Blocking P2RX7 Attenuates Ferroptosis in Endothelium and Reduces HG-induced Hemorrhagic Transformation After MCAO by Inhibiting ERK1/2 and P53 Signaling Pathways

Hyperglycemia is a risk factor for poor prognosis after acute ischemic stroke and promote the occurrence of hemorrhagic transformation (HT). The activation of P2RX7 play an important role in endotheliocyte damage and BBB disruption. Ferroptosis is a novel pattern of programmed cell death caused by the accumulation of intracellular iron and lipid peroxidation, resulting in ROS production and cell death. This study is to explore the mechanism of P2RX7 in reducing HT pathogenesis after acute ischemic stroke through regulating endotheliocyte ferroptosis. Male SD rats were performed to establish middle cerebral artery occlusion (MCAO) model injected with 50% high glucose (HG) and HUVECs were subjected to OGD/R treated with high glucose (30 mM) for establishing HT model in vivo and in vitro. P2RX7 inhibitor (BBG), and P2RX7 small interfering RNAs (siRNA) were used to investigate the role of P2RX7 in BBB after MCAO in vivo and OGD/R in vitro, respectively. The neurological deficits, infarct volume, degree of intracranial hemorrhage, integrity of the BBB, immunoblotting, and immunofluorescence were evaluated at 24 h after MCAO. Our study found that the level of P2RX7 was gradually increased after MCAO and/or treated with HG. Our results showed that treatment with HG after MCAO can aggravate neurological deficits, infarct volume, oxidative stress, iron accumulation, and BBB injury in HT model, and HG-induced HUVECs damage. The inhibition of P2RX7 reversed the damage effect of HG, significantly downregulated the expression level of P53, HO-1, and p-ERK1/2 and upregulated the level of SLC7A11 and GPX4, which implicated that P2RX7 inhibition could attenuate oxidative stress and ferroptosis of endothelium in vivo and in vitro. Our data provided evidence that the P2RX7 play an important role in HG-associated oxidative stress, endothelial damage, and BBB disruption, which regulates HG-induced HT by ERK1/2 and P53 signaling pathways after MCAO.


Introduction
Ischemic stroke is a common cerebrovascular disease caused by acute blockage of cerebral vascular circulation, and is the main cause of death in the world. Hemorrhagic transformation (HT) is a common complication of acute ischemic stroke (AIS), occurring in 10 to 40% of AIS patients. Diabetes is one of the most common clinical risk factors in patients with ischemic stroke, which may promote the pathological process of ischemic stroke and the occurrence of HT [1]. Hence, high glucose (HG) was used to induce the occurrence of HT after cerebral infarction [2]. The mechanisms of HG-induced HT involve oxidative damage and disruption of blood-brain barrier (BBB). Although many studies have tried to find ways of reducing HT damage, there are very few effective drugs or therapeutic strategies available to attenuate HG-enhanced HT in animal models. Hence, there is a greatly needed to investigate the mechanisms of HT and potential therapies that can reduce the risk of HT and improve outcomes in patients with AIS. Chengli Liu and Qi Tian contributed equally to the manuscript.
Ferroptosis is an iron-dependent programmed cell death triggered by lipid peroxides accumulation in the presence of increased production of reactive oxygen species (ROS) and inactivation of glutathione peroxidase 4 (GPX4) [3]. Many regulatory factors, such as SLC7A11, GSH, GPX4, P53, ferroportin, and transferrin, are thought to be involved in ferroptosis processes [4]. Some studies have found that ferroptosis is involved in some neurological disease, such as stroke, Alzheimer's disease and Parkinson's disease [5]. Furthermore, ischemic stroke has been revealed to be improved after treatment with the ferroptosis inhibitor [6,7]. Inhibition of ferroptosis in cerebral ischemia-reperfusion injury to treat stroke is a research hotspot. In addition, high glucose induces cytotoxicity and the accumulation of lipid peroxide, which can be reversed by ferrastatin-1 [8]. Some studies showed that ferroptosis plays an important role in the occurrence and development of diabetes complications [9]. Studying the mechanism of ferroptosis has important potential value for AIS patients with diabetes.
The P2X7 receptor is an ATP-gated, non-selective cation channel, belonging to the family of ionotropic P2X receptors [10]. The activation of P2RX7 causes the opening of the channel pore, allowing the passage of small cations (Ca 2+ , Na + , and K + ), and activating downstream cascade, such as MAPK signaling pathway [10]. P2RX7 is widely expressed in astrocytes, microglia, and brain capillary endothelial cells. Some evidence showed that the P2X7 receptor plays an important role in central nervous system disorder [10]. Genetic deletion and pharmacological blockade of the P2X7 receptor play neuroprotection in stroke [11][12][13]. Moreover, inhibition of P2X7 receptor can reduce neuroinflammation, oxidative stress, and endothelial dysfunction [14][15][16]. In spite Fig. 1 HG increased expression of P2RX7 and disruption of ZO-1 in ischemic brain tissue. A, B, C, D Protein expression levels of P2RX7, p-ERK1/2, ERK1/2, and P53 at 0 h, 6 h, 12 h, and 24 h after MCAO (n = 3 rats/group). E, F Protein expression levels of ZO-1 at 24 h after MCAO (n = 4 rats/group). E, G, H Protein and mRNA expression levels of P2X7 receptor at 24 h after MCAO (n = 4 rats/group). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test for multiple comparisons. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001 ◂ Fig. 2 P2X7 receptor agonist reduces neurologic deficit and encephaledema after MCAO. A Schematic diagram of the experimental design was exhibited in this study. B, C Bar graphs showed rat performance change by neurologic deficit scores and corner test scores at 1 day, 3 days, and 7 days after MCAO in rats (n = 15 rats/group). D Bar graphs show cerebral edema on the infarct side at 24 h after MCAO (n = 5 rats/group). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001 Fig. 3 P2X7 receptor agonist reduced HG-induced infarct volume and hemorrhagic transformation after MCAO in rats. A, B Bar graphs displayed infarct volumes at 24 h after MCAO (n = 5 rats/group). C Hemorrhage volume as evaluated by hemoglobin spectrophotometric assay (n = 5 rats/group). D Histopathological changes detected by HE staining in infarcted cerebral medulla (original magnification × 400) (n = 4 rats/group). Data were analyzed by one-way ANOVA followed by Tukey's test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001 of the fact that P2RX7 mediates neuroinflammation and neuronal excitability, its function in BBB has proven particularly challenging. The role of P2RX7 in astrocytes is controversial [17]. Some studies have shown the sensitization of P2X7 receptors in astrocytes for induction of ischemic tolerance [18]. Other studies revealed that blocking of P2RX7 can reduce mitochondrial stress in brain microvascular endothelial cells and protect the integrity of the BBB [19]. However, the relationship of P2RX7 and ferroptosis in brain microvascular endothelial cells remain unclear.
Therefore, we hypothesized that blockade of the P2RX7 protects against HT by limiting ferroptosis-mediated oxidative damage after cerebral ischemia/reperfusion in rat. P2RX7 signaling pathway may be a potential therapeutic strategy to reduce the risk of HT and improve the outcome of patients with AIS.

Animals Models of Focal Cerebral Ischemia
Adult male SD rats weighing 250-280 g were enrolled in this research. The rats were obtained from Animal Center of Wuhan University and housed under standard conditions at 22 °C and 50-60% relative humidity, alternating a light/dark cycle of 12:12 h. Food and water were supplied ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of Wuhan University. The investigators were blinded to the treatment of the animals during all surgical procedures.
Focal cerebral ischemia was conducted by intravascular occlusion of the left middle cerebral artery (MCAO) according to previously described protocol [20]. Briefly, SD rats were anesthetized by 4% pentobarbital sodium (50 mg/kg) and cut a midline incision in the neck. Then, the left common carotid artery was separated and ligated, the external carotid artery was ligated with 4-0 silk thread, and the internal carotid artery was temporarily clipped with an arterial clamp. A silicone-coated nylon monofilament (0.26 mm diameter, Beijing Sunbio Biotech, China) was inserted 18-20 mm into the internal carotid artery, occluding the origin of the MCA. After 3 h, the nylon monofilament was withdrawn to restore the blood flow of middle cerebral artery. To established the HT model, 50% glucose was injected intraperitoneally at 6 mL/kg before, pulling out the plug to induce acute hyperglycemia in HT group. After the operation, the revived rats were sent back to their cages and given free access to water and food.

Experimental Design and Drugs
All SD rats were randomly divided into four groups: Sham group-rats underwent the MCAO surgical procedure without the filament insertion; MCAO grouprats underwent 3 h of MCAO and received instant reperfusion; HT group-rats underwent 3 h of MCAO and received 50% D-glucose (6 ml/kg) intraperitoneally at 30 min before the filament is pulled out; and HT + BBG group-rats underwent MCAO and received 50% D-glucose (6 ml/kg) intraperitoneally, injected intravenous BBG (10 mg/kg, MCExpress, USA) at 60 min before the filament is pulled out.

Behavioral Tests
An investigator blinded to treatment status examined behavioral test at 21 h after reperfusion using neurologic deficit score and corner test. Neurologic deficit score was assessed as described previously [20]. The higher the neurologic deficit score implied more serious injury of neurologic function. For the corner test, SD rats were placed between splints at an angle of 30° and rats with unilateral brain injury tend to turn to the non-infarcted side. We tested 15 times for each rat and recorded the frequency of right turns.

Brain Water Content
Cerebral edema was measured by brain water content with the standard wet-dry method as previously description [21]. Briefly, the brain was separated to the ipsilateral and contralateral hemispheres and respectively weighed by an electronic analytic balance for wet weight. After dry at 100 °C for 24 h, the dry weight was weighted. Cerebral water content was calculated as: (wet weight-dry weight)/wet weight × 100%.

TTC Staining
Infarct volume was evaluated by 2,3,5-triphe-nyltetrazolium chloride staining (TTC, Sigma, MO) at 21 h after reperfusion. Fresh brain tissue was cut into seven coronal Sects. (2 mm thick), stained with the 1% TTC solution at 37 °C for 30 min, and then fixed in 4% paraformaldehyde for 24 h. TTC-stained sections were photographed, and the infarct volume was analyzed using ImageJ imageprocessing software (NIH, Bethesda, MD). The normal tissue was stained red, whereas white or pale represented the infarct area. The ratio of infarct volume = infarct 464 volume of the ipsilateral hemisphere/total volume of the ipsilateral hemisphere × 100%.

Measurement of MDA and SOD
The level of malondialdehyde (MDA) and superoxide dismutase (SOD) in brain tissue and in HUVECs were measured by MDA assay kit (Jiancheng Bioengineering Institute, China) and SOD assay kit (Jiancheng Bioengineering Institute, China) according to the manufacturer's specifications, respectively.

Spectrophotometric Assay of Hemoglobin Content
Concentration of hemoglobin in brain tissue was measured with a QuantiChrom Hemoglobin Assay Kit (Bio-Assay Systems, USA) according to the previous reported [21]. The result was showed as milligrams per deciliter.

Hematoxylin-Eosin (HE) Staining and Prussian Blue Dyeing
The left cerebral tissues of rats were fixed by 4% paraformaldehyde, embedded in paraffin, and then stained by HE (C0105, Beyotime, CA) according to the instructions. Brain sections was stained with Perls' Prussian blue to determine ferric (Fe3 +) iron content as described previously [22]. After the sections were sealed, the cerebral sections of each group were observed and photographed under a microscope.

Cell Culture and Oxygen-Glucose Deprivation/ Reperfusion (OGD/R) Model
Human umbilical vein endothelial cells (HUVECs) were cultured in complete medium that contained RPMI1640, 20% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Beyotime Biotechnology, Shanghai, China) at 37 °C under 5% CO 2 . Cells were cultured every 5-6 days until grown to 80-90% fusion. Culture medium was replaced 24 h after passage and every 2 days thereafter. In order to established OGD/R model in vitro, the normal culture medium of HUVECs was replaced by DMEM medium without glucose, and the cells were placed in an anaerobic chamber at 94% N 2 /5% CO 2 /1% O 2 and cultured at 37 °C for 5 h. After oxygen-glucose deprivation injury, cells were cultured in complete RPMI1640 medium or HG media containing 30 mM D-glucose without growth factors for another 19 h.

Cell Viability and Dead/Live Assay
Cell viability was determined by the CCK-8 assay according to the manufacturer's instructions (MCE, USA). HUVECs were seeded on 96-well plates at a density of 5000 cells/well. After treatment of HUVECs, 10 μM of CCK-8 was added to each well and incubated for 1 h. The absorbance at 450 nm was detected by enzyme labeling apparatus. Cell viability was exhibited as a percentage of the sham group. For dead/live experiment, HUVECs were washed twice with PBS, then cells were incubated with PI (4 μM) and calcein-AM (3 μM) for 30 min at room temperature darkness. Cells were washed again and detected under a fluorescence microscope. PI-bound HUVECs produces red fluorescence, indicating dead cells, and calcein-AM-bound cells produces green fluorescence for identifying live cells.

Transendothelial Electrical Resistance (TEER)
The 24-wells were coated with collagen before implanting. Then, 5 × 105/ml HUVECs were seeded on corning transwell inserts with a 0.4-µm pore size (Corning 3470) in the luminal side and grown in the complete culture medium as previously described [23]. Total resistance (Ω) of cultured epithelial cells was measured using Millicell-ERS (US) with a STX2 probe daily for 1 week. Group experiment after the resistance is stabilized. All TEER values were normalized to the area of the membrane (0.33 cm 2 ) and corrected for the resistance without cells.

Measurement of Intracellular Iron Ion
The level of iron ion in HUVECs were measured using Intracellular Iron Colorimetric Assay Kit (PPLYGEN, China) according to the instructions. P2RX7 receptor agonist reduced HG-induced BBB damage after cerebral ischemic in rat. A The level of albumin detected by immunofluorescent staining in infarcted cerebral medulla at 24 h after MCAO (original magnification × 400). B, C Western blotting images and quantitative data of albumin extravasation at 24 h after MCAO (n = 4 rats/group). D MPO level of infarcted cerebral tissue in each group at 24 h after MCAO (n = 4 rats/group). E, F, G, H Western blotting images and quantitative data of expression level of ZO-1 and occludin after MCAO in rats (n = 4 rats/group). Data were analyzed by one-way ANOVA followed by Tukey's test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001 F Western blotting images and quantitative data of relative expression level of ferroptosis-related protein at 24 h after MCAO (n = 4 rats/ group). Data were analyzed by one-way ANOVA followed by Tukey's test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001

Real-time Quantitative Reverse-Transcriptase PCR
Total RNA was extraced with the Trizol reagent (Invitrogen, USA). The concentration was determined by a spectrophotometer. One microgram of total RNA was reversely transcribed with the Hifair® III 1st Strand cDNA Synthesis SuperMix Kit (Yesen Biotechnology, Shanghai, China). The primers s for P2X7R and GAPDH were obtained from (Sangon Biotech, Shanghai, China) as follow: P2RX7, forward primer: AGG TGG CAG TTC AGG GAG GAATC, reverse primer: TGT ATT TGG GTT GAC AGC GAT GGG . GAPDH, forward primer: GGC ACA GTC AAG GCT GAG AATG, reverse primer: ATG GTG GTG AAG ACG CCA GTA. The amplification was performed in a LightCycler 480 system (Roche, Pleasanton, CA, USA) using the Hieff® qPCR SYBR Green Master Mix (Yesen Biotechnology, shanghai, China). The reaction system with a total volume of 20 μl was incubated at 95 °C for 5 min, then 40 cycles of 10 s at 95 °C and 30 s at 60 °C. Endogenous control GAPDH normalized candidate genes mRNA levels in the same sample. The 2 −ΔΔCt method was used to quantify relative gene expression.

Statistical Analysis
The study was conducted by researchers following the principle of randomization and blinding. All results were analyzed by the GraphPad Prism version 5.04 statistical package and were showed mean ± standard deviation (SD). Differences of multiple groups were assessed by one-way analysis of variance (ANOVA) followed by Tukey's test. P < 0.05 was considered statistically significant.

HG Increases P2RX7 Expression in Cerebral Ischemic/Reperfusion Area
The expression of P2RX7 in the ipsilateral cerebral cortex was assessed by western blots. There was a significant increase of P2RX7 level with p-ERK1/2 and P53 at 24 h after MCAO when compared to the sham group ( Fig. 1A-D). Immunofluorescence staining was used to detect the protein levels of ZO-1 in cerebral infarct tissue, which suggested BBB damage after MCAO (Fig. 1E-F). We found that the protein level of ZO-1 was reduced in MCAO group compared with in sham group (P < 0.01) and further decreased in HT group (P < 0.01). To study the changes of P2RX7 expression after HT, the brain tissues on the infarcted side were removed for western blot analysis and qPCR analysis at 24 h after MCAO in rats. The results indicated that P2RX7 expression gradually increase in MCAO group and HT group (Fig. 1E, G, and H) compared to Sham group. These results indicated P2RX7 may play an important role in BBB damage and HT pathology.

Effects of BBG on Behavioral Tests and Cerebral Edema After MCAO Treated with High Glucose
The schematic diagram of the experimental design was exhibited in Fig. 2A. According to the performance tested by neurologic deficit score and corner tests, MCAO group exhibited significant neurologic deficit (P < 0.01). Rats treated with hyperglycemia can worsen the abnormal behavior on these tests (P < 0.05). Rats conducted with administered BBG and HG performed better than did rats treated with HG alone (P < 0.01; Fig. 2B-C). Cerebral edema was detected by dry and wet method, rats treated with HG displayed a trend toward brain edema compared to MCAO-treated rats (P < 0.01). BBG treatment decreased cerebral edema in rats treated with HG (P < 0.05; Fig. 2D). P2X7 receptor agonist can reduce neurologic deficit and cerebral edema aggravated by HT after MCAO in rats.

Effect of BBG on Infarct Volume and Hemorrhagic Transformation After MCAO Treated with Hyperglycemia
Cerebral infarction area tested by TTC staining can directly reflect the degree of brain injury. Infarct volume in the HT group was significantly higher than that of the MCAO group (P < 0.05). Treatment of BBG reduced the infarct volume compared with that in the HT group (P < 0.05, Fig. 3A-B). Moreover, we detected the hemoglobin concentration in ipsilateral hemisphere. The increase in HG-enhanced bleeding was significantly inhibited by BBG administration (P < 0.01, Fig. 3C). HE staining also showed the degree of cerebral tissue injury and hemorrhage on the infarct side. In the brain tissue section, tissue edema, degree of blood, and nucleus pyknosis increased significantly in the HT group compared to in the MCAO group. BBG can significantly improve the degree of HT and reduce brain tissue damage compared to MCAO group (Fig. 3D).

Effect of BBG on BBB disruption after MCAO treated with HG
In this experiment, considering the color of the BBG, albumin was used as an indicator of BBB permeability, was mainly concentrated in the regions of the ischemic hemisphere. Immunofluorescent staining and western blotting showed that albumin extravasation in the HT group was greater than that in the MCAO group (P < 0.01, Fig. 4A-C) but was significantly reduced by BBG treatment in rats (P < 0.01, Fig. 4A-C). Increased albumin extravasation indicated more severe damage to the BBB. MPO level means the degree of leukocyte infiltration. Our results indicated that the level of MPO was increased in MCAO group than in sham group (P < 0.01), and significant higher in HT group than in MCAO group (P < 0.01), whereas BBG can reduced the levels of MPO after MCAO treated with HG (P < 0.01, Fig. 4D). We also explored the influence of HG on degradation of BBB. Western blotting showed that band density of tight junction protein zonula occludens-1 (ZO-1) and occludin was reduced in the ischemic hemisphere, whereas BBG treatment significantly prevented the HG-enhanced BBB disruption (Fig. 4E-H).

Effect of BBG on Ferroptosis After MCAO Treated with HG
Prussian dyeing was used to tested the accumulation of iron in cerebral infraction. The result found that the iron accumulation was significantly higher in HT group than in the MCAO group. Rats treated with BBG can decrease the levels of iron compared to BBG group (Fig. 5A). Reactive oxygen species were measured by MDA level and SOD level. The level of MDA was significant higher in HT group than in the MCAO group (P < 0.01) and the concentration of SOD of HT group was decreased compared with MCAO group (P < 0.01), whereas BBG can reduced the levels of MDA and increased the level of SOD after MCAO treated with HG ( Fig. 5B-C). To further explore the relation with ferroptosis, western blot analysis showed that the expression of TFR1, SLC7A11, and GPX4. The expression of SLC7A11 and GPX4 was downregulated, but the expression of TFR1 was increased in HT group compared to MCAO group. BBG treatment can upregulate SLC7A11 and GPX4 and reduce TFR1 expression (Fig. 5D-F), which indicate BBG may regulate ferroptosis in HG-induced HT. Fig. 6 P2RX7-ERK1/2 signaling pathway and MMP-9 played important role in HG-induced hemorrhagic transformation after cerebral ischemic in rat. A, B Immunoblot images and data of expression level of P2RX7, p-ERK1/2, and ERK1/2 at 24 h after MCAO (n = 4 rats/ group). C Representative images of P2RX7 and Claudin-5 co-expression in infarcted cerebral medulla (original magnification × 400). D, E Immunoblot images and quantitative data of the protein level of the MMP-9 at 24 h after MCAO (n = 4 rats/group). Data were analyzed by one-way ANOVA followed by Tukey's test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001

Effect of BBG on P2RX7-ERK1/2 Signaling Pathway After MCAO Treated with HG
To illustrate the underlying mechanisms of HG-enhanced ferroptosis after cerebral ischemia, we first determined the expression levels of P2RX7, p-ERK1/2, and ERK1/2 in the ipsilateral brain tissue. The expression of P2RX7 and p-ERK1/2 was increased in HT group compared to MCAO group. BBG treatment can significantly reduce P2RX7 and p-ERK1/2 expression compared to HT group (Fig. 6A-B). P2RX7 and Claudin-5 co-expression detected by immunofluorescence show similar changes (Fig. 6C). Moreover, we also found the expression of MMP-9 apparently upregulated in MCAO group (P < 0.01), and the treatment of hyperglycemia further induced the expression of MMP-9 (P < 0.01). BBG can effectively decrease the expression of MMP-9 to improve BBB damage (P < 0.01; Fig. 6D-E).

Effects of P2RX7 Inhibition on HUVECs Damage After OGD/R with High Glucose In Vitro
An in vitro BBB model was constructed using HUVECs and OGD model was used to mimic ischemia-reperfusion injury. For further analyzing the degree of HG-induced HUVECs injury after OGD/R, the stability of endothelial cell structure was measured using TEER. We observed the changes of TEER within 7 days after HUVECs inoculation, and found that it reached the maximum value about the sixth day. When the resistance is stable, TEER of each group is detected (Fig. 7A-C). We found that the inhibition of P2RX7 can improve HG-induced HUVECs damage and protect cell integrity (Fig. 7B-C). HG can aggravate HUVECs damage after OGD/R (P < 0.01), and inhibition of P2RX7 can improve the viability of HUVECs injury compared to HG + siNC group in vitro (P < 0.01; Fig. 7D). Dead/live assay implicated that P2RX7 inhibition can attenuate HG-induced HUVECs damage in vitro (Fig. 7E). To clarify the extent of the damage to the extracellular structure, the protein expression of ZO-1 and occludin was measured by western blot analysis at 24 h after OGD in vitro. The results found that the expression of ZO-1 and occludin was significantly decreased after OGD, and showed a decreasing trend after OGD with high glucose. While the inhibition of P2RX7 can reversed these damages. So, these data indicated that the inhibition of P2RX7 can improve HUVECs viability, reduce the damage of extracellular structure, and protect the integrity of BBB.

Effects of P2RX7 Inhibition on HUVECs Ferroptosis After OGD/R with HG IN VITRO
To clarify the protective mechanism of HUVECs, we further tested that the level of oxidative stress and iron concentration which were related to the ferroptosis. We found that the MDA level of control group was increased compared to sham group (P < 0.01), and HG can further aggravate oxidative stress, while the MDA level of HG + si-P2RX7 group was reduced after OGD/R in vitro compared to HG + si-NC group (P < 0.05; Fig. 8A). We also found that the concentration of iron ions was changed in HUVECs with different treatment. The results indicated that the level of iron ions showed an increased trend in HG group after OGD/R in vitro. The inhibition of P2RX7 can decrease the accumulation of iron ions in HUVECs after OGD/R in vitro (P < 0.01; Fig. 8B). In order to identify ferroptosis pathway of HUVECs, we detected ferroptosis-related protein by western blot analysis in HUVECs at 24 h after OGD in vitro. The results showed that the expression level of SLC7A11 and GPX4 was reduced and further decreased after OGD with high glucose. But the expression of P53 was increased after OGD and showed an increasing trend after OGD with high glucose. The inhibition of P2RX7 can significantly increase the expression of SLC7A11 and GPX4 and reduce the level of P53 (Fig. 8C-F). These results implicated that SLC7A11/GPX4 pathway play important role in HUVECs ferroptosis after OGD with high glucose. High expression of P53 may regulate ferroptosis by inhibiting SLC7A11 after OGD with high glucose. P2RX7 may regulate SLC7A11/GPX4 pathway by suppressing P53 in HUVECs after OGD with high glucose.

Effects of P2RX7 Inhibition on ERK1/2-HO-1 Signaling Pathway in HUVECs After OGD/R Treated with High Glucose In Vitro
To further clarify the potential relationship of P2RX7 and ferroptosis, we also detected the expression of P2RX7, Fig. 7 The inhibition of P2RX7 reduced the damage of HUVECs and extracellular structure after OGD/R with high glucose in vitro. A Diagram of the blood-brain barrier in vitro. B The TEER value of HUVECs within 7 days after inoculation (n = 6). C The TEER value of HUVECs at 24 h after OGD in vitro (n = 6/group). D The viability of HUVECs treated by different way was accessed by CCK-8 kit at 24 h after OGD in vitro (n = 5/group). E The viability of HUVECs tested by dead (red)/live (green) assay kit at 24 h after OGD in vitro; F, G immunoblot images and quantitative data of the expression of the ZO-1 and occludin at 24 h after OGD (n = 4/group). Data were analyzed by one-way ANOVA followed by Tukey's test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001 ◂ p-ERK1/2, ERK1/2, and HO-1. The data indicated that the expression level of P2RX7 was significantly suppressed by P2RX7 siRNA in OGD/R + HG + si-P2RX7 group compared with in OGD/R + HG group (P < 0.01). The level of p-ERK1/2 was upregulated in control group compared to in sham group, and further increased after OGD/R treated with high glucose. P2RX7 inhibition significantly downregulated the level of p-ERK1/2. Some studies have found that the activation of p-ERK1/2 can enhance the level of P53 [24]. (P < 0.01). Our result also implicated that P2RX7 may activate p-ERK1/2 to regulate P53 after OGD/R and HG. In addition, the Fig. 8 The inhibition of P2RX7 reduced HUVECs ferroptosis after OGD/R with high glucose in vitro. A The oxidative stress levels of HUVECs tested by MDA assay kit at 24 h after OGD in vitro (n = 4 / group). B The iron ions levels of HUVECs tested by intracellular iron colorimetric assay kit at 24 h after OGD in vitro (n = 4 /group). C, D, E, F Immunoblot images and quantitative data of the expression of the P53, SLC7A11, and GPX4 in HUVECs at 24 h after OGD (n = 4/ group). Data were analyzed by one-way ANOVA followed by Tukey's test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001 HO-1 is needed for the import of iron into the cell and is regulated by intracellular iron concentration [25]; therefore, we analyzed the expression of HO-1. We also found the expression of HO-1 apparently increased in OGD/R group (P < 0.05), and the treatment of hyperglycemia further induced the expression of TFR1 (P < 0.05). P2RX7 inhibition can significantly reduce the expression of HO-1 after OGD/R and HG treatment (P < 0.01; Fig. 9A-E). These results indicated that the high level of HO-1 may be involve in the process of HUVECs ferroptosis. We screened three kinds of si-P2RX7 to inhibit the expression of P2RX7, and the results showed that they had the same effect, and the effect of si-P2RX7-1 was more obvious (supplement figure). Fig. 9 The inhibition of P2RX7 regulated HUVECs ferroptosis by ERK1/2-HO-1 pathway after OGD/R with HG in vitro. A, B, C, D, E Immunoblot images and quantitative data of the expression of P2RX7, p-ERK1/2, ERK1/2, and HO-1 in HUVECs at 24 h after OGD (n = 4/group). Data were analyzed by one-way ANOVA followed by Tukey's test. A single asterisk (*) means P < 0.05, double asterisks (**) mean P < 0.01, triple asterisks (***) mean P < 0.001

Discussion
This study investigated the mechanism of P2RX7 in the pathogenesis of HT after MCAO treated with high glucose. Our results indicated that oxidative stress and iron accumulation were significantly increased after MCAO treated with high glucose, which indicated the existence of ferroptosis in endothelium. The inhibition of P2RX7 showed the protective effect on improving neurological deficits, brain edema, infarct volume, BBB disruption, and HT in MCAO model treated with HG. Endothelial cell is one of important component of the BBB. Endothelium damage and destruction of extracellular matrix are important causes of BBB breakdown. Moreover, the inhibition of P2RX7 can reverse the reduction of SLC7A11 and GPX4 and reduce endothelium ferroptosis. We also Fig. 10 The mechanism of endotheliocyte damage and HG-induced hemorrhagic transformation after ischemic stroke. HG, high glucose; ROS, reactive oxygen species; P2RX7, purinergic receptor P2X7; SLC7A11, solute carrier family 7 member 11; GSH, glutathione; GPX4, glutathione peroxidase-4; ERK1/2, extracellular regulated protein kinases; HO-1, heme oxygenase-1; P53, tumor protein 53; TFR1, transferrin receptor 1; MMP-9, matrix metalloproteinase-9 demonstrated a potential mechanism that P2RX7 may regulate ERK1/2-HO-1 and P53 pathway (Fig. 10).
As we all known, the mechanism of HT after ischemic/reperfusion (I/R) was complex, including oxidative stress, inflammation, and excitotoxicity. What the brain tissue lacks vital nutrients such as oxygen and glucose is very important reason. Multiple mechanisms of cell injury are activated after cerebral I/R [26]. A direct impact of lack of oxygen and glucose delivery is the change of mitochondrial function, which was recognized as the main source of ROS in reperfusion injury [27]. Some evidence suggested that iron is a risk factor in the development of cerebral I/R [28], and the iron content is increased in ischemic brains [29]. Iron-overloaded animals are more susceptible to MCAO, whereas iron chelation or iron deficiency reduces the damage of I/R [30]. In addition, we think that heme degradation and iron accumulation after HT of MCAO further aggravate the content of iron in brain tissue and promote ferroptosis. The injury mechanism may be partially similar to that of intracerebral hemorrhage. The injury effect of ferroptosis in intracerebral hemorrhage has been clearly reported [31,32]. Ferroptosis has been suggested to be involve in the process of endothelial cell damage and BBB disruption [30,33]. Some drugs were showed protective effect by inhibiting ferroptosis via SLC7A11/GPX4 signaling pathway after MCAO [30,34,35]. Our result showed the existence of ferroptosis in infract brain tissue after MCAO and in HUVECs after OGD/R, with lipid peroxidation, increased iron concentration and decreased expression of SLC7A11 and GPX4, which indicated that ferroptosis of endothelium is a potential pathogenic mechanism.
In addition, several studies showed that high glucose levels are related with increased HT in diabetic patients with acute ischemic stroke [36]. Hyperglycemia has been considered an independent predictor of stroke outcome. With the increase of patients with diabetes, it is more and more important to study the effect of hyperglycemia on HT after ischemic stroke. The mechanism of hyperglycemiainduced HT may involve oxidative stress. The accumulation of ROS is also an important hallmark of ferroptosis, which is reduced by the iron-dependent Fenton response and contributes to lipid peroxidation [37]. Some studies indicated that HG can mediate endothelial dysfunction by ferroptosis pathway [38,39], and some studies showed that glycemic control can significantly reduce HT [40,41]. Iron increases markers of ferroptosis and lipid reactive oxygen species (ROS) to a greater extent in BMVECs from diabetic animals, and endothelial ferroptosis and HT can be prevented by iron chelation [33]. Understanding the molecular mechanism of HG-induced ferroptosis holds promising applications for the protection of the BBB in the future. In our study, we found that HG-induced HUVECs damage, and the reduction in TEER value as well as in the expression of tight junction protein, such as ZO-1 and occludin, significantly decrease BBB integrity in vitro, which is associated to lipid peroxidation. MMP-9, as a key protease regulating the BBB, also plays an important role in BBB disruption. HG-induced ferroptosis is one of important way of endothelial cell death after MCAO.
Under pathological conditions, the purinergic P2RX7 can be activated by increased extracellular ATP concentrations, which can trigger ATP excitotoxic neuronal death caused by calcium (Ca 2+ ) overload and disturbance of mitochondrial membrane potential [42,43]. P2RX7-mediated oxidative stress may play a part in the development and progression of neurodegenerative diseases, such as AD and PD [44,45]. Some studies have found that activation of P2RX7 mediates the production of NOX2dependent ROS by activating extracellular ERK1/2 [16]. Some studies implicated that P2RX7 involves damage to vascular endothelium, leading to endothelial dysfunction [46]. Blocking P2RX7 activation can attenuate free radical production [47][48][49]. P2RX7 activation can regulate the expression of P53 [50][51][52]. Some studies have identified that p53 can promote ferroptosis by suppressing the expression level of SLC7A11/GPX4 [53][54][55][56][57], which indicated the relationship of P2RX7 and SLC7A11. SLC7A11 and GPX4 are one of key regulators of ferroptosis. Furthermore, there have found that P2X7R mediates ERK1/2 phosphorylation [58,59] and ERK1/2 can regulate P53 [60,61]. Some studies indicated that inhibition of ERK1/2 can reduce the apoptosis of hippocampal neuron after SAH [62]. We hypothesized that P2RX7 may act on SLC7A11 by regulating ERK1/2 phosphorylation and activating P53. Furthermore, heme derived from HT after MCAO can further stimulate the expression of HO-1 and TFR1. HO-1 initiate heme catabolism, which releases iron, carbon monoxide, and biliverdin [63]. However, the role of HO-1 in ferroptosis remain controversial. Some studies hold that the upregulation of HO-1 pathway can attenuate ferroptosis [64][65][66]. Other studies suggested that the overexpression of HO-1 has been identified to significantly promote ferroptosis for the increase in the labile iron pool [67][68][69][70][71]. In the brain, HO-1 and TFR1 are considered to be involved in oxidative stress, iron homeostasis, and cellular adaptive mechanisms [72]. Inhibition of HO-1 can improve cerebral ischemia-reperfusion injury [73]. HO-1 expression level may influence its role in acute injury. Our results showed that ischemia/reperfusion and HG promote ferroptosis in endothelium by excessively inducing HO-1 expression, resulted in the progression of BBB damage in vitro and in vivo, which can be reversed by P2RX7 block. These data elucidated that P2RX7 blocking can regulate SLC7A11/GPX4 by suppressing ERK1/2 and P53 pathway, and plays important role in ferroptosis pathways.

Conclusion
In conclusion, our study showed that the high glucose can promote oxidative stress and iron accumulation, resulting in endothelium ferroptosis and BBB damage, causing HT after MCAO. The P2RX7 play an important role in HGassociated oxidative stress, endothelial damage, BBB disruption, and neurological deficient, inhibition of which reduces HG-induced hemorrhagic transformation after MCAO by inhibiting ERK1/2 and P53. Upregulation of SLC7A11 and GPX4 can reduce the ferroptosis of endothelium. P2RX7 was a potential therapeutic target to reduce the adverse effect of high glucose on BBB through ferroptosis pathway, ultimately reducing neurovascular damage and improving stroke prognosis. These finding have important clinical implications and could be developed to potentially therapeutic strategy for patients with diabetes after AIS.

Acknowledgements
We are grateful to Wei Wang at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology for the experimental advice.
Author Contribution CL-L and QT designed and complete the study, conducted data analysis, and prepared the manuscript. JF-W, PB-H, YJ-G, CY, GJ-W, SM-H, and HW built MCAO models of rats and cultured HUVECs. MC-L reviewed and revised the manuscript.
Funding This article was supported by grants from the National Natural Science Foundation of China (Nos. 81971870 and No. 82172173).

Data Availability
The datasets supporting the conclusions of this article are included within the article and its additional files. All material used in this manuscript will be made available to researchers subject to confidentiality.

Declarations
Ethics Approval and Consent to Participate All institutional and national guidelines for the care and use of laboratory animals were followed during the experiments. All procedures performed in this study observed the ethical standards of the Renmin Hospital of Wuhan University.

Competing Interests
The authors declare no competing interests.