LC–MS-based quantitation of proteomic changes induced by Norcantharidin in MTB-Treated macrophages

Tuberculosis drug resistance contributes to the spread of tuberculosis. Immunotherapy is an effective strategy for treating tuberculosis, with the regulation of macrophage-mediated anti-tuberculosis immunity being crucial. Norcantharidin (NCTD), a drug used in tumor immunotherapy, has significant immunomodulatory effects. Thus, NCTD may have an anti-tuberculosis role by regulating immunity. Understanding how NCTD affects the proteome of Mtb-infected macrophages can provide valuable insights into potential treatments. This study aimed to investigate the impact of NCTD (10 μg/mL) on the proteome of macrophages infected with Mtb H37Ra using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 69 differentially regulated proteins (DRPs) were identified, with 28 up-regulated and 41 down-regulated in the NCTD-treated group. Validation of six DRPs (CLTCL1, VAV1, SP1, TRIM24, MYO1G, and WDR70) by Western blot analysis confirmed the accuracy of the LC-MS/MS method used in this study. NCTD modulates various protein expressions involved in chromatin-modifying enzymes, RHO GTPases activating PAKs, Fc gamma R-mediated phagocytosis, T cell receptor signaling pathway, and antigen processing and presentation. Overall, the research provides new insights into the effects of NCTD on the proteome of Mtb-infected macrophages. The identified changes highlight potential targets for future therapeutic interventions aimed at enhancing host immunity against Mtb infection or developing new anti-TB drugs based on these findings.