靶向PGK1-NRF2通路抗神经元氧化损伤的作用及机制研究

NF-E2-related factor 2 (NRF2) is an important transcription factor that regulates oxidative stress after acute ischemic stroke, controlling the expression level of a series of antioxidant proteins via identifying antioxidant response elements. Inhibition of phosphoglycerate kinase 1 (PGK1) leads to the dimerization of KEAP1, resulting in the dissociation of the KEAP1-NRF2 complex and facilitating NRF2 activation. We have found that the activation of NRF2 can inhibit oxidative injury in neurons. Our preliminary studies revealed that use of CBR-470-1, a small molecule PGK1 inhibitor, activated the NRF2 pathway in cultured SH-SY5Y cells and in primary murine hippocampal neurons. shRNA-mediated knockdown of PGK1 activated the NRF2 pathway in hippocampal neurons both in vitro and in vivo. We therefore hypothesize that PGK1 is a primary inhibitor protein of NRF2 pathway in neurons and targeting PGK1-NRF2 pathway can inhibit neuronal oxidative injury. In this proposal, we will use pharmacological (CBR-470-1), shRNA, and CRISPR/Cas9 strategies to inhibit neuronal oxidative injury via activating the PGK1-NRF2 pathway. The neuroprotective effects of CBR-470-1 in vivo will additionally be tested using a murine MCAO model. We will also explore the molecular mechanisms of CBR-470-1-mediated neuronal protection. The results of this study will provide a new therapeutic target for the treatment of oxidative injury in neurons.

核转录相关因子2（NRF2）是调控急性缺血性脑卒中后氧化应激的重要转录因子，通过识别抗氧化反应元件，调控一系列抗氧化蛋白表达。抑制磷酸甘油酸激酶1（PGK1）致KEAP1二聚化、KEAP1-NRF2解耦联，激活NRF2通路。前期研究证实激活NRF2可抑制神经元氧化损伤。预实验发现在SH-SY5Y细胞和小鼠海马神经元中，PGK1小分子抑制剂CBR-470-1激活NRF2通路；离体/在体敲减PGK1激活海马神经元NRF2通路。我们提出PGK1是神经元NRF2通路关键抑制蛋白，靶向PGK1-NRF2通路抗神经元氧化损伤。我们拟运用药理学（CBR-470-1），shRNA和CRISPR/Cas9等手段靶向激活PGK1-NRF2通路，达到抑制神经元氧化损伤的目的；拟建立小鼠MCAO模型，观察CBR-470-1在体神经保护作用；解析CBR-470-1神经元保护分子机制。为神经元氧化损伤干预提供新靶点。

目的：了解星形胶质细胞在炎症和氧化反应中的作用和机制，对于寻找减少脑缺血再灌注损伤（CIRI）中炎症和氧化损伤的治疗策略具有重要意义。研究发现，磷酸甘油酸激酶1（PGK1）在促炎症和氧化应激中发挥重要作用，但PGK1在CIRI中的具体机制尚不清楚。本研究旨在研究PGK1对大鼠CIRI后炎症和氧化反应的调节作用及其相关机制。..方法：雄性成年SD大鼠和原代星形胶质细胞（取自新生SD大鼠）作为实验对象。采用缝合线闭塞法建立大鼠大脑中动脉闭塞再灌注（MCAO/R）模型。通过无氧、无糖和无血清培养建立星形胶质细胞的氧-葡萄糖剥夺/再氧化（OGD/R）模型。在建模前24小时将AAV8-PGK1-GFP注射到左心室。进行实时定量聚合酶链反应（RT-qPCR）、酶联免疫吸附试验（ELISA）、共免疫沉淀（CoIP）、荧光原位杂交（FISH）和蛋白质印迹，以深入探讨PGK1在CIRI中的作用机制。..结果：PGK1的过度表达显著加重了MCAO/R后大鼠的神经功能缺损，增加了脑梗塞体积，加重了神经细胞损伤。通过FISH和CoIP测定验证了PGK1和Nrf2在原代星形胶质细胞中的定位。复苏实验进一步证实，Nrf2的敲除消除了CBR-470-1（PGK1抑制剂）对CIRI的保护作用。最后，我们证实PGK1通过抑制Nrf2/ARE通路而加重CIRI。..结论：PGK1的抑制通过激活Nrf2/ARE信号通路抑制星形胶质细胞炎性和氧化因子的释放来减轻CIRI。