Cholestasis represents pathophysiologic syndromes defined as impaired bile flow from the liver. As an outcome, bile acids accumulate and promote hepatocyte injury, followed by liver cirrhosis and liver failure. Glycochenodeoxycholic acid (GCDCA) is relatively toxic and highly concentrated in bile and serum after cholestasis. However, the mechanism underlying GCDCA-induced hepatotoxicity remains unclear. In this study, we found that GCDCA inhibits autophagosome formation and impairs lysosomal function by inhibiting lysosomal proteolysis and increasing lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to the death of L02 human hepatocyte cells. Notably, through tandem mass tag (TMT)-based quantitative proteomic analysis and database searches, 313 differentially expressed proteins were identified, of which 71 were increased and 242 were decreased in the GCDCA group compared with those in the control group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that GCDCA suppressed the signaling pathway of transcription factor E3 (TFE3), which was the most closely associated with autophagic flux impairment. In contrast, GCDCA-inhibited lysosomal function and autophagic flux were efficiently attenuated by TFE3 overexpression. Specifically, the decreased expression of TFE3 was closely related to the disruption of reactive oxygen species (ROS) homeostasis, which could be prevented by inhibiting intracellular ROS with N-acetyl cysteine (NAC). In summary, our study is the first to demonstrate that manipulation of ROS/TFE3 signaling may be a therapeutic approach for antagonizing GCDCA-induced hepatotoxicity.

中文翻译：

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甘草脱氧胆酸通过破坏 L02 细胞中的活性氧稳态来损害转录因子 E3 依赖性自噬溶酶体机制

胆汁淤积代表病理生理综合征，定义为来自肝脏的胆汁流动受损。结果，胆汁酸积聚并促进肝细胞损伤，继而导致肝硬化和肝功能衰竭。甘草脱氧胆酸 (GCDCA) 具有相对毒性，并且在胆汁淤积后高度集中在胆汁和血清中。然而，GCDCA 引起肝毒性的机制尚不清楚。在这项研究中，我们发现 GCDCA 通过抑制溶酶体蛋白水解和增加溶酶体 pH 值来抑制自噬体形成并损害溶酶体功能，从而导致自噬清除缺陷并随后导致 L02 人肝细胞死亡。值得注意的是，通过基于串联质量标签 (TMT) 的定量蛋白质组学分析和数据库搜索，鉴定了 313 种差异表达的蛋白质，其中GCDCA组与对照组相比增加71个，减少242个。京都基因和基因组百科全书 (KEGG) 通路分析显示，GCDCA 抑制转录因子 E3 (TFE3) 的信号通路，该通路与自噬通量损伤最密切相关。相比之下，TFE3 过表达有效减弱了 GCDCA 抑制的溶酶体功能和自噬通量。具体而言，TFE3 表达的降低与活性氧 (ROS) 稳态的破坏密切相关，这可以通过用 N-乙酰半胱氨酸 (NAC) 抑制细胞内 ROS 来预防。总之，我们的研究首次证明操纵 ROS/TFE3 信号可能是一种对抗 GCDCA 诱导的肝毒性的治疗方法。