在目前的研究中,我们旨在研究双硫仑(DSF)是否通过调节铁氧还蛋白1(FDX1)调节铜离子(Cu)水平和抑制炎症反应,在脑缺血再灌注(CI-RI)损伤中发挥神经保护作用.要模拟CI-RI,采用C57/BL6小鼠的短暂性大脑中动脉闭塞(tMCAO)模型。在tMCAO之前和之后,在有或没有DSF的情况下施用小鼠。使用TTC染色观察tMCAO后梗死体积的变化。采用Nissl染色和苏木精-伊红(HE)染色在显微水平上观察神经细胞的形态学变化。通过TUNEL法验证DSF对初始炎症的抑制作用,凋亡相关蛋白检测和铁浓度检测。FDX1是铜逝世亡的主要调控卵白,而铜逝世亡的发生会招致HSP70应激和炎症反响的加重。蛋白质印迹法检测细胞凋亡相关蛋白和下游炎症因子,免疫荧光染色,和免疫组织化学。使用特定的试剂盒检测铜离子的含量,而电子显微镜用于检查线粒体变化。我们发现DSF减少了脑梗死的体积,调节角化相关蛋白的表达,并通过下调FDX1的表达来调节铜含量。此外,DSF抑制HSP70/TLR-4/NLRP3信号通路。总的来说,DSF可以通过抑制FDX1调节Cu稳态,作用于HSP70/TLR4/NLRP3通路以减轻CI/RI。因此,DSF可以减轻炎症反应并保护线粒体完整性,为缺血再灌注损伤的临床治疗提供新的治疗靶点和机制。
In the current study, we aimed to investigate whether disulfiram (DSF) exerts a neuroprotective role in cerebral ischemiareperfusion (CI-RI) injury by modulating ferredoxin 1 (FDX1) to regulate
copper ion (Cu) levels and inhibiting inflammatory responses. To simulate CI-RI, a transient middle cerebral artery occlusion (tMCAO) model in C57/BL6 mice was employed. Mice were administered with or without DSF before and after tMCAO. Changes in infarct volume after tMCAO were observed using TTC staining. Nissl staining and hematoxylin-eosin (he) staining were used to observe the morphological changes of nerve cells at the microscopic level. The inhibitory effect of DSF on initial inflammation was verified by TUNEL assay, apoptosis-related protein detection and iron concentration detection. FDX1 is the main regulatory protein of
copper death, and the occurrence of
copper death will lead to the increase of HSP70 stress and inflammatory response. Cuproptosis-related proteins and downstream inflammatory factors were detected by western blotting, immunofluorescence staining, and immunohistochemistry. The content of
copper ions was detected using a specific kit, while electron microscopy was employed to examine mitochondrial changes. We found that DSF reduced the cerebral infarction volume, regulated the expression of cuproptosis-related proteins, and modulated
copper content through down regulation of FDX1 expression. Moreover, DSF inhibited the HSP70/TLR-4/NLRP3 signaling pathway. Collectively, DSF could regulate Cu homeostasis by inhibiting FDX1, acting on the HSP70/TLR4/NLRP3 pathway to alleviate CI/RI. Accordingly, DSF could mitigate inflammatory responses and safeguard mitochondrial integrity, yielding novel therapeutic targets and mechanisms for the clinical management of ischemia-reperfusion injury.