Research Paper Volume 15, Issue 20 pp 11184—11200

Figure 4. Effect of SIRT1 on the H₂O₂-induced pyroptosis of HAECs. (A) qRT-PCR results demonstrating the decreased mRNA expression of Sirt1 upon H₂O₂ treatment in a time-dependent manner. (B) Western blot demonstrating the decreased protein levels of SIRT1 upon H₂O₂ treatment in a time-dependent manner. (C) Western blot demonstrating the overexpression of the SIRT1 protein in HAECs treated with ov-SIRT1 via transient transfection. (D) LDH release from HAECs treated with or without ov-SIRT1 following H₂O₂ treatment. (E) PI staining results demonstrating the increased percentage of PI positive cells in H₂O₂-treated HAECs. Double staining of PI (red) and Hoechst 33342 (blue). The percentage of PI positive cells in H₂O₂-treated HAECs decreased after transfection with ov-SIRT1. Plotting scale = 200 μm. (F) Western blot demonstrating the increased ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, IL-18 in cells with H₂O₂ treatment compared with cells without H₂O₂ treatment. The ratio of NLRP3, GSDMD-N, C-caspase-1, ASC, IL-1β, and IL-18 decreased in cells transduced with ov-SIRT1 under H₂O₂ treatment compared with cells transduced with NC. (^*p<0.05 vs control or NC, ^**p<0.01 vs control or NC, ^***p<0.001 vs NC, ^#p<0.05 vs H₂O₂, ^##p<0.01 vs H₂O₂). Data are presented as mean ± SD (n=3). HAECs, human aortic endothelial cells; qRT-PCR, quantitative real-time polymerase chain reaction; LDH, lactate dehydrogenase; PI, propidium iodide; GSDMD, gasdermin-D; ASC, apoptosis-associated speck-like protein containing a CARD; NC, negative control.