PKC-δ isozyme gene silencing restores vascular function in diabetic rats
PKC-δ isozyme gene silencing restores vascular function in diabetic rats
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DOI：
10.1515/jbcpp-2013-0147
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发表时间：
2014-11-01
影响因子：
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通讯作者：
Soloviev, Anatoly
中科院分区：
文献类型：
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作者：
Klymenko, Kateryna;Novokhatska, Tetiana;Soloviev, Anatoly
背景：平滑肌细胞（SMCs）的内皮和K+通道功能调节血管功能，并暴露于糖尿病的损害中。众所周知，调节酶蛋白激酶C （PKC）在健康和疾病的血管张力调节中起关键作用。在这项研究中，我们评估了使用小干扰rna （sirna）沉默PKC-delta基因对糖尿病血管SMCs内皮功能障碍和获得性钾通道病变的影响。方法：采用链脲唑菌素（65 mg/kg）诱导大鼠糖尿病、RNA干扰、离体主动脉环收缩记录、全细胞膜片钳技术、活性氧（ROS）测定和实时聚合酶链反应技术进行实验设计。在糖尿病第3个月和静脉注射sirna后第7天，分别给予氯胺酮（45 mg/kg, i.p）和噻嗪（10 mg/kg, i.p）麻醉，以颈椎脱臼致死。结果：与对照组相比，糖尿病大鼠主动脉内皮依赖性松弛和整体SMCs向外K+电流明显降低。在第7天，PKC-delta基因沉默有效地恢复了K+电流，并将血管舒张幅度提高到控制水平。在靶向PKC-delta基因沉默后，糖尿病主动脉中PKC-delta mRNA水平的升高似乎有所降低。同样，在给药sinas后，糖尿病患者的ROS生成水平也恢复到控制值。结论：利用sirna沉默pkc - δ基因表达可使糖尿病大鼠血管舒张电位恢复。siRNA技术很可能成为糖尿病血管功能正常化的良好治疗工具。
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-delta 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-delta gene silencing effectively restored K+ currents and increased the amplitude of vascular relaxation up to control levels. An increased level of PKC-delta mRNA in diabetic aortas appeared to be reduced after targeted PKC-delta 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-delta 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.