基于调控UCP2及其介导分子事件的五酯片抵抗APAP肝毒性的护肝新机制

Acetaminophen (APAP) hepatotoxicity currently is the most frequent cause of drug-induced liver failure in humans. Formation of reactive oxygen species (ROS) and mitochondrial oxidant stress is one of the most commonly invoked cell death mechanisms in APAP-induced liver damage. Mitochondrial uncoupling protein 2 (UCP2) has been proved to have protective effects against oxidant stress theoretically by reducing the mitochondrial membrane potential, decreasing reactive oxygen species (ROS) generation, preventing mitochondrial Ca2+ overload, or a combination of the above. UCP2 also functions as a metabolic switch that enables the promotion of fatty acid metabolism. Our recent study demonstrates a convincing role of UCP2 in protective effects against APAP-induced hepatotoxicity through preservation of mitochondrial function during fatty acid β-oxidation. Our previous metabolomics results demonstrate significant hepatoprotection of Wuzhi Tablet (Schisandra sphenanthera extract, WZ) against APAP-induced liver injury, and Ucp2 mRNA level was significantly induced by WZ. These results indicate UCP2 may play an important role in hepatoprotection of WZ and the involved mechanisms need to be further elucidated. Here, for the first time, we assess the role of UCP2 in WZ-induced hepatoprotection against APAP toxicity and elucidate through which defense mechanism WZ regulate UCP2 to preserve mitochondrial function. In this study, UCP2 overexpression and UCP2 silencing Zhang's liver cell lines, Ucp2-null mice, and mice forced overexpression of UCP2 by receiving Ad-Ucp2 were used to confirm UCP2-mediated hepatoprotection of WZ against APAP toxicity. Mitochondrial membrane potential, ROS production, mitochondrial Ca2+ concentration, and mitochondrial swelling sensitivity of samples from the above UCP2 overexpression and silencing models treated with or without WZ were measured to determine through which defense mechanism WZ mediates UCP2 to maintain proper mitochondrial function and then processes cytoprotection against APAP-induced hepatotoxicity. The studies will provide a new insight into the importance of UCP2 in WZ-induced hepatoprotection against APAP liver toxicity.

解偶联蛋白2（UCP2）是线粒体阳离子转运体，由于其在线粒体功能保护中的重要作用备受关注。氧化应激导致的线粒体功能障碍是扑热息痛（APAP）肝毒性的重要机制。我们最新研究证实，中药五酯片对APAP肝损伤有显著的保护作用，UCP2在抵抗APAP肝毒性过程中起到了重要作用。该研究结果为五酯片抵抗APAP肝毒性的作用机制提供了新的线索。基于此，本项目拟应用Ucp2基因敲除小鼠、Ad-Ucp2腺病毒感染Ucp2过表达小鼠、pcDNA3.1-hUCP2转染和siRNA干扰手段构建UCP2高表达和低表达细胞系，结合常规分子生物学技术，论证五酯片对UCP2信号通路的调控作用，并深入探讨五酯片调控UCP2后对活性氧簇（ROS）、线粒体膜电位/游离钙/肿胀敏感性、细胞凋亡等方面的作用机制，明确UCP2在五酯片抵抗APAP肝毒性的重要作用，为五酯片对抗APAP肝毒性的护肝机制提供新思路和新观点。

扑热息痛（APAP）肝毒性是欧美国家导致急性肝衰竭的首要原因并得到广泛关注。抑制APAP代谢激活、降低氧化应激水平、促进代偿性肝再生是抵抗APAP所致肝损伤的重要机制，NRF2、UCP2、p53等被认为是干预APAP肝毒性的重要靶点。我们前期发现中药五酯片对APAP所致肝损伤有显著保护作用，但其分子机制尚不清楚。基于此，本研究开展了以下研究内容并取得了重要结果：证实了五酯片预防给药对APAP所致肝损伤具有显著的保护作用。在APAP造模前，提前给予五酯片能显著减轻APAP引起的小鼠肝脏组织坏死和阻止血清ALT、AST的异常升高，并呈现一定的剂量效应关系。基于APAP肝毒性产生的机制和五酯片的作用特点，本研究对五酯片保护作用的分子机制进行了探讨。发现此前我们假设的五酯片通过调控UCP2通路抵抗APAP肝毒性不成立，因此我们从其他氧化应激、代谢激活、肝再生等方面作了系统的考察。体外肝微粒体孵育实验的结果表明，五酯片可抑制APAP代谢激活相关的CYP450酶(CYP2E1、CYP1A2、CYP3A)活性，减少毒性代谢物NAPQI的生成；同时上调UGTs、SULTs及部分MRPs的mRNA表达，增加APAP的Ⅱ相代谢并促进代谢物排出。五酯片还能显著减轻APAP引起的氧化应激并激活NRF2-ARE通路，启动下游Ⅱ相解毒和抗氧化基因(Nqo1、Ho-1、Gclc、Gclm)的转录和翻译，从而减轻APAP肝损伤。同时，五酯片能降低p53和p21蛋白水平，显著上调细胞增殖相关蛋白(Cyclin D1、CDK4、PCNA、ALR)的表达，促进肝细胞增殖和损伤后肝脏组织的修复。综上所述，本研究证明了五酯片对APAP所致的肝损伤具有显著的保护作用，其分子机制与抑制APAP代谢活化，激活NRF2-ARE通路减轻氧化应激以及促进p53/p21介导的损伤后肝脏组织的恢复有关。本研究可为进一步阐释五酯片护肝作用特点和将其开发为APAP肝损伤的治疗药物提供新数据。