Ferrostatin-1 improves neurological impairment induced by ischemia/reperfusion injury in the spinal cord through ERK1/2/SP1/GPX4

Abstract

Spinal cord ischemia/reperfusion injury (SCIRI) induced by artificial aortic occlusion for a while during aortic surgery is a serious complication, leading to paraplegia and even death. Ferroptosis in the nervous system has been confirmed to contribute to neuronal death induced by SCIRI. Therefore, we investigated the therapeutic benefits of ferrostatin-1 (Fer-1, a ferroptosis inhibitor) and explored the mechanism and target of Fer-1 in SCIRI. Our results demonstrate that intrathecal injection of Fer-1 had a strong anti-SCIRI effect, improved ferroptosis-related indices, increased neurological function scores and motor neuron counts, and reduced BSCB leakage and neuroinflammation levels in the anterior horn. We found that SCIRI significantly elevated the levels of several important proteins, including SP1, p-ERK1/2/ERK1/2, COX2, TFR1, SLC40A1, SLC7A11, cleaved Caspase 3, GFAP, and Iba1, while reducing FTH1 and GPX4 protein expression, with no effect on ACSL4 expression. Fer-1 effectively ameliorated the ferroptosis-related changes in these proteins induced by SCIRI. However, for p-ERK1/2 and SP1, Fer-1 not only failed to reduce their expression but also significantly enhanced it. Fer-1 was injected into sham operation rats, abnormal increases in p-ERK1/2/ERK1/2 and SP1 were observed, along with an increase in GPX4. Fluorescent double labeling revealed that SP1 and GPX4 were expressed in neurons and astrocytes. Inhibitors of the ERK pathway (SCH772984) and siRNA against SP1 (AV-sh-SP1) significantly decreased the increase in SP1 and GPX4 protein levels, fluorescent density of SP1 and GPX4 in neurons, and the number of SP1-positive and GPX4-positive neurons induced by Fer-1. SCH772984 but not AV-sh-SP1 significantly reversed the decrease in GFAP and Iba1 induced by Fer-1. In conclusion, our results indicate that Fer-1 inhibited ferroptosis in spinal cord anterior horn neurons, improving neurological impairment and BSCB damage after SCIRI through the ERK1/2/SP1/GPX4 signaling pathway in rats.

Keywords: Ferroptosis; Ferrostatin-1; GPX4; Motor neuron; Rat; SP1; Spinal cord ischemia/reperfusion injury.