目的 探究石见穿来源外泌体（Salvia chinensis-derived nanoparticles，SDNPs）诱导SK-Hep-1肝癌细胞及其皮下移植瘤铁死亡和凋亡的作用机制。方法 分别采用超高速离心法和聚乙二醇（polyethylene glycol，PEG）沉淀法提取SDNPs，利用纳米颗粒跟踪分析技术和透射电子显微镜对SDNPs进行表征检测，BCA法测定SDNPs蛋白浓度；以SK-Hep-1肝癌细胞系为研究对象，将细胞分为对照组和SDNPs低、高剂量（15、30μg/mL）组以及索拉非尼（100 nmol/L）组，采用CCK-8法、EdU法和集落实验检测肝癌细胞增殖情况，通过划痕实验、Transwell小室实验检测细胞迁移、侵袭能力，流式细胞术检测细胞凋亡情况，透射电镜检测肝癌细胞超微结构变化，荧光法检测胞内活性氧（reactive oxygen species，ROS）水平，比色法检测细胞中亚铁离子（Fe²⁺）、谷胱甘肽（glutathione，GSH）和丙二醛（malondialdehyde，MDA）水平，Western blotting法检测胱氨酸转运蛋白系统（system xc^-，xCT）、谷胱甘肽过氧化酶4（glutathione peroxidase 4，GPX4）和酰基辅酶A合成酶长链家族成员4（acyl-CoA synthetase 4，ACSL4）蛋白表达；通过SK-Hep-1肝癌细胞系皮下移植构建裸鼠成瘤模型，将裸鼠随机分为对照组、石见穿（0.147 g/g）组和SDNPs ig给药组（SDNPs-ig，50 μg/g）、ip给药组（SDNPs-ip，50 μg/g）、尾iv给药组（SDNPs-tiv，50 μg/g）以及索拉非尼（30 μg/g）组，每组5只，观察SDNPs对SK-Hep-1肝癌细胞系皮下移植瘤的影响，苏木素-伊红（hematoxylin-eosin，HE）染色观察肿瘤以及心、肝、肺和肾组织病理变化，免疫组织化学染色（immunohistochemistry，IHC）检测SK-Hep-1肝癌细胞系皮下移植瘤细胞增殖情况。 结果 超高速离心法和PEG沉淀法均能提取SDNPs，但得到的SDNPs粒径峰度不统一，超高速离心法粒径集中在（144.8±79.7）nm，PEG沉淀法粒径集中在88、157和202 nm；SDNPs能够被肝癌细胞摄取，与对照组比较，SDNPs能显著抑制肝癌细胞的增殖（P＜0.01）、迁移（P＜0.01）和侵袭（P＜0.001），诱导肝癌细胞线粒体结构变化，导致细胞内ROS及Fe²⁺、MDA水平显著上升（P＜0.05、0.001），GSH水平下降（P＜0.01、0.001），并下调铁死亡相关蛋白xCT、GPX4（P＜0.05、0.01、0.001）表达，上调ACSL4蛋白的表达（P＜0.001），诱导肝癌细胞铁死亡；与对照组比较，石见穿组、索拉非尼组以及SDNPs-ip、SDNPs-tiv组的移植瘤体积明显减小（P＜0.05、0.01），而SDNPs-ig组的移植瘤体积则无明显差异；IHC结果表明，与对照组比较，SDNPs-ig组Ki-67和xCT蛋白表达无明显变化，其他组以上蛋白表达均显著下降（P＜0.01、0.001）；各组小鼠心、肝、肺和肾组织无明显病理改变。 结论 SDNPs能被肝癌细胞摄取，并抑制肝癌细胞的增殖、迁移和侵袭，限制肝癌细胞皮下移植瘤的生长，其机制可能与改变肝癌细胞线粒体功能、导致胞内过氧化物积累、诱发细胞铁死亡和凋亡有关。

Objective To investigate the mechanism of SK-Hep-1 hepatoma cells and their subcutaneous transplanted tumor ferroptosis and apoptosis induced by Salvia chinensis - derived nanoparticles (SDNPs). Methods SDNPs were extracted by ultra-high speed centrifugation and polyethylene glycol (PEG) precipitation, respectively. SDNPs were characterized and detected by nanoparticle tracking analysis technology and transmission electron microscopy, and SDNPs protein concentration was determined by BCA method. SK-Hep-1 hepatocellular carcinoma cells were divided into control group, SDNPs low-, high-dose (15, 30 μg/mL) groups and sorafenib (100 nmol/L) group. The proliferation of hepatocellular carcinoma cells was detected by CCK-8 assay, EdU method and colony experiment. Cell migration and invasion capacity were detected by scratch assay and Transwell assay. Cell apoptosis was detected by flow cytometry. Ultrastructural changes in hepatocellular carcinoma cells were detected by transmission electron microscopy. Intracellular reactive oxygen species were detected by fluorescence. Ferrous ion (Fe²⁺), glutathione (GSH) and malondialdehyde (MDA) levels were detected by colorimetric assays, and the cystine transporter system (system xc^-, xCT), glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase family member 4 (ACSL4) were detected by Western blotting. Nude mice were randomly divided into control group, S. chinensis group (0.147 g/g), SDNPs intragastric administration group (SDNPs-ig, 50 μg/g), intraperitoneal administration group (SDNPs-ip, 50 μg/g), tail intravenous administration group (SDNPs-tiv, 50 μg/g) and sorafenib (30 μg/g) group, five mice in each group, to observe the effect of SDNPs on subcutaneous transplantation of SK-Hep-1 hepatocellular carcinoma cell line, hematoxylin and eosin staining (HE) staining to detect tumors and pathological changes in heart, liver, lung and kidney tissues, and immunohistochemistry (IHC) to detect the proliferation of subcutaneous transplantation of SK-Hep-1 hepatocellular carcinoma cell line. Results Both ultracentrifugation and PEG precipitation methods can extract SDNPs, but the particle size kurtosis of SDNPs obtained is not uniform. The kurtosis of ultracentrifugation method is concentrated at (144.8 ±79.7) nm, and the kurtosis of PEG precipitation method is concentrated at 88, 157 and 202 nm. SDNPs can be absorbed by hepatoma cells. Compared with the control group, SDNPs can significantly inhibit the proliferation (P < 0.01, 0.001), migration (P < 0.01) and invasion (P < 0.001) of hepatoma cells, induce mitochondrial structural changes in hepatoma cells, resulting in significant increase in intracellular ROS, Fe²⁺ and MDA levels (P < 0.05, 0.001), decrease in GSH levels (P < 0.01, 0.001), down-regulate ferroptosis-related proteins xCT and GPX4 (P < 0.05, 0.01, 0.001), up-regulate the expression of ACSL4 (P < 0.001), and induce ferroptosis in hepatoma cells. Compared with the control group, the transplanted tumors volume in the S. chinensis, sorafenib, SDNPs-ip and tiv group were significantly reduced (P < 0.05, 0.01), while the transplanted tumor volume in the SDNPs-ig group was not significantly different; IHC results showed that compared with the control group, the expression of Ki-67 and xCT proteins in the SDNPs-ig group did not change significantly, and the expression of proteins in the other groups and above decreased significantly (P < 0.05, 0.01). There were no obvious pathological changes in the heart, liver, lung and kidney tissues of mice in each group. Conclusion SDNPs can be internalized by hepatoma cells, inhibit the proliferation, migration and invasion of hepatoma cells, and limit the growth of subcutaneous transplanted tumors of hepatoma cells. The mechanism may be related to changes in mitochondrial function of hepatoma cells, accumulation of intracellular peroxides, and induction of cell iron death and apoptosis.

R285.5

国家自然科学基金资助项目(82074425);湖南省自然科学基金资助项目(2023JJ30364，2023JJ30448，2023JJ30361);长沙市自然科学基金资助项目(kq2202264)