Protection against ischemia-reperfusion injury using a short interfering RNA that inhibits the activity of myeloperoxidase

为了研究使用短干扰RNA （siRNA）抑制人中性粒细胞髓过氧化物酶（MPO）活性对缺血再灌注损伤（IRI）的保护可行性，进行了以下研究。设计并合成了降低MPO活性的siRNA。制备了有效（阳性）和无效（阴性）sirna。然后，利用nucleoector系统将siRNA电转移到中性粒细胞中。成功地用阳性siRNA吸吸MPO mRNA的表达，用实时荧光定量PCR证实。然而，当中性粒细胞被活化的酶酵素激活时，与阴性siRNA结合的中性粒细胞相比，阳性siRNA结合的中性粒细胞的活性氧（ROS）的产生并未减少。也就是说，RNA干扰不能有效抑制中性粒细胞的MPO活性。作为另一种捕获方法，我们尝试使用人脐静脉内皮细胞（HUVECs）来抑制内皮细胞中ROS的产生。首先，揭示了一些刺激物，如脂多糖（LPS），诱导白细胞介素-8 （IL-8）的表达，而IL-8反过来又诱导huvec中ROS的产生。设计合成了抑制IL-8表达的siRNA。制备了阳性和阴性sirna。然后，利用nucleoector系统将siRNA电转移到HUVECs中。成功地用阳性siRNA吸吸IL-8 mRNA的表达，用实时荧光定量PCR证实。并且，当LPS刺激HUVECs时，与与负siRNA结合的HUVECs相比，与阳性siRNA结合的HUVECs中ROS的产生减少。我们知道，内皮细胞产生的ROS在IRI过程中会损伤自身。因此，我们可以说使用siRNA实现了对WI的保护。

In order to investigate the feasibility of protection against ischemia-reperfusion injury (IRI) using a short interfering RNA (siRNA) that inhibits the activity of myeloperoxidase (MPO) of human neutrophils, the following studies were Performed.The siRNA to diminish the MPO activity was designed and synthesized. Both effective (positive) and ineffective (negative) siRNAs were prepared. Then, the siRNA was electrically transferred into neutrophils using NucleofectorR system. The Suction of the expression of mRNA of MPO by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. However, when the neutrophils were activated by opsonized zymosan, the generation of reactive oxygen species (ROS) was not diminished in the neutrophils incorporated with a positive siRNA compared to that in the neutrophils incorporated with a negative siRNA. That is, the RNA interference was not effective to inhibit the MPO activity of neutrophils.As an alternative capture, we tried to inhibit the ROS generation in endothelial cells using human umbilical vein endothelial cells (HUVECs). First, it was revealed that some stimulants, such as lipopolysaccaride (LPS), induced the expression of interleukin-8 (IL-8) and that IL-8, in turn, induced ROS generation in HUVECs. The siRNA to diminish the expression of IL-8 was designed and synthesized. Both positive and negative siRNAs were prepared. Then, the siRNA was electrically transferred into HUVECs using NucleofectorR system. The Suction of the expression of mRNA of IL-8 by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. And, when the HUVECs were stimulated by LPS, the generation of ROS was diminished in the HUVECs incorporated with a positive siRNA compared to that in the HUVECs incorporated with a negative siRNA. It was known that the ROS generated by endothelial cells hurt themselves during IRI. So, we can say that protection against WI using a siRNA was achieved.