目的 探究缺氧条件下瑞戈非尼与伊立替康联合治疗肝细胞癌的作用及机制，为抗肿瘤的联合用药提供一种新的治疗策略。 方法 在常氧和缺氧条件下检测不同药物对肝癌细胞的半数抑制率（IC₅₀）并绘制量效曲线，评估不同氧浓度下肝癌细胞对奥沙利铂、瑞戈非尼及伊立替康的敏感性。应用CompuSyn和SynergyFinder 3.0软件分析不同浓度瑞戈非尼和伊立替康对肝癌细胞的联合效应。流式细胞术评估瑞戈非尼和伊立替康联合用药杀伤肝癌细胞的作用。EdU增殖实验评估瑞戈非尼和伊立替康联合对细胞增殖的影响。Transwell及划痕实验评估瑞戈非尼和伊立替康联合对肝癌细胞迁移的影响。Western blot实验检测瑞戈非尼和伊立替康联合对相关信号通路的影响。 结果 常、缺氧下，瑞戈非尼及伊立替康的IC₅₀值无明显差异。相较奥沙利铂，二者可以克服缺氧诱导治疗耐受。缺氧条件下瑞戈非尼与伊立替康具有协同效应，联合指数CI值< 1，ZIP协同分数> 10。与瑞戈非尼组及伊立替康组相比，联合组显著提高了SubG1期细胞比例（P < 0.000 1），对细胞迁移具有联合抑制作用（P < 0.000 1）。缺氧条件下伊立替康提高了增殖细胞比例（P < 0.000 1），联合瑞戈非尼后增殖细胞比例显著下降（P < 0.000 1），细胞内CyclinD1水平下降（P < 0.000 1）。缺氧状态下，伊立替康上调了HIF-1α的表达并活化ERK1/2信号（P < 0.000 1），联合瑞戈非尼能够调控伊立替康介导的二者表达或活化（P < 0.000 1），联合用药下调了AKT及ERK1/2信号的活性（P < 0.001）。 结论 缺氧条件下瑞戈非尼与伊立替康联合用药可以克服缺氧诱导的治疗耐受，促进肝癌细胞死亡，抑制肝癌细胞的增殖及迁移。该联合效应可能与肝癌细胞内HIF-1α和CyclinD1的表达抑制、AKT和ERK1/2的磷酸化水平下调相关。

Objective This study investigates the mechanism of combination therapy of Regorafenib and Irinotecan in hepatocellular carcinoma under hypoxic conditions and aims to provide a new therapeutic strategy for anticancer drugs combination to improve outcomes. Methods The median inhibitory concentration （IC₅₀） of drugs against hepatocellular carcinoma cells under normoxic and hypoxic conditions were determined， dose-response curves were plotted to evaluate the sensitivity to Oxaliplatin Regorafenib and Irinotecan. Combined effects of different concentrations of Regorafenib and Irinotecan on hepatocellular carcinoma cells were analyzed using CompuSyn and SynergyFinder 3.0 software. The impacts on cell death rates were determined via flow cytometry. The effects on cell proliferation were determined using EdU proliferation assays. The influences on cell migration were assessed through Transwell and wound healing assays. The alterations of relevant proteins in signaling pathways were detected by Western blot experiments. Results Hypoxia had no significant effect on the IC₅₀ values of Regorafenib and Irinotecan. Compared to Oxaliplatin， both agents can overcome hypoxia-induced treatment tolerance. Under hypoxic conditions， Regorafenib and Irinotecan exhibited synergistic effects with a combination index （CI values） < 1 and a ZIP synergy score > 10. Compared to regorafenib and irinotecan groups， the combination of Regorafenib and Irinotecan significantly increased the cells proportion in SubG1 phase （P < 0.000 1） and exhibited combined inhibitory effects on cell migration （P < 0.000 1）. Under hypoxic conditions， Irinotecan increased the proportion of proliferating cells （P < 0.000 1）， which significantly decreased upon combination with Regorafenib （P < 0.000 1）， accompanied by reduced intracellular CyclinD1 levels （P < 0.000 1） Under hypoxic conditions， Irinotecan upregulated HIF-1α expression and activated ERK1/2 signaling （P < 0.000 1）， combination with Regorafenib modulated Irinotecan-mediated expression or activation （P < 0.000 1）， and the combination therapy downregulated AKT and ERK1/2 signaling activity （P < 0.001）. Conclusions Under hypoxic conditions， the combination of Regorafenib and Irinotecan overcomes hypoxia-induced treatment tolerance， promotes hepatocellular carcinoma cell death， and inhibits hepatocellular carcinoma cell proliferation and migration. This synergistic effect may be associated with the suppression of HIF-1α and CyclinD1 expression， as well as the downregulation of AKT and ERK1/2 phosphorylation levels in hepatocellular carcinoma cells.