Abstract:[Background] Actinobacillus pleuropneumoniae (APP) causes severe inflammatory damage to the lungs. Cellular oxidative stress is closely linked to lung inflammatory damage, while the relationship between APP infection and cellular oxidative stress remains unclear. [Objective] To clarify the relationship between APP infection and cellular oxidative stress and explore its mechanism, thereby laying a foundation for elucidating the pathogenic mechanism of APP. [Methods] After infecting porcine alveolar macrophages (PAMs) with APP wild-type strain 5b (5b WT) and Adh-deleted strain (5bΔAdh), respectively, we observed the levels of intracellular reactive oxygen species (ROS) using fluorescence microscopy, and determined the expression of oxidative stress-related molecules by RT-qPCR and Western blotting. Cells were treated with the NLRP3 inflammasome inhibitor MCC950 and then infected with 5b WT and 5bΔAdh, respectively. Biochemical methods were employed to measure the intracellular lactate and pyruvate levels. Flow cytometry was employed to determine the ROS levels and changes in mitochondrial membrane potential. Additionally, TMT-labeled quantitative proteomics sequencing was conducted to analyze changes in cellular protein expression and detect the release of mitochondrial DNA (mtDNA). [Results] Results from immunofluorescence and flow cytometry indicated that the 5b WT group had higher levels of ROS than the 5b ΔAdh group, and this trend was reversed following MCC950 treatment. Biochemical analysis further revealed that the 5b WT group had significantly lower levels of pyruvate and lactate, which elevated again after NLRP3 was inhibited with MCC950. Additionally, the PAMs infected by 5b WT and 5bΔAdh showcased notable declined levels of Nrf2 and GCLM and up-regulated expression of NOX2, with a more pronounced up-regulation observed in the 5b WT group. Proteomics analysis indicated significant changes in the expression of 15 proteins. GO analysis revealed that these differentially expressed proteins were mainly involved in key biological processes such as ATP metabolism, purine metabolism, and inflammation. KEGG analysis predicted that these proteins were involved in inflammation regulation, energy transport, and oxidative phosphorylation. Notably, 5b WT and 5bΔAdh infections increased the proportions of PAMs with mitochondrial damage to 69.6% and 58.5%, respectively, with mtDNA release into the cytoplasm. [Conclusion] APP induced oxidative stress in PAMs via Adh, and the activation of NLRP3 inflammasome participated in the generation of cellular oxidative stress.
