目的 探究白花蛇舌草-半枝莲水提物（HSAE）抑制及驯化外泌体的作用机制。方法 采用MTT法、细胞划痕实验和流式细胞术实验考察不同质量浓度HSAE（0.5～5.0 mg·mL^-1）对A549细胞增殖、迁移和凋亡的作用，Western blotting检测HSAE对A549细胞中肾脏脑蛋白（KIBRA）表达的影响。成功提取并分离了含HSAE药效成分的内源性外泌体（HSAEExos），通过原子力显微镜（AFM）和透射电子显微镜（TEM）对外泌体形貌进行了表征，通过共聚焦显微镜和流式细胞术检测A549细胞对其摄取，Western blotting检测标志蛋白TSG101的表达；采用UPLC-Q-Exactive-MS/MS研究其药效学成分。通过外泌体（50 μg·mL^-1）与A549细胞共培养，评估对受体细胞增殖、迁移、凋亡及程序性死亡配体（PD-L1）蛋白表达水平的影响。HSAE Exos作用于人T淋巴细胞瘤Jurkat后与A549细胞共培养，结晶紫染色观察Jurkat细胞对A549细胞的杀伤效果。结果 MTT法、细胞划痕实验和流式细胞术实验表明，与对照组相比，HSAE显著抑制A549细胞增殖、迁移（P＜0.05、0.01、0.001），诱导A549细胞凋亡（P＜0.05、0.01），降低A549细胞中KIBRA的表达（P＜0.01、0.001），从而减少了外泌体的分泌量；与对照组比较，HSAE Exos标志蛋白TSG101的表达量较低（P＜0.05、0.01）； HSAE Exos可以逆转Exos促进受体细胞增殖、迁移并抑制凋亡的能力。TEM以及AFM结果表明，提取分离的Exos与外泌体的经典结构相符，粒径分布均匀，为30～150 nm。UPLC-Q-Exactive-MS/MS结果表明，HSAE Exos含有HSAE的关键药效成分。共聚焦显微镜表明受体细胞以时间相关性的方式对HSAE Exos进行摄取。Western blotting结果表明，HSAE Exos可以降低A549细胞核因子-κB（NF-κB）以及PD-L1蛋白表达从而抑制免疫逃逸。HSAE Exos增强Jurkat细胞杀伤A549细胞效果。结论 HSAE不仅可以抑制A549细胞外泌体的生物发生，还能驯化外泌体，使其从促癌功能逆转为抑癌功能，该发现从外泌体介导的细胞间通讯角度，探索了HSAE抑制肺癌的一种新途径。

Objective This study investigates the mechanism by which Hedyotis diffusa and Scutellaria barbata aqueous extract (HSAE) suppresses and reprograms extracellular vesicles. Methods The effects of different concentrations of HSAE (0.5-5.0 mg·mL^-1) on A549 cell proliferation, migration, and apoptosis were evaluated using MTT assays, scratch assays, and flow cytometry. Western blotting was used to examine the impact of HSAE on the expression of kidney and brain protein (KIBRA) in A549 cells. Endogenous extracellular vesicles containing bioactive components of HSAE (HSAE Exos) were successfully extracted and isolated. Their morphology was characterized using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Uptake of HSAE Exos by A549 cells was assessed via confocal microscopy and flow cytometry, and the expression of the marker protein TSG101 was analyzed by Western blotting. The pharmacologically active components were identified using UPLC-Q-Exactive-MS/MS. Co-culture experiments with 50 μg·mL^-1 of HSAE Exos and A549 cells were conducted to evaluate their effects on receptor cell proliferation, migration, apoptosis, and PD-L1 protein expression. After treating human T-cell leukemia Jurkat cells with HSAE Exos and coculturing them with A549 cells, crystal violet staining was performed to assess the cytotoxic effect of Jurkat cells on A549 cells. Results MTT, scratch, and flow cytometry assays showed that compared to the control group, HSAE significantly inhibited A549 cell proliferation and migration (P<0.05, 0.01, 0.001), induced apoptosis (P<0.05, 0.01), and reduced KIBRA expression (P<0.01, 0.001), thereby decreasing extracellular vesicle secretion. Compared to the control, the expression level of the HSAE Exos marker protein TSG101 was lower (P<0.05, 0.01). HSAE Exos reversed the ability of extracellular vesicles to promote receptor cell proliferation, migration, and inhibit apoptosis. TEM and AFM results confirmed that the isolated exosomes matched the classical structure of extracellular vesicles, with a uniform particle size distribution ranging from 30 to 150 nm. UPLC-Q-Exactive-MS/MS analysis revealed that HSAE Exos contained key bioactive compounds of HSAE. Confocal microscopy demonstrated time-dependent uptake of HSAE Exos by recipient cells. Western blotting showed that HSAE Exos significantly downregulated nuclear factor-κB (NF- κB) and PD-L1 protein expression in A549 cells, thereby suppressing immune evasion. HSAE Exos enhanced the cytotoxicity of Jurkat cells against A549 cells. Conclusion HSAE not only inhibits the biogenesis of A549-derived extracellular vesicles but also reprograms them, transforming their pro-tumorigenic function into an anti-tumorigenic one. This finding reveals a novel pathway through which HSAE suppresses lung cancer via extracellular vesicle-mediated intercellular communication.

R965

山东省自然科学基金资助项目（ZR2025MS1285）；山东省中医药科技项目（M20251207）；济南市“新高校20条”资助项目（202228085）