目的 阐明半边莲来源细胞外囊泡样纳米颗粒（Lobelia chinensis-derived extracellular vesicle-like nanoparticles，LC-EVLP）通过重塑谷氨酰胺代谢网络诱导肝癌细胞致死性自噬的分子机制，并系统评价其抗肿瘤活性与生物安全性。方法 采用超速离心法分离LC-EVLP并进行理化表征。结合网络药理学、分子对接及多维度细胞功能实验，评估LC-EVLP对人肝癌Mahlavu细胞增殖、迁移及侵袭的抑制作用。利用自噬流阻断剂与代谢中间体α-酮戊二酸（α-ketoglutaric acid，α-KG）开展回补实验，验证“代谢-自噬”调控轴；构建裸鼠皮下移植瘤模型，检测LC-EVLP的体内抗肿瘤作用与主要脏器组织病理学变化。结果 成功分离获得LC-EVLP，其呈典型囊泡结构，粒径主要分布于126.0～151.7 nm，并被Mahlavu细胞有效摄取。功能实验表明，LC-EVLP在≥12.5μg/mL时显著抑制Mahlavu细胞活力（P＜0.001），而对LO2正常肝细胞无显著影响。进一步研究发现，LC-EVLP显著抑制Mahlavu细胞克隆形成能力及DNA合成（P＜0.05、0.01、0.001），并显著降低细胞迁移和侵袭能力（P＜0.001）。自噬抑制剂巴佛洛霉素A1（bafilomycin A1，BafA1）可显著回补细胞活力（P＜0.001），表明自噬性细胞死亡是LC-EVLP发挥抗肝癌活性的主导方式。LC-EVLP显著上调Beclin 1蛋白表达（P＜0.05、0.01、0.001），提高微管相关蛋白1轻链3-II/I（microtubule associated protein 1 light chain 3-II/I，LC3-II/I）值（P＜0.05、0.01），并降低p62蛋白的表达（P＜0.01、0.001），且BafA1进一步增强LC3-II/I的累积并逆转p62表达下降（P＜0.01、0.001），证实其促进自噬流通畅。代谢方面，LC-EVLP显著抑制丙酮酸脱氢酶激酶1（pyruvate dehydrogenase kinase 1，PDK1）、丙酮酸激酶M型（pyruvate kinase M，PKM）及谷氨酰胺酶1（glutaminase 1，GLS1）表达（P＜0.01、0.001），α-KG回补可逆转自噬异常（P＜0.01）。体内实验结果显示，LC-EVLP显著抑制肿瘤生长（P＜0.05、0.001），下调Ki67和PCNA表达（P＜0.001），并诱导自噬相关蛋白变化（P＜0.05、0.01、0.001），且未见明显毒性。结论 LC-EVLP通过调控PDK1/PKM轴干扰细胞代谢平衡，并诱导自噬过程增强，从而发挥抗肿瘤作用。其较低的毒性特征提示其具有进一步作为天然纳米抗肿瘤制剂开展研究的潜在价值。

Objective To elucidate the molecular mechanism by which Lobelia chinensis-derived extracellular vesicle-like nanoparticles (LC-EVLP) induce lethal autophagy in hepatocellular carcinoma (HCC) by remodeling the glutamine metabolic network, and to systematically evaluate their antitumor efficacy and biosafety. Methods LC-EVLP were isolated by differential ultracentrifugation and physicochemically characterized. By integrating network pharmacology, molecular docking and multidimensional cell-based assays, the effects of LC-EVLP on the proliferation, migration and invasion of human HCC Mahlavu cells were assessed. Rescue experiments using an autophagic-flux inhibitor and the metabolic intermediate α-ketoglutarate (α-KG) were conducted to interrogate the “metabolism-autophagy” regulatory axis. A nude mouse subcutaneous xenograft model was established to evaluate the in vivo anti-tumor effects of LC-EVLP and histopathological changes in major organs. Results LC-EVLP was successfully isolated and characterized as typical vesicular structures with particle sizes mainly distributed between 126.0 and 151.7 nm, and were efficiently internalized by Mahlavu cells. Functional assays demonstrated that LC-EVLP significantly reduced the viability of Mahlavu cells at concentrations ≥ 12.5 μg/mL (P < 0.001), while exerting no significant cytotoxicity toward LO2 cells. Further analyses revealed that LC-EVLP significantly inhibited colony formation and DNA synthesis (P < 0.05, 0.01, 0.001), and significantly suppressed cell migration and invasion (P < 0.001). The autophagy inhibitor bafilomycin A1 (BafA1) significantly rescued cell viability (P < 0.001), indicating that autophagic cell death was the dominant mechanism underlying the anti-HCC activity of LC-EVLP. LC-EVLP significantly upregulated Beclin 1 expression (P < 0.05, 0.01, 0.001), increased microtubule associated protein 1 light chain 3-II/I (LC3-II/I) value (P < 0.05, 0.01), and reduced p62 protein expression (P < 0.01, 0.001). Moreover, BafA1 further enhanced LC3-II/I accumulation and reversed the decrease in p62 expression (P < 0.01, 0.001), confirming the promotion of a complete autophagic flux. In terms of metabolic regulation, LC-EVLP significantly downregulated the expressions of pyruvate dehydrogenase kinase 1 (PDK1), pyruvate kinase M (PKM) and glutaminase 1 (GLS1) (P < 0.01, 0.001), while α-KG supplementation reversed the abnormal autophagic phenotype (P < 0.01). The in vivo experimental results showed that LC-EVLP significantly inhibited tumor growth (P < 0.05, 0.001), downregulated Ki67 and PCNA expressions (P < 0.001), and induced changes in autophagy related proteins (P < 0.05, 0.01, 0.001), with no significant toxicity observed. Conclusion LC-EVLP exerts anti-tumor effects by regulating PDK1/PKM axis, disrupting cellular metabolic balance and inducing enhanced autophagy. Its low toxicity characteristics suggest its potential for further research as a natural nanomaterial-based antitumor agent.

R285.5

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