The Cytoprotective Role Of Sodium Nitrite Preconditioning Against The Peroxide-Induced Damage In PC12 Cells

Objective: To study the cytoprotective role of Low-dose sodium nitrite preconditioning against the peroxide-induced damage and to observe the differentiation of sodium nitrite in PC12 cells.Methods: PC12 cells were treated with different concentrations of sodium nitrite for different times, the cell viability was measured by MTT to find the minimum sodium nitrite dose and the optimum time which could promote the cell proliferation . The PC12 cells were treated with 400 mmol·L^-1 ethanol, 45 mmol·L^-1 NaNO₂ and 1.1 mmol·L^-1H₂O₂ for 2h to establish the peroxide-induced damaged cell model.The proliferation of PC12 cell was detected by MTT, cell counting and Hochest33258 staining. FITC-annexinâ…¤/PI flow cytometry , Hoechst33258 staining and Hoechst 33258/PI staining were used to detect the cells apoptosis. Colorimetric technique was performed to measure the SOD, CAT and GSH-Px , MDA. Western-blotting to detect the expression of the apoptosis-related protein. Wright's staining to observe the differentiation of PC12 cell.Results: Dose-responses results showed that NaNO₂ on PC12 cells proliferation was typical inverted U-shaped curve.The concentration of the minimum stimulatory response was 0.14 mmol·L^-1(more than 15% of the control group)and the maximum stimulatory response at the 24 h. The PC12 cells were treated with 400 mmol·L^-1 ethanol, 45 mmol·L^-1 NaNO₂ and 1.1 mmol·L^-1 H₂O₂ for 2 h, Flow cytometry assay show the apoptosis rate of the cells were (50.63±2.14)%, (52.06±8.32)%, (53.6±8.51)% respectively. The PC12 cells were pretreated with 0.14 mmol·L^-1 NaNO₂ for 24 h then treated with the oxygenants above-metioned for 2 h, the apoptosis rate of the cells were decreased as (19.71±3.19)%, (19.65±2.17)%, (18.23±4.19)% respectively(P﹤0.05). While NaNO₂ and nitric oxide specific scavenger c-PTIO combined to treat the cells for 24 h, then treated with the oxygenants above-metioned for 2 h, the apoptosis rate of the cells were (32.28±7.31)%, (38.63±5.08)%, (30.75±3.81)% respectively. The results of Hoechst33258 staining, Hoechst 33258/PI staining and cell counting are same as flow cytometry. The PC12 cells were treated with 400 mmol·L^-1 ethanol, the activities of SOD, CAT, GSH-Px and the levels of MDA were (20.95±1.68)U/mg.Prot, (11.45±0.93) U/mg.Prot, (41.17±2.72)μmol/mg.Prot, (98.38±11.5)μmol/mg.Prot respectively. The PC12 cells were pretreated with NaNO₂ then treated with ethanol, the activities of SOD, CAT and the levels of GSH-Px, MDA were (92.38±2.98) U/mg. Prot, (19.29±0.27 ) U/mg.Prot, (62.59±1.41)μmol/mg.Prot, (49.38±2.93)μmol/mg.Prot. While NaNO₂ and nitric oxide specific scavenger c-PTIO combined to treat the cells for 24 h, then treated with the oxygenants above-metioned for 2 h, the activities of SOD, CAT and the levels of GSH-Px, MDA were (50.23±1.57) U/mg.Prot, (15.98±1.9) U/mg.Prot, (51.21±1.52)μmol/mg.Prot, (73.58±14.7)μmol/mg.Prot. Western blotting showed that in ethanol treated cells pretreated with NaNO₂, the expression of Bax, Caspase-9, Caspase-3 were decreased, HIF-1αand Bcl-2 were increased(P﹤0.05). Add to c-PTIO could reverse this phenomenon. The results of 45 mmol·L^-1 NaNO₂ and1.1 mmol·L^-1 H₂O₂ were same as ethanol group. The PC12 cells were treated with 0.14 mmol·L^-1 NaNO₂ for 48h, the results of wright's staining showed that NaNO₂ have the same effects of NGF, including prolong the prominency of the cells, and increase the expressions of HIF-1 and VEGF .Conclusions:1. Dose-responses results showed that NaNO₂ on PC12 cells proliferation was typical inverted U-shaped curve.2. The PC12 cells were pretreated with low dose of NaNO₂ could increase their anti-oxidant activities and expressions of HIF-1α, resist the apoptosis induced by ethanol, high dose of NaNO₂ and H₂O₂. The mechanism might be related with the reduction of NaNO₂ to NO and the increased expressions of HIF-1α.3. NaNO₂ could promote the differentiation of PC12 cells.