Bie-Jia-Jian Pill: Inhibiting tumor glycolysis and promoting CD8 cell-mediated anti-tumor immunity by targeting HIF-1α-PI3K/AKT/mTOR and CCL20 in hepatocellular carcinoma.

[ETHNOPHARMACOLOGICAL RELEVANCE] The efficacy of immunotherapy for hepatocellular carcinoma (HCC) is often limited by an immunosuppressive tumor microenvironment (TME), largely driven by tumor metabolic reprogramming (the Warburg effect) that impairs CD8 T cells. Targeting this metabolism to restore immunity is a promising strategy. Bie-Jia-Jian Pill (BJJP), a traditional formula for "softening hardness and dissipating masses," conceptually aligns with modulating the TME. However, its role in reprogramming tumor metabolism to enhance immunotherapy remains unclear.

[AIM OF THE STUDY] We aimed to investigate if BJJP enhances anti-tumor immunity by inhibiting the Warburg effect, elucidate its molecular mechanism, and evaluate its synergy with PD-L1 blockade.

[MATERIALS AND METHODS] Using UHPLC-MS, the major components of BJJP were analyzed and its efficacy was evaluated in combination with the PD-L1 neutralizing antibody for HCC treatment. CD8 T cells and tumor markers were assessed in vitro and in vivo using flow cytometry. Transcriptomics, metabolomics, Seahorse metabolic assays, Western blotting, ELISA, and immunofluorescence staining were employed to identify and validate the mechanisms and targets of BJJP. Its targeting capabilities were confirmed by lentivirus and siRNA transfections, network pharmacology and molecular docking.

[RESULTS] BJJP suppressed HCC cell proliferation, growth and migration, promoted their apoptosis, reduced PD-L1 expression, and enhanced CD8 T cell cytotoxicity and infiltration in the tumor. BJJP and immunotherapy exerted synergistic effects, including inhibiting tumor cell proliferation, increasing immune cell infiltration, and improving treatment efficacy. BJJP suppressed glycolysis in HCC cells, alleviated the excessive uptake of energy substrates, and reduced lactate accumulation in the microenvironment, thereby mitigating CD8 T cell exhaustion. This glycolytic metabolic reprogramming was related to BJJP's effect on inhibiting the HIF-1α-PI3K/AKT/mTOR axis. Additionally, BJJP inhibited the downstream NF-κB activation, down-regulated CCL20, and alleviated the inhibitory effect of CCL20 against CD8 T cells.

[CONCLUSION] BJJP targets the HIF-1α-PI3K/AKT/mTOR axis to reprogram tumor glycolysis, reduce CCL20 secretion, enhance CD8 T cell immune response, and modulate the tumor microenvironment, thereby exerting anti-tumor effects against HCC. When used in combination with immunotherapy, BJJP can improve treatment efficacy, providing valuable evidence for clinical treatment.