基于肝窦内皮细胞外泌体和SphK1-S1P-S1PRs信号通路研究红景天苷抑制肝星状细胞迁移的抗肝纤维化机制

Salidroside have anti hepatic fibrosis function. Our studies have shown that Salidroside can inhibit hepatic stellate cell(HSC) migration induced by platelet-derived growth factor through blocking AKT phosphorylation. A recent study has show that exosomes derived from Liver Snusoidal Endothelial Cell(LSEC) express SphK1 which can upregulate AKT phosphorylation in HSC and promote cell migration. Base on these progress, we hypothesize that "Salidroside can inhibit the migration of HSC by regulate exosomes drived from LSEC and through regulating SphK1-S1P-S1PRs signaling pathway”. In this study, we will establish liver fibrosis model in SphK1 knockout mice, to investigate the effect of SphK1 on the development of liver fibrosis and detect the migration of HSC induced by exosomes derived from fibrotic mice serum and LSEC culture medium. In vitro, the mechanism study of salidroside inhibit migration of HSC from SphK1 content of LSEC-derived exosomes, exosomes-HSC adhesion endocytosis and S1P-S1PRs signal transduction in HSC. Finally our experiments were performed on fibrotic model mice in vivo, to observe the effect of salidroside on development of liver fibrosis and SphK1 signaling pathway. The project is beneficial to explore mechanism of anti hepatic fibrosis in Chinese Traditional Medicine.

课题组前期发现具有抗肝纤维化作用的红景天苷可阻断AKT磷酸化抑制血小板衍化生长因子诱导的肝星状细胞（HSC）迁移，结合肝窦内皮细胞（LSEC）来源的外泌体携带SphK1上调HSC内AKT磷酸化促进细胞迁移的文献报道，提出“红景天苷可通过肝窦内皮细胞外泌体调控SphK1-S1P-S1PRs信号通路抑制HSC迁移”科学假说。项目拟首先构建SphK1基因敲除小鼠，复制纤维化模型后提取血清及分离培养的原代LSEC培养液中的外泌体，验证SphK1在纤维化形成中的作用及不同来源外泌体对HSC迁移的影响，体外实验从LSEC外泌体SphK1含量、外泌体-HSC黏附内吞，以及HSC内S1P-S1PRs信号传导等环节研究红景天苷抑制HSC迁移的机制，最后体内实验复制肝纤维化模型小鼠，观察红景天苷对纤维化形成及外泌体SphK1信号通路的影响。项目将外泌体引入中药抗肝纤维化机制研究，为推动中医药现代化做有益探索。

肝窦内皮细胞（Liver sinusoidal endothelial cell，LSEC）和肝星状细胞（Hepatic stellate cell，HSC）是肝脏重要的间质细胞，研究表明，LSEC可通过分泌外泌体调控SphK1-S1P-S1PRs信号通路促进HSC的活化和迁移，在肝纤维化发生发展中发挥重要作用。红景天苷（Salidroside，Sal）是传统中药红景天的主要活性成分，可抑制HSC迁移，发挥抗肝纤维化作用，基于以上，提出“红景天苷可通过 LSEC 外泌体调控 SphK1-S1P-S1PRs 信号通路抑制 HSC 迁移”的科学假说。. 围绕这一假说，项目收集临床患者肝组织和血清，复制肝纤维化小鼠模型，观察不同来源外泌体携带SphK1对HSC迁移的影响；体外实验构建过表达SphK1细胞，明确Sal是否通过LSEC外泌体调控SphK1信号通路抑制HSC迁移；动物实验观察Sal对肝纤维化小鼠的干预作用及SphK1-S1P-S1PRs信号通路的影响，并提取CCL4模型及Sal给药小鼠肝组织蛋白进行了蛋白质组学分析，为项目的后续进一步深入研究探索可行的途径。. 结果证实：不同来源肝纤维化血清外泌体中SphK1高表达并促进HSC迁移作用； Sal能够抑制LSEC来源SphK1高表达外泌体介导的HSC活化和迁移，其机制可能与抑制HSC内AKT活化有关；Sal能够减轻CCl4模型小鼠肝纤维化，抑制CCl4模型小鼠肝细胞凋亡，其机制与调节肝内SphK/S1P/S1PRs信号通路和JNK信号通路有关； 经Sal干预后小鼠肝纤维化血清外泌体中SphK1表达及其促HSC迁移作用均较模型组减小；蛋白质组学分析显示，Sal抗肝纤维化的作用不仅与鞘脂代谢有关，而且与糖蛋白代谢、内质网中蛋白质的糖基化、泛素介导的蛋白质降解等途径相关。. 通过项目的实施，初步明确了Sal调控LSEC外泌体抑制HSC迁移的抗肝纤维化作用机制，并为后续深入研究打下良好的基础。