Neferine-enhanced Shenling Baizhu Tang potentiates anti-PD-1 therapy in colorectal cancer liver metastasis via CYP2E1-PPARα-mediated lipid reprogramming.

BACKGROUND

Immune checkpoint inhibitors elicit responses in merely 20-40% of patients with microsatellite instability-high or mismatch repair-deficient colorectal cancer (CRC), making immunotherapy resistance a formidable clinical challenge. The immunosuppressive tumor microenvironment, characterized by regulatory T-cell accumulation and metabolic reprogramming, substantially drives this treatment failure.

PURPOSE: To determine whether neferine, a bioactive alkaloid derived from the traditional Chinese medicine formulation Shenling Baizhu Tang (SLBZT), enhances anti-PD-1 (aPD-1) efficacy in CRC liver metastasis by modulating the CYP2E1-PPARα metabolic axis.

METHODS

A CRC liver metastasis model was established via intrasplenic injection of MC38-Luc cells into C57BL/6 J mice. The animals were administered aPD-1 alone or in combination with low- or high-dose SLBZT. Tumor burden was evaluated via in vivo imaging and histopathology. Integrated transcriptomics and metabolomics, CRISPR-Cas9-mediated CYP2E1 knockout, T-cell coculture assays, alongside computational and biophysical analyses, were employed to elucidate the active components and underlying mechanisms. Evaluated functional outcomes included tumor burden, immune phenotyping, CYP2E1/PPARα signaling, and homovanillic acid (HVA) levels.

RESULTS

High-dose SLBZT markedly augmented aPD-1-mediated suppression of CRC liver metastasis without compromising systemic tolerability. Multi-omics profiling coupled with genetic validation revealed that CYP2E1-driven lipid metabolic reprogramming and its downstream metabolite HVA serve as key mediators of regulatory T-cell expansion and CD8⁺ T-cell exhaustion. Both SLBZT and neferine diminished HVA accumulation, restored effector T-cell function, and potentiated aPD-1 efficacy. Furthermore, biophysical and computational analyses confirmed the direct inhibition of CYP2E1 by neferine.

CONCLUSION

Neferine sensitizes tumors to PD-1 blockade by reprogramming lipid metabolism and remodeling the immune microenvironment via the CYP2E1-PPARα axis, highlighting its translational potential as a metabolic immunoadjuvant to overcome immunotherapy resistance in CRC.