Fig. 3

ITA mediates the effect of MSC-exos on AMs. To investigate the role of ITA, IRG1 was knocked down in MH-S cells using three siRNA-IRG1 with different sequences. si-RNAs were subsequently transfected to the AMs. (A) The knockdown efficiency was validated by PCR and Western blot analysis, with the most efficient siRNA selected for further assays. After that, (B) the horseradish peroxidase (HRP) permeation assay was conducted and (C) western blot analysis revealed the relative levels of tight junction proteins Claudin-5 and ZO-1. Apoptosis rates were assessed using (D) Annexin V-FITC/PI staining and (E) TUNEL assays. (F) ELISA was performed for (F) IL-6 and (G) TNF-α concentrations. (H) For M1 polarization evaluation, iNOS and Arg-1 proteins were determined by Western blot analysis. (I) For M2 polarization assessment, the proportion of CD206-positive cells were calculated by flow cytometry. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with *p < 0.05, **p < 0.01, and ***p < 0.001. ITA: itaconic acid; MSC-exos: mesenchymal stem cell-derived exosome; AMs: alveolar macrophages; HRP: horseradish peroxidase