| Objective To investigate the effect of PIO on proliferation and NO and ET-1produced by the high glucose-induced vascular endothelial cell. To investigate the effects of PIO on cell apoptosis and reactive oxygen species produced by the high glucose-induced vascular endothelial cell. To investigate the changes of JNK expression and phosphorylation in high glucose-induced vascular endothelial cell under the action of PIO.Methods The vascular endothelial cells in the logarithmic growth were randomly divided into6groups, A group:normal culture groupn (Glu5.5mmol/l), B group: cultured in high glucose group (Glu33mmol/l), C group:high glucose+low concentrations PIO group (PIO1×10-8mmol/L), D group:high glucose+middle concentrations PIO group (PIO1×10-6mmol/L), E group:high glucose+high concentrations PIO group (1×10-4mmol/L), F group:10.0uM SP600125+PIO group (PIO1×10-4mmol/L). Among them F group was pretreated by SP600125for1hour and then cultivated by high glucose for18hours,and it only use in the Western-blot analysis of JNK, p-JNK protein expression.①Vascular endothelial cells were cultured in vitro low and high glucose medium with different concentrations of PIO for6h,12h,18h,24h, and to determine the optimal duration of PIO suppression of VEC proliferation by MTT assay.②The levels of NO and ET-1in each group were measured by nitrate reductase and Radioimmunoassay respectively.③The levels of ROS in each group were measured by fluorescent probe DCFH-DA.④The rate of apoptosis in each group were measured by Annexin V/PI double staining.⑤The protein expression of JNK and p-JNK in every group were detected by Western-Blot, and to observe the changes of JNK expression and phosphorylation in high glucose-induced vascular endothelial cell under the action of PIO.Results①Compared to the normal control group, VEC proliferation of high glucose group was significantly weakened. Compared to high glucose group, VEC proliferation can be significantly enhanced by PIO in the same time point (P<0.05), and in a dose-dependent manner. Compared to high glucose group in the same dose at different time points, VEC proliferation can be significantly enhanced by PIO,6h enhanced mild,18h enhanced the most obvious, culturing to24h proliferation capacity of VEC declined slightly.②Compared to the normal control group, the serum level of NO in cell declined and the level of ET-1increased obviously in high glucose group. Compared to high glucose group, PIO reduced the level of ET-1obviously in high glucose group as well as increased the level of NO (P<0.05), and into a dose-dependent manner.③Compared to the normal control group, the level of ROS increased obviously in high glucose group(P<0.05), Compared to high glucose group, PIO reduced the level of ROS obviously in high glucose group (P<0.05), and into a dose-dependent manner.④Compared to the normal control group, The rate of early apoptosis and late apoptosis increased obviously in high glucose group(P<0.05), Compared to high glucose group, middle concentrations PIO (1×10-6mmol/L) and high concentrations PIO (1×10-4mmol/L) reduced the apoptosis of VEC obviously (P<0.05), and into a dose-dependent manner.⑤Compared to the normal control group, the phosphorylation level of JNK was obviously increased in high glucose group, the differences were significant(P<0.05). PIO obviously decreased the phosphorylation level of JNK in high glucose group (P<0.05), and into a dose-dependent manner. Compared to high glucose group, JNK inhibitor SP600125declined the level of phosphrylation, the differences was significant (P<0.05). Conclusion PIO can obviously suppress VEC apoptosis induced by high glucose, increase the level of NO, as well as decrease the secretion of ET-1, the production of ROS and the expression of p-JNK. After the pretreatment of JNK inhibitor SP600125, the phosphorylation level of JNK declined obviously, the suppress VEC apoptosis effect of PIO may be achieved by affecting the JNK pathway. |
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