基于P53/xCT/GPX4通路探讨实脾消积方干预糖代谢异常肝癌细胞铁死亡的机制研究

Mechanism of Shipi-Xiaoji Formula intervening ferroptosis in liver can⁃cer cells with abnormal glucose metabolism based on P53/xCT/GPX4 pathway

目的:探讨实脾消积方(SPXJF)对糖代谢异常下肝癌细胞体外模型铁死亡的作用及机制。方法:使用高胰岛素干预HepG2细胞建立糖代谢异常肝癌细胞模型,并采用葡萄糖氧化酶法检测葡萄糖含量和CCK-8法检测抑制率验证模型是否成功;设立对照组、模型组、二甲双胍组和SPXJF低、中、高剂量组,检测各组葡萄糖消耗量、细胞活力和细胞增殖、迁移、侵袭能力;检测细胞谷胱甘肽(GSH)、活性氧簇(ROS)及丙二醛(MDA)水平以评估脂质过氧化水平;Western blot法检测铁死亡相关蛋白P53、溶质载体家族7成员11(SLC7A11/xCT)和谷胱甘肽过氧化物酶4(GPX4)的表达。结果:与对照组相比,糖代谢异常肝癌细胞葡萄糖消耗量显著降低(P<0.05),细胞活力和增殖能力无显著差异(P>0.05),但迁移和侵袭能力显著提高(P<0.05),而二甲双胍及SPXJF能明显抑制糖代谢异常下肝癌细胞活力以及增殖、迁移和侵袭能力(P<0.05);模型组GSH水平高于其他组,而ROS和MDA水平低于其他组,此外,模型组P53表达下调,xCT和GPX4表达下调,而加入SPXJF及二甲双胍处理后上述指标表达逆转(P<0.05)。结论:SPXJF能通过P53/xCT/GPX4通路诱导糖代谢异常下肝癌细胞铁死亡。

AIM:To investigate the effect of Shipi-Xiaoji Formula(SPXJF)on ferroptosis in an in vitro model of liver cancer cells with abnormal glucose metabolism and its mechanism.METHODS:High insulin was used to treat HepG2 cells to establish a liver cancer cell model with abnormal glucose metabolism.The glucose oxidase method was used to measure glucose content,and the cell counting kit-8(CCK-8)assay was used to evaluate the degree of inhibition to confirm the success of modeling.The cells were divided into control group,model group,metformin group,and low-,medium-and high-dose SPXJF groups.The glucose consumption,viability,proliferation,migration and invasion of the cells in each group were measured.Intracellular glutathione(GSH),reactive oxygen species(ROS)and malondialdehyde(MDA)levels were measured to assess the level of lipid peroxidation.Western blot was used to detect the expression of ferroptosis-related proteins P53,solute carrier family 7 member 11(SLC7A11/xCT)and glutathione peroxidase 4(GPX4).RESULTS:The results showed that glucose consumption was significantly reduced in HepG2 cells with abnormal glucose metabolism compared with normal HepG2 cells(P<0.05),while the cell viability remained unchanged.After treatment in each group,no significant difference in cell viability and proliferation was observed between control group and model group(P>0.05).However,the migration and invasion of the cells in model group were significantly higher than those of normal HepG2 cells(P<0.05).Metformin and SPXJF treatment significantly inhibited the viability,proliferation,migra‐tion and invasion of HepG2 cells with abnormal glucose metabolism(P<0.05).The GSH level was higher in model group than that in the other groups,while the ROS and MDA levels were lower.The expression of P53 was down-regulated in model group but up-regulated in metformin and SPXJF groups.The expression of xCT and GPX4 was down-regulated in model group but up-regulated in metformin and SPXJF groups(P<0.05).CONCLUSION:Shipi-Xiaoji Formula induces ferroptosis in liver cancer cells with abnormal glucose metabolism through P53/xCT/GPX4 pathway.