Unlocking the potential of Traditional Chinese Medicine (TCM): Shipi Xiaoji formula (SPXJF) as a novel ferroptosis inducer in hepatocellular carcinoma

Hepatocellular Carcinoma (HCC) is a major health concern with limited treatment options. Traditional Chinese Medicine (TCM) offers potential therapeutic approaches for HCC, and SPXJF, a TCM formula, has shown promise in clinical observations for prolonging the survival of liver cancer patients. To investigate the anti-tumor effects of SPXJF on HCC cells and explore its potential mechanism, focusing on ferroptosis induction. LC/Q-TOF-MS was used for compound identification. Cell viability assays, EdU proliferation assay, colony formation assay, wound healing assay, Transwell assay, and Western-blotting were conducted to evaluate the effects of SPXJF on HCC cell proliferation, migration, and invasion. Bioinformatics analysis and RT-PCR were employed to identify potential ferroptosis-related genes and validate the results. Ferroptosis induction was investigated using ferroptosis inhibitors, ROS and lipid peroxidation detection, and TEM. In vivo experiments using a subcutaneous xenograft tumor model confirmed the anti-tumor effects of SPXJF and its ability to induce ferroptosis in HCC. SPXJF effectively inhibited the proliferation, migration, and invasion of HCC cells in vitro . The mechanism of action was found to be related to the induction of ferroptosis, as evidenced by increased intracellular Fe 2+ and ROS levels, decreased GSH levels, altered mitochondrial morphology, and upregulation of ferroptosis-inducing proteins ACSL4 and LPCAT3, along with downregulation of ferroptosis-inhibiting proteins xCT and GPX4. Bioinformatics analysis and RT-PCR further identified GSTZ1, CDC25A, AURKA, NOX4, and CAPG as potential ferroptosis-related genes regulated by SPXJF. In vivo experiments confirmed the anti-tumor effects of SPXJF and its ability to induce ferroptosis in HCC. SPXJF exerts anti-tumor effects on HCC cells by inducing ferroptosis, and its mechanism of action involves the regulation of ferroptosis-related genes and proteins. This study provides a theoretical basis for the clinical treatment of HCC and the development of new anti-cancer drugs, offering a valuable contribution to the field of ethnopharmacology. • SPXJF, a Chinese medicine formula, exhibits significant anti-tumor effects against hepatocellular carcinoma (HCC) cells. • SPXJF induces ferroptosis, a unique programmed cell death, by regulating ferroptosis-related genes and proteins in HCC cells. • This study uses multi-omics and network pharmacology to explore SPXJF’s molecular mechanisms in inducing ferroptosis. • GSTZ1, CDC25A, AURKA, NOX4, and CAPG are identified as potential therapeutic targets for HCC treatment. • SPXJF’s anti-tumor effects and ferroptosis induction are validated in vitro with HCC cells and in vivo in a xenograft model.