p23 protects against ferroptosis of brain microvascular endothelial cells in ischemic stroke
- Authors:
- Published online on: February 19, 2025 https://doi.org/10.3892/ijmm.2025.5505
- Article Number: 64
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Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Stroke is among the most prevalent causes of mortality and disability worldwide (1), and can be classified into two types, ischemic stroke and cerebral hemorrhage. At present, the principal treatment of ischemic stroke lies in restoring the blood supply to the ischemic area without delay. However, while restoring blood perfusion to the ischemic area, brain tissue damage is aggravated and leads to more serious neurological dysfunction, known as cerebral ischemia/reperfusion (I/R) injury (CIRI) (2). A pathophysiological trait of ischemic stroke is the disruption of the blood-brain barrier (BBB), characterized by increased permeability due to the breakdown of tight junctions (TJs) between brain microvascular endothelial cells (BMECs) and the increased vesicular transport in BMECs, causing unregulated influx of blood-derived cells, macromolecules and fluid. This ultimately leads to destructive cytotoxicity and vascular-origin edema, as well as life-threatening hemorrhagic transformation (3-5). In contrast to patients with mild BBB injury, patients with severe BBB injury exhibit higher National Institutes of Health Stroke Scale scores, worse functional outcomes and a greater risk of mortality (6,7). Therefore, alleviating the BBB injury can improve the neurological prognosis of patients with stroke (8).
Ferroptosis was initially reported in 2012 as a new form of regulated cell death that is distinct from apoptosis during the processes of tumor and embryonic development (9). Research has indicated that ferroptosis is associated with BBB injury caused by CIRI (10). In 2017, research demonstrated that the inhibition of ferroptosis in a middle cerebral artery occlusion (MCAO) model protected mice from CIRI (11). Furthermore, dietary iron supplementation can enhance the rebuilding of the BBB and defend neurons following ischemic stroke (12,13). In addition, co-injection of ceruloplasmin and Fe2+ has been shown to significantly reduce BBB permeability, DNA damage, neuronal cell death and the degree of neurological deficits (14).
p23 is a protein with a molecular weight of 23 kDa, which is widely expressed in the heart, brain, kidney, lung and other tissues (15). Structural studies have reported that the N-terminus of the p23 protein contains a β-sheet domain consisting of two inverted β-sheets, and its C-terminus is an unfolded tail (16,17). Furthermore, yeast p23 has been reported to regulate the normal function of the Golgi apparatus, ribosome synthesis and DNA repair (18). Deletion of the p23 gene can lead to abnormal skin and lung tissue development, and perinatal death in mouse embryos (19). Several studies have shown that p23 can bind to a variety of proteins and serve a regulatory role in various diseases by regulating the biological functions of these target proteins. For example, the helical motif at the C-terminus of p23 can bind to the glucocorticoid receptor, thereby regulating its activity (20). In gastric cancer, p23 bound to gedunin is more efficiently cleaved by activated caspase 7, thereby promoting tumor cell apoptosis (21). However, to the best of our knowledge, the biological significance of p23 in ferroptosis caused by CIRI-induced BBB injury has not yet been studied.
The present study aimed to determine whether ferroptosis occurs in BMECs during ischemic stroke, and to investigate the regulation of p23 on BBB function and ferroptosis in BMECs during ischemic stroke, so as to explore its specific mechanism.
Materials and methods
Animals and ethics approval
Male Sprague-Dawley rats were acquired from the Experimental Animal Center of Central South University (Changsha, China). GPX4 FosCreERT2 mice (GPX4 FosCreERT2/− and GPX4 FosCreERT2/+, GPX4 FosCreERT2/− mice were used as controls for GPX4 FosCreERT2/+ mice to rule out the influence of the Cre recombinase or other experimental factors) were produced using GPX4-flox mice and FosCreERT2 mice (both obtained from GemPharmatech Co., Ltd.). It was confirmed that the expression levels of GPX4 were reduced in the brain tissues of GPX4 FosCreERT2/+ mice (Fig. S1). The rats that were aged 8 weeks and weighed 280±20 g underwent Ferrostatin-1 (Fer-1) or short hairpin (sh)RNA treatments, whereas the rats that were aged 6 weeks and weighed 130-150 g were treated with for adeno-associated virus. The mice were aged 8-12 weeks and weighed 22±25 g. All animals were kept in a room with a controlled temperature of 25°C and humidity at 40-60% under a 12-h light/dark cycle, with free access to food and water.
A total of 78 rats and 30 GPX4 FosCreERT2 mice were used in the present study. For the Evans Blue extravasation test in vivo, 3 rats were allocated to the sham group, and 6 rats were allocated to the I/R 12-h group and the I/R 24-h group. For the ZO-1 and p23 immunofluorescence assay in vivo, 3 rats were allocated to the sham group and 6 rats were allocated to the I/R 12-h group. To assess the effects of Fer-1 on ZO-1 expression in vivo, 3 rats were allocated to the sham + vehicle group and the sham + Fer-1 group, whereas 6 rats were allocated to the I/R + vehicle group and I/R + Fer-1 group. For the p23 shRNA test in vivo, the sham + shNC group and sham + p23 shRNA group were each allocated 3 rats, and the I/R + shNC group and I/R + p23 shRNA group were each allocated 6 rats. For the p23 overexpression (OE) test in vivo, the sham + vector group and sham + OE p23 group were each allocated 3 rats, and the I/R + vector group and I/R + OE p23 group were each allocated 6 rats. For the GPX4 FosCreERT2 immunofluorescence assay in vivo, 3 GPX4 FosCreERT2/− and 3 GPX4 FosCreERT2/+ mice were allocated to the sham + OE-vector group, and the I/R + OE-vector group and I/R + OE-p23 group were each allocated 6 GPX4 FosCreERT2/− mice and 6 GPX4 FosCreERT2/+ mice. All experiments and animal procedures were approved by the Experimental Animal Center of Central South University (approval no. 2018sydw0222).
Cell culture and treatment
The mouse BMEC line bEnd.3 was procured from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Gen-View Scientific, Inc.) at 37°C and 5% CO2. bEnd.3 cells were used to establish an in vitro model of the BBB, and the oxygen-glucose deprivation/reoxygenation (OGD/R) experiment was used to simulate I/R injury in vitro. As for the administration of Fer-1 (Sigma-Aldrich; Merck KGaA), bEnd.3 cells were exposed to 10 µM Fer-1 at 37°C for 2 h before OGD/R. For the cycloheximide (CHX) chase assay, CHX (10 µM; cat. no. NSC-185; Selleck Chemicals) was applied in OGD/R-insulted b.End 3 cells for 3-12 h at 37°C to inhibit the proteasome pathway for the degradation of TJ proteins. The GPX4 agonist PKUMDL-LC-101-D04 (200 µM; CAS no. 2143896-83-5; MedChemExpress) was used to treat the cells for 1 h at 37°C after OGD/R and p23 siRNA transfection, whereas the GPX4 inhibitor RAS-selective lethal 3 (RSL3; 200 nM; CAS no. 1219810-16-8; MedChemExpress) was used to treat the cells for 16 h at 37°C after OGD/R and p23 oe.
Small interfering RNA (siRNA) transfection
siRNA transfection was performed using the riboFECT™ CP Transfection Kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Briefly, 1×106 cells/well were plated in 6-well plates and cultured for 12-24 h until the cell density reached 50-60%. The transfection complex was prepared by diluting 5 µl 20 µM siRNA storage solution with 120 µl 1X riboFECT CP Buffer, adding 12 µl riboFECT CP Reagent and incubating at room temperature for 15 min. The transfection complex was then added to the medium and cultured in a CO2 incubator at 37°C for 24 h. A negative control (NC) siRNA that does not target any known human, mouse or rat genes, and is not chemically modified, was used as a NC (cat. no. siN0000001-1-10; Guangzhou RiboBio Co., Ltd.). The of p23 siRNA sequence was as follows: 5′-GGTCCTGTTTGAACAAAGA-3′. The cell samples were subjected to OGD. After 3 h of OGD exposure, the cells were cultured in normal medium containing 10% FBS within a cell incubator containing 5% CO2 at 37°C for 12 h.
Plasmid transfection
The p23 OE plasmid, truncated p23 plasmids and blank vector plasmid (GV486; both from Shanghai GeneChem Co., Ltd.) were transfected into cells using the Neofect™ DNA transfection reagent (Neofect Biotech Co., Ltd.) according to the manufacturer's protocol. The truncated p23 sequences are provided in Table I. A total of 1×106 cells/well were plated in 6-well plates and cultured for 12-24 h until they reached a cell density of 60-80%. The DNA diluent was prepared by adding 100 µl Opti-MEM serum-free (Gibco; Thermo Fisher Scientific, Inc.) to 2 µg plasmid. Subsequently, 2 µl Neofect was added to the DNA diluent and incubated at room temperature for 15-30 min. Finally, the complex was added to the cells in 6-well plates and cultured in a CO2 incubator at 37°C for 48 h.
Cell viability assay
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; cat. no. GK3607; Gen-View Scientific, Inc.). A total of 5,000 cells were seeded in a 96-well plate and were cultured for ~24 h. Following treatment, CCK-8 was added to the medium in each well at a 1:10 ratio and the cells were incubated at 37°C for 1-4 h. Subsequently, the colorimetric absorbance of each well was measured at 450 nm using a microplate reader and a ratio was calculated compared with the control group.
Reverse-transcription quantitative PCR (RT-qPCR)
Following extraction with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), 1 µg RNA from cells or animal brain samples was reverse transcribed using 5X Evo M-MLV RT Reaction Mix (Accurate Biology) according to the manufacturer's protocol. Subsequently, qPCR was carried out according to the manufacturer's protocol using the SYBR Premix Ex Taq II kit (Accurate Biology) with the following amplification conditions: Pre-denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 34 sec. The primer sequences used are presented in Table II. The relative quantification of mRNA was analyzed using the 2−ΔΔCq method (22).
Western blotting
Proteins from cells or animal brain samples were extracted using radioimmunoprecipitation assay lysis buffer (Guangzhou Dingguo Biotechnology Co., Ltd.) containing 1% PMSF, and were quantified using the BCA method. Subsequently, 20-30 µg cell proteins or 50-80 µg animal proteins were transferred to a PVDF membrane following SDS-PAGE on 10-15% gels. Subsequently, the membranes were blocked with TBS-1% Tween 20 (TBST) containing 5% skim milk powder at room temperature for 2 h. The membranes were then incubated with primary antibodies against β-actin (cat. no. A1978; dilution, 1:5,000; Sigma-Aldrich; Merck KGaA), p23 (cat. no. 160150; dilution 1:200; Cayman Chemical Company), zonula occludens-1 (ZO-1; cat. no. 402200; dilution 1:2,000; Invitrogen; Thermo Fisher Scientific, Inc.), occludin (cat. no. 404700; dilution 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.), GPX4 (cat. no. ab125066; dilution 1:3,000; Abcam), cyclooxygenase (COX)-2 (cat. no. 160106; dilution 1:200; Cayman Chemical Company), heat shock protein 90 (HSP90; cat. no. 60318-1-lg; dilution 1:2,000; Proteintech Group, Inc.) and Flag (cat. no. 20543-1-AP; dilution 1:2,000; Proteintech Group, Inc.) at 4°C overnight. The membranes were washed with TBST three times at 10-min intervals and were then incubated with the HRP-conjugated goat anti-rabbit IgG (cat. no. BA1055; dilution 1:5,000; Boster Biological Technology) or goat anti-mouse IgG (cat. no. BA1056; dilution 1:5,000; Boster Biological Technology) secondary antibodies at room temperature for 1 h, followed by three further washes with TBST at 10-min intervals. The color reaction was developed using the Clarity Western ECL Substrate (cat. no. 170-5061; Bio-Rad Laboratories, Inc.), and band density was measured using ImageJ software V 1.8.0 (National Institutes of Health) and was normalized to β-actin.
Immunofluorescence
After fixing with 4% paraformaldehyde for 10 min at room temperature, 5,000 bEnd.3 cells grown in 96-well plates were washed three times with PBS (5 min each) and were blocked with BSA (Gen-View Scientific, Inc.) at 37°C for 1 h. The cells were then incubated with a primary antibody against ZO-1 (cat. no. 61-7300; dilution 1:50; Invitrogen; Thermo Fisher Scientific, Inc.), p23 (dilution 1:20; cat. no. 160150; Cayman Chemical Company), CD31 (cat. no. ab9498; dilution 1:1,000; Abcam) and GPX4 (dilution 1:100; cat. no. ab125066; Abcam) at 4°C overnight. The cells were then incubated with a TRITC-conjugatedsecondary antibody (cat. no. AS040; dilution 1:1,000; ABclonal Biotech Co., Ltd.) at 37°C in the dark for 1 h. Nuclei were stained with 1X DAPI working solution at room temperature for 5 min, washed three times with PBS-1% Tween 80 (PBST) and images were captured using a fluorescence microscope (Olympus Corporation).
Experimental MCAO model
The rats or GPX4 FosCreERT2 mice were anaesthetized through an intraperitoneal injection of 50 mg/kg sodium pentobarbital (China National Pharmaceutical Group). An incision was made in the neck to expose the external carotid, internal carotid, right common carotid and common carotid arteries, and the internal carotid artery was clamped. Following the ligation of the external carotid artery, a small incision was made into the external carotid artery. Monofilaments coated with poly-L-lysine were inserted via the incision, through the internal carotid artery and ultimately to the middle cerebral artery. Following occlusion (2 h for rats and 1 h for mice), the nylon monofilament was withdrawn for reperfusion for 24 h. The sham group underwent the same procedure as the MCAO group without insertion of the monofilament. The survival and health status of the animals was observed every 3 h. The cortex of the infarcted hemisphere was collected for subsequent experiments at 12 or 24 h post-reperfusion. The animals were euthanized by rapid dislocation of the head and neck when it was time to collect the specimens, or when the animals reached the following humane endpoints: They were too weak to eat or drink for up to 24 h; they were in a moribund state, i.e., without anesthesia or sedation, they were showing mental depression accompanied by hypothermia (<37°C). The loss of respiratory movement (the rise and fall of the chest and abdomen), and reflex actions of the eyes, nose and feet were used to verify the death of the animals. Notably, after MCAO treatment, 12 rats and 8 mice were sacrificed before sample collection, and 6 rats and 4 mice died spontaneously.
Lateral ventricle injection
After anesthetizing with 50 mg/kg sodium pentobarbital and preserving the skin on the top of the head, the rats were positioned horizontally and prone on a stereotaxic frame (RWD Life Science). Subsequently, 40 nmol/kg Fer-1, with saline used as a vehicle control; or p23 shRNA [forward (5′-3′): CCG GGA TCC TAG ACC ATG GAT TTA ACT CGA GTT AAA TCC ATG GTC TAG GAT CTT TTT G, reverse (5′-3′): AAT TCA AAA AGA TCC TAG ACC ATG GAT TTA ACT CGA GTT AAA TCC ATG GTC TAG GAT C; pRNAT-U6.1/Neo, Guangzhou RiboBio Co., Ltd.], with shNC [forward (5′-3′): TAC GAT ACA AGG CTG TTA GAG AG, reverse (5′-3′): TAG AAG GCA CAG TCG AGG used as the control] was infused into the right lateral ventricle at a flow rate of 0.5 µl/min using a syringe pump (KD Scientific) in conjunction with a Hamilton syringe. The stereotaxic coordinates for the right lateral ventricle were -1.5 mm laterally, -1.2 mm anteroposteriorly and -4.5 mm dorsoventrally, with the bregma point serving as the origin. The p23 OE adeno-associated virus (AAV-9; Shanghai GeneChem Co., Ltd.) was injected into the right lateral ventricle -1.2 mm laterally, -0.8 mm anteroposteriorly and -3.5 mm dorsoventrally, with empty AAV-9 used as the vector control. Following the injection, the skull was sealed using bone wax. OE was induced 2-3 weeks after injection. Similarly, the lateral ventricle injection of GPX4 FosCreERT2 mice was performed as that performed in rats; the stereotaxic coordinates were as follows: -1.2 mm laterally, -0.8 mm anteroposteriorly and -3.5 mm dorsoventrally. Subsequently, the animals underwent MCAO modeling and the brain tissue was collected for testing.
In vivo BBB permeability assay
In rats, the permeability of the BBB was evaluated through Evans Blue extravasation. A 2% Evans Blue solution was prepared using normal saline prior to the experiment. A total of 1 h before the conclusion of reperfusion, 2% Evans Blue (2 ml/kg) was injected into the tail vein of the rats. After 1 h, the rats were anesthetized with 50 mg/kg sodium pentobarbital and underwent cardiac perfusion with PBS. The brains were then collected and 2-mm thick coronal sections were made.
OGD/R
Cells were subjected to a 3-h culture in glucose- and serum-free medium in an anaerobic incubator (37°C, 95% N2, 5% CO2). After 3 h of OGD exposure, the cells were cultured in normal medium containing 10% FBS within a cell incubator containing 5% CO2 at 37°C for 12 and 24 h. The control group cells were cultured under normoxic condition, while the 0 h OGD/R group underwent OGD without reperfusion. The cell samples were then collected for subsequent experiments.
In vitro permeability assay
The bEnd.3 cells were cultured on the upper chamber of gelatin-coated Transwell inserts (0.4 µm; Costar; Corning, Inc.) and were exposed to hypoxic conditions (0% O2) at 37°C for 3 h. Subsequently, 165 µg/ml Evans Blue -0.1% BSA (Sigma Aldrich; Merck KGaA) was added to the upper chamber 1 h prior to measurements. The intensities of the diffused Evans Blue -0.1% BSA in the lower chamber were subsequently measured at 650 nm using a microplate reader. The results are presented as the ratio of the Evans Blue -0.1% BSA concentration in the lower chamber to the total concentration of Evans Blue -0.1% BSA added to the upper chamber at the onset of the experiment.
Reactive oxygen species (ROS) assay
Using the fluorescent probe diacetyldichlorofluorescein (DCFH-DA) ROS detection kit (cat. no. S0033S; Beyotime Institute of Biotechnology), DCFH-DA was diluted at a ratio of 1:1,000 with serum-free medium to a final concentration of 10 µM. Subsequently, the cell culture medium was removed from the cells, an appropriate volume of diluted DCFH-DA was added and incubation was carried out in a cell incubator at 37°C for 20 min. The cells were then washed with PBS three times, and a 488-nm excitation wavelength and 525-nm emission wavelength were used to detect the optical density using a microplate reader.
Malondialdehyde (MDA) assay
The MDA assay was conducted using a Lipid Peroxidation MDA Assay Kit (cat. no. S0131S; Beyotime Institute of Biotechnology). Following homogenization, the brain or cell samples were centrifuged at 13,000 × g and 4°C for 10 min to obtain the supernatant for detection. Subsequently, the prepared thiobarbituric acid working solution was added and heated at 100°C for 15 min. Eventually, the mixtures were centrifuged at 13,000 × g and 4°C for 10 min to obtain the supernatant, which was transferred to a 96-well plate to measure absorbance at 532 nm using a microplate reader. The content of MDA was normalized with respect to the protein amount of the samples, which was determined using the BCA method.
Glutathione (GSH) assay
The GSH assay was conducted using a GSH and reduced glutathione (GSSG) assay kit (cat. no. S0053; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. After OGD/R treatment, the cells were collected and resuspended with protein removal agent M. After rupturing the cell membrane through two freeze-thaw cycles, the cells were centrifuged at 10,000 × g at 4°C for 10 min and the supernatant was used for the determination of total GSH. Part of the supernatant was taken for GSSG detection: GSH removal auxiliary solution and GSH removal reagent working solution were added, and the reaction was performed at 25°C for 60 min. The protein removal reagent M solution and total GSH detection working solution were then added to the samples for total GSH or GSSG detection, and were incubated at 25°C for 5 min, before the addition of NADPH. The absorbance of the sample was detected at 512 nm with a microplate reader. The GSH content was calculated as follows: GSH=total GSH-GSSG x2. The GSH content was normalized with respect to the protein amount of the samples, which was determined using the BCA method.
Molecular docking
The structures of HSP90, p23 and GPX4 were downloaded from the Protein Data Bank (PDB) database (https://www.rcsb.org/); HSP90 and p23 are in the same protein file (PDB ID: 7L7J), whereasGPX4 (PDB ID: 6HN3) has its own protein structure file. The interaction modes between GPX4, HSP90 and p23 were investigated using the ZDOCK (https://zdock.wenglab.org/) server.
Transmission electron microscopy
Briefly, 5,000 cells/well were plated on a 96-well slide. After OGD/R, the cells were washed with PBS and successively fixed with 2.5% glutaraldehyde and 1% osmium acid at room temperature for 6 h. Following dehydration using varying concentrations of acetone, the samples were embedded in acetone and embedding solution at room temperature for 12 h, and solidified in an oven at 60°C for 24 h. Subsequently, the slides were double-stained with 3% uranium acetate and lead nitrate at room temperature for 2-3 min. Finally, the samples were observed and images were captured using a Hitachi HT7700 transmission electron microscope (Hitachi, Ltd.).
Co-immunoprecipitation assay
This assay was performed using agarose beads (Thermo Fisher Scientific, Inc.). Proteins from cell samples were extracted using radioimmunoprecipitation assay lysis buffer (Guangzhou Dingguo Biotechnology Co., Ltd.). After extraction, 300 µg cellular protein was incubated with the p23 (cat. no. 160150; dilution 1:200; Cayman Chemical Company), GPX4 (cat. no. ab125066; dilution 1:3,000; Abcam), and Flag (cat. no. 20543-1-AP; dilution 1:2,000; Proteintech Group, Inc.) antibodies at 4°C for 1 h with agitation. Subsequently, 20 µl suspended agarose beads were added and incubated at 4°C overnight with agitation. The immunoprecipitation products were collected and centrifuged at 2,500 × g for 5 min at 4°C, and the supernatant was discarded. The agarose beads were washed with PBST and centrifuged again; this was repeated four times. Finally, the agarose beads were resuspended with 40 µl electrophoretic loading buffer, boiled at 85°C for 5 min, and 20 µl samples underwent SDS-PAGE and western blotting.
Statistical analysis
All experiments were independently repeated at least three times. The results, presented as the mean ± SD, were analyzed using one-way or two-way ANOVA along with the multiple-comparison Bonferroni correction test. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software Inc.). P<0.05 was considered to indicate a statistically significant difference.
Results
Increased permeability of BMECs in ischemic stroke
To investigate the effect of the MCAO model on BBB dysfunction, the Evans Blue extravasation test was used to measure cerebral microvascular permeability. As shown in Fig. 1A, the ischemia group had markedly higher Evans Blue leakage than the sham group. TJ proteins are transmembrane proteins located between endothelial cells of the BBB, which serve critical roles in regulating the paracellular permeability of the BBB (23). The TJ protein ZO-1 was markedly decreased after 12-h I/R (Fig. 1B). To investigate the effects of OGD/R on BBB permeability during hypoxia in vitro, a Transwell assay was used to generate an in vitro BBB model, and BBB permeability was measured in BMECs using Evans Blue staining. The content of Evans Blue in the lower Transwell chamber was higher in the 0 and 12 h OGD/R groups than in the normoxia group (Fig. 1C). Subsequently, western blotting was performed to determine whether OGD/R affected the protein levels of ZO-1 and occludin. The expression levels of ZO-1 and occludin were decreased in BMECs in the OGD/R group at 12 h after reoxygenation compared with those in the control group (Fig. 1D-F). In addition, the expression of the TJ ZO-1 was detected in OGD/R-treated BMECs using immunofluorescence. The results showed that the expression of ZO-1 in BMECs in the OGD/R group was significantly decreased and exhibited a discontinuous distribution (Fig. 1I and J).
Ferroptosis in BMECs is involved in BBB damage in ischemic stroke
Increasing evidence has indicated that ferroptosis in BMECs is the main cause of BBB injury induced by I/R (24,25). Pretreatment with Fer-1 was performed via intraventricular injection for 2 h, followed by MCAO modeling, and the results showed that, as compared with in the sham + vehicle (saline) group, ZO-1 expression in brain tissues was decreased in the I/R + vehicle group, whereas it was increased in the I/R + Fer-1 group compared with that in the I/R + vehicle group (Fig. 2A). In addition, BMECs were pretreated with Fer-1, and it was revealed that the reduction in cell viability induced by OGD/R was significantly suppressed (Fig. 2B). Following OGD/R, BMECs exhibited mitochondrial swelling, and a decrease or disappearance in cristae (Fig. 2C), as well as decreased levels of the antioxidant molecules GSH and GPX4, and increased levels of MDA and ROS (Fig. 2D-J). This indicated that intracellular lipid peroxidation levels were increased. In addition, the expression levels of COX-2, another biomarker of ferroptosis, were significantly increased in the 12 h OGD/R group (Fig. 2K-M). These results suggested that ferroptosis may be involved in OGD/R-induced BMECs injury. Inhibiting ferroptosis in BMECs could protect against BBB damage caused by cerebral I/R.
p23 protects against BMEC injury in ischemic stroke
p23 can bind to client proteins and serves a regulatory role in numerous diseases by regulating their biological functions. In the present study, the in vitro results showed that p23 expression was significantly induced by OGD/R in BMECs (Fig. 1D, G and H) Subsequently, the permeability of BMECs was examined following OE or knockdown of p23 in BMECs. First, in the OGD/R model, siRNA was used to knockdown p23 expression (Fig. 3A), the results showed that p23 and ZO-1 expression levels were inhibited by p23 knockdown (Fig. 3C and D), and the permeability of BMECs was clearly increased (Fig. 3B), which indicated that the injury of BMECs induced by OGD/R was aggravated by p23 knockdown. Transfection with the p23 OE plasmid significantly increased the expression of p23 in the OGD/R model (Fig. 4A-C). Compared with in the vector + OGD/R group, the content of Evans Blue in the p23 + OGD/R group was significantly decreased (Fig. 4D and E), and the expression of ZO-1 was increased (Fig. 4F), which indicated that the OE of p23 reversed the OGD/R-induced damage of BMECs. In vivo, p23 knockdown by shRNA reduced the expression of ZO-1 in the MCAO group compared with that in the MCAO + shNC group (Fig. 3E), while p23 OE in the MCAO group could rescue the decrease in ZO-1 compared with in the MCAO + vector group (Fig. 4G); CD31 was used to indicate the location of blood vessels.
p23 protects against OGD/R-induced ferroptosis in BMECs
Given that ferroptosis is involved in BBB damage in ischemic stroke, the effect of p23 on OGD/R-induced ferroptosis in BMECs was determined. Compared with in the control group, the mortality of BMECs increased by p23 knockdown (Fig. 5A). In addition, p23 was overexpressed at the cellular level, and p23 could rescue cell death induced by OGD/R (Fig. 5B). In the OGD/R group, transfection with the p23 siRNA further decreased the contents of GSH and GPX4, and increased the levels of ROS, MDA, and acyl-CoA synthetase long-chain family member 4 (ACSL4) and subunit solute carrier family 7 member 11 (SLC7A11; Fig. 5C-I) compared with in the OGD/R + NC group, indicating that the increase of lipid peroxidation in BMECs promoted OGD/R-induced ferroptosis. After OGD/R, p23 OE decreased the levels of ROS, MDA, ACSL4 and SLC7A11, and increased those of GSH and GPX4 compared with those in the vector group (Fig. 5J-P), indicating that the levels of antioxidants in BMECs were increased, inhibiting OGD/R-induced ferroptosis. These results suggested that the regulatory role of p23 in OGD/R-induced BMECs may be achieved through ferroptosis.
p23 enhances GPX4 stability to suppress ferroptosis
Notably, p23 can function as an independent molecular chaperone or as a co-chaperone of HSP90 to exert its biological functions (26). To further explore the mechanism of p23-induced inhibition of ferroptosis, protein docking showed that p23 formed a complex with HSP90 and GPX4 through its N-terminal domain (1-90aa) (Fig. 6A). Co-IP experiments demonstrated that anti-GPX4 antibodies could precipitate p23 and HSP90 proteins in cells. Similarly, anti-p23 antibodies could precipitate GPX4 and HSP90 proteins in cells. (Fig. 6B). Full-length and truncated p23 plasmids were then constructed: Flag-p23(1-160), N-terminal Flag-p23(1-90) and C-terminal Flag-p23(91-160) fragments. After transfecting the BMECs with the truncated plasmids, immunoprecipitation was performed with anti-Flag antibody. The results showed that GPX4 could be precipitated by anti-Flag antibody, and the most important binding region was p23(1-90), which indicated that p23 formed a complex with HSP90 and GPX4 to inhibit GPX4 degradation and stabilize the expression of GPX4 through the N-terminal domain (1-90aa) of p23, thus inhibiting ferroptosis (Fig. 6C). In addition, immunofluorescence co-localization verification showed that p23 and GPX4 were co-located in cells (Fig. 6D). The effect of p23 on the stability of GPX4 protein was then tested. The results showed that the stability of GPX4 protein was markedly increased following p23 OE (Fig. 6E). To further verify the experimental results, GPX4 FosCreERT2 mice were used to construct a model of MCAO. The results showed that the reduction of ZO-1 in GPX4 FosCreERT2/− mice in the MCAO model was partially reversed by p23 OE, while p23 OE did not have this effect on GPX4 FosCreERT2/+ mice (Fig. 6F); CD31 was used to indicate the location of blood vessels, indicating that p23 may have a protective role by regulating ferroptosis through GPX4. The present findings demonstrated that p23 may form complexes with HSP90 and GPX4 through its N-terminal domain (1-90aa), thus enhancing the stability of GPX4, inhibiting the degradation of GPX4 and ultimately inhibiting BMEC ferroptosis, thereby protecting against cerebral I/R-induced BBB damage.
Discussion
The present study illustrated the crucial role of p23 as a key regulator of BBB injury following CIRI. First, p23 was revealed to be markedly upregulated under stroke-like conditions in both in vitro and in vivo models. p23 also formed a complex with HSP90 and GPX4 through its N-terminal structure (1-90aa), which improved their stability, inhibited GPX4 degradation, and ultimately inhibited ferroptosis in BMECs, thereby protecting against I/R-induced BBB injury. The aforementioned results are shown in Fig. 7. These findings may have important implications for potential future stroke treatment or prevention.
Ischemic stroke is the leading cause of death and long-term disability worldwide, and reperfusion after ischemia can trigger a series of harmful events, among which BBB disruption is one of the most critical indicators (27). TJs are complexes formed between microvascular endothelial cells, and their function is especially crucial for maintaining the integrity of the BBB. When TJs open, the integrity of the BBB is undermined. TJs are composed of a complex network of proteins, such as occludin, claudin and ZO-1, -2 and -3. Among them, occludin and ZO-1 have been discovered to be particularly significant in the assembly of endothelial cell connections that sustain the BBB, and their loss is associated with disruption of the BBB (28,29). Ferroptosis is a novel iron-dependent form of regulated cell death that differs in genetics, morphology and biochemistry from apoptosis, autophagy and necrotic cell death (9). Notably, it has been discovered to serve an important role in BBB dysfunction (10,14,30). In line with previous reports, the present results showed that cerebral I/R may lead to BBB injury and increased BBB permeability. In vitro, OGD/R led to BMEC damage and decreased the expression of TJ proteins ZO-1 and occludin. In addition, rats were pretreated with an intracerebroventricular injection of Fer-1 and a MCAO model was established. The results indicated that Fer-1 markedly enhanced ZO-1 expression. In vitro, the level of lipid peroxidation in BMECs rose significantly, and the antioxidant content was markedly decreased following OGD/R treatment, indicating that ferroptosis may participate in OGD/R-induced BMEC injury.
Ferroptosis has become a global research focus. The co-chaperone p23 is also known as cytosolic prostaglandin E (PGE) synthase (15), and the COX-2/PGE2 pathway has been reported to be closely associated with CIRI (31). p23 is involved in PGE2 synthesis, and there is evidence suggesting a connection between the COX-2/PGE2 pathway and ferroptosis (32). Recent studies have shown that PGE2 can prevent CIRI-induced ferroptosis (13,33), but the specific mechanism requires further exploration. In the present study, in vitro experiments showed that p23 knockdown promoted OGD/R-induced ferroptosis in BMECs, and further aggravated I/R-induced BBB damage. After OE of p23, the occurrence of ferroptosis in BMECs could be inhibited and BBB damage could be improved. These results indicated that p23 may exert a protective effect by regulating GPX4.
The biochemical mechanism of ferroptosis involves the iron-dependent formation of lipid-ROS, and consumption of GSH or inactivation of GPX4 (34,35). Phospholipid hydroperoxide is an enzyme that reduces phospholipid hydroperoxides to the corresponding phospholipid alcohol, and its expression or activity is regulated by selenium and GSH (36). In the course of the present study, it was discovered that the expression levels of GPX4 were regulated by p23, suggesting that p23 may regulate ferroptosis by targeting GPX4. To further prove this, the effect of p23 on GPX4 protein stability was assessed, and it was demonstrated that GPX4 protein stability was markedly increased following p23 OE. In GPX4 FosCreERT2/+ mice, p23 almost lost the ability to increase the expression of ZO-1, suggesting that GPX4 is an important site for p23 to serve a protective role in BBB. p23 often plays a unique biological function as a molecular chaperone and serves a regulatory role in a variety of diseases by binding to a various protein to regulate their biological function. First, it is an important co-molecular chaperone of HSP90, assisting HSP90 in completing its molecular chaperone function through its C-terminal co-chaperone complex (37). Genome and proteomics screening in yeast has revealed a network showing that p23 may have an HSP90-independent effect in intracellular receptor-regulated gene expression, and shows the characteristics of ribosomal biogenesis and vesicle-mediated transport (18,38). Through immunofluorescence co-localization experiments, the present study revealed that p23 co-localizes with GPX4 in cells. Further verification by protein docking and co-immunoprecipitation experiments showed that p23 forms a complex with HSP90 and GPX4 through its N-terminal domain (1-90aa) and enhances the stability of GPX4, inhibits the degradation of GPX4 and ultimately inhibits ferroptosis in BMECs, protecting against I/R-induced BBB injury. To the best of our knowledge, the present study was the first to explore the mechanism of p23 regulating ferroptosis in BMECs to protect against BBB dysfunction, providing hope for future clinical applications and laying the foundation for more effective stroke treatment by protecting against BBB damage.
There are some limitations in the current study. Although the OE of p23 did not significantly change protein expression levels in the control group, there was a significant difference in the OGD/R group, as well as in the mRNA levels of p23 in the control group. It could be hypothesized that this may be due to the fact that in the control group, after p23 is overexpressed, cells may counteract functional activation by downregulating the protein level of p23, thus achieving homeostasis of the body, while in the OGD/R group, the destruction of homeostasis led to effective OE.
In conclusion, p23 exerts a protective effect against cerebral I/R-induced BBB injury by suppressing the ferroptosis of BMECs through enhancing the stability of GPX4, offering a potential therapeutic target for ischemic stroke.
Supplementary Data
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
YL and JZ designed and guided the project. YZ and YX conducted the experiments. YZ, YX, QX and NH analyzed the data. All authors discussed the results and contributed to the final version of the manuscript. YZ and YX wrote the manuscript. YL and JZ revised the manuscript. YL is the principal corresponding author and is responsible for the integrity of the work in its entirety from its initiation to the publication of the article. YL and JZ confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
All the procedures carried out in studies involving animals were in line with the ethical standards of the institution or practice where the studies were conducted, and were approved by the Experimental Animal Center of Central South University (Changsha, China; approval no. 2018sydw0222).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Not applicable.
Funding
This work was funded by the National Natural Science Foundation of China (grant nos. 82172147, 81571880, 81373147, 30901555 and 30972870) and the Natural Science Foundation of Hunan Province (grant nos. 2021JJ30900 and 2016JJ2157). All these funding bodies supported the study design, data collection, analysis, interpretation and manuscript writing.
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