Chlorogenic acid mitigates ferroptosis in macrophages induced by pneumolysin and streptococcus pneumoniae through activation of NRF2/GPX4 pathway.

BACKGROUND: Drug-resistant Streptococcus pneumoniae (SP) infections pose a significant global health threat, with a limited availability of effective treatments to prevent disease progression. Chlorogenic acid (CGA) exhibits potent antioxidant and antimicrobial properties, though its exact mechanisms in combating bacterial infections remain not yet fully elucidated. Pneumolysin (PLY), a pivotal virulence factor of SP, specifically contributes to the pathogenesis of these infections. PURPOSE: This study aimed to investigate the role of SP-derived PLY in triggering macrophage ferroptosis and to elucidate the potential mechanism underlying CGA-mediated regulation of this process. METHODS: A macrophage-SP co-culture model was established to investigate PLY-induced ferroptosis and the mechanisms underlining CGA-mediated antimicrobial effects. Hemolysis assays were conducted to assess PLY activity, and macrophage apoptosis was evaluated using an Annexin V/PI apoptosis detection kit and CCK-8 assay. Key biomarkers, including intracellular ROS levels, mitochondrial membrane potential, MDA content, and total iron levels, were measured using commercial assay kits. Transmission electron microscopy (TEM) was utilized to analyze mitochondrial ultrastructural alterations, particularly morphological changes in the mitochondrial membrane and cristae. Additionally, the expression of key ferroptosis-related factors was analyzed via qRT-PCR, Western blotting, and immunofluorescence staining to delineate the underlying molecular pathways. RESULTS: CGA markedly suppressed SP proliferation and attenuated PLY activity. Co-culture with SP or PLY exposure significantly decreased macrophage viability and triggered apoptotic cell death, whereas CGA treatment markedly attenuated apoptosis. CGA upregulated the expression of Nrf2, SOD1, and HO-1 while substantially decreasing intracellular ROS accumulation. Additionally, CGA preserved mitochondrial membrane integrity and significantly lowered MDA content and total iron levels in macrophages. Furthermore, CGA significantly upregulated the expression of key ferroptosis-related factors, including GPX4 and SLC7A11. CONCLUSIONS: CGA effectively attenuates SP-induced macrophage ferroptosis by activating the Nrf2/GPX4 signaling axis. Notably, PLY was identified as a critical mediator of SP-induced macrophage ferroptosis, and further investigations are warranted to elucidate the exact molecular mechanisms.