自噬在肝再生增强因子保护重型肝炎肝衰竭中的新作用及机制

It has been documented that autophagy has a protective effect on liver injury induced by alcohol and drug. We previously discovered the protective effect of augmenter of liver regeneration (ALR) on severe hepatitis and liver failure, but whether the protective effect relates to autophagy is still not clear. Data also show that ALR can regulate autophagy via mitochondrial apoptosis. Based on our work that: ① In mouse model with acute liver injury, serum ALR level rose firstly and then declined in the ensuing 12 hours to near-basal level (F=30.495, p<0.01). The ALR level was high during liver injury, and back to normal lever with the recovery of liver injury. ② Serum ALR level was higher in acute-on-chronic liver failure (ACLF) than in normal control (2.68±1.95 vs. 0.74±0.31, p<0.01). Serum ALR level of patients with ACLF was more significant in survival group than in dead group (7.83±1.77 vs. 2.14±1.58, t=7.576, p<0.01).③ ALR can promote autophagy in HepG2 cells in serum free medium. Therefore we propose a hypothesis that autophagy may play an important role in the protective effect of ALR on severe hepatitis and liver failure. In this proposal we will extend our work to address the role of autophagy in the protective effect of ALR on severe hepatitis and liver failure. First, we will determine the effect of ALR on autophagy by transfecting ALR plasmid or siRNA to primary hepatocytes or HepG2 cells in serum free medium. Second, we will explore the expression of autophagy in LPS/D-GalN induced mouse model treated by ALR plasmid, to determine whether autophagy plays an important role in the protective effect of ALR on liver failure. Third, using clinical specimens, we will detect the expression of ALR and autophagy and theirs relativity during the development of severe hepatitis. Finally, we will explore hepatocyte apoptosis and inflammation in LPS/D-GalN induced mouse model treated by ALR plasmid and inducement or inhibition of autophagy. In summary, through this study we will unveil the new mechanisms of the protective effect of ALR on severe hepatitis and liver failure, which may be valuble for new therapeutic treatment.

最新研究表明，细胞自噬对酒精、药物诱导的肝损伤和肝衰竭具有保护作用。我们前期发现，肝再生增强因子（ALR）与重型肝炎肝衰竭预后相关，ALR水平升高提示良好预后，但其保护作用是否与自噬有关尚不清楚。有证据显示ALR可能通过线粒体凋亡途径参与了细胞自噬。据此提出本研究假说：ALR可能通过自噬对重型肝炎肝衰竭起保护作用。本课题首先利用饥饿诱导细胞自噬模型，明确ALR对自噬的调控作用；其次，应用脂多糖/D-氨基半乳糖诱导小鼠急性肝衰竭模型，观察ALR处理后该模型中自噬的表达，探讨ALR是否通过自噬对肝衰竭起保护作用；随后，利用乙型肝炎及重型肝炎肝衰竭的患者标本，探讨ALR和自噬在重型肝炎肝衰竭疾病过程中的作用及相互关系；最后，应用上述动物模型，分别激活或者抑制自噬，研究干预自噬对ALR调控肝细胞凋亡和炎症因子的影响。本课题将为重型肝炎肝衰竭保护性机制和治疗的研究提供新的线索。

最新研究表明，细胞自噬对酒精、药物诱导的肝损伤和肝衰竭具有保护作用。我们前期发现，肝再生增强因子（ALR）与重型肝炎肝衰竭预后相关，ALR水平升高提示良好预后，但其保护作用是否与自噬有关尚不清楚。有证据显示ALR可能通过线粒体凋亡途径参与了细胞自噬。据此提出本研究假说：ALR可能通过自噬对重型肝炎肝衰竭起保护作用。. 本课题首先利用饥饿诱导细胞自噬模型，明确ALR对自噬的调控作用；其次，应用小鼠急性肝衰竭模型，观察ALR处理后该模型中自噬的表达，探讨ALR是否通过自噬对肝衰竭起保护作用；随后，利用乙型肝炎及重型肝炎肝衰竭的患者标本，探讨ALR和自噬在重型肝炎肝衰竭疾病过程中的作用及相互关系；最后，应用上述动物模型，抑制自噬，研究干预自噬对ALR调控肝细胞凋亡和增殖的影响。. 我们研究发现，肝再生增强因子（augmenter of liver regeneration, ALR）对重型肝炎肝衰竭具有保护性作用，重型肝炎肝衰竭患者血清ALR水平与预后相关，ALR水平升高提示良好预后，ALR持续低水平提示预后不良。我们进一步研究发现，ALR可以促进HepG2细胞内自噬流的发生，应用自噬抑制剂（3-MA）抑制HepG2细胞自噬后，细胞凋亡增加，ALR对HepG2细胞的保护性作用减弱或消失，提示自噬可能参与到ALR抗凋亡作用中。. 我们应用小鼠急性肝损伤模型和肝衰竭患者病例，深入探讨自噬在ALR保护重型肝炎肝衰竭中的作用及机制。研究结果提示ALR可以促进急性肝损伤小鼠中自噬流的表达。应用自噬抑制剂（3-MA）尾静脉注射抑制细胞自噬后，细胞凋亡增加，肝细胞增殖减少，ALR对急性肝损伤小鼠的保护性作用减弱或消失，提示自噬可能在ALR抗凋亡及促增殖作用中发挥重要的作用。我们最后在人体标本上进一步验证我们的研究结果，发现在肝硬化和急性肝衰竭患者中，肝组织中ALR的表达降低，自噬流减弱，提示肝硬化和急性肝衰竭患者中ALR表达的降低与自噬流减弱有一定的相关性。. 本研究明确了ALR对自噬信号通路的调控作用，并阐明了ALR保护重型肝炎肝衰竭中的作用机制，从而为重型肝炎肝衰竭保护性机制和治疗的研究提供了新的线索和依据。