四君子汤上调 RDH5 介导 Hippo/YAP 通路逆转 EMT 抑制肝癌 CSCs 干性维持的机制研究

Mechanistic study of Sijunzi Decoction in reversing EMT and inhibiting stemness maintenance of hepatocellular carcinoma CSCs through RDH5 upregulation-mediated activation of the Hippo/YAP pathway

摘要

目的 通过观察四君子汤对小鼠肝癌的作用及其对视黄醇脱氢酶 5（RDH5）、Hippo/YAP 通路、上皮-间质转化（EMT）以及肿瘤干细胞（CSCs）干性维持的影响，探讨四君子汤抑制肝细胞癌（肝癌）生长的机制。方法 将 C57BL/6 小鼠随机分为 A 组（非脾虚肝癌 RDH5 过表达组）、B 组（脾虚肝癌 RDH5 过表达组）、C 组（脾虚肝癌 RDH5 空载组）、D 组[脾虚肝癌四君子汤低剂量组，4.6 g/（kg·d）]、E 组[脾虚肝癌四君子汤高剂量组，18.2 g/（kg·d）]。利血平制备脾虚模型，经慢病毒转染 RDH5 过表达或空载，原位移植 Hepa1-6 细胞构建肝癌模型，D、E 组给予四君子汤灌胃。测量肿瘤体积和质量，苏木精-伊红（HE）染色观察病理，蛋白免疫印迹法检测相关蛋白表达。结果 与 C 组相比，B、D、E 组肿瘤体积和质量均降低（均 P ＜ 0.05）；与 B 组相比，D、E 组进一步降低（均 P ＜ 0.05），呈一定剂量相关性。四君子汤干预后，D、E 组 RDH5 表达较 C 组升高（均 P ＜ 0.05）。Hippo/YAP 通路检测显示，与 C 组和 B 组相比，D、E 组磷酸化哺乳动物不育 20 样激酶 1（p-MST1）、Salvador 家族 WW 结构域包含蛋白 1（SAV1）、Mps1 结合蛋白 1（MOB1）、磷酸化大肿瘤抑制因子 1（p-LATS1）及磷酸化 Yes 相关蛋白（p-YAP）表达升高，YAP 总蛋白降低（均 P ＜ 0.05）。EMT 检测显示，与 C 组和 B 组相比，D、E 组上皮钙黏蛋白（E-cadherin）表达升高，神经钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）降低（均 P ＜ 0.05）。CSCs 干性标志物 Nanog、性别决定区 Y 框蛋白 2（Sox2）、八聚体结合转录因子 4（Oct-4）及上皮细胞黏附分子（EpCAM）较 C 组和 B 组均降低（均 P ＜ 0.05）。上述 Hippo/YAP 通路激活、EMT 逆转及抑制 CSCs 干性维持效应，四君子汤干预组（D、E 组）均优于 B 组，呈一定剂量相关性。结论 四君子汤可有效抑制脾虚肝癌生长，其机制与恢复 RDH5 表达、激活 Hippo/YAP 通路，逆转 EMT 并抑制肝癌 CSCs 干性维持有关。四君子汤的抗肝癌效应并非单纯依赖 RDH5 单一靶点，而体现为多成分、多靶点协同调节的整体作用模式。

Abstract

Objective To investigate the mechanism by which Sijunzi Decoction inhibits the growth of hepatocellular carcinoma (HCC) by observing its effects on HCC in mice and its influence on retinol dehydrogenase 5 (RDH5), the Hippo/YAP pathway, epithelial-mesenchymal transition (EMT), and stemness maintenance of cancer stem cells (CSCs). Methods C57BL/6 mice were randomly divided into group A (non-spleen-deficiency HCC with RDH5 overexpression), group B (spleen-deficiency HCC with RDH5 overexpression), group C (spleen-deficiency HCC with empty vector), group D [spleen-deficiency HCC treated with low-dose Sijunzi Decoction, 4.6 g/(kg·d)], and group E [spleen-deficiency HCC treated with high-dose Sijunzi Decoction, 18.2 g/(kg·d)]. A spleen-deficiency model was established using reserpine. RDH5 overexpression or empty-vector transfection was performed using lentiviral transduction, and an orthotopic HCC model was constructed by transplanting Hepa1-6 cells. Groups D and E were administered Sijunzi Decoction by gavage. Tumor volume and weight were measured, pathological changes were observed by hematoxylin-eosin (HE) staining, and the expression of related proteins was detected by western blot. Results Compared with group C, tumor volume and weight were reduced in groups B, D, and E (all P ＜ 0.05). Compared with group B, tumor volume and weight were further reduced in groups D and E (all P ＜ 0.05), showing a certain dose-related trend. After intervention with Sijunzi Decoction, RDH5 expression was increased in groups D and E compared with group C (both P ＜ 0.05). Detection of the Hippo/YAP pathway showed that, compared with groups C and B, the expression levels of phosphorylated mammalian sterile 20-like kinase 1 (p-MST1), Salvador family WW domain-containing protein 1 (SAV1), Mps1 binder protein 1 (MOB1), phosphorylated large tumor suppressor 1 (p-LATS1), and phosphorylated Yes-associated protein (p-YAP) were increased in groups D and E, while total YAP protein expression was decreased (all P ＜ 0.05). EMT analysis showed that, compared with groups C and B, epithelial cadherin (E-cadherin) expression was increased in groups D and E, whereas neural cadherin (N-cadherin) and vimentin expression were decreased (all P ＜ 0.05). The expression levels of CSC stemness markers, including Nanog, sex-determining region Y-box 2 (Sox2), octamer-binding transcription factor 4 (Oct-4), and epithelial cell adhesion molecule (EpCAM), were lower in groups D and E than in groups C and B (all P ＜ 0.05). The effects of Hippo/YAP pathway activation, EMT reversal, and inhibition of CSC stemness maintenance were more pronounced in the Sijunzi Decoction intervention groups (groups D and E) than in group B, showing a certain dose-related trend. Conclusions Sijunzi Decoction can effectively inhibit the growth of spleen-deficiency HCC. Its mechanism may be associated with restoration of RDH5 expression, activation of the Hippo/YAP pathway, reversal of EMT, and inhibition of stemness maintenance in HCC CSCs. The anti-HCC effect of Sijunzi Decoction does not depend solely on RDH5 as a single target, but reflects an integrated mode of action involving synergistic regulation by multiple components and multiple targets.

关键词

四君子汤 / 肝癌 / RDH5 / Hippo/YAP 通路 / EMT / CSCs 干性维持

Key words

Sijunzi Decoction / HCC / RDH5 / Hippo/YAP pathway / EMT / CSC stemness maintenance

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基金资助

国家自然科学基金(82304944)

中西医协同“旗舰”科室建设项目(国中医药综结合函〔2024〕221 号)