The role of KCa3.1 activating NLRP3 inflammasome in paraquat treated-alveolar epithelial cells / 中华急诊医学杂志

ABSTRACT

Objective:To explore the effect of KCa3.1 activating NLRP3 inflammasome in paraquat PQ treated-alveolar epithelial cells.Methods:The A549 cells were cultured in vitro and divided into the control group, TRAM-34 (specific inhibitor of KCa3.1) group, PQ group and PQ+TRAM-34 group. The expression of KCa3.1 was detected by immunofluorescence in A549 cells. Western blot was used to detect the level of the proteins related with the NLRP3 infammasome and NEK7 protein. And the level of cell potassium was detected by cell potassium concentration kit.Results:The level of KCa3.1 was significantly increased in A549 cells after PQ treatment by immunofluorescence. The expressions of NLRP3 infammasome-related proteins (NLRP3, ASC and Caspase-1) and NEK7 protein were increased after PQ treatment, and the expressions of NLRP3 infammasome-related proteins and NEK7 protein were decreased after inhibition of KCa3.1, and the difference was statistically significant [NLRP3/β-actin (control group vs TRAM-34 group vs PQ group vs PQ+TRAM-34 group): [ (0.02±0.00) vs (0.03±0.00) vs (0.74±0.00) vs (0.32±0.01) , ASC/β-actin (control group vs TRAM-34 group vs PQ group vs PQ+TRAM-34 group): [ (0.12±0.01) vs (0.11±0.03) vs (0.46±0.02) vs (0.17±0.03) ];Caspase-1/ β-actin (control group vs TRAM-34 group vs PQ group vs PQ+TRAM-34 group): [ (0.05±0.00) vs (0.04±0.00) vs (0.34±0.03) vs (0.15±0.01) ]; NEK7/ β-actin (control group vs PQ group vs PQ+TRAM-34 group);[ (0.38±0.03) vs (0.83±0.02) vs (0.51±0.01) , P<0.01]. The potassium level was decreased after PQ treatment and the degree could be declined by the KCa3.1 inhibitor by colorimetric detection with statistically significant difference (control group vs PQ group vs PQ+TRAM-34 group:[ (1.00±0.00) vs (0.60±0.05) vs (0.86±0.02) , P<0.01]. Conclusions:The KCa3.1 could promote the outflow of intracellular potassium and up-regulate the expression of NEK7, thereby activate the NLRP3 inflammatory in PQ-induced pulmonary fibrosis.