GSDMD-miR-223-NLRP3 axis involved in B(a)P-induced inflammatory injury of alveolar epithelial cells.

ABSTRACT

Benzo(a)pyrene [B(a)P], a ubiquitous environmental pollutant, causes lung inflammatory damage. Pyroptosis，a new inflammation-dependent programmed cell death, happened when pyroptosis-related GSDMD is activated mediated by NLRP3 inflammasome. microRNA-223 (miRNA-223) is involved in inflammatory diseases by regulating NLRP3. However, whether GSDMD regulate NLRP3 inflammasome through miR-223 in B(a)P induced lung inflammatory injury remain unknown. In this study, alveolar epithelial cells (A549) were stimulated with 0, 2, 4, 8 µM B(a)P for 12 h or 24 h. The inflammatory injury and pyroptosis were determined. And the activation of NLRP3 inflammasomes and the level of miRNA-223 were detected. Then, the change of inflammatory injury and activation of NLRP3 inflammasomes in B(a)P-induced A549 cells were detected after inhibiting of GSDMD or miR-223 using siRNA-GSDMD (siGSDMD) or miR-223 inhibitor, respectively. Our results indicated that after B(a)P exposure, TNF-α and IL-6 in the supernatant were increased. Transmission electron microscope (TEM) results showed that A549 cells were obviously swollen and the cell membrane ruptured. Hoechest33342/PI staining showed that pyroptosis occurred. NLRP3, IL-1ß, IL-18, GSDMD, GSDMD-N, pro caspase-1 and cleaved caspase-1 were significantly increased. Additionally, after transfecting with siGSDMD in B(a)P-induced A549 cells, the expression level of miR-223 was significantly increased. But IL-6 and TNF-α in the supernatant, the expression of NLRP3, IL-1ß, IL-18 and cleaved caspase-1 protein were also decreased. And after inhibiting miR-223 in B(a)P-induced A549 cells, the expression of TNF-α and IL-6 in the supernatant, the protein expression of NLRP3, IL-1ß, IL-18 and cleaved caspase-1 were increased. In conclusion, GSDMD may regulate NLRP3 inflammasome through miR-223, which is involved in B(a)P induced inflammatory damage in A549 cells.