Cytoprotective role of low-dose sodium nitrite preconditioning against H_2O_2 induced damage in PC12 cells / 中国病理生理杂志

ABSTRACT

AIM: To study the cytoprotective role of NaNO_2 preconditioning against H_2O_2 induced damage in PC12 cells. METHODS: PC12 cells were treated with different concentrations of NaNO_2 for 6 h, 12 h, 24 h and 48 h, respectively. The viability of the cells was measured by MTT method and cell counting. The apoptotic rate of PC12 was determined by Hoechst 33258 staining to calculate the ratio of the cells between concentrated and broken nucleus in the total cell count. PC12 cells were pretreated with NaNO_2 at concentration of 3 mmol/L for 24 h. The cytoprotective role to the toxicity of H_2O_2 at concentration of 1.1 mmol/L for 6 h was observed by MTT. The cell apoptosis was measured by flow cytometry and staining with Hoechst 33258 and PI. The activities of catalase (CAT), superoxide dismutase (SOD), and the changes of glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured by the method of colorimetry. RESULTS: The dose-response results showed that the effect of NaNO_2 on PC12 cell proliferation was a typical β-shape curve. The maximal stimulatory response was at 24 h, and the concentration of the maximum stimulatory response was 1.4 mmol/L. The maximal stimulation of the concentration-responses was 156% above the control. No observable adverse effect level (NOAEL) was 6 mmol/L. IC_(50) was 45 mmol/L. When the cells were pretreated by NaNO_2 at concentration of 3 mmol/L for 24 h, and then exposed to H_2O_2 at concentration of 1.1 mmol/L for 6 h, the proliferation rate was increased as compared to the cells treated with H_2O_2 alone. Under the conditions of treating the cells with NaNO_2 at concentration of 3 mmol/L to induce the adaptive response, then exposing the cells to H_2O_2 at concentration of 1.1 mmol/L, the apoptosis rate in non-preconditioning group was 44.9%, the apoptosis rate in preconditioning group was 19.1%, the difference was significant (P<0.05). The cytoprotective effect of NaNO_2 was inhibited by nitric oxide (NO) scavenger ferrohemoglobin. The activities of SOD, CAT and the level of GSH-Px were markedly increased, the content of MDA decreased in preconditioning group. CONCLUSION: Exposure of NaNO_2 at concentration of <6 mmol/L induces hormesis on PC12 cells. Low dose of nitrite plays an important role in cytoprotection by reducing nitrite to NO, indicating that decrease in lipid peroxidation and increase in endogenous antioxidants may play a key role in cytoprotection induced by preconditioning with low dose of NaNO_2 in PC12 cells.