RANBP3 promotes mitophagy through the CCAR2/SIRT1 pathway to alleviate pyroptosis in macrophages in TBTB

Abstract

Abstract Mycobacterium tuberculosis infection of the trachea causes tracheobronchial tuberculosis (TBTB), a chronic inflammatory illness, and its pathological features include macrophage pyroptosis. RanBP3, as a potential regulator, participates in cell stress, but its specific mechanism of action in TBTB has not been clarified. The mouse and bone marrow-derived macrophages (BMDMs) models were constructed by infecting with M. smegmatis . RanBP3 protein level and the levels of pyroptosis-related proteins were measured by Western blotting (WB). The levels of inflammatory factors were measured by ELISA. Pearson’s correlation analysis was employed to evaluate their correlation with RanBP3, and the interaction of RanBP3/CCAR2/SIRT1 was verified by Co-immunoprecipitation (Co-IP). The detection of macrophage pyroptosis and mitophagy was done using flow cytometry, and mitophagy was further assessed by transmission electron microscopy. Bacterial survival in BMDMs was evaluated by colony-forming unit assays. Hematoxylin and eosin (H&E) and Masson staining were used to assess lung tissue damage. RanBP3 was down-regulated in TBTB mucosa. At the cellular level, RanBP3 overexpression alleviated BMDM’s pyroptosis by promoting mitophagy and reduced intracellular bacterial survival, whereas RanBP3 knockdown exacerbated pyroptosis and increased bacterial survival. Mechanistically, RanBP3 competitively binds to CCAR2, disrupts the CCAR2-SIRT1 interaction, and promotes SIRT1 release, thereby activating mitophagy. At the animal level, RanBP3 overexpression promoted mitophagy, alleviated macrophage pyroptosis, and alleviated TBTB, but the SIRT1 inhibitor reversed these effects. RanBP3 mediates the release of SIRT1 through the CCAR2-SIRT1 axis, thereby activating mitophagy and inhibiting pyroptosis, providing a new target for TBTB treatment.