复方叶下珠（CP）是一种传统中药，具有很强的抗癌作用，广泛应用于肝细胞癌（HCC）的临床治疗。虽然乐伐替尼和其他口服酪氨酸激酶抑制剂已被授权作为晚期不可切除肝癌的初始治疗，但由于耐药性的逐渐发展，患者的生存最终受到限制。幸运的是，CP和乐伐替尼的联合给药有望在抗癌应用中发挥作用。我们的目的是了解CP中生物活性植物化合物的分子水平机制，以探索CP和乐伐替尼联合治疗的抗HCC效果，以及揭示潜在机制。此外，我们还发现了与自噬相关的新 miRNA，这些 miRNA 在 HepG2 衍生的外泌体和 HepG2 细胞中都很常见。这些 miRNA 在 HCC 的进展中发挥作用，并通过使用 CP 和乐伐替尼进行鉴定。为了评估 CP 与乐伐替尼联合使用的抗 HCC 效果，我们进行了体外 CCK-8 测定和体内异种移植模型测定进行了。采用 TEM、NTA 和纳米流式细胞术来鉴定分离的外泌体。为了确定 HepG2 细胞和 HepG2 衍生的外泌体中的 miRNA 表达模式，进行了 miRNA 测序分析。进一步的研究涉及使用实时PCR、检查融合蛋白GFP-mRFP-LC3、TEM分析和蛋白质印迹。在体外和体内，CP和乐伐替尼的组合对HCC表现出更强更强的影响与单独使用 CP 或乐伐替尼相比。细胞和外泌体 miRNA 测序证明，CP 和乐伐替尼的组合对 HepG2 衍生的外泌体和 HepG2 细胞中自噬相关的 miRNA 有显着影响。其他测试表明，经过 CP 处理以及 CP 和乐伐替尼联合治疗的 HepG2 细胞中自噬的抑制作用增强，是通过调节 Beclin-1、LC3-II 和 P62 的表达来实现的。 总之，我们的结果表明CP和乐伐替尼联合可以通过促进外泌体介导的自噬抑制来有效抑制HCC。这种新颖的治疗选择高效且持久，使其成为 HCC 的一种有前途的治疗方法。此外，本研究中发现的差异表达且与外泌体介导的自噬相关的 miRNA 可能成为 HCC 临床治疗的目标。版权所有 © 2023。由 Elsevier GmbH 出版。

Compound Phyllanthus urinaria (CP), a traditional Chinese herbal remedy, possesses strong anti-cancer effects and is extensively employed in the clinical management of hepatocellular carcinoma (HCC). While lenvatinib and other oral tyrosine kinase inhibitors have been authorized as initial treatments for advanced unresectable HCC, the survival of patients is ultimately restricted due to the gradual development of drug resistance. Fortunately, the co-administration of CP and lenvatinib holds promise for anti-cancer applications.Our objective was to understand the molecular-level mechanisms of bioactive phytocompounds in CP, in order to explore the anti-HCC effects of combining CP and lenvatinib treatment and reveal the underlying mechanisms. Furthermore, we discovered new miRNAs associated with autophagy that are common to both HepG2-derived exosomes and HepG2 cells. These miRNAs play a role in the advancement of HCC and were identified through the utilization of CP and lenvatinib.To assess the anti-HCC effects of CP in combination with lenvatinib, both an in vitro CCK-8 assay and an in vivo xenograft model assay were performed. TEM, NTA, and nano-flow cytometry were employed for the identification of isolated exosomes. To ascertain the miRNA expression patterns in HepG2 cells and HepG2-derived exosomes, miRNA-sequencing analysis was conducted. Further investigation involved the use of real-time PCR, examination of the fusion protein GFP-mRFP-LC3, TEM analysis, and western blotting.In vitro and in vivo, the combination of CP and lenvatinib showed a stronger and more powerful impact on HCC compared to either CP or lenvatinib alone. The combination of CP and lenvatinib had a significant impact on autophagy-related miRNAs in HepG2-derived exosomes and HepG2 cells, as demonstrated by cellular and exosomal miRNA sequencing. Additional tests indicated that the increased inhibition of autophagy in HepG2 cells subjected to CP treatment, as well as the combination of CP and lenvatinib, was accomplished through the regulation of Beclin-1, LC3-II, and P62 expression.In conclusion, our results indicate that the combination of CP and lenvatinib can effectively inhibit HCC by promoting the exosome-mediated suppression of autophagy. This novel therapeutic option is highly efficient and durable, making it a promising treatment for HCC. Moreover, the miRNAs that are differentially expressed and associated with exosome-mediated autophagy, which have been discovered in this study, could potentially be targeted for clinical treatment of HCC.Copyright © 2023. Published by Elsevier GmbH.