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.