谷胱甘肽修饰携载siRNA-PARP1的纳米颗粒调控ROS介导小胶质细胞极化治疗缺血性脑损伤的机制研究

The primary goal of therapy for ischemic brain injury is to inhibit oxidative stress and inflammation damage in the ischemic penumbra. Many studies have shown that excessive production of oxygen free radicals (ROS), the increased number of M1 microglia (M1-MG) and decreased number of M2 microglia (M2-MG) induced by ROS can result in oxidative stress and inflammatory injury. Polyadenosine diphosphate ribose polymerase-1 (PARP1) plays an important role in microglial polarization. Our experiments have showed that glutathione (GSH)-modified nanoparticles carrying siRNA-PARP1 (GSH-NP/siRNA-PARP1) could clear ROS, increase the expression of Sirtuin 1 (SIRT1) and nuclear factor erythroid-derived factor 2 (Nrf2), inhibit the M2a-MG to M1-MG polarization, and reduce the number of M1-MG in microenvironment. To investigate whether PARP1/SIRT1/Nrf2 signaling is the key mechanism that ROS mediates the polarization of M2a-MG to M1-MG, we enhance or inhibit the expression of SIRT1 and Nrf2. To explore the mechanism of inhibiting oxidative stress injury, we analyze the changes of superoxide dismutase and glutathione peroxidase in ischemic penumbra. To investigate the mechanism of inhibiting inflammatory cascade injury, we analyze the change of M2a-MG, M1-MG cells and inflammatory cytokines in ischemic penumbra. We analyzed the relationship between GSH-NP/siRNA-PARP1 treatment, protection of dying neurons and improvement of neurological function, which provides a new treatment for ischemic brain injury.

抑制缺血半暗带的氧化应激反应和炎症损伤是治疗缺血性脑损伤的关键。研究表明：过量氧自由基（ROS）和ROS介导的小胶质细胞（MG）M1型极化是氧化应激、炎症损伤的主要原因。我们预实验发现：聚腺苷二磷酸核糖聚合酶-1(PARP1)调控ROS介导的M1型MG极化，谷胱甘肽修饰的携载siRNA-PARP1的纳米颗粒（GSH-NP/siRNA-PARP1）能清除ROS，能增加MG的SIRT1、Nrf2表达，能抑制M2a型MG向M1型MG极化。本项目拟干预SIRT1、Nrf2的表达，探讨PARP1/SIRT1/Nrf2信号调控ROS介导的M2a型MG极化为M1型MG的机制；分析氧化应激相关分子、M2a-MG、M1-MG细胞、炎症细胞和因子数量的变化，研究GSH-NP/siRNA-PARP1抑制氧化应激和炎症损伤的机制，分析其与保护濒死神经元、改善神经功能的关系，为缺血性脑损伤提供新的治疗方法。

针对缺血性脑损伤急性期，抑制氧化应激反应、减轻炎症损伤是保护濒死神经元的关键，也是本研究的主要内容。在体外实验，我们利用小胶质细胞的吞噬特性，将携载聚腺苷二磷酸核糖聚合酶-1（poly ADP-ribose polymerase，PARP1）的小分子干扰RNA片段（poly ADP-ribose polymerase siRNA, siRNA-PARP1）的纳米颗粒递送到小胶质细胞内调控其极化；我们证实：PARP1是调控ROS介导的M2a-MG向M1-MG极化的关键信号分子，SIRT1/Nrf2是PARP1下游的关键信号。在体内实验，我们使用谷胱甘肽（Glutathione, GSH）修饰纳米载体携载siRNA-PARP1的纳米颗粒（GSH modified nanoparticles loaded with siRNA-PARP1, GSH-NP/siRNA-PARP1）。我们的结果表明，GSH-NP/siRNA-PARP1治疗①能够有效抑制氧化应激、炎症级联损伤的作用，能够有效保护濒死神经元；②能够减少脑梗死的体积、减少水肿体积，能够有效改善神经功能。