The Mechanism Of Apoptosis Of NCI-H292 Cells Induced By Poly-L-arginine Via ERK1/2 Signaling Pathway

ObjectiveHerein,we aimed to study the signaling mechanism whereby Poly-L-arginine(PLA)induces the apoptosis of airway epithelial cell and the expression on Bcl-2/Bax and Caspase-3 in NCI-H292 cells to reveal the mechanism that PLA plays an important role in airway injury.Methods 1.Cell cultureNCI-H292 cells were cultured and propagated in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2/95% air at 37°C.The cells were subcultured every 3 to 4 days according to their growth status.Optimal growth state of the cells were selected and seeded into 6-well culture plates for subsequent experiments.2.The apoptosis of NCI-H292 cells was observed by transmission electron microscopy NCI-H292 cells were grouped into control group and PLA(60 mg/L)group.The cellswere digested by trypsin and collected.After gradient dewatering,the embedding tissuewas sliced to thin sections.After dipped in the citrate for 15 min,thin sections wereobservated by ultrastructural electron microscopy.3.The apoptosis rate of NCI-H292 cells was detected by Annexin V-FITC/PI The cells were grouped into control,10 mg/L,20 mg/L,40 mg/L and 60 mg/L group.The others were grouped into control,PD,PLA and PLA+PD group.The apoptotic rate was measured by flow cytometry.Flowering software was used to analyze the apoptotic results.4.The expression of the Bax,Bcl-2,Casepase-3 and signal pathway ERK1/2 was observed by Western BlotNCI-H292 cells were grouped into control,10 mg/L,20 mg/L,40 mg/L and 60 mg/L group.The others were grouped into control,PD,PLA and PLA + PD group.The levels of Bax,Bcl-2,Casepase-3 and ERK1/2 in different concentrations of PLA and the effects of ERK signal pathway inhibitor PD98059(20 μmol/L)on Bcl-2/Bax,Casepase-3 and ERK1/2 phosphorylation were detected by chemiluminescence substrate of Western Blot.5.Statistical processingAll the experiments were repeated for three times.Statistical analyses were perfored using SPSS version 16.0.All values were displayed as means ± standard error of the mean.One-way Analysis of Variance(ANOVA)was applied for comparisons of more than two groups,and LSD was used when equal variance was assumed between groups.P-values less than 0.05 were considered to denote statistically significant differences.Result1.The NCI-H292 cells in PLA group changed in apoptosis significantly.The nuclear membrane shrinked and the chromatin on the nuclear membrane concentrated.The arrangement of the cristae of mitochondria was irregular,and the number was reduced significantly,even disappeared in some cases.2.Annexin V-FITC/PI double staining for apoptosis detection showed that in the control group,10 mg/L group,20 mg/L group,40 mg/L group,60 mg/L group,the apoptosis rate of NCI-H292 cells were(4.97±0.17)%,(7.82±0.21)%,(11.99± 0.21)%,(29.52±0.55)% and(55.23±0.67)%.Compared with the control group,the difference was statistically significant(P<0.001).3.The expression level of Bcl-2/Bax in the 10 mg/L group,20 mg/L group,40 mg/L group and 60 mg/L group showed decreased compared with the control group(P<0.01).The activity of protein expression of actived Caspase-3 in the 10 mg/L group,20 mg/L group,40 mg/L group and 60 mg/L group were increased compared with the control group(P<0.001).4.Compared with the control group,the phosphorylation of ERK increased statistically in the 10 mg/L group,20 mg/L group,40 mg/L group and 60 mg/L group.And the peak of P-ERK/ERK was advanced in the 20 mg/L group(P<0.01).5.The apoptosis rate of NCI-H292 cells in the control group,PD group,PLA group,PLA+PD group were(5.04±0.26)%,(4.99±0.19)%(P=0.801),(20.72±1.37)%(P<0.001),(8.67±0.18)%(P<0.001).The apoptosis rate of PLA+PD group was significantly lower than that in the PLA group(P<0.001).6.Compared with group PLA,the activity of Bcl-2/Bax increased significantly in the PLA+PD group and the activity of Caspase-3 was lower significantly(P<0.001).Compared with the control group,the expression of P-ERK/ERK in the PLA group increased significantly(P<0.001).Compared with PLA group,the activity of P-ERK/ERK decreased significantly in PLA+PD group(P<0.001).Conclusion 1.PLA can induce apoptosis of airway epithelial cells in asthma.2.PLA induced apoptosis of airway epithelial cells by activating Bax,Bcl-2 and Caspase-3.3.PLA can regulate the expression of Bax,Bcl-2 and Caspase-3 to induce apoptosis of airway epithelial cells by ERK signaling pathway.