Ferrostatin-1 facilitated neurological functional rehabilitation of spinal cord injury mice by inhibiting ferroptosis

Spinal cord injury (SCI) is a devastating neurological disorder with high mortality and disability rates [1]. For 30 years, its global prevalence has increased from 236 to 1298 cases per million in different countries [2, 3]. Despite its relatively small total numbers compared to cancer and coronary artery disease, this condition places a significant economic burden on individuals, families, and society. The annual costs associated with it are estimated at 7.7 billion dollars, while the lifetime costs for each patient exceed 3 million dollars [4, 5]. The psychological impact of adapting from a healthy individual to a paraplegic one can be devastating, especially since SCI typically affects young people. Those patients with SCI are two to five times more likely to die than those without SCI, especially in low-income countries. In clinical practice, SCI treatment usually includes methylprednisolone therapy, surgical decompression, and physical rehabilitation [6]. Despite numerous basic research studies on SCI, the complexity of pathophysiological changes makes it challenging to control the development of cascades, leading to poor treatment outcomes [7]. Therefore, knowledge of SCI pathophysiology and event sequences, including primary and secondary injury, is important for designing a suitable intervention for SCI.

It has been demonstrated that regulated cell death plays a crucial role in SCI, and the impact of different types of lethal cascades varies in their effect on SCI [8, 9]. Ferroptosis is a type of cell death characterized by iron requirement and reactive oxygen species (ROS) accumulation [10]. The process involves activating, expressing and regulating a series of genes. It was first studied in cancer and then found in many other diseases [11]. Recent studies discovered that ferroptosis plays a vital role in SCI, and a ferroptosis inhibitor can effectively protect neurons and promote motor function recovery [12, 13]. In another similar study, zinc activated the Nrf2 pathway to inhibit ferroptosis occurrence and positively affected nervous system recovery [14].

Ferrostatin-1 is the first ferroptosis inhibitor, which can bind with lipid ROS and protect cells against lipid peroxidation [12]. In this study, we mainly analyzed the function of ferrostatin-1 in SCI both in vitro and in vivo. Therefore, this new direction may provide a new therapeutic theory and approach for recovering neurological function after SCI.

SCI is a major medical challenge facing humanity, and its injury process can be divided into primary and secondary injuries. The bottleneck of treatment lies in effectively dealing with the massive death of nerve cells caused by secondary injury [16]. Therefore, it is crucial to effectively control secondary injury after SCI, promote remaining neurons’ survival, protect neurological function, and promote SCI repair. Although the secondary injury mechanism is currently unclear, the generation of ROS and lipid peroxidation after injury are important reasons for secondary injury. Previous studies have shown that ferroptosis plays a vital role in SCI [17, 18]. In this study, the ferroptosis inhibitor, ferrostatin-1, contributed to the rehabilitation of SCI in vivo, with BMS scores in mice being higher than the NC group within 28 days’ post-injury, and the area of SCI being smaller than the NC group. In vitro experiments showed that ferrostatin-1 could reduce the local Fe²⁺ concentration after SCI and improve the neuronal status after SCI to ensure the survival of more neurons and inhibit scar formation. Further mechanism studies showed that ferrostatin-1 regulated ferroptosis in secondary injury after SCI by inhibiting the Nrf2/HO-1 signaling pathway. This is the first study of the role of the ferroptosis inhibitor ferrostatin-1 in SCI and provides new ideas for future SCI recovery.

Ferroptosis was first defined in 2012 as a regulated form of iron-catalyzed necrosis, which occurs through excessive peroxidation of polyunsaturated fatty acids and is distinct from previously discovered apoptosis, necrosis, and autophagy [10]. Initially, apoptosis and necrosis were considered the two primary types of cell death after SCI [3]. With the discovery of ferroptosis, more and more research has shown its important role in secondary damage after SCI. After SCI, primary injury causes vascular rupture, cell damage, and disruption of the blood–spinal cord barrier, which triggers a cascade of injury, including massive bleeding, release of inflammatory and apoptotic factors in the acute phase, red blood cell aggregation, cell fragmentation, and hemolysis, all of which can lead to Fe²⁺ overload at the site of injury [19, 20]. In addition, emergency injury causes a large amount of ROS, which increases the toxic excitability of glutamate and excessive lipid peroxidation. These factors interact to cause ferroptosis. In 2019, Yao et al. preliminary confirmed the existence of ferroptosis in the early stage of SCI through a rat model of SCI. They found that the iron content, HNE, GPX4, xCT protein levels, and mitochondrial morphology all changed in SCI. Intervention with iron chelator in SCI rats could inhibit the occurrence of ferroptosis, increase neuron survival, alleviate morphological changes in spinal cord tissue, and promote spinal cord function recovery [21]. Zhang et al. found through electron microscopy that ferroptosis-related manifestations appeared 15 min after injury. By using the iron-specific inhibitor SRS16-86 to intervene in SCI rats, they reduced lipid peroxide products, enhanced endogenous antioxidant capacity, and inhibited ferroptosis, thereby promoting spinal cord function recovery [12]. Our research was similar to previous results. Within 28 days after SCI, Fe²⁺ and MDA levels were significantly elevated. On the first day after modeling, GPX4 was significantly downregulated and 4HNE was significantly upregulated, indicating the occurrence of ferroptosis after SCI.

With the discovery of ferroptosis in SCI, more and more scholars have tried to inhibit this process from observing the recovery of spinal cord function. (−)-Epigallocatechin-3-gallate could reduce the death rate of cerebellar granule neurons by increasing the phosphorylation level of PKD1, while inhibiting ferroptosis to promote recovery after SCI [22]. Another study investigated the role of proanthocyanidin in which it was found that the proanthocyanidin-treated group significantly reduced the level of iron accumulation, while also reducing TBARS, ACSL4, and Alox15B and increasing the levels of GSH, GPX4, Nrf2, and HO-1 in the injured spinal cord, which led to the improvement of spinal cord function [23]. These reports demonstrated that iron inhibitors could inhibit ferroptosis from promoting recovery in SCI.

The core of ferroptosis is that damage to the GSH peroxidase system in cells leads to large amounts of lipid ROS accumulation in the cell, inducing cell death. GPX4 is the second largest GSH peroxidase in mammals and is an important regulatory upstream factor in ferroptosis. It can reduce lipid peroxides to their corresponding alcohol forms and interrupt the chain reaction of lipid peroxidation [24, 25]. Chen et al. induced motor neuron degeneration and paralysis in mice by conditionally knocking out the GPX4 gene in neurons, suggesting that ferroptosis could accelerate the progression of SCI [26]. Nrf2 is a key regulator of cellular antioxidant response and plays a key role in preventing lipid peroxidation and ferroptosis. Under normal conditions, Nrf2 in the cell nucleus induces the transcription of the HO-1 gene and upregulates HO-1 protein expression. There is evidence that ferroptosis-related diseases may result from abnormal Nrf2 signaling. A large number of experimental evidence suggests that the Nrf2/HO-1 pathway is involved in the occurrence and development of ferroptosis, and when the Nrf2/HO-1 signaling pathway is activated, ferroptosis in cells is inhibited, and damaged cells and abnormally metabolized lipids are improved [27, 28].

Ferrostatin-1 is a selective iron inhibitor that can remove iron-generated lipid ROS by inhibiting iron-catalyzed oxidation and enzyme-mediated iron-dependent catalysis. Through the intervention of ferrostatin-1, we found that it could significantly promote the recovery of nerve function after SCI. Firstly, the specimens obtained after modeling showed that the injury area of the SCI group formed a cavity, with a large number of cell infiltrations and scar formation. In contrast, the cavity, scar area, and neuron survival in the Ferrostatin-1 group were significantly smaller than those in the SCI group. Next, the BMS scores of mice in the Ferrostatin-1 group at different time points were higher than those in the pure SCI group. All of this indicated that the ferroptosis inhibitor plays a certain role in SCI. Further experimental results suggested that in GPX4, an important upstream regulator of ferroptosis, and key signaling pathway Nrf2/HO-1 of ferroptosis, through the introduction of ferrostatin-1 and the small molecule activator Spinosin, it was found that the inhibitory effect of ferrostatin-1 and the local Fe²⁺ concentration can be restored by small molecule activator, and histologically and behaviorally also have partial recovery effect.

There are some limitations in the present study. First, there is no direct evidence that ferrostatin-1 inhibits neuronal apoptosis after SCI in vitro, and second, there is no specific mechanism of inhibiting scar hyperplasia by ferrostatin-1. However, the article also provided a new direction in the treatment of SCI.

In summary, this study provides in vivo evidence that the ferroptosis inhibitor ferrostatin-1 could improve the rehabilitation neurological functional rehabilitation after SCI, by interaction with the classical Nrf2/HO-1/GPX4 signaling pathway (Fig 6).