Glutathione peroxidase 4 participates in secondary brain injury through mediating ferroptosis in a rat model of intracerebral hemorrhage

Accepted Manuscript Research report Glutathione peroxidase 4 participates in secondary brain injury through mediating ferroptosis in a rat model of intracerebral hemorrhage Zhuwei Zhang, Yu Wu, Shuai Yuan, Peng Zhang, Juyi Zhang, Haiying Li, Xiang Li, Haitao Shen, Zhong Wang, Gang Chen PII: DOI: Reference:

S0006-8993(18)30469-4 https://doi.org/10.1016/j.brainres.2018.09.012 BRES 45944

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Brain Research

Received Date: Revised Date: Accepted Date:

5 March 2018 5 September 2018 7 September 2018

Please cite this article as: Z. Zhang, Y. Wu, S. Yuan, P. Zhang, J. Zhang, H. Li, X. Li, H. Shen, Z. Wang, G. Chen, Glutathione peroxidase 4 participates in secondary brain injury through mediating ferroptosis in a rat model of intracerebral hemorrhage, Brain Research (2018), doi: https://doi.org/10.1016/j.brainres.2018.09.012

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Glutathione peroxidase 4 participates in secondary brain injury through mediating ferroptosis in a rat model of intracerebral hemorrhage

Zhuwei Zhang1#, Yu Wu1#, Shuai Yuan1, Peng Zhang1, Juyi Zhang1, Haiying Li1, Xiang Li1, Haitao Shen1*, Zhong Wang1*, Gang Chen1

1 Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China #These authors contributed equally to this work.

*Corresponding author: Haitao Shen or Zhong Wang, Department of Neurosurgery, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, China. E-mail: [email protected].

Abbreviations: GPX4: glutathione peroxidase 4 ICH: intracerebral hemorrhage SBI: secondary brain injury ROS: reactive oxygen species SD: Sprague–Dawley BBB: blood-brain barrier CNS: central nervous system SOD: superoxide dismutase HO-1: hemeoxygenase-1 GST: glutathione-S-transferase GSH: glutathione NQO1: nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1 TRX: thioredoxin RCD: regulated cell death CSF: Cerebro-Spinal Fluid

LDH: Lactate Dehydrogenase

Abstract Oxidative stress plays an important role in secondary brain injury (SBI) after intracerebral hemorrhage (ICH), but the underling mechanism has not been fully elucidated. Recently, the antioxidant enzyme glutathione peroxidase 4 (GPX4), has attracted increasing attention due to its ability to degrade reactive oxygen species (ROS) which are the major indicator of oxidative stress; However, the role of GPX4 in ICH has not been reported. This study was designed to investigate the changes in protein levels, as well as potential role and mechanism of GPX4 in SBI following ICH using a Sprague-Dawley (SD) rat model of ICH induced by autologous blood injection into the right basal ganglia. Firstly, GPX4 protein levels in the brain were reduced gradually and bottomed out at 24 h after ICH, compared with the Sham group. Secondly, genetic-overexpression of GPX4 effectively increased level of GPX4 in the brain, and clearly relieved neuronal dysfunction, brain edema, blood brain barrier (BBB) injury, oxidative stress and inflammation after ICH. In contrast, inhibiting GPX4 with a specific pharmacological inhibitor or genetic knockdown exacerbated SBI after ICH. Finally, Ferrostatin-1, a chemical inhibitor of ferroptosis, was used to explore the role of ferroptosis in brain injury after ICH. The results suggest that inhibiting ferroptosis can significantly alleviate SBI after ICH. In summary, our work indicated that GPX4 contributes to SBI following ICH by mediating ferroptosis. Therefore, inhibiting ferroptosis with specific inhibitors or upregulation of GPX4 may be a potential strategy to ameliorate brain injury induced by ICH.

Keywords: Glutathione peroxidase 4; Reactive oxygen species; Ferroptosis; Secondary brain injury; Intracerebral hemorrhage

1. Introduction Intracerebral hemorrhage (ICH) is a common acute central nervous system (CNS) disease with high rates of mortality and disability, accounting for about 15% of all

types of stroke in patients (van Asch et al., 2010). Currently, no effective treatment approaches (drug or surgical therapies) exits for ICH, some patients undergo hematoma evacuation, but it is often not an adequate intervention. More than half of all surviving ICH patients have poor neurological outcomes, including motor, sensory, and language deficits (Schlunk and Greenberg, 2015). Previous research has confirmed that brain injury after ICH encompass not only the initial mechanical injury induced by hematoma mass effect and potential hematoma expansion, but also subsequent damage called secondary brain injury (SBI) (Keep et al., 2012). The mechanisms involved in SBI mainly include oxidative stress (especially generation of reactive oxygen species), inflammation, mitochondrial dysfunction, and cell death (including necrosis and apoptosis). These mechanisms cause multiple pathological events in the brain after ICH, such as blood-brain barrier (BBB) damage and brain edema, which eventually lead to more comprehensive brain injury (Zhou et al., 2014). Due to the complicated nature of these often-intertwined mechanisms, they have not been fully elucidated. Reactive oxygen species (ROS) are highly reactive and short-lived small molecules, which include non-radical oxidants, such as hydrogen peroxide (H2O2) and singlet oxygen (1O2), and free radicals, such as the superoxide anion radical (O2•) and hydroxyl radical (•OH) (Zorov et al., 2014). Under normal conditions, ROS are generated as a part of cellular metabolism and physiological defense. ROS are maintained at stable levels by a homeostatic balance of ROS production and elimination performed by mitochondrial oxidative phosphorylation and antioxidant mechanisms, respectively (Ray et al., 2012). However, greater amounts of ROS are rapidly generated in the brain after ICH, which destroys the natural homeostasis between ROS production and elimination systems. Excessive ROS causes lipid peroxidation, protein oxidation, DNA damage and mitochondrial injury, eventually resulting in cell and tissues damage (Qu et al., 2016). In addition, ROS can promote neuronal apoptosis, necroptosis of astrocytes, BBB disruption, and brain edema; therefore, ROS plays a critical role in brain injury after ICH (Duan et al., 2016). At the same time, many antioxidant enzymes are activated in the brain after ICH, such as

superoxide dismutase (SOD), hemeoxygenase-1 (HO-1), glutathione-S-transferase (GST), glutathione (GSH), nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1(NQO1), catalase, and thioredoxin (TRX). These antioxidant enzymes can provide neuroprotective benefits to the brain after ICH by removing excess ROS (Qu et al., 2016). Glutathione peroxidase 4 (GPX4) is an antioxidant enzyme with an essential role in many diseases due to its ability to reduce phospholipid hydroxide (PL-OOH) and prevent lipoxygenase (LOX) overactivation and lipid peroxidation (Seiler et al., 2008). In a previous study, decreasing GPX4 by genetic knockdown increased the cytotoxicity (lipid peroxidation and apoptosis) mediated by glutamate-induced oxytosis in the retina (Sakai et al., 2015). Another study indicated that, GPX4 played an essential role in protecting mitochondria from oxidative damage in gut epithelial cells (Cole-Ezea et al., 2012). It was also reported that, altered GPX4 expression and distribution were associated with pathological changes in the brains of Parkinson’s disease (PD) patients, indicating that increased GPX4 levels could protect neurons against oxidative stress (Bellinger et al., 2011). However, the role of GPX4 in ICH has not been elucidated; therefore, this study specifically examined the role of GPX4 in SBI after ICH. Reflecting the amount of bleeding and the site of occurrence in clinical cases of ICH, an ICH model of controlled autologous whole blood injection in adult male rats was utilized in this study. Recently, ferroptosis, as a newly discovered form of regulated cell death (RCD), has attracted increasing attention (Galluzzi et al., 2015). Interestingly, GPX4 is one of the major upstream regulators of ferroptosis (Dixon et al., 2012; Maiorino et al., 2017). According to prior research and our previous investigations, the forms of cell death involved in SBI after ICH are apoptosis and necroptosis (Shen et al., 2017; Zhou et al., 2014); however, it is not clear whether other forms of RCD occur in neural cells after ICH. Therefore, this study also investigated whether ferroptosis occurs in the brains of rats injured by ICH and its potential role in SBI after ICH. The insights gained from this work will contribute to our understanding of cell death involved in SBI after ICH.

2. Results 2.1 General observations We observed significant differences in the body weights of rats in the Sham and ICH groups. Meanwhile, we found no significant differences in body weight, body temperature, or blood pressure of rats in the different experimental ICH groups (data not shown). No rats died (0/30 rats) in the Sham group, and the mortality rate of the rats with ICH was 20% (42/210 rats). Representative, coronal brain sections of rats in the Sham, ICH, ICH + Vehicle, ICH + RSL-3, and ICH + Ferrostatin-1 groups are shown in Fig. 1A. We were able to measure stable hematoma volumes in ipsilateral hemispheres of rats in the ICH, ICH + Vehicle, ICH + RSL-3, and ICH + Ferrostatin-1 groups 24 h after ICH induction; however, rats in the Sham group only had a hemorrhage points induced by the insertion of the microsyringe. 2.2 GPX4 protein levels were significantly reduced in rats after ICH To test the protein levels of GPX4 in the brain tissue of rats after ICH, the western blot analysis was conducted. Our results revealed that the expression of GPX4 was significantly reduced as early as 12 h post-ICH and reached the lowest point at 24 h after ICH (Fig. 2A and B, P < 0.01). In contrast, the protein levels of GPX4 were increased at 48 h after ICH, compared with its expression level at 24 h after ICH (Fig. 2A and B, P < 0.01). Additionally, we found no statistical difference in GPX4 expression between the Sham group and the Normal group (Fig. 2A and B, P > 0.05). To determine the cellular distribution of GPX4 in brain tissue after ICH, we performed double-immunofluorescence staining with antibodies for GPX4 and the neuron-specific marker (NeuN). Our results suggest that the ICH-induced decrease in the protein level of GPX4 was mainly true in neurons (Fig. 2C and D, P < 0.01). Based on these findings, the following experiments were performed 24 h after ICH. 2.3 Inhibiting GPX4 by pharmacological inhibition or genetic-knockdown aggravated brain injury in rats after ICH To determine whether GPX4 is involved in brain injury after ICH, we used RSL-3 (a specific inhibitor of GPX4), short-interfering RNA (siRNA) knockdown of GPX4 (Si-GPX4), as well as adenovirus-mediated overexpression of GPX4 (Ad-GPX4) in

rats with ICH.

The controls for Si-GPX4 and Ad-GPX4 were scrambled siRNA

(negative control siRNA; Si-NC) and an adenovirus expressing an empty vector (Ad-Vector), respectively. Using western blotting and double-immunofluorescence staining, we confirmed that, RSL-3 and Si-GPX4 treatment markedly reduced the protein levels of GPX4, and transfection with recombinant adenovirus (Ad-GPX4) significantly increased the expression of GPX4 in rats after ICH (all P < 0.01, Fig. 3A-D). The levels of IL-1β and TNF-α in the serum and Cerebro-Spinal Fluid (CSF) were detected by ELISA. We found that, IL-1β and TNF-α levels were significantly increased in ICH-injured rats treated with RSL-3 (compared with Vehicle) and Si-GPX4 (compared with Si-NC). In contrast, IL-1β and TNF-α levels were clearly decreased in the ICH + Ad-GPX4 group compared with the ICH + Ad-Vector group. These results suggest that inhibiting GPX4 can promote inflammation in the brain tissue of ICH-injured rats (all P < 0.01, Fig. 4A-D). The results of Nissl staining indicated that there were fewer surviving neurons in the temporal cortex and the hippocampal CA2 region of RSL-3 and Si-GPX4 -treated rats compared with Vehicle- and Si-NC-treated rats, respectively; On the contrary, the number of the surviving neurons was significantly greater in the ICH + Ad-GPX4 group compared with those in the ICH + Ad-Vector group. These results demonstrated that inhibiting GPX4 aggravated neuronal loss in ICH-injured rats (all P < 0.01, Fig. 4E-G). Additionally, we also measured the levels of ROS, an indicator of oxidative stress, and Lactate Dehydrogenase

(LDH), as an index of necrosis. The results indicated

that, ROS and LDH levels were both significantly increased in the ICH + RSL-3 group and the ICH + Si-GPX4 group compared with the ICH + Vehicle group and the ICH + Si-NC group respectively. However, ROS and LDH levels were significantly lower in the ICH + Ad-GPX4 group than in the ICH + Ad-Vector group. These results indicated that inhibiting GPX4 could exacerbate oxidative stress and necrosis in rats injured by ICH (all P < 0.01, Fig. 5A and C). The neurological scores of rats in the ICH + RSL-3 group and the ICH + Si-GPX4 group were higher than those in the ICH

+ Vehicle group and the ICH + Si-NC group respectively; but scores were markedly lower in the ICH + Ad-GPX4 group compared with the ICH + Ad-Vector group. These results indicated that inhibiting GPX4 exacerbated neurological defects in rats after ICH (all P < 0.01, Fig. 5B). BBB permeability was measured by albumin extravasation, and subsequent western blotting for albumin protein in the brain tissue of rats in different experimental groups. We found that, compared with the corresponding controls, treatment with RSL-3 and Si-GPX4 promoted albumin extravasation and BBB injury in ICH-injured rats, while treatment with Ad-GPX4 produced the opposite result (all P < 0.01, Fig. 5D and E). Brain water content was calculated by the wet/dry weight method. The results showed that while brain water content was significantly increased in the ICH + RSL-3 group and the ICH + Si-GPX4 group compared with their corresponding controls, it was clearly decreased in the ICH + Ad-GPX4 group compared with the ICH + Ad-Vector control (all P < 0.01 in Ipsi-CX and Ipsi-BG, Fig. 5F). Taken together, our findings indicated that GPX4 participated in numerous processes of brain injury after ICH, including inflammation, neuronal loss, BBB injury, neuronal dysfunction, brain edema, oxidative stress and cell death. 2.4 Blockade of ferroptosis by Ferrostatin-1 can improve brain injury in rats after ICH Previous studies have indicated that GPX4 mediated ferroptosis participates in the progression of various diseases. Thus, to further determine whether GPX4 mediated ferroptosis participates in brain injury after ICH, Ferrostatin-1, a specific inhibitor of ferroptosis, was employed. As shown in Fig. 6, administration of Ferrostatin-1 effectively suppressed the level of GPX4 in the brain at 24 h after ICH. Moreover, treatment with Ferrostatin-1 markedly reduced the levels of IL-1β and TNF-α in the serum and CSF compared with the levels in the corresponding Vehicle groups. These results indicated that inhibiting ferroptosis with Ferrostatin-1 can alleviate inflammation in ICH-injured rats (all P < 0.01, Fig. 7A-D). We also assessed the BBB permeability of Ferrostain-1- and Vehicle-treated ICH rats and revealed that treatment with Ferrostatin-1 significantly decreased albumin extravasation and BBB injury after

ICH injury (P < 0.01, Fig. 7E and F). The results of the ROS and LDH assays indicated that, the levels of ROS and LDH were both significantly reduced in the ICH + Ferrostatin-1 group compared with levels in the ICH + Vehicle group (both P < 0.01, Fig. 8A and D). Similarly, brain water content was also significantly reduced in the ICH + Ferrostatin-1 group compared with that in the ICH + Vehicle group (P < 0.01 in Ipsi-CX and Ipsi-BG, Fig. 8B). We observed significantly higher neurological scores in rats in the ICH + Ferrostatin-1 group compared with the ICH + Vehicle group (P < 0.01, Fig. 8C). Nissl staining showed that inhibition of ferroptosis by Ferrostatin-1 caused an increasing number of surviving neurons in the temporal cortex and hippocampal CA2 region compared with the ICH + Vehicle group (Fig. 8E-G). Taken together, these results supported the involvement of ferroptosis in brain injury after ICH, including processes of inflammation, neuronal loss, BBB injury, neuronal dysfunction, brain edema, and oxidative stress. 2.5 Effects of RSL-3, Si-GPX4, Ad-GPX4, and Ferrostatin-1 treatments on cognitive behavior after ICH in rats To further confirm that inhibiting GPX4 chemically or genetically aggravates brain injury in rats after ICH, we used the Morris water maze to assess cognitive function. Rats in the ICH group showed severe impairments in cognitive behavior compared with those in the Sham group (P < 0.01, Fig. 9A, D, and E). As expected, there was no statistical difference in the behavior of ICH-injured rats and ICH rats treated with the various controls (drug Vehicles, Si-NC, and Ad-Vector) (all P > 0.05, Fig. 9A-C). After Ad-GPX4 or Ferrostain-1 treatments, the impairment of cognitive behavior induced by ICH was noticeably improved (both P < 0.05), while the RSL-3 and Si-GPX4 treatments produced the opposite effects (both P < 0.01, Fig. 9A, D-G).

3. Discussion Cell death plays an essential role in SBI after ICH. About 20 years ago, two studies reported that both apoptotic and necrotic cells existed in the perihematomal region after surgical evacuation of hematomas in patients and in the brains of animals

injured by ICH (Hickenbottom et al., 1999; Qureshi et al., 2001). Nonetheless, almost all previous investigations concluded that apoptosis is the only form of cell death that occurs in brain tissue after ICH; few studies noticed that necrosis also contributed to SBI after ICH (Salihu et al., 2016). Oxidative stress is a major cause of brain injury after ICH, and its primary trigger is excessive ROS production (Duan et al., 2016). Superfluous ROS can damage the outer mitochondrial membrane, lead to cytochrome-c release, and activate caspase-dependent pathways, thereby contributing to apoptosis in brain injury after ICH (Qu et al., 2016). As an antioxidant enzyme, GPX4 inhibits lipid peroxidation by degrading H2O2, some small-molecule peroxides, and complex lipid peroxides, as well as GSH (Ursini et al., 1995). In this study, we explored the role of GPX4 in an autologous blood injection model of ICH for the first time. Our results showed that protein levels of GPX4 gradually decreased and bottomed out at 24 h after ICH, suggesting that GPX4 may be involved in SBI induced by ICH. These encouraging results led us to investigate the effects of these changes in GPX4 protein levels on SBI using pharmacological methods (a specific inhibitor of GPX4, RSL-3), as well as genetic knockdown and overexpression techniques. Our results showed that reduced GPX4 levels could aggravate the degree of brain injury following ICH by decreasing the number of surviving neurons, exacerbating brain edema, disrupting the BBB, inducing neurological dysfunction, and promoting inflammation and oxidative stress. However, upregulation of GPX4 by viral-mediated overexpression produced the opposite effects. Collectively, these results suggest that reduced GPX4 can promote SBI and amplify the pathophysiological progression of ICH. Ferroptosis is a recently discovered form of regulated necrosis that has attracted increasing attention in recent years. It is mediated by GPX4 and is characterized by metabolic dysfunction that leads to excessive ROS generation via an iron-dependent pathway (Cardoso et al., 2017). Dolma and colleagues discovered erastin, a chemical compound that can lead to cell death of RAS-mutated tumor cells in a manner different from apoptosis (Dolma et al., 2003). In 2012, they coined the term ‘ferroptosis’ to describe this form of cell death induced by the accumulation of

iron-dependent lipid peroxides (Dixon et al., 2012). Ferroptosis is caused by the inactivation of GPX4 via GSH depletion and accumulation of ROS from lipid peroxidation. The primary morphological features of ferroptosis include cell volume shrinkage, lack of rupture and blebbing of the plasma membrane, and small mitochondria with condensed membrane densities lacking the traditional apoptotic and necrotic phenotypes (Yang et al., 2014). Normally, the cystine–glutamate antiporter, system Xc−, exchanges one molecule of extracellular cystine for one molecule of intracellular glutamate. Once inside the cell, cystine is reduced by reduced glutathione (GSH) to cysteine, which is subsequently used for protein and GSH synthesis. GPX4 preferentially reduces phospholipid hydroperoxide (PL‑ OOH) to its corresponding alcohol phospholipid hydroxide (PL‑ OH) by using two molecules of GSH. GPX4 is one of the central upstream regulators of ferroptosis; it prevents lipoxygenase (LOX) overactivation and lipid peroxidation. Concerted action of these pathways is essential to control the formation of oxidized phospholipids (Conrad et al., 2016). Based on these facts, we hypothesized that, following ICH injury, excessive amounts of glutamate disrupt system Xc− and inhibit the transfer of cysteine. This would, in turn, reduce GSH synthesis and decrease GPX4 activity. As a result, excessive ROS and oxidative lipids would not be cleared up and would eventually induce ferroptosis in the brain (Fig. 10). To validate this hypothesis, we performed a preliminary investigation to test whether ferroptosis is involved in the brain injury induced by ICH by using a specific inhibitor of ferroptosis, ferrostatin-1. Our results confirmed that ferrostatin-1 treatment significantly increased the protein level of GPX4 in brain tissue after ICH and attenuated neuronal dysfunction, brain edema, BBB injury, oxidative stress, and inflammation induced by ICH. These findings suggest that ferroptosis is indeed involved in SBI after ICH and can be considered as a potential target for the treatment of ICH. As previous studies from our and other groups reported, the forms of cell death involved in SBI after ICH include both apoptosis and necroptosis (Shen et al., 2017; Zhou et al., 2014). In this study, we showed for the first time that ferroptosis, another type of regulated necrosis, existed in the ICH-injured brain and was involved in

ICH-induced brain injury. Taking all the above findings together, we can conclude that multiple forms of cell death coexist in the brain following ICH. However, whether these types of cell death associate with each other to jointly promote the progression of SBI in ICH needs further investigation. Marietta et al. reported that treatment with some chemical inhibitors of ferroptosis (including cycloheximide, actinomycin D, ferrostatin-1, deferoxamine, N-acetylcysteine, and Trolox) alleviated cellular toxicity induced by ICH in vitro (Zille et al., 2017). Another recent study also showed that administration of Ferrostatin-1 reduced neuronal death and iron deposition induced by hemoglobin treatment in organotypic hippocampal slice cultures, exhibited clear neuroprotective effects, and improved neurologic function in a collagenase-induced ICH model in mice (Li et al., 2017). Our results corroborate these findings; however, due to the correction of necrosis and inflammatory reactions (Linkermann et al., 2014b), the side effect of collagenase-induced exaggerated inflammation that is directly toxic to neurons should not be ignored in such necrosis-related investigations (MacLellan et al., 2008; MacLellan et al., 2010; Participants, 2005). As a type of regulated necrosis, ferroptosis can also induce inflammation by plasma membrane rupture and subsequent release of cytoplasmic contents (Linkermann et al., 2014); thus, we used the autologous blood injection-induced ICH model in our study. We observed that treatment with Ferrostatin-1 significantly reduced the levels of inflammatory factors (IL-1β and TNF-α) in the CSF of rats after ICH. These results suggest that ferroptosis can induce inflammatory reactions in the brain after ICH; however, the downstream effects of this occurrence need to be investigated. In addition, we noticed that the autologous blood injection model had some limitations in the study of long-term functional outcomes following ICH (Kirkman et al., 2011). In light of this finding, our future work will explore the suitability of the collagenase-induced ICH model for the long-term study of functional outcomes after ICH and ferroptosis inhibitor treatment. Another limitation of our current study was the sole use of adult, male rats, while clinical scenarios often include female and/or older ICH patients (some with cardiovascular diseases). In addition, some previous

studies suggested that iron overload plays a key role in brain injury after ICH (Zheng et al., 2015), and we know that ferroptosis is induced by accumulation of iron-dependent lipid peroxides; however, whether there is an association between iron overload and ferroptosis in ICH is uncertain. In summary, our study confirmed that GPX4 plays an important role in brain injury following ICH and that the ICH-induced decrease in GPX4 may facilitate the role of ferroptosis in SBI after ICH. We also demonstrated that there are multiple types of cell death involved in brain injury after ICH and Ferrostatin-1 may present a potential therapeutic target for the treatment of SBI following ICH.

4. Materials and methods 4.1 Ethics and animals All experimental procedures were approved by the Institutional Animal Care Committee of Soochow University and were performed in accordance with the guidelines of the National Institutes of Health on the care and use of animals. Male Sprague Dawley (SD) rats weighing 280-300 g were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. All rats were housed at a constant temperature of 22°C, under a 12 h light/dark cycle (light switched on at 6:00 am) with free access to food and water. All rats were placed under general anesthesia before fixation-perfusion and euthanasia procedures. All data from animal experiments were reported in accordance with ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Sample sizes were determined by power analyses that were also approved in the animal ethics application for this study. All efforts were made to minimize the number of animals used and any pain or suffering they experienced.

4.2 Experimental grouping The same method of random assignment was used to create all the experimental groups; rats were assigned using a random-number table by a technician who was blinded to the experimental groups. First, all 238 rats in the study cohort were

numbered and randomly divided into three groups: 6 rats went to the Normal group, 30 rats went to the Sham groups, and the rest (202 rats) went to the different ICH groups. Next, the 30 sham-operated rats (all survived the surgical procedure) were further divided into groups for Experiments 1, 2, and 3. Similarly, following the induction of ICH, the 162 rats that survived the surgical procedure were divided into different groups for Experiments 1, 2, and 3 (details shown below). Experiment 1 was comprised of nine groups: the Normal group (6 rats), Sham group (6 rats randomly assigned from among the sham-operated animals), and seven experimental groups ordered by time: 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 168 h after ICH (6 rats per group; randomly assigned from among the ICH-injured animals). At the indicated time points after ICH, the rats were deeply anesthetized with chloral hydrate, and cerebral tissue samples were collected for analysis following transcardial perfusion with PBS. Whole-brain coronal sections (including temporal lobe tissue) were taken from 3 rats in each group for immunofluorescence analysis, and the other 3 rat brains were frozen in liquid nitrogen for western blot analysis (Fig. 1B). Experiment 2 was comprised of eight groups (12 rats per group; randomly assigned from among the sham-operated or ICH-injured animals): the Sham group, ICH group, ICH + Vehicle group, ICH + RSL-3 group, ICH + Negative Control siRNA (Si-NC) group, and ICH + GPX4 SiRNA (Si-GPX4) group, ICH + Ad-Vector group, and the ICH + Ad-GPX4 group (Fig. 1C). Experiment 3 was comprised of four groups (12 rats per group; randomly assigned from among the sham-operated and ICH-injured animals): the Sham group, ICH group, ICH + Vehicle group, and ICH + Ferrostatin-1 group (Fig. 1D). At 24 h after ICH, all rats assigned to groups in Experiments 2 and 3 were examined for behavioral impairment, then immediately euthanized to collect cerebral tissue, blood, and Cerebro-Spinal fluid (CSF) samples for further analysis. In each of these groups, 6 of 12 rats were randomly assigned for western blot analysis, immunofluorescence analysis, Nissl staining, ROS and LDH analysis, and ELISA, and the other 6 rats were used to assess brain edema. The “n” is defined in every figure legend as the sample size for each independent experiment. Detailed

information about each group is included in the individual method descriptions below. 4.3 ICH model in rats As described in our previous study (Wang et al., 2017), an autologous whole-blood injection in adult male rats was utilized as the ICH model in this study. Briefly, SD rats were anesthetized with an intraperitoneal (i.p.) injection of 4% chloral hydrate (10 ml/kg) and secured on a stereotaxic apparatus (ZH-Lanxing B type stereotaxic frame, Anhui Zhenghua Biological Equipment Co. Ltd., Anhui, China). Then, the scalp was exposed and a hole drilled above the right basal ganglia (0.2 mm anterior and 3.5 mm lateral to bregma). A microsyringe was affixed to the stereotaxic frame and a needle slowly inserted to a depth of 5.5 mm. Then, 100 μl autologous blood collected from the heart was slowly injected (20 μl/min) into the right basal ganglia. The needle was kept in place for an additional 5 min at the end of the injection. Sham-operated rats were intracerebrally injected with 100 μl physiological saline solution. Then, the burr hole was sealed with bone wax, and the skin incision was disinfected and sutured. During the ICH surgical procedure, the room temperature was maintained at 23 ± 1°C, the animal’s heart rate monitored, and rectal temperature maintained at 37 ± 0.5°C. 4.4 Drug administration Based on previous research, RSL-3 (a chemical inhibitor of GPX4) and Ferrostatin-1 (a specific inhibitor of ferroptosis) were used in this study. These two inhibitors (both purchased from Selleckchem, Shanghai, China) were dissolved in DMSO and diluted to their final concentrations with 0.9% normal saline. Both were injected i.p. at a dose of 2 mg/kg 2 h before induction of ICH (Linkermann et al., 2014a; Yang et al., 2014). Vehicle-treated rats received an equal volume of Vehicle, which was also injected i.p. at the same timepoint. 4.5 Transfection of siRNA and adenoviruses in vivo As described in our previous study (Shen et al., 2017), two kinds of small interfering RNAs (siRNA) were applied in this experiment: siRNA with a specific sequence recognizing rat GPX4 mRNA (Si-GPX4), which silences GPX4 transcription; and scrambled siRNA (Si-Negative Control, both from Genescript, Nanjing, China). According to the manufacturer instructions for Entranster-in vivo RNA transfection

reagent (Engreen, Shanghai, China), 500 pmol scrambled siRNA and 500 pmol GPX4 siRNA were separately dissolved in 5 μl RNase-free water. Subsequently, 10 μl Entranster-in vivo RNA transfection reagent was added and mixed for 15 min. Finally, the Entranster-in vivo-siRNA mixture was injected intracerebroventricularly (i.c.v.) 24 h before ICH induction. The GPX4 siRNA sequences are shown here: Sense: 5’ UGGUUUGCCUGGAUAAGUA dTdT 3’ Antisense: 3’ dTdT ACCAAACGGA CCUAUUCAU5’ Two kinds of recombinant adenoviruses were also used in this study: an adenovirus expressing rat GPX4 (Ad-GPX4; Genbank ID: 90903248), which was used to overexpress GPX4 protein; and an adenovirus expressing only the empty vector (Ad-Vector) as a negative control for Ad-GPX4. Ad-GPX4 and Ad-Vector (both at 3×109 pfu/ml titer) were produced by Genescript (Nanjing, China). Both were stored at −80°C and diluted to 1×109 pfu/ml in enhanced transfection solution (Genescript, Nanjing, China) before being injected i.c.v. in rats. 4.6 Western blot analysis As described previously (Shen et al., 2015), brain tissue samples were mechanically lysed in a lysis buffer containing phenylmethylsulfonyl fluoride (PMSF). The bicinchoninic acid (BCA) method (Enhanced BCA Protein Assay Kit from Beyotime Institute of Biotechnology, Shanghai, China) was used to detect the protein concentration of each sample. A molecular weight marker (5 μl/lane; Thermo Fisher Scientific, Waltham, MA, USA) and the protein samples (30 μg/lane) were loaded on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, separated, and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA), which was subsequently blocked with 5% skim milk for 1 h at room temperature. Then, the membrane was incubated overnight at 4°C with primary antibodies; anti-GPX4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted at 1:5000, and anti-β-tubulin served as the loading control. Next, the membrane was incubated with a horseradish peroxidase (HRP)-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 37°C and washed three times with PBST (PBS + 0.1% Tween 20). Finally, an

enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for signal detection. The density of protein bands was analyzed using Image J software (NIH, Bethesda, MA, USA) and normalized to that of the corresponding loading controls. 4.7 Immunofluorescence and microscopy The immunofluorescence procedure was performed as described previously (Dou et al., 2017). Briefly, the brain was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. Following antigen retrieval and blocking against non-specific-binding, the brain sections were incubated with primary antibodies for GPX4 and NeuN (a neuronal marker; Abcam, Cambridge, UK, 1:300 dilution) overnight at 4°C. Subsequently, the brain sections were washed three times with PBST (PBS + 0.1% Tween 20) and incubated with appropriate secondary antibodies (Alexa Fluor 488 donkey anti-rabbit IgG antibody and Alexa Fluor 555 donkey anti-mouse IgG antibody, Life Technologies, Carlsbad, CA, USA, 1:500 dilution) at 37°C for 1 h. Next, these brain sections were washed three times with PBST and coverslipped with anti-fading mounting medium containing 4,6-diamino-2-phenylindole (DAPI, SouthernBiotech, Birmingham, AL, USA). Finally, the brain sections were observed under a fluorescence microscope (Olympus BX50/BX-FLA/DP70, Olympus Co., Tokyo, Japan) and analyzed using Image J software (NIH, Bethesda, MA, USA) by a technician who was blinded to the experimental groups. We obtained at least three photomicrographs per section, and at least two sections from each rat were used for final quantitative analysis of each group. 4.8 ELISA At 24 h after ICH, blood and Cerebro-Spinal fluid (CSF) samples were collected from each rat under terminal anesthesia by puncturing the heart and foramen magnum, respectively. The CSF samples were immediately centrifuged (12000g) for 30 min at 4°C, whereas the blood samples were centrifuged (1000g) for 5 min at 4°C. Subsequently, the supernatants were collected to detect the levels of TNF-α and IL-1β using specific ELISA kits (Bio-Swamp, Hubei, China) according to the manufacturers’

instructions. 4.9 Reactive oxygen species (ROS) detection assay The levels of ROS in the brain served as an index of oxidative stress and were detected by the Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). The collected brain tissue was homogenized and then centrifuged at 12000g for 10 min at 4°C, and the supernatants were used for the ROS assay. ROS levels were measured using the oxidant-sensitive probe 2,7-dichlorofluorescein diacetate (DCF-DA) as per manufacturer instructions. A fluorometric microplate reader (FilterMax F5, Molecular Devices, Sunnyvale, CA, USA) was used to measure the fluorescence intensity of each sample. The levels of ROS were expressed as fluorescence intensity/mass of total protein (mg) and the data from each group was normalized to the Sham group. 4.10 Brain water content and behavioral tests As described in previous research (Wang et al., 2016), rats were injected i.p. with 4% chloral hydrate 24 h after ICH induction, and the intact brain removed immediately. The brain was divided into two hemispheres along the midline, and each hemisphere was further dissected into two parts containing the cortex and basal ganglia. The resulting tissue was then subdivided into five samples: contralateral basal ganglia (Cont-BG), contralateral cortex (Cont-CX), cerebellum (CB), ipsilateral basal ganglia (Ipsi-BG), and ipsilateral cortex (Ipsi-CX). These brain samples were immediately weighed with an electronic analytical balance and their wet weights recorded. Then, the samples were dried in a thermostatic drier at 100°C for 72 h (or until the dried sample weights were consistent) to obtain the dry weights. Brain water content was calculated as (wet weight − dry weight)/(wet weight) × 100%. Behavioral tests for neurological scoring were performed 1 h prior to euthanasia. All rats in the experimental groups were tested using a previously published scoring system and monitored for appetite, activity, and neurological defects (Shen et al., 2017). More details are shown in Table 1. 4.11 BBB Injury BBB permeability was assessed on the basis of albumin extravasation. Under normal

conditions, the existence of the BBB keeps the albumin level in the brain very low; however, the level of albumin significantly increases once the BBB is damaged. Thus, a change in albumin levels in the brain is considered an indicator of the degree of BBB injury (Fleegal-DeMotta et al., 2009). Accordingly, western blotting was performed to measure the levels of albumin in the brain tissue of rats in each group. Briefly, the tissue samples were mechanically lysed in a lysis buffer containing PMSF. The BCA method (Enhanced BCA Protein Assay Kit from Beyotime Institute of Biotechnology, Shanghai, China) was used to detect the protein concentration of each sample. The molecular weight marker (5 μl/lane; Thermo Fisher Scientific, Waltham, MA, USA) and protein samples (30 μg/lane) were loaded on a 10% SDS-polyacrylamide gel, separated, and electrophoretically transferred to a PVDF membrane (Millipore Corporation, Billerica, MA, USA), which was subsequently blocked with 5% skim milk for 1 h at room temperature. Next, the membrane was incubated overnight at 4°C with primary antibodies; anti-albumin (Abcam, Cambridge, MA, USA) was diluted at 1:5000 and anti-β-tubulin served as the loading control. Next, the membrane was incubated with an HRP-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 37°C and washed three times with PBST (PBS + 0.1% Tween 20). Finally, an ECL kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for signal detection. The density of the protein bands was analyzed using Image J software (NIH, Bethesda, MA, USA) and normalized to that of the corresponding loading control. 4.12 Nissl staining Nissl staining was performed to measure neuronal loss in rats from each group as described previously (Tian et al., 2017). Paraffin-embedded sections were dewaxed and then hydrated with distilled water. Next, sections were stained with 0.5% toluidine blue for 40 min at 37°C, dehydrated with graded concentrations of alcohol, and then cleared in xylene. Finally, the sections were covered in glass coverslips with neutral resins and evaluated under a light microscope (Olympus CX53, Olympus Co., Tokyo, Japan). The perihematomal temporal cortex and hippocampal CA2 region were observed. Surviving neurons with pale nuclei and large cell bodies were counted

and averaged. Dark-stained neurons and those with shrunken cell bodies were considered dead and were not counted. 4.13 LDH assay The concentration

of

LDH in the CSF was

detected using a specific LDH

assay kit (Jiancheng Biotech, Nanjing, China). This assay was performed according to the manufacturers’ instructions, and all data were expressed relative to corresponding standard curves. 4.14 Morris water maze Cognitive capacity was tested using the Morris water maze as previously described (Guo et al., 2017). In brief, the water maze (divided into four quadrants) had a total diameter of 120 cm, and the circular target platform was 10 cm in diameter and 40 cm in height. The target platform was wrapped with black corded fabric and placed in the middle of the third quadrant, with the platform height 2 cm below the water surface. The water temperature was maintained at 25 ± 2°C, and the water was blackened with ink. The visual points of reference around the pool remained unchanged during the training and testing phases. During the training phase, the rats were placed in the pool from all four quadrants and given 60 s to find the target platform. If the rats could not find the target platform within 60 s, we used a rod to guide them to the platform. After reaching the target platform, rats were allowed to stay there for 10 s to solidify their learning and memory, and then they were removed from the maze. This method was applied for five consecutive days of training, and the ICH surgery was performed on the sixth day. The time taken from the starting point to the target platform was recorded and defined as the escape latency (EL). In the testing phase, the rats were placed in the pool from a randomly chosen quadrant and allowed to find the target platform. Over the next four days of testing, their ELs and swimming distances were recorded. The mean EL and distance were used as indicators of learning ability and cognitive function. The parameters of the entire experiment were recorded on a computer. 4.15 Statistical Analysis All data are expressed as the mean ± SEM. GraphPad Prism 5.0 software (San Diego,

USA) was used for statistical analysis. The data sets in each group were tested for normality of distribution with a Kolmogorov-Smirnov test. The two data groups with normal distributions were compared using the two-tailed, unpaired, Student’s t- test, and the Mann-Whitney U test was used for the non-parametric data. Values of P < 0.05 were considered statistically significant.

Conflict of Interest Statement: The authors declare that they have no competing interest.

Role of Authors: All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Gang Chen, Zhong Wang and Haitao Shen. Acquisition of data: Zhuwei Zhang and Yu Wu. Analysis and interpretation of data: Shuai Yuan, Peng Zhang, Juyi Zhang, Haiying Li, and Xiang Li. Drafting manuscript: Zhuwei Zhang and Yu Wu. Statistical analysis and critical revision of the manuscript for important intellectual content: Zhuwei Zhang and Haitao Shen. Administrative, technical, and material support: Haiying Li, and Xiang Li. Study supervision: Gang Chen, Zhong Wang and Haitao Shen.

Acknowledgments: This work was supported by a grant from the National Natural Science Foundation of China (No. 81601007).

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Figure legends Figure 1 Intracerebral hemorrhage (ICH) model and experimental design. (A) Coronal sections from rats in various groups at 24 h after ICH. (B) Experiment 1 was designed to detect the changes in GPX4 protein levels in rats after ICH and to determine a time point for subsequent experiments. (C) Experiment 2 was designed to explore the roles of GPX4 in brain injury after ICH in rats. (D) Experiment 3 was designed to investigate the role of ferroptosis in brain injury after ICH in rats.

Figure 2 The protein levels of GPX4 were decreased in the brain tissue of rats after ICH. (A) Western blots show that GPX4 protein levels progressively decrease and bottom out at 24 h after ICH. (B) Quantitative analysis of the relative protein levels of GPX4 normalized to the mean value of the Normal group. All values are expressed as means ± SEM.

@@

P < 0.01 compared with the Sham group;

&&

P < 0.01 and

$$

P<

0.01 compared with the ICH group evaluated at 24 h; NS, no significant difference compared with the Normal group; n = 6. (C) Double-immunofluorescence analysis was performed with antibodies against GPX4 (green) and NeuN (neuronal marker, red), and nuclei were fluorescently labeled with DAPI (blue). Arrows point to GPX4-positive neurons. Scale bar = 32 μm. (D) Quantification of the relative fluorescence intensities normalized to the mean fluorescence intensity of the Normal group. All values are expressed as means ± SEM. **P < 0.01 compared with the Sham group; NS, no significant difference compared with the Normal group; n = 6.

Figure 3 Effects of a pharmacological inhibitor, genetic knockdown, or overexpression on the GPX4 protein levels in rats at 24 h after ICH. (A) Western blots show that GPX4 protein levels are significantly decreased in the RSL-3 and Si-GPX4 treatment groups but are increased in the ICH + Ad-GPX4 group. (B) Quantification of relative protein levels of GPX4 in rats in various groups. Data are presented as means ± SEM. **P < 0.01 compared with the Sham group; with the ICH + Vehicle group; and

$$

@@

P < 0.01 compared

&&

P < 0.01 compared with the ICH + Si-NC group;

P < 0.01 compared with the ICH + Ad-Vector group; NS, no significant

difference compared with the ICH group; n = 6. (C) Double-immunofluorescence analysis was performed with antibodies for GPX4 (green) and NeuN (neuronal marker, red), and nuclei were fluorescently labeled with DAPI (blue). Arrows point to GPX4-positive neurons. Scale bar = 32 μm. (D) Quantification of the relative fluorescence intensity of GPX4, where all values are normalized to the mean fluorescence intensity of the Sham group. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

@@

P < 0.01 compared with the ICH +

Vehicle group;

&&

P < 0.01 compared with the ICH + Si-NC group;

$$

P < 0.01

compared with the ICH + Ad-Vector group; NS, no significant difference compared with the ICH group; n = 6.

Figure 4

Inhibiting GPX4 by pharmacological blockade or genetic knockdown

aggravated inflammation and neuronal loss in the brain tissue of rats after ICH. (A–D) The levels of TNF-α and IL-β in the serum and Cerebro-Spinal fluid (CSF), measured by ELISA. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

##

P < 0.01 compared with the ICH + Vehicle group;

@@

P < 0.01

compared with the ICH + Si-NC group; $P < 0.05 and $$P < 0.01 compared with the ICH + Ad-Vector group; NS, no significant difference compared with the ICH group; n = 6. (E) Nissl staining in the hippocampal CA2 region and temporal cortex shows that the overexpression of GPX4 significantly inhibited neuronal loss in rats after ICH. Arrows point to surviving neurons. Scale bar = 32 μm. (F and G) The number of surviving neurons in the hippocampal CA2 region and temporal cortex. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

##

P < 0.01

compared with the ICH + Vehicle group; @@P < 0.01 compared with the ICH + Si-NC group;

$$

P < 0.01 compared with the ICH + Ad-Vector group; NS, no significant

difference compared with the ICH group; n = 6.

Figure 5 Overexpression of GPX4 by transfection with recombinant adenoviruses expressing GPX4 attenuated brain injury in rats after ICH. (A) The levels of ROS in the brain tissue of rats in various groups. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group; ##P < 0.01 compared with the ICH + Vehicle group; @@P < 0.01 compared with the ICH + Si-NC group; $$P < 0.01 compared with the ICH + Ad-Vector group; NS, no significant difference compared with the ICH group; n = 6. (B) The neurological scores of rats in the different groups. All data are expressed as individual values. **P < 0.01 compared with the Sham group; #P < 0.05 compared with the ICH + Vehicle group; @P < 0.05 compared with the ICH + Si-NC

group; $P < 0.05 compared with the ICH + Ad-Vector group; NS, no significant difference compared with the ICH group; n = 6. (C) The levels of LDH in the CSF of rats in each group. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

##

P < 0.01 compared with the ICH + Vehicle group;

compared with the ICH + Si-NC group;

$$

@@

P < 0.01

P < 0.01 compared with the ICH +

Ad-Vector group; NS, no significant difference compared with the ICH group; n = 6. (D) The level of albumin (index of BBB injury) in the rat brain increased after ICH, whereas it significantly reduced in Ad-GPX4-treated rats. (E) Quantification of the relative protein levels of albumin in the different groups. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group; NS, no significant difference compared with the ICH group; Vehicle group;

@@

##

P < 0.01 compared with the ICH +

P < 0.01 compared with the ICH + Si-NC group;

$$

P < 0.01

compared with the ICH + Ad-Vector group; n = 6. (F) Brain water content was partially reduced by overexpression of GPX4. All data are expressed as means ± SEM. **P < 0.01 (both in Ipsi-CX and Ipsi-BG) compared with the Sham group; NS, no significant difference compared with the ICH group; #P < 0.05 compared with the ICH + Vehicle group; @P < 0.05 compared with the ICH + Si-NC group; $P < 0.05 compared with the ICH + Ad-Vector group; n = 6.

Figure 6 The effects of Ferrostatin-1 on GPX4 protein levels in rats at 24 h after ICH. Ferrostatin-1, a specific inhibitor of ferroptosis, is used in this study to explore whether ferroptosis participates in brain injury after ICH. (A) Western blots show that the protein level of GPX4 is significantly increased in the Ferrostatin-1 treatment group. (B) Quantification of relative GPX4 protein levels in rats in various groups. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group; &&P < 0.01 compared with the ICH + Vehicle group; NS, no significant difference compared with the ICH group; n = 6. (C) Double-immunofluorescence analysis was performed with antibodies for GPX4 (green) and NeuN (neuronal marker, red), and nuclei were fluorescently labeled with DAPI (blue). Arrows point to GPX4-positive neurons. Scale bar = 32 μm. (D) Quantification of the relative fluorescence intensity

of GPX4, where all values are normalized to the mean fluorescence intensity of the Sham group. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

&&

P < 0.01 compared with the ICH + Vehicle group; NS, no significant

difference compared with the ICH group; n = 6.

Figure 7 Inhibition of ferroptosis by Ferrostatin-1 treatment reduced inflammation and neuronal loss in rats after ICH. (A–D) The levels of TNF-α and IL-β in the serum and CSF, measured by ELISA. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

&&

P < 0.01 compared with the ICH + Vehicle group;

NS, no significant difference compared with the ICH group; n = 6. (E) Western blots show that albumin was increased in brain tissue after ICH in rats, but this increase was inhibited by treatment with Ferrostatin-1. (F) Quantification of relative protein levels of albumin in rats in the various groups. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group; &&P < 0.01 compared with the ICH + Vehicle group; NS, no significant difference compared with the ICH group; n = 6.

Figure 8 Ferrostatin-1 treatment attenuated brain injury after ICH in rats. (A) The levels of ROS in the brains of rats in various groups. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

##

P < 0.01 compared with the

ICH + Vehicle group; NS, no significant difference compared with the ICH group; n = 6. (B) Brain water content was partially decreased by Ferrostatin-1 treatment. All data are expressed as means ± SEM. **P < 0.01 (both in Ipsi-CX and Ipsi-BG) compared with the Sham group; NS, no significant difference compared with the ICH group; &P < 0.05 compared with the ICH + Vehicle group; n = 6. (C) The neurological scores of rats in the various groups. All data are expressed as individual values. **P < 0.01 compared with the Sham group; &P < 0.05 compared with the ICH + Vehicle group; NS, no significant difference compared with the ICH group; n = 6. (D) The relative levels of LDH in the CSF of rats in the various groups. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group; &P < 0.05 compared with

the ICH + Vehicle group; NS, no significant difference compared with the ICH group; n = 6. (E) Nissl staining in the hippocampal CA2 region and temporal cortex shows that Ferrostatin-1 treatment decreased neuronal loss induced by ICH. Scale bar = 32 μm. (F and G) The number of surviving neurons in the hippocampal CA2 region and temporal cortex. All data are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

##

P < 0.01 compared with the ICH + Vehicle group; NS, no

significant difference compared with the ICH group; n = 6.

Figure 9 The cognitive behavioral impairment of rats was detected by the Morris water maze. (A, B, D, and F) Representative images of the swimming trajectories of rats in each group. Motor learning was assessed by the escape latency (time, s). (C, E, and G) Spatial learning was assessed by the length of the swimming track of each rat. The values are expressed as means ± SEM. **P < 0.01 compared with the Sham group;

@@

P < 0.01 compared with the ICH + Vehicle group;

&&

P < 0.01 compared

with the ICH + Si-NC group; $P < 0.05 compared with the ICH + Ad-Vector group; &

P < 0.01 compared with the ICH + Vehicle group; n = 6.

Figure 10 Hypothesized model of the molecular mechanism of GPX4-mediated ferroptosis contributing to brain injury in rats after ICH. Normally, the cystine–glutamate antiporter, system Xc−, exchanges one molecule of extracellular cystine for one molecule of intracellular glutamate. After entering the cell, cystine is reduced by reduced glutathione (GSH) to cysteine, which is subsequently used for protein and GSH synthesis. GPX4 preferentially reduces phospholipid hydroperoxide (PL‑ OOH) to its corresponding alcohol phospholipid hydroxide (PL‑ OH), by using two molecules of GSH. GPX4 is one of the central upstream regulators of ferroptosis; it prevents lipoxygenase (LOX) overactivation and lipid peroxidation. The concerted action of these pathways is essential to control the formation of oxidized phospholipids. After ICH occurs, excessive amounts of glutamate inhibit system Xc−, inhibiting the intracellular transfer of cystine. Thus, GSH synthesis is reduced and GPX4 activity is decreased. As a result, excessive ROS and oxidative lipids are not

cleared up and eventually induce ferroptosis in the brain.

Table 1 Behavior and activity scores Category

Behavior

Score

Appetite

Finished meal

0

Left meal unfinished

1

Scarcely ate

2

Walk and reach at least three

0

Activity

corners of the cage

Deficits

Walk with some stimulations

1

Almost always lying down

2

No deficits

0

Unstable walk

1

Impossible to walk

2

Highlights 1. The protein level of GPX4 in brain tissues was decreased at 24 h after ICH. 2. Over-expression of GPX4 can alleviate brain injury induced by ICH. 3. Blockade ferroptosis by Ferrostatin-1 improved brain injury after ICH. 4. Decreased GPX4 may contribute to SBI after ICH by mediating ferroptosis.

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