Development Of Ischemic Precondition Model By Oxygen-glucose Deprivation On PC12 Cell Line And Investigate The Signal Transduction Pathway

Objective To develop the ischemic precondition (IPC) model of PC12 cell line in vitro and explore the effect of nitric oxide (NO) on the IPC cerebral protection. Methods PC12 cells were cultured and utilized for developing the model of ischemic 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 incubated with low glucose (< 1g/L) and 2 % FBS medium in normal oxygen; in IPC group, the cells were underwent OGD for 6 h, and then subjected with reperfusion before OGD15 h; in NIPC group, the cells were treated as control group for 6 h, and then subjected with reperfusion before OGD15 h; in L-NAME group, the cells received L–NAME (1 mmol/L) and cocultured for 30 min before OGD6 h, and then underwent as the IPC group. Metabolic rate of MTT, LDH releasing were measured and the apoptosis rate was detected by flow cytometry following OGD15 h to test whether the model was established. The activity of nitric oxide synthases (NOS) was assessed by biochemical assay. One-way ANOVA and LSD multiple comparison test were used to evaluate differences among different groups and the differences were considered significant when P < 0.05. Results Compared with NIPC group, the metabolic rate of MTT increased (94.9 %±15.1 %, P <0.05) while LDH release and the cell apoptotic rate decreased significantly in IPC group (279.1 %±28.1 %, P <0.01). The activities of NOS increased both in NIPC and IPC groups (126.1 %±10.6 %, P <0.01; 189.9 %±14.6 %, P <0.01). Moreover, the flow cytometry showed that the apoptosis rates in each group (control group, IPC group, NIPC group and L-NAME group ) were 5.90, 8.71, 18.62 and 11.73% respectively. Conclusions IPC reduces the death and apoptosis rate of PC12 cell after oxygen-glucose deprivation injury, in which NO might also be involved. However, it is not the sole factor.Part two Investigate the HIF-1αsignal transduction pathway in the IPC protectionObjective To investigate the expression of hypoxia-inducible-1α(HIF-1α) in ischemic tolerance induced by the way of oxygen-glucose deprivation. Methods PC12 cells were cultured and utilized for developing the model of ischemic precondition by the way of oxygen-glucose deprivation. Fifteen dishes of cells were randomly divided into three groups (5 dishes for each group): control group, ischemic precondition group (IPC), non-ischemic precondition group (NIPC). RT-PCR were performed to analyze the mRNA levels of HIF-1α,VEGF in cells. One-way ANOVA and LSD multiple comparison test were used to evaluate differences among different groups and the differences were considered significant when P < 0.05. Results The HIF-1αmRNA expression in IPC was increased (1.66±0.11 , P < 0.01) compared with control group(1.05±0.07) and was also significantly enhanced compared with NIPC group (1.26±0.07,P<0.05). There was a significant increasing of VEGF mRNA in IPC group (1.33±0.07) compared with control group(1.00±0.06,P<0.01) and the NIPC group(1.19±0.02,P<0.05). VEGF mRNA in NIPC group increased significantly compared with control group (P<0.05) as well. Conclusions Up-regulation of HIF-1αmRNA might be a key event in the molecular mechanism of cerebral ischemic preconditioning.