Sodium nitroprusside (SNP)-treated PC12 cell line is being used in our laboratory as a cell model of nitric oxide (NO)-mediated damage for in vitro evaluation of potential neuroprotective compounds, thus cell response to SNP must be standardized to gain reproducible data. The NO-donor SNP has been shown to induce cell death at high concentrations in undifferentiated PC12 cells. Differences were found in sensitivity to SNP between cells from short- and long-term cultured cells. After 24-h exposure to 100-500 microM SNP, a decrease of cell viability was observed in both short- (17, 21 and 23rd passages) and long-term cultures (46, 49 and 50th passages), with IC(50) values of 312.72 and 462.90 microM, respectively. In cells from early passages, SNP-induced cell death was accompanied by significant increases of LDH leakage, nitrite production, malondialdehyde (MDA) levels, catalase (CAT) activity, cleavage of poly(ADP-ribose)polymerase (PARP) and caspase-3 activation in comparison with those from late passages. Furthermore, untreated and SNP-treated cells from long-term cultures displayed an increase of the stress protein Hsp70 levels when compared with those from short-term cultures. Up-regulated levels of Hsp70 may be associated with cell survival. Therefore, cells may acquire a certain resistance to SNP-induced toxicity associated with an increase in cell passage-dependent Hsp70. The protein Hsp70 might modulate the cellular response to the toxic insult by increasing CAT and GSH-Px activities and decreasing caspase-3 activation. Finally, it is crucial for the standardization of this cell model of neurotoxicity, at least in part, the use of PC12 cells in an optimum and reliable range of passages.
Hypericum perforatum is a medicinal herb possessing ability for protecting neurons from oxidative stress. Since nitric oxide (NO) may be protective against oxidative stress-induced cell death as occurs in glucose deprivation (GD)-induced neurotoxicity, whether a standardized extract of H. perforatum (HP) increases the NO-mediated neuroprotective effect in GD-PC12 cells was investigated. Induced death in PC12 cells by GD exposure for 18 h was partially prevented by cell incubation with sodium nitroprusside (SNP), a NO-donor. SNP increased survival and nitrite production in GD-cells in a concentration-dependent manner. Co-incubation of cells with 10 μM SNP plus 50-100 μg/ml HP under GD insult significantly prevented GD-induced cell death to a higher extent than SNP alone as shown by an augmentation of cell survival and intracellular bcl-2 levels and a decrease of lipid peroxidation, caspase-3 activation and PARP cleavage. Cytoprotection by the NO-donor was almost abolished by the use of a NO scavenger and potentiated by the presence of superoxide dismutase. SNP and/or HP neuroprotection on GD-cells was significantly reversed by rotenone treatment. These results suggest that: (1) SNP could protect PC12 cells from GD-induced cytotoxicity through NO generation and (2) the enhancement of the SNP-mediated neuroprotective effect on GD-cells by HP might arise in part through scavenging of reactive oxygen species (ROS) and inhibition of mitochondrial dysfunction associated with the hypoglycemic episode. This current finding might highlight the development of therapeutic strategies aimed at manipulating NO-donors in combination with herb supplements containing ROS scavenger compounds for prophylaxis from brain ischemia.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.