Klymenko Kateryna, Novokhatska Tetiana, Kizub Igor, Parshikov Alexander, Dosenko Viktor, Soloviev Anatoly
J Basic Clin Physiol Pharmacol. 2014 Jan 27:1-9. doi: 10.1515/jbcpp-2013-0147.
Abstract Background: Endothelium and K+ channel functionality in smooth muscle cells (SMCs) regulates vascular function and is exposed to damage in diabetes. The regulatory enzyme protein kinase C (PKC) is known to play a key role in vascular tone regulation in health and disease. In this study, we evaluated the effect of PKC-δ gene silencing using small interfering RNAs (siRNAs) on endothelial dysfunction and acquired potassium channelopathy in vascular SMCs in diabetes. Methods: The experimental design comprised diabetes induction by streptozotocin (65 mg/kg) in rats, RNA interference, isolated aortic ring contractile recordings, whole-cell patch-clamp technique, measurements of reactive oxygen species (ROS), and real-time polymerase chain reaction technique. Animals were killed by cervical dislocation following ketamine (45 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia administration on the third month of diabetes and on the seventh day after intravenous injection of siRNAs. Results: The aortas of diabetic rats demonstrated depressed endothelium-dependent relaxation and integral SMCs outward K+ currents as compared with those of controls. On the seventh day, PKC-δ gene silencing effectively restored K+ currents and increased the amplitude of vascular relaxation up to control levels. An increased level of PKC-δ mRNA in diabetic aortas appeared to be reduced after targeted PKC-δ gene silencing. Similarly, the level of ROS production that was increased in diabetes came back to control values after siRNAs administration. Conclusions: The silencing of PKC-δ gene expression using siRNAs led to restoration of vasodilator potential in rats with diabetes mellitus. It is likely that the siRNA technique can be a good therapeutic tool to normalize vascular function in diabetes.
摘要 背景：内皮细胞和平滑肌细胞（SMC）中的钾通道功能调节血管功能，且在糖尿病中易受损伤。已知调节酶蛋白激酶C（PKC）在健康和疾病状态下的血管张力调节中起关键作用。在本研究中，我们评估了使用小干扰RNA（siRNA）沉默PKC-δ基因对糖尿病血管SMC中内皮功能障碍和获得性钾通道病的影响。方法：实验设计包括用链脲佐菌素（65mg/kg）诱导大鼠糖尿病、RNA干扰、分离主动脉环收缩记录、全细胞膜片钳技术、活性氧（ROS）测量以及实时聚合酶链反应技术。在糖尿病第三个月以及静脉注射siRNA后第七天，给予氯胺酮（45mg/kg，腹腔注射）和赛拉嗪（10mg/kg，腹腔注射）麻醉后，通过颈椎脱臼处死动物。结果：与对照组相比，糖尿病大鼠的主动脉表现出内皮依赖性舒张和SMC整体外向钾电流降低。在第七天，PKC-δ基因沉默有效恢复了钾电流，并使血管舒张幅度增加至对照水平。糖尿病主动脉中PKC-δ mRNA水平升高在靶向PKC-δ基因沉默后似乎降低。同样，糖尿病中升高的ROS产生水平在给予siRNA后恢复到对照值。结论：使用siRNA沉默PKC-δ基因表达可使糖尿病大鼠的血管舒张潜能恢复。siRNA技术很可能是使糖尿病血管功能正常化的良好治疗工具。
