Glutamine Induced Alveolar Type ¢ò Cells On Hsp70 Expression And H <sub> 2 </ Sub> The O <sub> 2 </ Sub>-induced Apoptosis

Objective To investigate the effect of glutamine on heat shock protein (HSP) 70 expression in the pulmonary alveolar type II epithelial cell line A549 cells.Methods Human pulmonary alveolar type II epithelial cell line A549 were cultured in Dulbecco' s Modified Eagle's Medium(DMEM) without fetal bovine serum over night after cells grew to 80% confluence. Then cells were cultured in DMEM with different concentrations(0 2 4 8 12 and 16mmol/L) of glutamine for 24 hours. Cells were incubated in DMEM with 8mmol/L glutamine for different times (1 2 6 12 24 and 48h). The cells incubated in DMEM without glutamine for 24 hours were seen as control group(C group). The cells incubated at 43℃ for 1 hour and harvested 4 hours later as positive control group(G group). RT-PCR and Western blotting were utilized to investigate the expression of HSP70 mRNA and protein.Results (1) Compared with the C group, the expression of HSP70 mRNA and protein increased significantly in glutamine groups and reached the highest level in DMEM with 8mmol/L glutamine (HSP70 mRNA: 293 ± 17.74, P<0. 01; HSP70 protein: 10. 42 ± 2. 51, P<0. 01); Compared with the G group, the expression of HSP70 mRNA in DMEM with 8mmol/L glutamine increased (P<0. 01) ; (2) Compared with the C group, the expression of HSP70 mRNA and protein began to increase after cells were incubated 1h, and increased to the highest level at 24h. They still maintain high level at 48h(HSP70 mRNA: 191 ± 17.45, P<0.01, HSP70 protein: 7.33±1.67, P<0.01). Conclusion Glutamine induces the expression of HSP70 mRNA and protein obviously in pulmonary alveolar type II epithelial cell line A549 under basal conditions.Part IIThe protective effect of glutamine pretreatment against cell apoptosis in pulmonary alveolar type II epithelial cellsinduced by hydrogen peroxide.Objective To investigate the protective role of glutamine pretreatment against cell apoptosis in pulmonary alveolar type II epithelial cell line A549 induced by hydrogen peroxide. Methods Human pulmonary alveolar type II epithelial cell line A549 were cultured in DMEM without fetal bovine serum over night after cells grew to 80% confluence. The cells incubated in DMEM without glutamine and then stimulated by physiologic saline(PS) for 5 hours were seen as C group. The cells incubated in DMEM with 8mmol/L glutamine for 12 hours and then stimulated by physiologic saline for 5 hours were seen as glutamine group (Gln group). The cells incubated in DMEM without glutamine for 12 hours and then stimulated by 400μmol/L hydrogen peroxide for 5 hours were seen as hydrogen peroxide stimulated group(H₂O₂ group). The cells incubated in DMEM with 8mmol/L glutamine for 12 hours and then stimulated by 400μmol/L hydrogen peroxide for 5 hours were seen as glutamine pretreated and hydrogen peroxide stimulated group (Gln+H₂O₂ group). Western blotting was utilized to investigate the expression of HSP70 protein and Flow cytometer was utilized to investigate the apoptosis of A549. Results Compared with the C group (1.87 ± 0.55) , the expression of HSP70 increased significantly in the Gln group (8. 42 ± 1. 38) H₂O₂ group (9.35 ± 1.57) and Gln+H₂O₂ group (13.12 ± 1.85) (P<0.01). Compared with the H₂O₂ group, the expression of HSP70 increased significantly in the Gln+H₂O₂ group (P<0.01). Compared with the C group, the apoptosis of cells in Gln group had not changed (P>0. 05), while the apoptosis of cells in H₂O₂ and Gln+H₂O₂ group increased significantly (P<0. 01). Compared with the H₂O₂ group(7. 48 ± 0. 53%), the apoptosis of cells in Gln+H₂O₂ group(6. 06 ± 0. 39%) decreased significantly (P<0. 01) . Conclusion Human pulmonary alveolar type II epithelial cell line A549 pretreated in DMEM with 8mmol/L glutamine for 12 hous can enhance the expression of HSP70 and inhibit the apoptosis induced by 400μmol/L hydrogen peroxide .