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.