Effect Of Silicon Dioxide On Expression Of Aquaporin-1and Aquaporin-4 In A549 Cells

Objectives To study the expression and significance of aquaporin-1(AQP-1) and aquaporin-4(AQP-4) in Human alveolar type II epithelial line A549 cells stimulated by silica(Si O2).Methods A549 cells were divided into 7 groups: the 0.5,1.0,2.0,4.0 and 8.0 hour-silicastimulated groups stimulated with silica(mass concentration 50mg/L) in the corresponding time; the inhibitor group was pretreated with Hg Cl2(specific channel inhibitor of AQP-1,concentration was 200μmol/L) for 3min or TGN-020(specific channel inhibitor of AQP-4,concentration was 100μmol/L) for 2.0 hours and then stimulated with the same concentration of silica for 1.0 hour; the control group was untreated. The expression of AQP-1and AQP-4 m RNA was detected by real-time fluorescent quantitative polymerase chain reaction(Real-time PCR). At the same time, A549 cells were divided into 6 groups:the 3.0, 6.0, 12.0, and 24.0 hour-silica-stimulated groups were stimulated with silica( mass concentration 50mg/L) in the corresponding time; the inhibitor group was pretreated with Hg Cl2(specific channel inhibitor of AQP-1, concentration was 200μmol/L) for 3min or TGN-020(specific channel inhibitor of AQP-4, concentration was 100μmol/L) for 2.0hours and then stimulated with the same concentration of silica for 6.0 hour; the control group was untreated. Both of western blot and immunocytochemistry were used to detect the expression of AQP-1and AQP-4 protein.Results Real-time PCR analysis showed that, compared with that of the control, the relative expression of AQP-1 and AQP-4 m RNA in the silica-stimulated groups increased first and then decreased as the silica stimulated time prolonged(P<0.01), with the peak vale of relative expression of AQP-1 and AQP4 m RNA both at 2.0 hour after silica stimulated(P<0.01) and return to normal 8.0 hours after silica stimulated(P>0.05). Western blot and immunocytochemistry assay demonstrated that, compared with that of the control, the expression of AQP-1 and AQP-4 protein in the silica-stimulated groups increased first and then decreased as the silica stimulated time prolonged(P<0.01), with the peak vale of relative expression of AQP-1 and AQP4 protein at 6.0 hour after silica stimulated(P<0.01), but it did not returned to normal 24.0 hours after silica stimulated(P<0.01).AQP-1 and AQP4 both exist in the cytoplasm and cell nucleus of A549 cells. The relative expression of AQP-1 and AQP-4 m RNA and the expression of AQP-1 and AQP-4 protein in the inhibitor group were higher than the control, but lower than those in the silicastimulated group which was stimulated by silica at the same time point(P<0.01).Conclusions The expression of AQP-1 and AQP4 m RNA and protein of A549 cells was changed regularly when exposed to silica. The specific channel inhibitor of AQP-1 and AQP-4 can inhibit the increase of AQP-1 and AQP-4 expression stimulated by silica. We speculate that AQP-1 and AQP4 maybe participate the process of the occurrence anddevelopment of acute lung injury of silicosis.