目的了解不同浓度吡格列酮(pioglitazone,PIO)对高糖诱导下血管内皮细胞(vascular endothelial cell,VEC)凋亡的保护作用,并探讨其机制.方法将处于对数生长期的VEC随机分为6组,分别为A组:正常对照组(Glu 5.5 mmol/L);B组:高糖组(Glu 33 mmol/L);C组:高糖+低浓度PIO组(PIO 1×10^-8mmol/L);D组:高糖+中浓度PIO组(PIO 1×10^-6mmol/L);E组:高糖+高浓度PIO组(PIO 1×10^-4mmol/L);F组:高糖+SP600125组(SP600125,10.0μmmol/L):(1)采用Annexin V/PI双染法检测各组细胞凋亡率;(2)Western-blot检测各组血管内皮细胞JNK、p-JNK蛋白的表达,观察不同浓度PIO作用下JNK磷酸化水平的变化.结果 (1)与高糖组相比,中浓度PIO(1×10^-6mmol/L)及高浓度PIO(1×10^-4mmol/L)均能使VEC凋亡均明显减少(P〈0.05),呈浓度-效应依赖关系;(2)PIO可明显抑制VEC内的p-JNK表达(P〈0.05),且呈浓度-效应依赖关系.与高糖组比较,JNK抑制剂SP600125干预后p-JNK表达减少,差异有统计学意义(P〈0.05).结论 PIO能够明显抑制高糖诱导引起的VEC凋亡.PIO抑制血管内皮细胞凋亡的作用有可能是通过影响JNK通路来实现的.
Objective To investigate the effect of pioglitazone(PIO) on vascular endothelial cell(VEC)apoptosis induced by the high glucose and its mechanism. Methods The VECs in the logarithmic growth were randomly divided into 6 groups: group A,normal control group(Glu 5.5 mmol/L);group B,cultured in high glucose group(Glu 33 mmol/L);group C,high glucose +low concentrations PIO group(PIO 1×10^-8 mmol/L);group D,high glucose + middle concentrations PIO group(PIO 1×10^-6 mmol/L);group E, high glucose + high concentrations PIO group(1×10^-4 mmol/L);group F,10.0 μM SP600125+PIO group(PIO 1×10-4 mmol/L).The apoptosis rate in each group was measured by Annexin V/PI double staining. The protein expressions of JNK and p-JNK in each group were detected by Western-Blot, and the change of JNK phosphorylation in high glucose-induced vascular endothelial cell under the action of PIO was observed. Results(1) Compared to the high glucose group,middle concentrations PIO(1×10^-6 mmol/L) and high concentrations PIO(1×10^-4 mmol/L)reduced the apoptosis of VEC obviously(P〈0.05),in a dose-dependent manner.(2) PIO obviously decreased the phosphorylation level of JNK in high glucose group(P〈0.05),and in a dose-dependent manner. Compared to high glucose group, JNK inhibitor SP600125 declined the level of phosphrylation, and the difference was significant(P〈0.05). Conclusion PIO can obviously inhibit VEC apoptosis induced by high glucose, the inhibition effect of PIO may be achieved by affecting the JNK pathway.