基于ASGPR介导的肝细胞自噬效应研究苦柯胺B在脓毒症中的肝保护作用及机制

Sepsis is a common and serious complication among critical ill patients. Hepatic injury is the major pathophysiological features of sepsis and an independent predictor for poor prognosis with no effective treatment available. Kukoamine B(KB) is our own new drug with anti sepsis efficacy.Recently, we found KB possessed indenpendent liver protective effects. Such effects are due to its binding to ASGPR receptors in hepatocytes to induce hepatic autophagy, without releations with its known mechanisms of LPS and CpG DNA neutralization. Therefore, we speculate that KB may activate the lysosome calcium-TFEB pathway via ASGPR internalization, which thereby induces autophagy in hepatocytes and exerts hepatic protection. To verify the hypothesis, we will first evaluate whether the ASGPR is the potential drug target of KB and identify the binding pattern. Then we will assess the involving patterns of lysosome calcium efflux induced by ASGPR internalization. Last, we will investigate whether TFEB is activated by lysosome calcium efflux induced by KB and then mediate autophagy induction and hepatic protection in hepatocytes.We wil also evaluate the mechanism of TFEB in regulating autophagy and mediate hepatic protection induce by KB. Overall, we will illustrate a new molecular mechanism of autophagy induction and hepatic protection in hepatocytes triggered by Kukoamine B, which may also provide new clues for new drug research in sepsis based on autophagy related liver protection.

脓毒症是临床常见和严重的感染并发症。肝功能障碍是导致脓毒症患者死亡的独立危险因素，尚无有效治疗措施。苦柯胺B（KB）是我们课题组研发的抗脓毒症新药。新近我们发现，KB具有独立的肝保护作用，作用机制与其经去唾液酸化糖蛋白受体（ASGPR）介导肝细胞自噬增强有关，与原有中和LPS和CpG DNA机制无关。为此，我们提出“KB经ASGPR内化途径进入溶酶体、触发Ca2+外流并激活Ca2+-TEFB信号通路、进而上调肝细胞自噬并发挥肝保护作用”的假说。本项目拟在体内外脓毒症肝损伤模型中开展研究：首先明确ASGPR是KB的作用靶点并阐明可能结合位点；进而研究KB经ASGPR内化进入溶酶体后对Ca2+外流的影响，明确介导Ca2+外流的通道蛋白；最后探讨KB对转录因子TFEB的激活作用，明确TFEB在KB介导肝细胞自噬和肝保护作用中的调控机制。为阐明KB肝保护效应的分子机制和发现新药物靶点提供依据。

肝功能障碍是导致脓毒症患者死亡的独立危险因素，尚无有效治疗措施。苦柯胺B是我们原研的抗脓毒症新药。新近发现，苦柯胺B具有独立的肝保护作用，且与其经去唾液酸化糖蛋白受体（ASGPR）介导肝细胞自噬增强有关，但机制不明，为此，本项目主要围绕苦柯胺B经由ASGPR调控溶酶体Ca2+外流并活化自噬关键调控分子TFEB、进而启动肝细胞保护效应的调控机制展开研究，以期阐为基于自噬调控的脓毒症肝保护药物研发提供新思路。经项目研究，我们首先明确了ASGPR是苦柯胺B介导肝细胞自噬和肝保护作用的靶点，初步探明了其结合位点。其次明确了苦柯胺B结合ASGPR后可选择性介导溶酶体Ca2+外流进而上调自噬，最后阐明了苦柯胺B通过溶酶体Ca2+-Calcineurin-TFEB通路，介导TFEB核转位活性，进而上调肝细胞自噬和介导脓毒症肝保护作用的分子机制。与此同时，我们还拓展了有关自噬参与脓毒症免疫调控相关研究，证实ATP阻断P2X7与MyH9形成促吞噬复合物，引发巨噬细胞吞噬抑制和炎症小体活化，介导脓毒症肝损伤的作用机制，发现了胞浆Ca2+超载介导AMPK抑制以及转录因子ATF3负调控TFEB，进而导致巨噬细胞自噬障碍并介导脓毒症免疫抑制发生的相关作用及机制，为基于TFEB调控的脓毒症防治新策略提供了新的实验证据。我们还拓展了有关环境-微生物因素协同介导脓毒症肝损伤的作用研究，阐明了纳米金协同细菌脂多糖促进巨噬细胞凋亡或介导中性粒细胞胞外诱捕网生成，进而介导巨噬细胞与肝细胞的交互作用，引发脓毒症急性肝损伤的作用，发现了环境污染物聚苯乙烯纳米粒子单独诱导巨噬细胞坏死性凋亡，引发巨噬细胞与肝细胞的交互作用并介导急性肝损伤的作用机制，进一步丰富和完善了细胞交互作用参与脓毒症肝损伤发生的研究证据。