葡萄糖神经酰胺合成酶调控iNKT细胞功能对免疫介导肝损伤的作用及分子机制

Immune-mediated liver injury is one of the important reasons for the occurrence and development of many liver diseases. It has been confirmed that sphingolipids play a pleiotropic role in liver injury. However, the role of glycosphingolipids anabolism in liver injury induced by immune response remains poorly understood now. Our previously clinical and basic studies indicated that glycosphingolipids contributed to the liver injury in viral hepatitis, and glucosylceramide synthase(GCS), the key enzyme in the first step of glycosphingolipids biosynthesis, regulated the apoptosis of hepatocytes in vitro. Therefore, based on our previous findings, the present study will focus on the effects of GCS on the outcome of immune-mediated liver injury by regulating the function of iNKT cells. We will use in vivo and in vitro models of the concanavalin A- and alpha galactoceramide-induced immune-mediated liver injury. The inhibition of the gene expression and activity of GCS will be employed. The high performance liquid chromatography/mass spectrometry will be performed to detect the alteration of hepatic glycosphingolipids, liver histology will be used to evaluate the hepatic necrosis or inflammation grade, flow cytometry will be used to detect changes of iNKT cells, Western blot will be used to detect the protein of nuclear factor κb, qRT-PCR and ELISA methods will be used to detect expression of cytokines. The effects of GCS on the outcome of liver injury and its mechanisms in regulating iNKT cell function in immune-mediated liver injury will be investigated from multiple perspectives. The present study will provide a new theoretical basis and therapeutic targets for mechanisms of liver injury in future.

免疫介导的肝损伤是众多肝脏疾病发生发展的重要原因之一。研究发现鞘脂在肝损伤中发挥多效性作用，但糖鞘脂合成代谢在免疫性肝损伤中的功能目前尚不清楚。我们前期临床和基础研究提示糖鞘脂参与病毒性肝炎肝损伤过程，且糖鞘脂合成首步关键酶葡萄糖神经酰胺合成酶（GCS）在体外调节肝细胞凋亡。本研究在我们前期基础上进一步关注GCS可能通过调控iNKT细胞功能影响免疫性肝损伤结局。本研究拟利用刀豆蛋白A和α-半乳糖神经酰胺构建免疫性肝损伤体内和体外模型，干预GCS基因表达和活性，主要用高效液相色谱/质谱法检测肝内糖鞘脂变化、肝组织学评估肝坏死及炎症、流式细胞术观察iNKT细胞的功能改变、Western blot检测核因子κb的表达、qRT-PCR和ELISA检测细胞因子水平等，旨在阐明GCS对免疫性肝损伤结局的影响及其对iNKT细胞功能调控机制。研究结果将为肝损伤的病理机制提供新的理论基础和治疗靶点。

糖鞘脂在免疫性肝损伤中的功能及其分子机制目前还不清楚。本项目在刀豆蛋白A和α-半乳糖神经酰胺诱导的免疫性肝损伤小鼠和细胞模型基础上，从基因及蛋白水平干预葡萄糖神经酰胺合成酶（GCS），展开GCS调控iNKT细胞功能对免疫性肝损伤的作用及机制的研究。本项目的成果首先阐述了iNKT细胞在免疫性肝损伤发病机制中的重要地位，同时综述了GCS与肝脏疾病之间的重要关系。研究结果发现抑制GCS能够降低免疫性肝损伤血清学指标的升高，改善肝脏病理变化，影响肝细胞的凋亡，减轻肝内免疫反应引起的IL-4、IL-6和IL-10等炎症因子的释放，通过调节基质金属蛋白酶-1和基质金属蛋白酶组织抑制因子-1表达，影响肝损伤的修复过程。同时，电子显微镜显示肝细胞存在明显的线粒体损伤、内质网应激和凋亡小体形成，进一步转录组学和蛋白组学分析表明，GCS可能通过NF-κb通路介导iNKT细胞功能，影响肝损伤结局。随后本项目通过创新性方法分离和培养肝细胞及iNKT细胞等多种细胞。给予α-半乳糖神经酰胺刺激iNKT细胞后，细胞活化标志物的表达明显升高，同时IL-4、IFN-γ和TNF-α等水平上升，在此基础上，抑制NF-κb后，iNKT细胞活化明显减少。然后将原代iNKT细胞与原代肝细胞共培养，α-半乳糖神经酰胺刺激的iNKT细胞与肝细胞共培养组的肝细胞增殖活性降低。抑制GCS后肝细胞损伤减轻，同时观察到内质网应激蛋白GRP78和pNF-κb明显升高，表明抑制GCS后增加了内质网应激介导的肝细胞自噬，从而保护肝细胞免于严重的肝损伤。本项目的研究结果首次证实GCS通过调节糖鞘脂的表达，干预NF-κb通路的激活，影响iNKT细胞活化、相关炎症因子的释放和肝脏免疫微环境，最终影响免疫性肝损伤发生发展。本项目将有助于明确免疫性肝损伤的发病机制，为以免疫介导的肝损伤为关键机制的肝脏疾病提供新的治疗靶点。