甘草查尔酮A靶向Nrf2/ARE通路抗D-GalN/LPS诱导急性肝损伤的机制研究

Intestinal endotoxemia-induced acute liver injury is a common inflammatory disease leading to acute liver failure and death in clinic. Oxidative stress, caused by a combination of increased production of reactive oxygen species (ROS), nitric oxide and inflammatory cytokines impaired antioxidant capacity, has attracted considerable attention because of their potential role in the pathogenesis of acute liver injury. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway plays an imperative role in cellular redox homeostasis and activation of this pathway is one of the main defense mechanisms against oxidative or electrophilic stress. Activation of the Nrf2 anti-oxidant signalling pathway has been identified as a major target in cellular defense against oxidative stress of acute liver injury. Bioactive compounds derived from natural products that are activators of the Nrf2/ARE pathway and recapitulate molecular mechanisms for inducing Nrf2 levels to provide protective effects in acute liver injury. In our previous study, we screened several bioactive compounds which has antioxidant potential. Licochalcone A, the major bioactive polyphenol present in liquorice, has been suggested to inhibit NLRP3 inflammasome activation induced by ATP and LPS and inhibit oxidative stress via activating Nrf2/ARE signalling pathway in our previous study. But its effectiveness for the treatment of acute liver injury is still unknown. Based on the inhibitory effect of licochalcone A on lethality and histological change of acute liver injury mouse in pre-experiment, we speculate that licochalcone A may protect the liver against D-GalN/LPS-induced oxidative stress, inflammation and apoptosis via activating Nrf2/ARE pathway. Furthermore, using wild type and Nrf2 knockout mice injected with D-GalN/LPS in vivo and LPS/H2O2 stimulated HepG2 cell line in vitro, we aimed to investigate the effect of licochalcone A on oxidative stress, inflammation, apoptosis and further demonstrated that Licochalcone A targeting the transcription factor Nrf2 to ameliorate oxidative stress, inflammation, apoptosis multiple signalling axes to suppress acute liver injury. These findings support a novel therapeutic value for licochalcone A in the treatment of acute liver injury and ALF.

肠源性内毒素介导的急性肝损伤是肝功能衰竭的重要因素之一，临床尚缺乏有效防治手段。氧化应激在急性肝损伤发生发展中发挥重要作用，Nrf2作为抑制氧化应激的重要转录因子，其激活剂的应用成为防治急性肝损伤的重要策略。课题组前期筛选了大量的Nrf2小分子激活剂，其中甘草查尔酮A体外可显著抑制氧化应激损伤及ATP和LPS介导的炎症小体活化，改善内毒素急性肝损伤小鼠生存率。因此我们提出甘草查尔酮A作用于Nrf2/ARE通路，保护内毒素急性肝损伤这一假说。本研究拟采用药理学及分子生物学方法，构建Nrf2敲除细胞模型及Nrf2敲除小鼠急性肝损伤模型，分别研究甘草查尔酮A对Nrf2/ARE抗氧化通路、炎症和凋亡信号轴、以及敲除Nrf2后其对氧化应激、炎症和凋亡的作用，层次阐明甘草查尔酮A以Nrf2为靶标保护内毒素急性肝损伤的调节机制，以期为以激活Nrf2为靶标防治急性肝损伤及肝衰竭的创新药物研发奠定基础。

肠源性内毒素介导的急性肝损伤是肝功能衰竭的重要因素之一，临床尚缺乏有效的防治手段。氧化应激在急性肝损伤发生发展中发挥重要作用，Nrf2作为抑制氧化应激的重要转录因子，其激活剂的应用拟成为防治急性肝损伤的重要策略。课题组前期筛选大量Nrf2小分子激活剂，其中甘草查尔酮A体外可显著抑制氧化应激损伤及ATP或LPS介导的炎症小体活化，改善内毒素急性肝损伤小鼠生存率。因此我们提出甘草查尔酮A可作用于Nrf2/ARE通路，保护内毒素急性肝损伤这一假说。本研究拟采用药理学及分子生物学方法，构建Nrf2敲除细胞模型及Nrf2敲除小鼠急性肝损伤模型，分别研究甘草查尔酮A对Nrf2/ARE抗氧化通路、炎症和凋亡信号轴、以及敲除Nrf2后其对氧化应激、炎症和凋亡的作用。我们的研究结果发现，甘草查尔酮A通过降低内毒素急性肝损伤小鼠的致死率、减轻肝组织病理改变、降低丙氨酸转氨酶和天冬氨酸氨基转移酶水平、减少炎性细胞因子的分泌、调节氧化标记物等几方面显著降低了小鼠的肝毒性。此外,甘草查尔酮A通过抑制TLR4-MAPK、NF-κB和Txnip-NLRP3信号通路有效缓解炎症反应。同时，甘草查尔酮A诱导Nrf2和QSTM1(P62)信号通路的激活，促进自噬参与AMPK-转录因子EB (TFEB)信号通路，从而发挥保肝活性。进一步的机制研究评估了甘草查尔酮A对Nrf2敲除小鼠的肝脏保护作用，发现缺乏Nrf2促进了甘草查尔酮A诱导的自噬，从而促进了甘草查尔酮A对Nrf2敲除小鼠的肝脏保护作用。此外，与自噬抑制剂（3-甲基ladenine,3-MA）联合治疗减弱了但未消除甘草查尔酮A的保肝作用，这可能与甘草查尔酮A激活Nrf2的能力有关。综上所述我们的研究表明，甘草查尔酮A对内毒素性急性肝损伤具有保护作用，可能与Nrf2激活和自噬密切相关。