MLKL介导肝细胞程序性坏死在肝衰竭中的作用及机制研究

Liver failure is still a major health problem with aggressive progression and poor prognosis. The underlining pathogenesis of liver failure remains unclear. Recently, necroptosis, a novel demonstrated programmed cell death has been implicated in mediating multiple inflammation associated diseases. Our preliminary studies found that the expression of the key regulator of necroptosis, MLKL and its upstream molecules were up-regulated in the serum of liver failure patients and liver tissue of liver failure mice. Blocking the kinase activity of RIPK1, an up-stream regulator of MLKL in the necroptosis pathway, by necrostatins could inhibit the activation of necroptosis and ameliorate liver injury in the presence of LPS/D-GalN. The results suggested the involvement of necroptosis in the progression of liver failure. Therefore, we proposed to utilize murine liver failure model to further investigate the role of key regulators of the necroptosis pathway in the progression of liver failure and its relationship with hepatic inflammation, oxidative stress and apoptosis, and to further determine whether MLKL plays a vital role in the progression of liver failure and its relative mechanism by using MLKL over-expressed or knock-down murine model. Furthermore, in ex-vivo experiment, focusing on the mitochondrial, we proposed to isolate primary hepatocytes of mice to investigate the regulatory mechanism of MLKL in mediating hepatocyte necroptosis. Meanwhile, liver failure patient cohort would be prospectively established to validate the impact of necroptosis in liver failure patients and to determine its prognostic value. The study will provide new evidences for a deeper understanding of the pathophysiology mechanisms of liver failure and would also provide new therapeutic strategies to lower the high mortality of liver failure.

肝衰竭病程进展快，病死率高，发病机制仍不明。最新研究报道，程序性坏死是一种可调控的细胞坏死，在多种炎症损伤相关的疾病中发挥重要作用。本课题组前期发现程序性坏死关键调控分子MLKL等在肝衰竭患者血清及小鼠肝组织中表达上调，抑制MLKL上游的激酶活性具肝脏保护效应，提示MLKL可能通过介导肝细胞程序性坏死参与肝衰竭病程进展。本项目拟采用肝衰竭小鼠，研究程序性坏死信号通路关键分子在肝衰竭病程中的作用，探讨其与炎症损伤、氧应激、凋亡等的内在联系；过表达/低表达MLKL，观察其表达水平对小鼠肝衰竭病程的影响并探讨机制；利用细胞体外诱导程序性坏死，针对线粒体损伤环节，阐明肝衰竭中MLKL介导肝细胞程序性坏死的调控机制；同时，建立前瞻性肝衰竭临床队列，明确程序性坏死及关键调控分子MLKL与肝衰竭发生及预后转归的关系，为深入理解肝衰竭发病机制提供新证据，也为降低肝衰竭死亡率提供新的干预靶点和治疗策略。

肝衰竭病程进展快，病死率高，发病机制仍不明。其中MLKL介导的肝细胞程序性坏死可能参与肝衰竭病程进展。本课题按计划，首先从临床层面论证肝细胞总体死亡水平在肝衰竭疾病进展过程中的变化情况与意义。利用前期已验证过的肝细胞死亡标志物：细胞角蛋白18（Keratin-18）的完整片段（M65）及凋亡裂解片段（M30），进一步构建临床队列证实了（1）肝内细胞死亡程度与肝内炎症反应水平呈正相关性；（2）短期内急性发生的大量肝细胞死亡会促进肝内局部炎症反应演变为全身性炎症反应，导致肝衰竭进展至全身多器官衰竭。在这些工作的基础上，课题组进一步聚焦肝细胞程序性坏死，首先从临床角度，明确了其关键分子调控蛋白受体相互作用蛋白激酶3 （Receptor Interacting Protein Kinase 3, RIPK3）及混合系列蛋白激酶样结构域蛋白（Mixed Lineage Kinase Domain-like, MLKL）在肝衰竭患者肝组织中表达水平显著高于其在肝炎及正常人中的表达。其中游离的RIPK3可在外周血中被检测到，且其浓度与肝病的严重程度和预后正相关，浓度越高患者预后越差。同时课题组利用脂多糖联合D-氨基半乳糖（LPS/D-GalN）腹腔注射成功构建肝衰竭小鼠模型并通过腺病毒转染技术顺利构建mlkl基因过表达shRNA。在这两项工作基础上，针对程序性坏死信号通路关键调控基因mlkl进行在体干预，发现mlkl过表达小鼠较对照组更难耐受LPS/D-GalN引起的肝损伤，有更强烈的氧化应激反应，更早形成肝衰竭，存活时间更短。这一现象可被mlkl上游靶分子RIPK1的抑制剂NEC-1逆转。进一步选取小鼠正常肝细胞系（AML12）进行细胞学实验，联合利用TNF-α/ SM-164/z-VAD-fmk（TSZ）诱导成功构建程序性坏死细胞模型。在前期已获得mlkl过表达腺病毒基础上，进一步构建mlkl-shRNA干扰腺病毒，并发现程序性坏死关键基因mlkl的过表达或干扰会相对应地加重或减缓程序性坏死的形成。本项目的研究成果为进一步明确程序性坏死及关键调控分子MLKL与肝衰竭发生及预后转归的关系，为深入理解肝衰竭发病机制提供新证据，也为降低肝衰竭死亡率提供新的干预靶点和治疗策略。