Enriched environment attenuates ferroptosis after cerebral ischemia/reperfusion injury by regulating iron metabolism

Preventing neuronal death after ischemic stroke (IS) is crucial for neuroprotective treatment, yet current management options are limited. Enriched environment (EE) is an effective intervention strategy that promotes the recovery of neurological function after cerebral ischemia/reperfusion (I/R) injury. Ferroptosis has been identified as one of the mechanisms of neuronal death during IS, and inhibiting ferroptosis can reduce cerebral I/R injury. Our previous research has demonstrated that EE reduced ferroptosis by inhibiting lipid peroxidation, but the underlying mechanism still needs to be investigated. This study aims to explore the potential molecular mechanisms by which EE modulates iron metabolism to reduce ferroptosis. The experimental animals were randomly divided into four groups based on the housing environment and the procedure the animals received: the sham-operated + standard environment (SSE) group, the sham-operated + enriched environment (SEE) group, the ischemia/reperfusion + standard environment (ISE) group, and the ischemia/reperfusion + enriched environment (IEE) group. The results showed that EE reduced IL-6 expression during cerebral I/R injury, hence reducing JAK2-STAT3 pathway activation and hepcidin expression. Reduced hepcidin expression led to decreased DMT1 expression and increased FPN1 expression in neurons, resulting in lower neuronal iron levels and alleviated ferroptosis. In addition, EE also reduced the expression of TfR1 in neurons. Our research suggested that EE played a neuroprotective role by modulating iron metabolism and reducing neuronal ferroptosis after cerebral I/R injury, which might be achieved by inhibiting inflammatory response and down-regulating hepcidin expression.


Introduction
Stroke is the leading cause of permanent disability, with ischemic stroke (IS) being the most common type (Campbell et al., 2019).The majority of stroke survivors experience neurological dysfunction to varied degrees after the acute phase, which significantly lowers their life quality and places a severe burden on their families and society (Powers et al., 2018).Currently, post-stroke rehabilitation is one of the few available treatment options that can effectively help patients recover from stroke-induced deficits (Rothwell and Buchan, 2021;Stinear et al., 2020;Wei et al., 2001).Multisensory stimulation and motor training have been found to accelerate recovery after IS (Linder et al., 2019;Rosenfeldt et al., 2019;Särkämö et al., 2008).As a stroke rehabilitation strategy, enriched environment (EE) contains multiple stimuli.By providing the housing animals with larger space, novel play props, and more social partners, the animals housed in the EE have more sensory, cognitive, motor, and social stimulation than in the standard environment (SE) (Pusic and Kraig, 2014;Zhang et al., 2022).EE has been shown to effectively promote neurological recovery after IS by inducing neurogenesis and angiogenesis, enhancing synaptic plasticity, as well as saving ischemic penumbra neurons from different types of cell deaths, such as apoptosis, pyroptosis, and ferroptosis (Kempermann, 2019;Zhou et al., 2022;Deng et al., 2021;Zhou et al., 2020;Young et al., 1999).
Ferroptosis is a new form of regulated cell death characterized by iron-dependent lipid peroxide accumulation (Dixon et al., 2012;Alim et al., 2019).It has been shown that inhibition of ferroptosis reduces infarct volume, alleviates ischemia/reperfusion (I/R) injury, and offers neuroprotection (Zhan et al., 2023;Zhang et al., 2021a;Bu et al., 2021).Although the regulatory mechanism of ferroptosis is still being explored and refined, iron metabolism is unquestionably at the center of ferroptosis (Stockwell et al., 2017;Tang et al., 2018).The labile iron pool (LIP) is composed of redox-active iron in the cell, which is the central link in cellular iron metabolism (Kakhlon and Cabantchik, 2002;Worwood, 1990).Ferritin is composed of ferritin heavy chain (FTH1) and ferritin light chain (FTL), which is the main form of cellular iron storage and can reflect cellular iron levels (Knovich et al., 2009;Fleming and Joshi, 1987).One of the main ways by which cells absorb iron is through the transferrin receptor 1 (TfR1) binding to transferrin (Willson, 2020;DeGregorio-Rocasolano et al., 2018).Divalent metal transporter 1 (DMT1) mainly plays a role in the uptake of various divalent metal ions such as iron (Skj Ã¸rringe et al., 2015).The transmembrane protein ferroportin (FPN1) can transfer intracellular iron ions out of the cell to keep intracellular iron levels stable (Bao et al., 2021).These molecules are crucial in maintaining compatible iron metabolism in cells.However, IS alters the expression of TfR1, DMT1, and FPN1, which disrupts iron metabolism, leading to increased LIP and triggering ferroptosis (Ding et al., 2011;Selim and Ratan, 2004).
Hepcidin is a small peptide, which plays a crucial role in iron metabolism by regulating FPN1, and recent research indicates that it can also regulate DMT1 (Sangkhae and Nemeth, 2017;Nemeth et al., 2004;Li et al., 2011;Zhang et al., 2021b).Neuroinflammation has also been shown to disrupt iron metabolism via hepcidin, which ultimately results in elevated intracellular LIP (Ward et al., 2022;Ganz and Nemeth, 2015).On the other hand, EE has been proven to suppress the expression of IL-6, which has been shown to promote the transcription of hepcidin (Ganz, 2013;Guo et al., 2022).Our previous research has demonstrated that EE could prevent ferroptosis and promote functional recovery following IS (Liu et al., 2023a).However, whether EE inhibits ferroptosis by affecting iron metabolism needs further exploration.In this study, we hypothesized that EE inhibits hepcidin secretion by decreasing neuroinflammation after IS, thereby regulating the expression of FPN1, and DMT1, and ultimately modulating iron metabolism to alleviate neuronal ferroptosis after cerebral I/R injury.

Animals and middle cerebral artery occlusion and reperfusion
All procedures in this experiment were approved by the Animal Care and Use Committee of Zhongnan Hospital of Wuhan University (ZN2022014) and the welfare of experimental animals was protected during the experiment.Male Sprague-Dawley rats (200-220 g, 6-7 weeks old) were obtained from Hubei Beiente Laboratory Animal Technology Company.After 3 days of acclimation, the rats were randomly divided into four groups: the sham-operated + standard environment (SSE) group, the sham-operated + enriched environment (SEE) group, the ischemia/reperfusion + standard environment (ISE) group, and the ischemia/reperfusion + enriched environment (IEE) group.
Middle cerebral artery occlusion and reperfusion were used to simulate ischemic reperfusion injury.Animals were anesthetized with isoflurane and a 2 cm incision was made in the neck.The right common carotid artery and vagus nerve were carefully separated and the proximal end of the common carotid artery was ligated.The internal carotid artery was separated and ligated at the bifurcation, and a suture was left on the external carotid artery for future use.A notch was cut in the common carotid artery and a monofilament nylon filament (Beijing Cinontech Biotech Co., Ltd., China) was inserted to pass through the internal carotid artery and occlude the middle cerebral artery.After min, the filament was removed to restore blood perfusion.The sham operation was identical except that no filament was inserted.After recovery from anesthesia, rats were scored for neurological deficits on a five-point scale, with rats scoring 0 or 4 being excluded and rats scoring 1-3 being retained.After three days of acclimation in a standard environment, rats were placed in their respective spaces according to their group assignment.The experimental procedure and timeline are shown in Fig. 1A.

Enriched environment and standard environment settings
The SSE and ISE groups were housed in a standard environment (SE), while the SEE and IEE groups were housed in an enriched environment (EE).The SE cage was 44 cm long, 32 cm wide, and 20 cm high, with rats per cage.The EE cage was 90 cm long, 75 cm wide, and 55 cm high, with 8-12 rats living in it.In the EE cage, there were various toys such as running wheels, platforms, ladders, and tunnels to ensure sensory stimulation.The environment was changed every three days to ensure novelty.The settings of EE and SE are shown in Figs.1B and 1C respectively.

Behavioral tests
The Modified Neurological Severity Scores (mNSS) test includes assessments of motor function, sensory function, reflexes, and balance, and is commonly used to evaluate neurological deficits.The total score is 18 points, with higher scores indicating more severe neurological injury (Bieber et al., 2019).The mNSS was used to evaluate neurological deficits in rats on postoperative days 3, 10, 17, and 24.Balance and coordination deficits of rats were assessed using a rotarod test that employed an accelerating velocity.The rotarod apparatus used was the LE8200 Panlab from Harvard Apparatus in the United States.The velocity was gradually increased from 4 to 40 rpm over a period of 260 s, with a maximum testing time of 300 s.The duration until the rats dropped from the rotarod was recorded.Each test was repeated twice, and the average values were used for statistical analysis.The Morris Water Maze (MWM) test is an objective measure of spatial learning and memory in rats and was performed on postoperative days 24-29.The MWM test was conducted in a circular water tank (diameter: 150 cm; height: 60 cm) with the water temperature maintained at 24 ± 2 • C. A black circular platform (diameter: 10 cm) was submerged 2 cm below the water surface in a randomly chosen quadrant.The MWM test procedure and operation are described below.The spatial learning phase was performed on the first five days.During this phase, the platform was fixed in one quadrant.Rats were placed in the water from a different location and were trained to swim to find the place.The training was performed four times a day.Once the rat found the platform or after 60 s had elapsed, the single trial was over.If the rat could not find the platform within 60 s, it was guided to the platform.Regardless of whether or not the rat found the platform, it was allowed to rest on the platform for 15 s to ensure that all rats had an equal amount of time to observe and memorize their surroundings.On day 6, the platform was removed and rats were allowed to swim freely for 60 s to test their spatial learning and memory abilities.Throughout the entire process, an animal video tracking analysis system (n = 10/group) (Anilab Scientific Instruments Co., Ltd., China) was used to record swimming trajectories, speed, time to reach the platform, and the number of platform crossings.

3,3-Diaminobenzidine tetrahydrochloride (DAB) enhanced Perl's staining and Nissl staining
Prussian blue staining was used to detect iron deposition (n = 5/ group).Section preparation and imaging were performed as previously described.In brief, paraffin sections were deparaffinized and rehydrated and then rinsed with distilled water.The sections were blocked with 0.3% hydrogen peroxide solution for 15 min and then washed three times with TBS (5 min each time).The sections were immersed in Perl's blue staining solution (a mixture of equal parts 2.5% potassium ferrocyanide solution and 2.5% hydrochloric acid) and incubated at room temperature for 12 h.After washing with TBS, the samples were treated with DAB to enhance the color reaction.For Nissl staining, paraffin sections were deparaffinized and rehydrated and then rinsed with distilled water.The sections were then soaked in cresyl violet solution (Servicebio, China) for 3 min, and rinsed with running water.Images were taken from four randomly selected areas in the peri-infarct cortex of each brain using a BX53 microscope (Olympus Corporation, Tokyo, Japan), with three sections counted per rat.To eliminate bias, the researchers were blinded to group assignments during image analysis.

Iron and Malondialdehyde (MDA) level assays
The iron content and malondialdehyde (MDA) levels in rat brains (n = 5/group) were measured using a tissue iron assay kit (Nanjing Biotechnology Co. Ltd., China) and an MDA assay kit (Beyotime Biotechnology, China), respectively, according to the manufacturer's protocols.

TdT-mediated dUTP nick-end labeling (TUNEL) assay
Section preparation and imaging were performed as previously described.TUNEL staining (n = 5/group) was performed using a Onestep TUNEL Apoptosis Detection Kit according to the manufacturer's Q.Luo et al. protocol (Beyotime Biotechnology, China).The nuclei were stained with DAPI (Beyotime Biotechnology, China).Images were taken from four randomly selected areas in the peri-infarct cortex of each brain using a BX53 microscope (Olympus Corporation, Tokyo, Japan), with three sections counted per rat.To eliminate bias, the researchers were blinded to group assignments during image analysis.

Statistical analysis
Statistical analyses were conducted using IBM SPSS Statistics 23.0 and GraphPad Prism 9.0 software.The results are presented as the mean ± standard deviation (SD), with a P-value of less than 0.05 considered statistically significant.The mNSS, rotarod test, and escape latency data from the MWM test were analyzed using two-way repeated-measures ANOVA, followed by Tukey's post hoc test for multiple comparisons.Group differences were assessed using a two-tailed Student's t-test and one-way ANOVA, followed by Tukey's post hoc test for multiple comparisons.

Enriched environment reduced neurological deficits after cerebral I/R injury
Compared with the SE group, the animals in EE group showed more significant activity.The rats in EE group exhibited autonomous exploration behaviors, such as climbing ladders, shuttling through pipes, jumping on different platforms and munching on wooden toys.When the setting of the EE was changed each time, the rats explored in the new environment.The rats in SE group showed no such behavior.The modified Neurological Severity Score (mNSS), rotarod test and Morris Water Maze (MWM) test were used to evaluate the effect of EE on neurological impairments caused by cerebral I/R injury.The results of the mNSS and the rotarod test at 3, 10, 17, and 24 days showed that EE significantly reduced neurological deficits (Fig. 2A,B; P < 0.05, P < 0.001).The spatial learning memory ability of rats was assessed by the MWM test at 24-29 days.The results showed that there was no difference in swimming speed between the different groups (Fig. 2C).Rats in the ISE and IEE groups took longer time to find the platform than rats in the sham-operated group, but rats in the IEE group had a shorter escape latency than rats in the ISE group (Fig. 2E; P < 0.05, P < 0.01 and P < 0.001).On day 29, the spatial probe test was performed and the results showed that rats in the IEE group stayed in the correct quadrant Q. Luo et al. longer than the ISE group (Fig. 2F; P < 0.001) and traversed the platform more frequently (Fig. 2G; P < 0.05), whereas there was no significant difference between IEE group and sham-operated group.These results indicate that EE can improve neurological function after cerebral I/R injury.

Enriched environment attenuated neuronal ferroptosis after cerebral I/R injury
Nissl and TUNEL staining were used to evaluate neuronal death and further examined the changes in ferroptosis-related indicators including changes in iron levels, malondialdehyde (MDA) levels, cyclooxygenase-2 (COX2) and glutathione peroxidase 4 (GPX4) protein levels.Nissl staining and TUNEL staining showed that EE significantly reduced neuronal death after cerebral I/R injury (Fig. 2 B, C, D, E; P < 0.001).Perl's staining (Fig. 2 A) and tissue iron assay kit results (Fig. 2 G) showed that compared with the sham-operated group, rats in the ISE and IEE group had increased iron levels (P < 0.001), while rats in the IEE group had a lower iron level than the ISE group (P < 0.001).The same trends were observed in the MDA levels (Fig. 2 F; P < 0.01 and P < 0.001).Western blot results showed that COX2 expression was higher in the ISE group than IEE group (Fig. 2H, J; P < 0.001), while GPX4 expression was lower in the ISE group than in the IEE group (Fig. 2H, I; P < 0.001).These results showed that EE attenuated neuronal ferroptosis after cerebral I/R injury..

Enriched environment reduced ferritin levels after cerebral I/R injury
In order to detect the stored iron levels in different groups, we examined the expression levels of ferritin.As the ferritin is composed of ferritin heavy chain (FTH1) and ferritin light chain (FTL), the changes in FTH1 and FTL expression were examined separately.Western blot results showed that compared with the sham-operated group, FTH1 was significantly increased in the ISE group (Fig. 4A, B; P < 0.001), while there was no significant difference in the IEE group.Compared with the ISE group, the FTH1 level was lower in the IEE group (P < 0.01).In addition, the immunofluorescence of FTH1 also showed the same trend as the western blot results (Fig. 4C, D; P < 0.001).Western blot results of FTL showed that compared with the sham-operated group, FTL was increased in the ISE group (Fig. 4E, F; P < 0.001) and the IEE group (P < 0.05).Compared with the ISE group, the FTL level was lower in the IEE group (P < 0.01).The immunofluorescence of FTL showed the same trend as the western blot results (Fig. 4G, H; P < 0.001).These results showed that EE indicated the stored iron levels after cerebral I/R injury.

Enriched environment decreased neuronal DMT1 expression after cerebral I/R injury
To investigate the changes in DMT1 expression, we first used western blot to identify the changes in DMT1 protein.According to the findings, DMT1 expression was increased in the ISE group (Fig. 5A, B; P < 0.001) and the IEE group (P < 0.01) compared with the sham-operated group.Compared with the ISE group, DMT1 expression was decreased in the IEE group (Fig. 5A, B; P < 0.01).We further co-localized DMT1 with the neuronal marker NeuN in different groups to more precisely define the changes in DMT1 levels in neurons.The results showed that most DMT1positive cells co-localized with NeuN-positive cells (Fig. 5C), and the trend of change was consistent with the western blot results (Fig. 5D; P < 0.001).

Enriched environment decreased neuronal TfR1 expression after cerebral I/R injury
To investigate the changes in TfR1 expression, we used western blot as well as immunofluorescence co-localization to identify the changes in neurons.Western blot results showed that compared with the shamoperated group, cerebral I/R injury elevated TfR1 expression in the ISE group (Fig. 6A, B; P < 0.001) and the IEE group (P < 0.05).Compared with the ISE group, TfR1 expression was decreased in the IEE group (P < 0.05).Immunofluorescence results showed that most TfR1positive cells co-localized with NeuN-positive cells (Fig. 6C)), and the trend of change was consistent with the western blot results (Fig. 6D; P < 0.01 and P < 0.001).

Enriched environment increased neuronal FPN1 expression after cerebral I/R injury
To investigate the changes in FPN1 expression, we used western blot and immunofluorescence co-localization to identify the changes in neurons.Western blot results showed that compared with the shamoperated group, FPN1 expression was decreased in the ISE group (Fig. 7A, B; P < 0.05).In contrast, FPN1 expression was increased in the IEE group compared with the ISE group (P < 0.01).In addition, immunofluorescence results showed that FPN1 and NeuN doublepositive cells were significantly increased in the IEE group compared with the ISE group (Fig. 7C, D P < 0.001).

Enriched environment reduced astrocyte secretion of hepcidin after cerebral I/R injury
To investigate the hepcidin expression level, western blot and immunofluorescence staining were applied.As shown in Figs.8A and  8B, the level of hepcidin protein was significantly upregulated in the ISE group compared with the sham surgery group and the IEE group (P < 0.001 and P < 0.05).There was no significant difference between the IEE group and the sham-operated group.Immunofluorescence staining results were consistent with the western blot (Fig. 8C, D; P < 0.001).Next, we detected the co-localization of hepcidin with astrocytes (GFAP), neurons (NeuN), and microglia (Iba-1) by immunofluorescence staining.The results showed that hepcidin was mainly expressed in astrocytes (Fig. 8E).

Enriched environment reduced neuroinflammation after cerebral I/R injury
As neuroinflammation is the main culprit of secondary injury after I/ R damage and IL-6 has been shown to activate the transcription of hepcidin through the JAK2/STAT3 signaling pathway, we further examined IL-6 and JAK2/STAT3 expressions by western blot.The results showed that cerebral I/R injury increased the expression of IL-6 and the phosphorylation of JAK2 and STAT3 (Fig. 9A, B, C, and E).However, EE effectively reduced the IL-6 production and inhibited the phosphorylation of JAK2 and STAT3 (P < 0.05, P < 0.01, and P < 0.001)..

Discussion
As a key experimental paradigm commonly used to study the interaction between genes and the environment, EE shows significant therapeutic effects.EE is considered to restore the body to normal homeostasis from various stimuli, not only has it shown therapeutic effects in other neurological diseases such as epilepsy, Alzheimer's, and Parkinson's disease, but it has also been proven to promote recovery from cancer and myocardial infarction recently (Zhou et al., 2022;Jankowsky et al., 2005;Bezard et al., 2003;Song et al., 2017;Bai et al., 2022).EE can augment synaptic plasticity by modulating epigenetic mechanisms (Fischer et al., 2007;Nithianantharajah and Hannan, 2006).And this cognitive enhancement has the potential to be transmitted to subsequent generations via sperm (Benito et al., 2018).More importantly, as a non-invasive and convenient intervention, EE can be used in combination with other post-stroke treatments or drugs to accelerate patient recovery (Yt et al., 2021;Zhai and Feng, 2019).Some researchers have already tried to apply EE to clinical work and achieved encouraging results (White et al., 2015;Janssen et al., 2012;White et al., 2014).Researchers are also endeavoring to develop appropriate scales for evaluating metrics of EE in order to quantify its effects (Flores-Ramos et al., 2022).While there is still a distance to traverse before EE can be fully integrated into clinical practice, we hold the belief that its clinical application is within reach in the foreseeable future.Therefore, it is imperative to thoroughly investigate the therapeutic mechanisms underlying EE prior to its implementation in clinical practice.Our previous study showed that EE inhibited ferroptosis by decreasing lipid peroxidation production via the HIF-1α-ACSL4 pathway (Liu et al., 2023a).In this study, we further demonstrated that EE suppresses neuroinflammation and modulates iron metabolism, ultimately reducing neuronal ferroptosis.Our results demonstrate that the elevation of IL-6 after cerebral I/R injury led to elevated expression of hepcidin.Hepcidin elevation boosted the expression of neuronal DMT1 while decreasing the expression of FPN1, resulting in increased iron intake and decreased iron excretion, ultimately leading to neuronal ferroptosis.EE reduced the IL-6 production, which inhibited hepcidin expression and in turn modified the expression of DMT1 and FPN1, ultimately reducing neuronal ferroptosis.The results of behavioral tests including the mNSS, rotarod test, and MWM test showed that EE can reduce neurological deficits after cerebral I/R injury, which is consistent with others and our previous experiments (Janssen et al., 2010;Liu et al., 2021).EE contains multiple stimuli such as more sensory, cognitive, motor, and social stimulation than in the standard conditions.EE could provide extensive cognitive and motor rehabilitation training for cerebral I/R injury rats, so rats in the IEE group could end up with better functional recovery than the ISE group.IS can lead to multiple forms of cell death, including apoptosis, parthanatos, pyroptosis, necroptosis, autophagic cell death, and ferroptosis (Mao et al., 2022).In cell experiments, it was observed that, unlike other types of cell death, ferroptosis can spread in a wave-like manner among cell populations, suggesting that ferroptosis originating from the infarct area may accelerate infarct expansion (Kim et al., 2016;Riegman et al., 2020;Linkermann et al., 2014).Therefore, inhibiting ferroptosis may be more meaningful than other types of cell death.Nissl staining and TUNEL staining from our study showed that EE could inhibit neuronal death in the peri-infarct area.
To further verify that EE can inhibit neuronal ferroptosis, we examined the changes in ferroptosis-related indicators.We found that EE reduced cyclooxygenase-2 (COX2) expression after cerebral I/R injury, which is a well-recognized hallmark of ferroptosis (Xu et al., 2022(Xu et al., , 2023)).Glutathione Peroxidase 4 (GPX4) is often utilized to reflect ferroptosis because it prevents cells from ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols (Bersuker et al., 2020;Friedmann Angeli et al., 2014).In addition, the level of malondialdehyde (MDA) can indicate the extent of membrane lipid peroxidation, which is one of the characteristics of ferroptosis (Wang et al., 2021;Liu et al., 2020).Our results also showed that EE increased GPX4 expression and decreased MDA levels after cerebral I/R injury.Iron is a key driving factor for ferroptosis, and studies conducted on animals and patients indicate that IS disrupts brain iron homeostasis, resulting in elevated iron levels in brain tissue (Kondo et al., 1995;Bishop and Robinson, 2001;Chen et al., 2020a).Disruption of brain iron homeostasis was previously considered a significant factor in acute neuronal injury following IS (Dávalos et al., 1994;Erdemoglu and Ozbakir, 2002).However, elevated iron deposition, lipid peroxidation, and neuronal death could also be detected in rat brain tissue 3-6 weeks after experimental stroke (Kondo et al., 1995;Palmer et al., 1999), which is consistent with our results.These results suggest that the imbalance in cerebral iron homeostasis resulting from cerebral I/R injury is not limited to the acute phase.Our data show that EE also decreased the levels of FTH1 and FTL, which are reliable indicators reflecting changes in iron levels.In combination, our results suggested that EE alleviated neuronal ferroptosis after cerebral I/R injury.
Cells have a tightly regulated system that regulates iron uptake, neutralization, storage, and output to ensure appropriate levels of intracellularly available iron (Dusek et al., 2022;Mietto et al., 2021).Cerebral I/R injury can interfere with iron metabolism, leading to cellular LIP elevation (Carbonell and Rama, 2007;Guo, 12 et al., 2023;DeGregorio-Rocasolano et al., 2019).Neurons absorb iron via TfR1 and DMT1, and cerebral I/R injury will lead to elevated TfR1 and DMT1 expression, resulting in increased iron uptake (Ryan et al., 2019;Li et al., 2021;Liu et al., 2023b;Guan et al., 2019).Non-transferrin-bound iron (NTBI) in cerebrospinal fluid is much higher than in plasma (Mills et al., 2010).Therefore, when DMT1 is elevated, neurons directly uptake Fe 2+ Fig. 10.A schematic illustration of the proposed mechanism for enriched environment attenuates ferroptosis after cerebral ischemia/reperfusion injury by regulating iron metabolism.
Q. Luo et al. from NTBI (Knutson, 2019).Moreover, TfR1-mediated iron uptake requires the help of DMT1 (Hémadi et al., 2006;Kawabata, 2019).Thus, upregulation of DMT1 can increase iron uptake directly or through the TfR1 pathway.It has been shown that increasing FPN1 expression could increase cell resistance to ferroptosis (Chen et al., 2020b;Zou et al., 2022;Song et al., 2022).In addition, amyloid precursor protein and ceruloplasmin can also reduce ferroptosis by increasing the stability of FPN1 (Tuo et al., 2017).The results from our study showed that EE intervention led to a reduction in iron uptake in neurons by downregulating the expression of DMT1 and TfR1.Additionally, it increased iron excretion by upregulating FPN1 expression in neurons.As a result, the iron homeostasis in neurons was reestablished following cerebral I/R injury.
Hepcidin is mainly secreted by astrocytes in the central nervous system and regulates iron metabolism by mediating FPN1 internalization and degradation (Wang et al., 2019;Xiong et al., 2016).Brain ischemia leads to the upregulation of hepcidin (Ding et al., 2011).The upregulation of hepcidin leads to an increase in cellular LIP and induces ferroptosis (Bao et al., 2023;Zhao et al., 2022).Hepcidin has been shown to operate not only on FPN1 but also on DMT1 (Nguyen et al., 2006).After intracerebroventricular injection of hepcidin, downregulation of FPN1 and upregulation of DMT1 were observed in the rat cerebral cortex (Li et al., 2011).In a rat model of cerebral hemorrhage, hepcidin has also been shown to upregulate DMT1 expression, and the use of hepcidin inhibitors could inhibit DMT1 expression and ferroptosis (Zhang et al., 2021b).Our results showed that hepcidin expression was elevated after IS, which boosted the expression of neuronal DMT1 and decreased the expression of FPN1.These results were in line with the previous studies.On the other hand, EE down-regulated hepcidin expression after IS which might in turn modify the expression of DMT1 and FPN1 and eventually inhibit neuronal ferroptosis.
Neuroinflammation leads to iron metabolism disorders, iron metabolism disorders lead to ferroptosis, and cell death exacerbates the inflammatory response, forming a vicious cycle that further exacerbates cerebral I/R injury.Accordingly, inhibiting the inflammatory response may break the cycle and reduce neuronal ferroptosis (Cui et al., 2021a;Wang et al., 2022;Cui et al., 2021b).Our data have shown that EE downregulated the expressions of IL-6.These results tie well with previous studies that EE could significantly reduce neuroinflammatory cytokines expressions such as IL-1β, IL-18, TNF-α, and IL-6 (Zhou et al., 2022;Liu et al., 2023a;Zhang et al., 2023).The inflammatory cytokine IL-6 has been found to upregulate hepcidin expression in ischemic brain tissue via the JAK2-STAT3 pathway (Zhao et al., 2018).IL-6 promotes the phosphorylation of JAK2 and STAT3, and phosphorylated STAT3 promotes the transcription of hepcidin antimicrobial peptide gene (You et al., 2017;Hepcidin, 2023).Besides, other inflammatory cytokines may also affect the expression of DMT1 and FPN1.For example, TNF-α has also been shown to enhance neuronal DMT1 expression and inhibit FPN1 expression, while EE has been shown to reduce its expression (Urrutia et al., 2013).Taken together, EE inhibited inflammatory response and disrupted the vicious cycle.EE reduced the IL-6 expression after cerebral I/R injury, which could modulate iron metabolism by up-regulating hepcidin expression and eventually inhibit ferroptosis.
In summary, our research has shown that EE alleviated the imbalance of iron homeostasis in the brain after I/R injury, inhibited the occurrence of neuronal ferroptosis, and promoted post-stroke neurological recovery.The potential mechanism might be that EE reduced neuroinflammation and reduced the expression of hepcidin, which in turn downregulated DMT1 and upregulated FPN1 expression, thereby restoring iron metabolism homeostasis in neurons.Our experiment reveals a new mechanism for EE treatment which may broaden the understanding of environmental interventions in ischemic stroke.However, there are still limitations of this study that need to be addressed.We focused on the ferroptosis of neurons, without paying attention to the other types of cells such as astrocytes and microglia, which also play an important role in reprogramming neural function after IS.Moreover, the regulatory mechanism of iron metabolism after stroke is complex, further research is needed to explore the dynamic evolution of iron metabolism after stroke and the effect of EE on this process.

Fig. 1 .
Fig. 1.Flow chart of the experimental protocol and enriched environment settings.(A) Timeline of the experiment.Neurological functional test was performed at 3, 10, 17, and 24 days after the stroke.The Morris water maze (MWM) test was performed from day 24 to day 29.(B) The setting of enriched environment.(C) The setting of standard environment.

Fig. 2 .
Fig. 2. Enriched environment attenuated neurological deficits after cerebral I/R injury.(A) The results of the modified Neurological Severity Score (mNSS).# P < 0.05, ## P < 0.01, ### P < 0.001 compared with IEE group.(B) The results of the rotarod test.# P < 0.05, ## P < 0.01, ### P < 0.001 compared with IEE group.(C) During the spatial learning phase, there was no significant difference in the swimming speed among different groups.(D) Representative swimming trajectories of each group of rats in the spatial probe test.(E) The mean escape latency time of each group during the spatial learning phase.# P < 0.05, ## P < 0.01, ### P < 0.001 compared with IEE group.(F) Duration of stay in the correct quadrant for each group of rats in the spatial probe test.(G) The number of times each group of rats crossed the platform in the spatial probe test.n = 10.Data are expressed as mean ± SD.*P < 0.05, * *P < 0.01, * **P < 0.001.

Fig. 3 .
Fig. 3. Enriched environment attenuated neuronal ferroptosis after cerebral I/R injury.(A) Representative images for Perls' staining.Scale bar = 50 µm.(B, D) Representative images of Nissl staining and quantitative analysis for the number of viable neurons.Scale bar = 50 µm.(C, E) Representative images of TUNEL staining for evaluation of the cell death.Scale bar = 50 µm.(F) Quantitative analysis of MDA levels.(G) Quantitative analysis of iron levels.(H) Representative images for western blotting of COX2 and GPX4 expressions.(I) GPX4 protein expression analysis, normalized to β-actin.(J) COX2 protein expression analysis, normalized to β-actin.(K) Schematic brain with a highlight of the peri-infarct area (Relevant areas for cell counting and tissue sampling).n = 5.Data are expressed as mean ± SD.*P < 0.05, * *P < 0.01, * **P < 0.001.