急性肝衰竭小鼠模型的肝脏损伤，也改变了肝脏或血液单核细胞/巨噬细胞群比例，并增加肝脏CD11b(+)单核细胞源性巨噬细胞的自噬水平。总之，本项目探讨sTim-3血浆水平与肝脏炎症疾病程度的关系，为使用该指标为肝衰竭提供潜在的诊断价值，本项目发现sTim-3通过促进自噬水平与单核细胞/巨噬细胞功能调节来减轻炎症应答和肝脏损伤，表明其对肝衰竭的潜在治疗作用。