凋亡和自噬已被证明在自我消除过程中具有合作和对抗作用。一方面，凋亡和自噬可以作为合作伙伴以协调或协同的方式诱导细胞死亡；另一方面，自噬作为拮抗物通过促进细胞存活来阻断细胞凋亡。我们之前的研究表明，川芎嗪可以诱导PLC/PRF/5细胞凋亡，但川芎嗪对自噬的影响以及与凋亡的功能关系尚未阐明。因此，本研究旨在研究川芎嗪对肝细胞癌（HCC）细胞自噬的功能和分子机制。本研究的目标是探究川芎嗪对HCC细胞自噬的分子机制。通过Western印迹检测自噬标志物beclin1，LC3B和p62的蛋白水平。使用6-羟基黄酮和stattic检测川芎嗪通过酪氨酸激酶（AKT）/细胞外调节蛋白激酶（ERK）1/2/哺乳动物雷帕霉素靶蛋白（mTOR）/信号转导与转录激活子（STAT3）信号通路调节自噬的作用。流式细胞仪被用来检测PLC/PRF/5细胞经过24小时川芎嗪处理后是否伴有拉帕霉素、stattic和6-羟基黄酮，以及其对半胱天冬酶3活性和细胞凋亡的影响。川芎嗪处理导致自噬标志物beclin1蛋白水平下降，而p62蛋白水平显著增加，表明川芎嗪处理抑制了HCC细胞的自噬。川芎嗪处理可以降低p-AKT和p-ERK1/2的蛋白水平，但增加mTOR和p-mTOR的蛋白水平，表明川芎嗪可以抑制AKT/ERK而不是mTOR。AKT/ERK激活剂6-羟基黄酮可以逆转川芎嗪引起的AKT和ERK1/2磷酸化损失，暗示川芎嗪通过激活mTOR而不是AKT/ERK来影响自噬。川芎嗪处理显著上调STAT3和p-STAT3的蛋白水平，表明自噬抑制是通过川芎嗪通过激活STAT3信号传导来介导的。STAT3抑制剂stattic能够显著逆转川芎嗪引起的STAT3酪氨酸705位点磷酸化增加。mTOR信号抑制剂拉帕霉素逆转了川芎嗪引起的mTOR磷酸化增强，但对mTOR总水平没有影响，表明川芎嗪处理通过激活mTOR/STAT3通路抑制了HCC细胞的自噬。此外，川芎嗪处理增加了半胱天冬酶3活性和细胞凋亡的总率，而这种增加被拉帕霉素、stattic和6-羟基黄酮逆转，证明川芎嗪通过激活mTOR/STAT3信号传导来促进细胞凋亡。川芎嗪通过激活mTOR/STAT3信号传导诱导PLC/PRF/5细胞自噬抑制和促进细胞凋亡。川芎嗪有望成为HCC治疗的可行选择。© 2023年。作者，独家授权给施普林格出版社德国部分，属施普林格自然出版集团的一部分。

Apoptosis and autophagy have been shown to act cooperatively and antagonistically in self-elimination process. On the one side, apoptosis and autophagy can act as partners to induce cell death in a coordinated or cooperative manner; on the flip side, autophagy acts as an antagonist to block apoptotic cell death by promoting cell survival. Our previous research indicated that trillin could induce apoptosis of PLC/PRF/5 cells, but the effects of trillin on autophagy as well as its functional relationship to apoptosis have not been elucidated. Here, the running study aims to investigate the function and molecular mechanism of trillin on autophagy with hepatocellular carcinoma (HCC) cells. The objective of this study is to investigate the molecular mechanism of trillin on autophagy in HCC cells. Protein levels of autophagy markers beclin1, LC3B, and p62 were detected by western blotting. 6-Hydroxyflavone and stattic were used to test the role of trillin regulation of autophagy via serine threonine kinase (AKT)/extracellular-regulated protein kinases (ERK) 1/2/mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Flow cytometry was used to detect caspase 3 activity and apoptosis in PLC/PRF/5 cells treated with trillin for 24 h with or without rapamycin, stattic, and 6-hydroxyflavone. The protein level of autophagy marker beclin1 was decreased, whilst the protein level of p62 was significantly increased by trillin treatment, indicating trillin treatment led to inhibition of autophagy in HCC cells. Trillin treatment could reduce the protein levels of p-AKT and p-ERK1/2, but enhance the protein levels of mTOR and p-mTOR, suggesting that trillin could inhibit AKT/ERK rather than mTOR. The AKT/ERK activator 6-hydroxyflavone could reverse the loss of AKT and ERK1/2 phosphorylation induced by trillin, implying that trillin impairs autophagy through activated mTOR rather than AKT/ERK. STAT3 and p-STAT3 were significantly upregulated by the trillin treatment with an increase in dose from 0 to 50 μM, suggesting that autophagy inhibition is mediated by trillin via activation of STAT3 signaling. The STAT3 inhibitor stattic significantly reversed the increased STAT3 phosphorylation at tyrosine 705 induced by trillin. The mTOR signaling inhibitor rapamycin reversed the trillin-induced mTOR phosphorylation enhancement but exerted no effects on total mTOR levels, suggesting trillin treatment led to inhibition of autophagy in HCC cells through activating mTOR/STAT3 pathway. Furthermore, caspase 3 activities and the total rate of apoptosis were increased by trillin treatment, which was reversed by rapamycin, stattic, and 6-hydroxyflavone, proving that trillin promotes apoptosis via activation of mTOR/STAT3 signaling. Trillin induced autophagy inhibition and promoted apoptosis in PLC/PRF/5 cells via the activation of mTOR/STAT3 signaling. Trillin has the potential to be a viable therapeutic option for HCC treatment.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.