The Role Of GSDMD-miR-223-NLRP3 Axis In B(a)p-induced Inflammatory Injury Of Alveolar Epithelial Cells

Benzo(a)pyrene [B(a)p] is a common environmental pollutant that causes oxidative stress and inflammatory damage.Pyroptosis is a new inflammation-dependent programmed cell death.NLRP3 is a member of the intracellular NLR receptor family,and NLRP3 inflammasome can participate in the inflammatory response.It has been reported that the activation of NLRP3 inflammasome could further activate the pyroptosis-related GSDMD protein to induce pyroptosis.However,it remains unclear whether the pyroptosis-related GSDMD could regulate NLRP3 inflammasomes in alveolar epithelial cells.miR-223 could affect the inflammatory disease process by regulating NLRP3.It has not been reported whether GSDMD may affect NLRP3 inflammasome expression and activation through miR-223.ObjectiveThis study aims to explore the role of GSDMD-miR-223-NLRP3 axis in B(a)p-induced inflammatory injury of alveolar epithelial cell,which provides a new idea to elucidate the mechanism of B(a)p induced pulmonary inflammatory injury.Methods1.Alveolar epithelial A549 cell was treated with B(a)p(0,2,4,8μM)for 12 h or24h.The changes in cell morphology were observed by inverted microscope.Inflammatory mediators TNF-α and IL-6 were detected in the cell supernatant.The dose and time of exposure to B(a)p-induced inflammatory damage were determined.The morphological changes of cells were observed by transmission electron microscope(TEM).Hoechest33342/PI staining was used to examine the cell death status.The expression of NLRP3 mRNA and miR-223 were detected by qRT-PCR.NLRP3,IL-1β and IL-18 protein expressions were detected by immunohistochemistry.Western blot assay was used to detect the protein expression levels of GSDMD,GSDMD-N,caspase-1 and caspase-4.2.Small interfering RNA(siRNA)were used to inhibit GSDMD expression.The inflammatory factors TNF-α and IL-6 were detected in the cell supernatant.NLRP3 Expression and activation of NLRP3 inflammasomes and expression of miR-223 were detected.miR-223 Inhibitor were used to inhibit miR-223 expression.TNF-αand IL-6 in the cell supernatant were detected by ELISA.Expression and activation of NLRP3 inflammasomes were detected.3.Statistical analysis: Statistical results were expressed in the form of mean ±standard deviation(±s).The independent sample t test was used to compare the measurement data between the two groups,and one-way ANOVA analysis was used to compare the measurement data between more than two groups.The pairwise comparison between groups was analyzed by LSD method.SPSS 21.0 was used for statistical analysis,and the difference was statistically significant when α=0.05.Results1.After treated with 8μM B(a)p for 12 h and 2μM,4μM,8μM B(a)p for 24 h,the cell volume of A549 cells was increased,the cell membrane was swollen and deformed,and the edge was blurred.With the increase of exposure time and concentration,the expression of TNF-α or IL-6 increased in cell supernatant(P<0.05)and NLRP3 mRNA and miR-223 expression were also increased(P<0.05).TEM results showed that A549 cells were obviously swollen and the cell membrane ruptured after stimulated by 8μM B(a)p for 24 hours.Hoechest33342/PI staining showed that the rate of PI-positive cells increased with the increase of the concentration of B(a)p for 24 h.Compared with DMSO group,the protein expressions of NLRP3,IL-1β and IL-18 were significantly increased when A549 cells were exposed to 2μM,4μM and 8μM B(a)p for 24h(P<0.05).Western blot analysis showed that the protein expression levels of GSDMD,GSDMD-N,pro caspase-1,cleaved caspase-1 pro caspase-4 and cleaved caspase-4 were increased after B(a)p treatment for 24h(P<0.05).2.After transfected with si GSDMD,the expression levels of TNF-α and IL-6were significantly decreased in the supernatant of A549 cells(P<0.05).Compared with SINC group,the mRNA expression level of NLRP3 was significantly decreased(P<0.01),while the expression level of miR-223 was significantly increased(P<0.01).Immunohistochemical results showed that the expression levels of NLRP3,IL-1β and IL-18 in si GSDMD group were significantly lower than that in si NC group(P<0.05).Western blot analysis showed that cleaved caspase-1 expression was significantly decreased in the si GSDMD group compared with the si NC group(P<0.01).3.The expression levels of TNF-α and IL-6 protein in miR-223 Inhibitor group were significantly higher than those in Inhibitor NC group(P<0.05).The mRNA expression levels of NLRP3 and GSDMD were increased in in miR-223 Inhibitor group(P<0.05).Immunohistochemistry showed that the protein expression levels of NLRP3,IL-1β and IL-18 in miR-223 Inhibitor group were significantly increased compared with the control group(P<0.05).Western blot results showed that the expression levels of cleaved caspase-1 were significantly increased after transfection with miR-223 Inhibitor(P<0.05).Conclusion1.Pyroptosis occurred simultaneously when B(a)p-induced inflammatory injury of alveolar epithelial A549 cell,and both classical caspase-1 and non-classical caspase-4 pathways were activated.2.GSDMD may regulate NLRP3 inflammasomes through miR-223,which is involved in B(a)p induced inflammatory damage in A549 cells.