Introduction: Nitric oxide (NO) is a free radical and a signaling molecule which controls many cellular and physiological mechanism such as; blood vessel contraction, blood pressure and immunological response. In previous studies it was shown that the short time treatment of the mesenchymal stem cells (MSCs) with sodium nitroprusside (SNP) as NO releasing agent at low concentration did not affect the cell viability, but caused metabolic imbalance. The aim of this study was to investigate the effect of low concentration of SNP for long time on cell viability, proliferation and cell cycle of these cells.
Methods: MSCs after 3re passage was treated every 1 hour in each 72 hours with 100 μM of SNP. After 5, 10, 15 and 20 day, the viability and proliferation of the cells was estimated using MTT (dimethyl thiazol-diphenyl tetrazolium bromide) and CFA (colony forming assay) method. The expression of the Raf1, Cdk2 (Cyclin - dependent kinase 2 ),Cdk4 (Cyclin-dependent kinase 4) and p53 genes involve in cell cycle was determined using RT-PCR (reverse transcription polymerase chain reaction). In addition the cell cycle was studied with the help of flow cytometry.
Results: cell treated with SNP at day 5, 10, 15 and 20 the viability reduced significantly. The same treatment caused the number of the colony to reduce significantly at all treatment time, but no changes were observed in diameter of the colonies. It was also observed that the SNP arrest the cell cycle at G1 in a time dependent manner, where at the day 20 the cell cycle arrest was estimated to be 93%. The treatment caused the down regulation of Cdk2,Cdk4 and up regulation of P53 gen, whereas no changes were observed in the expression of Raf1,In addition the Gapdh gene no changes were expression as the house kepping gene.
Conclusion: this study showed that the NO at long time treatment caused cell viability and proliferation reduction. It was also observed that the cell cycle also was arrested at G1 due to down regulation Cdk2,Cdk4 and up regulation of P53.