Figure 6

NETs amplified the pyroptosis of HG-treated rat CCSMCs. (A,B) Assessment of the viability and apoptosis in rat CCSMCs using CCK-8 assay and flow cytometry, respectively. (C) Assessment of the expression levels of H3Cit and MPO in CCSMCs using immunofluorescence (scale bar = 25 μm). (D) Observation of CCSMC pyroptosis under a microscope (scale bar = 50 μm). (E) Assessment of the protein expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, and ASC in CCSMCs using western blotting; the uncropped protein blots can be seen in Supplementary file 3. (F) Assessment of IL-1β and IL-18 levels in CCSMCs using ELISA. CCSMCs were subjected to HG (25 mM) treatment or/and co-cultured with 1 × 10⁴ neutrophils (N) for 24 h. CCSMCs cultured in 5 nM glucose served as controls. Data were presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. NET neutrophil extracellular trap, HG high glucose, CCSMCs corpus cavernosum smooth muscle cells, CCK-8 cell counting kit-8, H3Cit citrullinated histone H3, MPO myeloperoxidase, NLRP3 NOD-like receptor family, pyrin domain-containing protein 3, GSDMD gasdermin D, GSDMD-N N-terminal proteolytic fragment of gasdermin D, ASC apoptosis-associated speck-like protein containing a CARD, IL interleukin, ELISA enzyme-linked immunosorbent assay.