Integrated network pharmacology and metabolomics to investigate the effect and mechanism of nitidine chloride against cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive malignancy with a poor prognosis. Nitidine chloride (NitC), a bioactive alkaloid derived from Zanthoxylum nitidum (Roxb.) DC., exhibits potential anti-cancer activity against CCA. However, the metabolic mechanism underlying the anti-cancer effect of NitC remains poorly understood and requires further elucidation. This study integrated metabolomics and network pharmacology to systematically investigate the anti-cancer activity and underlying mechanisms of NitC in treating human cholangiocarcinoma cells. First, The effects of NitC on human cholangiocarcinoma cells were assessed by cell proliferation, apoptosis, and cycle. Then, potential mechanisms and targets were investigated using a combination of cell metabolomics and network pharmacology and verified by molecular docking. Finally, we measured the protein levels of potential targets in TFK1 cells using enzyme-linked immunosorbent assay (ELISA). Our results indicated that NitC treatment induced the proliferation inhibition, G2/M arrest and apoptosis of TFK1 cells in a concentration dependent manner. The metabolomics analysis identified forty differential metabolic biomarkers and five key metabolic pathways of NitC in treating CCA. Network pharmacology found 36 potential targets for NitC intervention on CCA. The integration of network pharmacology and metabolomics constructed the "compound-reaction-enzyme-gene" association and revealed that NitC exerts its efficacy on CCA through four key targets, eleven metabolic indicators, and glycine, serine and threonine metabolism, and tyrosine metabolism. Molecular docking further confirmed robust binding interactions between NitC and these key targets. Moreover, ELISA results showed that NitC treatment significantly attenuated the protein levels of PIK3CA, PTGS2, and PRKACA in TFK1 cells. This study demonstrates that combining metabolomics and network pharmacology provides a powerful strategy to elucidate the pharmacological mechanisms of natural compounds, also offering new insights into the therapeutic potential of NitC for CCA.