| Objective①To investigate the protective effects of different concentrations of PIO on high glucose injured vascular endothelial cell and to explore the optimum protective concentration;②To investigate the effect of PIO on the NO and ET-1 produced by the high glucose injured vascular endothelial cell;③To investigate the changes of JNK expression in high glucose injured vascular endothelial cell under the action of PIO;④To investigate the effect of JNK inhibitor SP600125 on the protective function of PIO in vascular endothelial cell.Methods①Vascular endothelial cells were cultured in vitro complete DMEM medium with different concentrations of PIO, and the optimum concentration of PIO played a protective role on cells was assessed by MTT assay. Then the mature vascular endothelial cells were randomly divided into 7 groups: normal culture group(A group of Glu 5.5mmol/l),cultured in high glucose group(B group of Glu 37.5mmol/l),PIO(1×10- 8mmol/L)(C group), PIO(1×10-6mmol/L)(D group), PIO(1×10- 4mmol/L)(E group) ,1.0uM SP600125+PIO(1×10- 4mmol/L)(F group), and 10.0uM SP600125+PIO(1×10- 4mmol/L)(G group). Among them E group and F group were pretreated by different concentrations of SP600125 for 1 hour and then they were cultivated by high concentration of PIO for 24 hours continuously; from C group to G group,they were cultivated by different concentrations of drugs and high glucose.②The serum levels of NO and ET-1 in each group were measured by nitrate reductase and Radioimmunoassay respectively;③The protein expression of JNK and p-JNK in every group were detected by Western-Blot . The effects of different concentrations of PIO on the phosphorylation level of JNK were changed dynamically at 15min, 30min, 1h, 2h, 4h, and 8h. The effect of different concentrations of SP600125 on the phosphorylation level of JNK of vascular endothelial cells which affected by high concentrations of PIO were observed.Results①PIO had protective effect on high glucose injured vascular endothelial cell at the concentrations of 10- 8mmol/L -1×10- 4mmol/L and in a dose-dependent manner.②Under the action of high glucose,the serum level of NO in cell declined and the level of ET-1 increased obviously.Compared to the normal culture group, there was significant differenc(eP<0.01). PIO reduced the level of ET-1 obviously in high glucose group as well as increased the level of NO, and into a dose-dependent manner. Compared to the high glucose culture group, the differences were significant(P<0.05).SP600125 pretreatment could reduce the level of ET-1 and promote the release of NO. There was significant difference(P<0.05)compared with without pretreatment group and it also showed significant differences between SP600125 intervention groups(P<0.05).③Under the action of high glucose, the phosphorylation level of JNK in vascular endothelial cell was obviously increased. Compared to control group, the differences were significant (P<0.01). PIO decreased the phosphorylation level of JNK in high glucose group, and developed the dependence of dosage. Compared to high glucose group, there was significant difference (P<0.05). JNK inhibitor SP600125 declined the level of phosphrylation further and developed the dependence of concentration. Compared to the PIO group , the differences was significant (P<0.05).④The phosphorylation level of JNK in high glucose group declined obviously with the high concentration of PIO(1×10- 4mmol/L) for 15 minutes, which lasted for 8 hours and the most obvious decline happened in 2-4 hours. There was significant difference(P<0.05). The phosphorylation level of JNK in high glucose group declined obviously with the PIO at the concentration of 1×10- 8mmol/L. It lasted for 8 hours and the most obvious affection happened in 4-8 hours. There was significant difference(P<0.05).Conclusion PIO can improve the survival rate of vascular endothelial cells in high-sugar damage, increase the level of NO, as well as decrease the secretion of ET-1 and the expression of JNK and p-JNK. The effect of PIO on vascular endothelial cells could be enhanced by the JNK inhibitor SP600125 and the protective effect of PIO may be achieved by affecting the JNK pathway. |
