健脾养正汤（JPYZ）在胃癌中具有潜在的抗肿瘤活性。然而，JPYZ对肿瘤相关巨噬细胞（TAM）源性外泌体调节胃癌的潜在作用仍不清楚。我们旨在阐明肿瘤相关巨噬细胞源性外泌体（TAM-exos）在胃癌浸润和转移中的作用，以及JPYZ调节TAM-exos对胃癌的机制。使用流式细胞术证明JPYZ是否参与TAM极化。在JPYZ处理后，胃癌细胞与TAM培养基（TAM-CM）/TAM-exos共培养，并通过伤口愈合和穿膜实验进行检测。转录组测序和生物信息学分析预测了JPYZ干预TAM后的外泌体miRNA。使用miRNA模拟物和抑制物验证外泌体中miRNA对胃癌细胞的影响。利用荧光定量PCR和荧光素酶报告基因分析明确miRNA与靶基因之间的靶向关系。通过Western blot assay检测表皮-间质转化（EMT）标志物和相关信号通路蛋白的表达水平。我们首次证明JPYZ干预的TAM-CM显著抑制了胃癌的侵袭和迁移。此外，在TAM上清液中的外泌体在胃癌的迁移中起着关键作用。同时，转录组测序和荧光定量PCR揭示，JPYZ干预后TAM-exos中miR-513 b-5p的表达显著降低。miR-513 b-5p在TAM-exos介导的胃癌细胞侵袭和迁移中起加重作用，miR-513 b-5p的抑制剂逆转了TAM-exos介导的促进作用。生物信息学分析和荧光素酶报告基因分析证实，在胃癌中，PTEN是miR-513 b-5p的直接靶标。miR-513 b-5p通过抑制PTEN激活AKT/mTOR信号通路，从而促进胃癌的侵袭和转移。重要的是，JPYZ抑制了TAM源性外泌体miR-513 b-5p的表达，并以PTEN为依赖方式减轻了AKT/mTOR的激活。TAM-exos含有miR-513 b-5p导致胃癌的侵袭和迁移。我们的发现阐明了JPYZ抑制胃癌进展的一种新型TAM-exos机制。版权所有©2023年。Elsevier B.V.出版。

Jianpi Yangzheng decoction (JPYZ) possesses a potential anti-tumor activity in gastric cancer. However, potential effect of JPYZ on regulating tumor-associated macrophage (TAM)-derived exosomes to affect gastric cancer is still unclear.We aimed to clarify the role of tumor-associated macrophage derived exosomes (TAM-exos) in invasive and metastasis of gastric cancer and the mechanism of JPYZ regulate TAM-exos against gastric cancer.Flow cytometry was performed to demonstrate whether JPYZ involved in TAM polarization. After JPYZ treatment, TAM conditioned medium (TAM-CM)/TAM-exos were co-cultured with gastric cancer cells and were detected by wound healing and transwell assay. Transcriptome sequencing and bioinformatics analysis predicted the exosomal miRNA after JPYZ intervention in TAM. miRNA mimic and inhibitor were used to verify the effect of miRNA in exosomes on gastric cancer cells. Q-PCR and luciferase reporter assay were employed to clarify the targeting relationship between miRNA and target gene. Western blot assay detected the expression levels of epithelial-mesenchymal transition (EMT) markers and related signaling pathways proteins.We firstly demonstrated that TAM-CM intervened by JPYZ significantly inhibited the invasion and migration of gastric cancer. Furthermore, exosomes in TAM supernatants play a key role in migration of gastric cancer. Meanwhile, transcriptome sequencing and q-PCR revealed that miR-513 b-5p expression was significantly reduced in TAM-exos intervened by JPYZ. And miR-513 b-5p in TAM aggravated TAM-exos mediated invasion and migration of gastric cancer cells, the inhibitor of miR-513 b-5p reversed TAM-exos mediated promotion. Bioinformatics analysis and luciferase reporter assay confirmed that PTEN was a direct target of miR-513 b-5p in gastric cancer. MiR-513 b-5p inhibited PTEN to activate AKT/mTOR signaling pathway thus promoting gastric cancer invasion and metastasis in vivo and in vitro. Importantly, JPYZ inhibited TAM derived exosomal miR-513 b-5p, and alleviated AKT/mTOR activation by PTEN depended manner in gastric cancer.TAM-exos containing miR-513 b-5p lead to gastric cancer invasion and migration. Our findings clarify a novel TAM-exos mechanism of JPYZ for inhibiting gastric cancer progression.Copyright © 2023. Published by Elsevier B.V.