Nrf-2 modulates excitability of hippocampal neurons by regulating ferroptosis and neuroinflammation after subarachnoid hemorrhage in rats

Excitability of hippocampal neurons in subarachnoid hemorrhage (SAH) rats has not been well studied. The rat SAH model was applied in this study to explore the role of nuclear factor E2-related factor (Nrf-2) in the early brain injury of SAH. The neural excitability of CA1 pyramidal cells (PCs) in SAH rats was evaluated by using electrophysiology experiments. Ferroptosis and neuroinflammation were measured by ELISA, transmission electron microscopy and western blotting. Our results indicated that SAH induced neurological deficits, brain edema, ferroptosis, neuroinflammation and neural excitability in rats. Ferrostatin-1 treatment significantly decreased the expression and distribution of IL-1 β , IL-6, IL-10, TGF-β and TNF-α . Inhibiting ferroptosis by ferrostatin-1 can attenuate neural excitability, neurological deficits, brain edema and neuroinflammation in SAH rats. Inhibiting the expression of Nrf-2 significantly increased the neural excitability and the levels of IL-1 β , IL-6, IL-10, TGF-β and TNF-α in Fer-1-treated SAH rats. Taken together, inhibiting the Nrf-2 induces early brain injury, brain edema and the inflammatory response with increasing of neural excitability in Fer-1-treated SAH rats. These results have indicated that inhibiting ferroptosis, neuroinflammation and neural excitability attenuates early brain injury after SAH by regulating the Nrf-2.


Introduction
Subarachnoid hemorrhage (SAH) is a subtype of stroke and has been reported to be related to high rates of mortality and poor prognoses (Calviere et al., 2019).Many studies have shown that ferroptosis plays a critical role in the development of early brain injury including neurological deficits, brain edema, and neuroinflammation after SAH (Cao et al., 2021).The early brain injury period was considered as the first 72 h after bleeding of SAH.Moreover, neuroinflammation contributes to neurological deficits and brain edema in early brain injury after SAH (Lai et al., 2020)., and inhibiting ferroptosis attenuates neurological deficits and neuroinflammation after SAH (Cao et al., 2021).However, the molecular mechanisms of the effects of inhibiting ferroptosis on neurological deficits, brain edema and neuroinflammation in the early brain injury of SAH remain unclear.
Ferrostatin-1 is widely applied to inhibit ferroptosis in various models, and its effects on inhibiting ferroptosis are more effective than those of phenolic antioxidants (Liu et al., 2020).Cao et al. illustrated that inhibiting ferroptosis attenuated neuroinflammation and neurological deficits without exploring the molecular mechanism (Cao et al., 2021).Thus, this study aims to explore the mechanisms of ferroptosis and neuroinflammation in early brain injury after SAH.
This study aimed to explore the role of the Nrf-2/HMGB1/TLR4 signaling pathway in the early brain injury process of ferroptosis, neuroinflammation and excitability of hippocampal neurons after SAH.Therefore, our findings might provide a new molecular mechanism for the comprehensive treatment of early brain injury after SAH.

Experimental animals, subarachnoid hemorrhage models and drug treatment
Adult male Sprague-Dawley (SD) rats (2 months old) were purchased from Guangdong Provincial Medical Experimental Animal Center.All animal studies were carried out according to protocols approved by the Laboratory Animal Ethics Committee of Southern Medical University (permit number: LAEC-2020-142.).
The subarachnoid hemorrhage (SAH) rat model was generated according to a previous study (Cao et al., 2021).Briefly, the rats were anesthetized with 1.5% isoflurane, and a 5-0 monofilament was inserted to the internal carotid artery and the bifurcation of the anterior and middle cerebral arteries via the left external carotid artery.Ferrostatin-1 (Fer-1, HY-100579, MedChemExpress) was dissolved in a working solution containing 10% DMSO, 40% PEG300, 5% Tween-80% and 45% saline by intraperitoneal injection (i.p.) 30 min after surgery.For the vehicle, the working solution was applied to SAH rats 30 min after surgery.

Measurement of subarachnoid hemorrhage grade and neurological score
The damage of the surgery was blindly measured at 6 h, 12 h, 24 h, 48 h and 72 h after SAH according to a previously reported SAH grading system.Briefly, the rats were anesthetized with 1.5% isoflurane, and the brain basal cistern was cut into six equal slices, each sample was scored from 0 to 3 and determined by presenting blood clotting.The SHA grades were the results from the sum scores of the six parts.Neurological function was measured by using the modified Garcia test (Garcia et al., 1995).

Brain water content
The rats were anesthetized with 1.5% isoflurane and then sacrificed after measuring neurological score.The rat brain was quickly removed after decapitation, and the brain was weighed immediately to measure its wet weight (WW).Then, the brains were dried at 105 ℃ for 72 h, and their dry weight (DW) was determined.The brain water content was calculated by the following formula: brain water content = [(WW-DW)/ WW] × 100%.

Electrophysiology
The rats were anesthetized with 1.5% isoflurane and then sacrificed after measuring neurological score, and coronal hippocampal slices were collected by using a vibratome slicer (VT1200S; Leica).The electrophysiology experiments of coronal hippocampal slices in rat SAH were performed according to a previous study (Wang et al., 2023).To measure the excitatory spontaneous synaptic transmission and intrinsic excitability of pyramidal cells (PCs) in SAH rats, whole-cell patch clamp including current-clamp and voltage recordings were applied in this study.The coronal hippocampal slices were incubated with an oxygenated recording solution containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 ,1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 25 mM D-glucose,1.3 mM sodium ascorbate, and 3.0 mM sodium pyruvate (pH 7.4) for 30 min.The brain slices were viewed by an upright microscope with a 40X water-immersion objective.To measure the neuronal excitability of PCs, APs were recorded by injecting a series of depolarizing currents (from 0 pA to 200 pA at a step of 10 pA) under the current-clamp model by holding resting membrane potential (RMP) at − 70 mV.And spontaneous excitatory postsynaptic currents (sEPSCs) were measured by holding the membrane potential at − 70 mV under the voltage recordings.

Measurement of Fe 3+ and Fe 2+ contents
The rats were anesthetized with 1.5% isoflurane and then sacrificed after measuring neurological score.The rat brain was quickly removed after decapitation.All the brains were ground with a glass mortar, and the lysate was diluted to 1/10 of the primary concentration by using PBS.The contents of Fe 3+ and Fe 2+ in the rat brain were measured by using an iron colorimetric assay kit (E-BC-K139-S, Elabscience, Wuhan, China) and ferrous iron colorimetric assay kit (E-BC-K773-M, Elabscience, Wuhan, China) according to the manufacturer's instructions.Rat brain was lysed with PBS and loaded with 1 μmol/1 calcein-AM for 30 min at 37 ℃.The calcein of the uncomplexed irons were measured by using a fluorescence microscope (Zeiss microscope) with quantify the fluorescence values.

Measurement of ROS
The rats were anesthetized with 1.5% isoflurane and then sacrificed after measuring neurological score.The rat brain was quickly removed after decapitation.All the brains were ground with a glass mortar and lysed with PBS buffer.The Dichloro-dihydro-fluorescein diacetate (DCFH-DA)-based reactive oxygen species (ROS) method was applied to explore the relative ROS level in the rat brain (Sun et al., 2021).A DCFH-DA solution was prepared with a centration of 10 mM in DMSO.All the brains were ground with a glass mortar, and the lysate was diluted to 1/10 of the primary concentration by using PBS.

Measurement of MDA and GSH-PX
The levels of malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) were measured according to a previous study (Jin et al., 2022).The rat brains were harvested and diluted with PBS and applied to the MDA (ab118970, Abcam) and GSH-PX ELISA kits (ab239727, Abcam) according to the manufacturer's instructions.

Western blotting
Western blotting was performed according to previous study (Wang et al., 2023).The rats were anesthetized with 1.5% isoflurane and then sacrificed after measuring neurological score.The rat brain was quickly removed after decapitation.All the brains were ground with a glass mortar and lysed with RIPA buffer (Sigma Chemical Co., St. Louis MO, USA) containing protease and phosphatase inhibitors, and the samples were centrifuged at 12,000 rpm for 20 min at 4 ℃.The denatured samples with loading buffer at the temperature of 90-100 ℃ were separated on 10% SDS-PAGE gels.Proteins from the SDS-PAGE gels were transferred to a polyvinylidene difluoride (PVDF) membranes and blocked with 3% bovine serum albumin (BSA), and incubated with the following primary antibodies: HO-1 (diluted 1:1000; 43966, Cell Signaling Technology), Nrf-2 (diluted 1:1000; 20733 S, Cell Signaling Technology), TLR4 (diluted 1:1000; ab22048, Abcam), HMGB1 (diluted 1:1000; 6893 S, Cell Signaling Technology) and β-actin (diluted 1:1000; 3700 T, Cell Signaling Technology) were applied in this study.Then, the PVDF membranes were incubated with HRP-conjugated goat anti-rabbit or rat IgG (diluted 1:5000, Cell Signaling Technology) at room temperature for 1 h.The target proteins were revealed by using the Image Quant™ RT ECL System (GE Healthcare) and analyzed by using ImageJ software (ImageJ 1.5, National Institutes of Health, Bethesda, MD,

Hematoxylin and eosin (H&E) staining
The brain samples were fixed with 4% paraformaldehyde and embedded in paraffin and were cut into 5 µm-thick sections.Paraffinembedded rat right brains were stained with hematoxylin and eosin (H&E).The results of H&E staining were visualized using an optical microscope equipped with a charge-coupled device camera (Pulnix, Sunnyvale, CA, USA).

Transmission electron microscopy
The ultrastructure of mitochondria in the rat brain was observed by using transmission electron microscopy (TEM).Briefly, brains were collected and fixed in 2.5% glutaraldehyde for 2 h at 4 • C.Then, all the samples were transferred to 1% osmium tetroxide for 1 h at 4 • C.Then, the samples were dehydrated and embedded in epoxy resin.All the samples were sliced by using an ultramicrotome (RMC/MTX; Elexience).Ultrathin sections of 60-80 nm were mounted on copper grids and then applied to a Philips CM100 transmission electron microscope equipped with a TENGRA 2.3 K × 2.3 K TEM camera.

Immunohistochemical staining
The brain samples were fixed with 4% paraformaldehyde and embedded in paraffin and were cut into 5 µm-thick sections.Paraffinembedded rat right brains were stained with IHC.IHC was applied to explore the expression and distribution of Iba1, HO-1, Nrf-2, TLR4 and HMGB1 in the ipsilateral cortex after SAH.All slices were incubated overnight with the following primary antibody: anti-Iba1 (diluted 1:200, 17198 T, Cell Signaling Technology) Then, all slices were washed with PBS 3 times and incubated with biotinylated goat anti-rabbit IgG (H+L) (diluted 1:100; ab64256, Abcam).Then, enzyme-labeled streptavidin was applied and incubated according to the manufacturer's protocol.

Intracerebroventricular injections of siRNA
Nrf2 siRNA with the sense strand 5′-GTC TTC AGC ATG TTA CGT GAT GAG GAT GG-3′ and its control siRNA were synthesized by Sangon Biotech (Shanghai, China) and dissolved in RNase-free resuspension buffer at a concentration of 500 pmol/μL.Intracerebroventricular (i.c.v) injection was applied to administer siRNAs to the right ventricle according to a previous study (Wilmes et al., 2022).

Statistical analysis
GraphPad Prism (Version 7.0, San Diego, CA) was applied to analyze the data in this study.All the data were presented as the mean ± standard error (SEM).The statistical significance of differences between two groups was assessed by using Student's t test, and the statistical significance of multiple groups was calculated by using two-way analysis of variance (2ANOVA) followed by Tukey multiple comparisons test, as shown in the results.P < 0.05 was considered to indicate statistical significance.

SAH induced neurological deficit and brain edema
The number of rats used in this study was 394, including the rats that died or were excluded from each group.Behavioral tests were performed 6 h, 12 h, 24 h, 48 h and 72 h after SAH (Fig. 1A).Typical photos of the brains from the Sham and SAH groups are shown in Fig. 1B.Our results showed that the SAH grade score was significantly increased in SAH group rats at different time points compared to the sham group, and it peaked at 24 h after SAH (Fig. 1C).The modified Garcia test showed that the SAH rats exerted a significant neurological deficit compared to the sham group, and the modified Garcia score was lowest at 24 h after SAH (Fig. 1D).The brain water content was significantly increased in SAH group rats compared to the sham group and peaked at 24 h after SAH (Fig. 1E).

SAH induced ferroptosis in rats
ELISA was performed 6 h, 12 h, 24 h, 48 h and 72 h after SAH (Fig. 2A), total Fe iron, Fe 2+ and Fe 3+ , reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) levels were measured to explore the effects of SAH on ferroptosis in the brain.The results showed that the brain Fe 2+ (Fig. 2B), Fe 3+ (Fig. 2C) and total Fe iron level (Fig. 2D) were significantly increased in SAH group rats at different time points compared to the sham group and peaked at 24 h after SAH.Moreover, the levels of ROS (Fig. 2E), MDA (Fig. 2F) and GSH-PX (Fig. 2G) were significantly increased in SAH group rats at different time points compared to the sham group and peaked at 24 h after SAH.

Neurological deficits, brain edema and ferroptosis were attenuated by ferrostatin-1 in SAH rats
To explore the role of ferroptosis in SAH rats, three doses (1, 2 and 4 mg/kg) of ferrostatin-1 (Fer-1) were applied at 0.5 h after SAH, and ELISA and behavioral tests were performed at 24 h after SAH (Fig. 3A).Our results showed that the SAH grade score (Fig. 3B) and brain water content (Fig. 3D) were significantly decreased in the Fer-1-treated  groups at doses of 2 mg/kg or 4 mg/kg compared to the SAH group.Meanwhile, the modified Garcia score was significantly increased in the Fer-1-treated groups at doses of 2 mg/kg or 4 mg/kg compared to the SAH group (Fig. 3C).

Ferrostatin-1 suppressed ferroptosis in SAH rats
Meanwhile, we found that the brain Fe 2+ (Fig. 3E), Fe 3+ (Fig. 3F) and total Fe iron (Fig. 3G) levels were significantly decreased in the ferrostatin-1 treatment group compared to the SAH group.The ROS level (Fig. 3H) and MDA (Fig. 3I) content were significantly decreased in the Fer-1-treated groups at doses of 2 mg/kg or 4 mg/kg compared to the SAH group.Moreover, the content of GSH-PX was significantly increased in the Fer-1-treated groups at doses of 2 mg/kg or 4 mg/kg compared to the SAH group (Fig. 3J).The results of H&E staining showed that cavities and tissue damage were increased in SAH group rats compared to sham group rats, and 2 mg/kg ferrostatin-1 treatment decreased the cavities and tissue damage after SAH treatment (Fig. 3K).Furthermore, we found that a reduction in volume and ruptured mitochondria occurred in SAH group rats compared to sham group rats, and 2 mg/kg ferrostatin-1 treatment reversed the reduction in volume and ruptured mitochondria in SAH group rats (Fig. 3L).

Ferrostatin-1 suppressed excitatory spontaneous synaptic transmission and intrinsic excitability of PCs in SAH rats
Here, 2 mg/kg of ferrostatin-1 was applied at 0.5 h after SAH and electrophysiology experiments were performed at 24 h after SAH to explore the role of excitatory spontaneous synaptic transmission and intrinsic excitability of PCs in SAH rats (Fig. 5A).Our results have shown that frequency (Fig. 5B, C) and amplitude (Fig. 5B, D) of sEPSCs in PCs were significant increased in SAH+Fer-1 group compared to SAH+Vehicle group.the number of spontaneous APs (Fig. 5E, F) and current-evoked APs (Fig. 5G, H) were increased in SAH+Fer-1 group compared to SAH+Vehicle group.

Inhibiting the Nrf-2 increases excitatory spontaneous synaptic transmission and intrinsic excitability of PCs in SAH rats
Our results have shown that injections of Nrf-2 siRNA significantly increased frequency (Fig. 6G, H) and amplitude (Fig. 6G, I) of sEPSCs in PCs in SAH+Fer-1 group.And the number of spontaneous APs (Fig. 6J,  K) current-evoked APs (Fig. 6M, N) was significantly increased after treatment with Nrf-2 siRNA.Taken together, our results have shown that inhibiting the Nrf-2/HMGB1/TLR4 signaling pathway increase neural excitability of PCs in Fer-1-treated SAH rats.

Discussion
Recent studies have revealed the involvement of ferroptosis in various neurological conditions, specifically its connection to neurological deficits, brain edema, and neuroinflammation (Cao et al., 2021).Inhibiting ferroptosis has been shown to alleviate early brain injury by reducing lipid peroxidation (Li et al., 2021) and iron-dependent oxidative cell injury (Chen-Roetling et al., 2018).Our study conducted time-point experiments to establish a subarachnoid hemorrhage (SAH) rat model, focusing on early brain injury.We observed peak levels of neurological deficits, ferroptosis, brain edema, and neural excitability in CA1 pyramidal cells (PCs) at 24 h post-SAH.Consequently, we selected the SAH-24 h group for further exploration of ferroptosis's molecular mechanisms in SAH rats.
Nuclear factor E2-related factor (Nrf-2), a leucine zipper transcriptional activating factor in the cap-n-collar (CNC) family (Kim et al., 2013), plays a crucial role in protecting the hemorrhagic brain.It reduces lesion volume, neurologic deficits, brain edema, and reactive oxygen species (Chang et al., 2014).The Nrf-2/high mobility group box-1 (HMGB1) and Nrf-2/HMGB1/toll-like receptor 4 (TLR4) signaling pathways are implicated in neurodegeneration and cognitive deficits across various neurological disorders.This led us to hypothesize that the Nrf-2/HMGB1/TLR4 pathway might be key in the molecular mechanism of early brain injury in SAH rats.We inhibited this pathway using intracerebroventricular injections of Nrf-2 siRNA in the right lateral ventricle.Our results indicated that these injections significantly altered the protein expression associated with this pathway and worsened SAH severity and brain water content while reducing the modified Garcia score.This suggests that suppressing the Nrf-2/HMGB1/TLR4 pathway may worsen neurological deficits and brain edema.Furthermore, this inhibition noticeably increased the levels of various inflammatory markers in Fer-1-treated SAH rats.
In conclusion, our findings demonstrate that ferrostatin-1 effectively inhibits ferroptosis, which in turn reduces neurological deficits, brain edema, and neuroinflammation in early SAH brain injury.Conversely, inhibiting the Nrf-2/HMGB1/TLR4 pathway aggravates early brain injury, increases brain edema, and triggers an inflammatory response.These insights highlight the potential of targeting the Nrf-2/HMGB1/ TLR4 pathway as a treatment strategy for early brain injury in SAH (Fig. 8).
Although our findings illustrated that inhibiting ferroptosis, neuroinflammation and neural excitability attenuates early brain injury after SAH by regulating the Nrf-2, there are still some challenges to be settled in our future study, such as 1) Nrf-2 gene knock mice should be applied to verify the role of Nrf-2 in the brain injury, brain edema of SAH rats, 2) try to explore the role of Nrf-2 IN regulating the neural excitability of PCs by using photogenetics and chemical genetics, 3) try to confirm these findings and explore the Nrf-2 for treating early brain injury with reducing brain edema and the inflammatory response in SAH.

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Fig. 1 .
Fig. 1.SAH induced neurological deficits, brain edema and neural excitability in rats.(A) Experimental design and animal groups.(B) Typical photos of the brain from the Sham and SAH groups.(C) SAH grades for each group.(D) The modified Garcia score for each group.(E) Quantification of brain water content in each group.n = 6 mice for each group.(F) Representative current trace of sEPSCs of PCs in all six groups was recorded.Frequency (G) and amplitude (H) of sEPSCs were measured in all six groups.(I) Representative traces of spontaneous APs were presented.(J) Mean firing rate of spontaneous APs were calculated.(K) Representative traces of evoked APs of PCs were presented.(L) Number of evoked APs against the injected current in PCs were measured in all six groups.Data shown are means ± SEMs, n = 20 cells for each group.*P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs. sham.2ANOVA with Tukey test was applied in this section.

Fig. 5 .
Fig. 5. Ferrostatin-1 decreased neural excitability in SAH rats.(A) Experimental design and animal groups.(B) Representative current traces of sEPSCs of PCs in all four groups were recorded.Frequency (C) and amplitude (D) of sEPSCs were measured in all four groups.(E) Representative traces of spontaneous APs were presented.(F) Mean firing rate of spontaneous APs were calculated.(G) Representative traces of evoked APs of PCs in all four groups was presented.(H) Number of evoked APs against the injected current in PCs were measured in all four groups.Data shown are means ± SEMs, n = 20 cells for each group.**** P < 0.0001 vs. sham, #### P < 0.0001 vs. SAH+Vehicle, 2ANOVA with Tukey test was applied in this section.SAH, subarachnoid hemorrhage.

Fig. 6 .
Fig. 6.Inhibiting the Nrf-2 increases neural excitability in SAH rats.(A) Experimental design and animal groups.(B) Western blotting analysis of the indicated proteins was performed, and β-actin was used as the loading control.Nrf-2 (C), HO-1 (D), TLR4 (E) and HMGB1 (F) were measured.Data shown are means ± SEMs (n = 4), (*P < 0.05, ** P < 0.01 and *** P < 0.001 vs. SAH + Fer-1).(G) Representative current trace of sEPSCs of PCs in the two groups was recorded.Frequency (H) and amplitude (I) of sEPSCs were measured in two groups.(J) Representative traces of spontaneous APs were presented.(K) Mean firing rate of spontaneous APs were calculated.(M) Representative traces of evoked APs of PCs in two groups was presented.(N) Number of evoked APs against the injected current in PCs were measured in two groups.**** P < 0.0001 vs. sham, n = 20 cells for each group, 2ANOVA with Tukey test was applied in this section.