Establishment of ischemic precondition model and the protective effect of nitric oxide on PC12 cell line

Abstract

Objective To establish the ischemic precondition ([PC) model of PC12 cell line in vitro, and to explore the effect of nitric oxide (NO) on the IPC cerebral protection. Method PC12 cells were cultured and used for producing the model of ischemie precondition by the way of oxygen-glucose deprivation. Twenty dishes of cells were randomly divided into four groups (5 dishes for each group): control group, ischemic precondition group (IPC),non-ischemic precondition group (NIPC) and L-NAME treatment group (L-NAME). In control group, the cells were in-cubated with low glucose (<1 g/L) and2% FBS medium in normal oxygen; in IPC group, the cells were administrated with oxygen-glucose deprivation (OGD) for 6 hours, and then subjected with reperfuaion before OGD 15 hours; in NIPC group, the cells were treated the same as control group for 6 hours, and then subjected with reperfusion before OGD 15 hours; in L-NAME group, the cells received L-NAME (1 mmol/L) and cocultured for 30 minutes before OGD 6 hours, and then received the same treatment as the IPC group. To test whether the model was established, metabolic rate of MIT, LDH release were measured and the apoptosis rate was detected by flow cytometry following oxygen-glucose deprivation 15 hours. The activity of nitric oxide synthases (NOS) was as-sessed by biochemical assay. One-way ANOVA and LSD multiple comparison test were used to analyze differences among different groups, and P<0.05 was considered different. Results Compared with NIPC group, the metabolic rate of MTT increased (94.9%±35.1%, P<0.05), while LDH release and the cell apoptotic rate decreased significantly in IPC group (279.1%±28.1%, P<0.03). Compared with control group(100.0%± 13.5%),the activities of NOS increased both in NIPC and IPC groups (390.0%±14.6%, P<0.01;126.3% ±10.6%, P<0.01). Moreover, the apoptosis rates in each group (control group, IPC group, NIPC group and L-NAME group) were 5.90, 8.73, 38.62 and 11.73%,respectively. Conclusions IPC reduces the death and apoptosis rate of PC12 cell after oxygen-glucose deprivation injury. NO might be involved, but it is not the only factor. Key words: Ischemic precondition; Nitric oxide; b-NAME; PC12 cell line