Juglans mandshurica-Eleutherococcus senticosus herb pair inhibits hepatocellular carcinoma growth by inducing immunogenic cell death via the HIF-1α/STAT3 pathway.

[BACKGROUND] Immunogenic cell death (ICD)-induced M1 polarization of macrophages represents a promising therapeutic strategy for hepatocellular carcinoma (HCC). Although the herb pair of Juglans mandshurica Maxim. and Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. exhibits anti-tumor activity, its specific efficacy against HCC and the underlying mechanisms remain to be fully elucidated.

[METHODS] The orthotopic HCC model was established through intraperitoneal transplantation of H22 mouse hepatoma cells, followed by interventions with varying doses of the herb pair and a positive control drug. The major constituents of the herb pair were identified using liquid chromatography-tandem mass spectrometry (LC-MS/ MS). Therapeutic efficacy was assessed through measurements of liver weight, liver function tests, and histopathological evaluation via H&E staining of both liver and spleen tissues. The expression levels of macrophage-related markers, including F4/80, iNOS, and CD206, as well as ICD markers CRT and HMGB1, were evaluated using immunofluorescence and immunohistochemistry. Macrophage-derived cytokines were quantified by RT-qPCR and ELISA. Network pharmacology was employed to predict the binding affinity of the active compounds in the herb pair to key target proteins HIF-1α and STAT3, and the underlying mechanism was further validated at both the gene and protein levels.

[RESULTS] The herb pair significantly reduced liver weight and serum levels of AST and ALT in HCC-bearing mice, ameliorated hepatic pathological damage, and promoted the proliferation of splenic lymphoid follicles. Concurrently, it down-regulated the expression of M2 macrophage markers (CD206, IL-4, CCL2) and immunosuppressive cytokines (IL-10, TGF-β), while up-regulating M1-associated markers (iNOS, TNF-α, IFN-γ) and pro-inflammatory cytokines (TNF-α, IL-6, IL-12), and enhanced the expression of key ICD molecules (CRT and HMGB1). Network pharmacology analysis indicated that HIF-1α and STAT3 are central targets, and experimental validation confirmed that the herb pair suppresses both mRNA and protein expression of these signaling molecules. Notably, the therapeutic effect of the high-dose herb pair group was comparable to that of the positive control drug.

[CONCLUSION] The herb pair composed of Juglans mandshurica and Eleutherococcus senticosus may exert anti-HCC effects through the inhibition of the HIF-1α/STAT3 signaling pathway, induction of ICD in HCC cells, and promotion of tumor-associated macrophage polarization toward the M1 phenotype, thereby reshaping the tumor immune microenvironment.