对乙酰氨基酚（acetaminophen，APAP）是常见的解热镇痛药，但其过量使用可导致严重的药物性肝损伤。人跨膜蛋白121（transmembrane protein121，TMEM121）是一种进化保守的跨膜蛋白，在肝脏组织中有较显著的表达，但其在肝脏损伤中的功能未知。为探讨TMEM121基因在APAP诱导的肝癌细胞损伤中的作用及其分子机制，本研究综合运用生物信息学预测、细胞功能实验和焦亡通路关键分子检测等方法进行了分析。首先，通过分子对接分析，发现TMEM121蛋白与APAP存在直接结合能力；GSE104601数据集分析及细胞实验证实，APAP负调控TMEM121基因的表达。在APAP刺激下，TMEM121过表达增加了Huh7细胞对APAP的敏感性，且逆转了TMEM121在未受刺激时对细胞增殖与迁移的促进作用。在机制探讨方面，利用蛋白质免疫印迹、ELISA等方法对焦亡通路关键分子进行检测，结果显示在APAP刺激下，TMEM121过表达可上调NLRP3和caspase-1的表达，促进GSDMD发生剪切生成活性片段GSDMD-N，同时显著增加炎症因子IL-1β与IL-18的释放，加剧细胞焦亡的发生。乳酸脱氢酶释放实验及透射电镜观察进一步证实，TMEM121过表达加重了APAP引起的细胞膜损伤，并使细胞呈现细胞膜气泡状凸起、破裂及内容物外溢等典型焦亡形态特征。综上所述，本研究揭示了TMEM121是APAP肝损伤中的一个关键调控因子，通过激活NLRP3/caspase-1/GSDMD信号通路加剧APAP诱导的肝癌细胞焦亡，为深入解析APAP药物性肝损伤的分子机制提供了新的理论依据和潜在靶点。

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but overdose causes severe acute liver injury. TMEM121 is an evolutionarily conserved transmembrane protein with high expression in liver tissue, yet its role in liver injury remains unknown. To investigate the function and molecular mechanism of the TMEM121 gene in APAP-induced injury in hepatoma carcinoma cells, we comprehensively employed three analytical approaches: bioinformatics prediction, cellular functional assays, and detection of key molecules in the pyroptosis pathway. Firstly, molecular docking analysis revealed that TMEM121 protein can directly bind to APAP. Analysis of the GSE104601 dataset and cellular experiments confirmed that APAP negatively regulates TMEM121 gene expression. Upon APAP stimulation, TMEM121 overexpression increased the sensitivity of Huh7 cells to APAP and reversed the promoting effects of TMEM121 on cell proliferation and migration observed under unstimulated conditions. Mechanistically, Western blotting and ELISA were used to assess key components of the pyroptosis pathway. The results demonstrated that, upon APAP stimulation, TMEM121 overexpression upregulated the expression of NLRP3 and caspase-1, promoted the cleavage of GSDMD to generate the active fragment GSDMD-N, and significantly increased the release of the inflammatory cytokines IL-1β and IL-18, thereby exacerbating pyroptosis. Furthermore, the lactate dehydrogenase release assay and transmission electron microscopy observation confirmed that TMEM121 overexpression aggravated APAP-induced cell membrane damage, leading to characteristic morphological features of pyroptosis, including membrane ballooning, rupture, and leakage of cellular contents. In summary, this study reveals that TMEM121 gene acts as a critical regulator of APAP-induced injury in Huh7 cells by activating the NLRP3/caspase-1/GSDMD signaling pathway and aggravating APAP-induced pyroptosis, providing novel mechanistic insights and potential therapeutic targets for APAP-induced hepatotoxicity.