LncRNA-MEG3 functions as ferroptotic promoter to mediate OGD combined high glucose-induced death of rat brain microvascular endothelial cells via the p53-GPX4 axis

Background Individuals with diabetes are exposed to a higher risk of perioperative stroke than non-diabetics mainly due to persistent hyperglycemia. lncRNA-MEG3 (long non-coding RNA maternally expressed gene 3) has been considered as an important mediator in regulating ischemic stroke. However, the functional and regulatory roles of lncRNA-MEG3 in diabetic brain ischemic injury remain unclear. Results In this study, RBMVECs (the rat brain microvascular endothelial cells) were exposed to 6 h of OGD (oxygen and glucose deprivation), and subsequent reperfusion via incubating cells with glucose of various high concentrations for 24 h to imitate in vitro diabetic brain ischemic injury. It was shown that the marker events of ferroptosis and increased lncRNA-MEG3 expression occurred after the injury induced by OGD combined with hyperglycemic treatment. However, all ferroptotic events were reversed with the treatment of MEG3-siRNA. Moreover, in this in vitro model, p53 was also characterized as a downstream target of lncRNA-MEG3. Furthermore, p53 knockdown protected RBMVECs against OGD + hyperglycemic reperfusion-induced ferroptosis, while the overexpression of p53 exerted opposite effects, implying that p53 served as a positive regulator of ferroptosis. Additionally, the overexpression or knockdown of p53 signi�cantly modulated GPX4 expression in RBMVECs exposed to the injury induced by OGD combined with hyperglycemic treatment. Furthermore, GPX4 expression was suppressed again after the introduction of p53 into cells silenced by lncRNA-MEG3. Finally, ChIP assay uncovered that p53 was bound to GPX4 promoter. Conclusions Altogether, these data revealed that, by modulating GPX4 transcription and expression, the lncRNA-MEG3-p53 signaling pathway mediated the ferroptosis of RBMVECs upon injury induced by OGD combined with hyperglycemic treatment.


Background
With the increasing trend of population aging in the Chinese mainland, the incidence of perioperative stroke is gradually increased.Perioperative stroke can cause signi cant morbidity and mortality, thus bringing heavy economic burden to the society and the families of patients [1,2].Epidemiological studies have identi ed DM (diabetes mellitus) as an independent factor in the increased risk of perioperative stroke in diabetic patients [2][3][4].Therefore, it is of a prime interest to develop new therapeutic strategies for the management of diabetic perioperative stroke.
As a class of non-coding RNAs with a length of larger than 200 nucleotides, lncRNAs (long non-coding RNAs) are involved in many critical physiological and pathophysiological processes, such as cell differentiation, cell proliferation, immunity, in ammation and apoptosis, through modulating their target genes [5,6].Accumulative experimental evidence has revealed that dysregulation of lncRNAs is closely associated with neurologic functional disorders upon brain stroke [7][8][9].In addition, several studies have demonstrated that lncRNAs, recognized as novel regulators, participated in the pathogenesis of DM and its complications [10,11].Therefore, it is very important to explore the relevance between lncRNAs and diabetic brain stroke.lncRNA-MEG3 (lncRNA maternally expressed gene 3), a lncRNA located in human chromosome 14q32, is consisted of 1.6 kb of nucleotides.Previous studies have shown that lncRNA-MEG3 acted as a tumour suppressor in cancer initiation, progression, metastasis and chemo-resistance [12,13].Furthermore, some reports have indicated that lncRNA-MEG3 mediated DM-associated micro-vascular dysfunction and the development of diabetic retinopathy [14,15].More strikingly, it is now believed that the inhibition of lncRNA-MEG3 confers robust neuroprotection against cerebral ischemic reperfusion injury both in vivo and in vitro [9,[16][17][18].However, up to now, the exact pathophysiological role of lncRNA-MEG3 in the development of diabetic brain stroke still remains obscure.
De ned as cell death triggered by generation of excessive lipid ROS (reactive oxygen species) dependent on iron, ferroptosis is different from other classical modes of programmed cell death [19].Iron accumulation was previously observed in stroke patients [20], while iron inhibitors and chelators such as ferrostatin-1 and deferoxamine presented signi cant protective bene ts against focal cerebral ischemiareperfusion injury in mice [21], indicating that ferroptosis attributes to several pathophysiological contexts of brain stroke.Accordingly, recent studies have reported that lncRNAs regulated oxidative stress-triggered ferroptosis in carcinoma cells [22,23].However, whether lncRNA-MEG3 mediates diabetic brain ischemia through ferroptosis remains to be clari ed.P53, once considered as a downstream target of lncRNA-MEG3, is involved in initiating cellular responses to endogenous or exogenous stress [16, 24,25].The functions of p53 in mediating the cellular response to oxidative stress-induced ferroptosis have been well established [23,26].Mechanistically, it appears plausible that the lncRNA-MEG3-p53 signaling pathway mediates ferroptotic cell death.However, the mechanistic details of the lncRNA-MEG3-p53 axis and diabetic brain ischemia-induced ferroptosis have not yet been elucidated.
Based on available data, brain microvascular endothelial cells are affected by hyperglycemia in the early stages of diabetes to cause functional impairment, which is one of the initiating factors of diabetic cerebrovascular diseases [27].In addition, brain microvascular endothelial cell damage can lead to microvascular occlusion and micro-thrombosis, which are closely related to the signi cantly increased incidence of perioperative stroke in diabetic patients.Therefore, in this current study, RBMVECs (rat brain microvascular endothelial cells) were exposed to 6 h of OGD (oxygen and glucose deprivation), and subsequently exposed to 24 h of reperfusion performed by incubating cells with various high concentrations of glucose to simulate in vitro diabetic brain ischemic injury.Furthermore, the regulatory roles of the lncRNA-MEG3-p53 axis in ferroptosis were studied by using an OGD + hyperglycemic reperfusion model to identify potential therapeutic strategies for diabetic brain stroke.

Cell separation and treatment
As described previously [28], Sprague Dawley (SD) rats aged three weeks were purchased from the Experimental Animal Center of Xiangya Hospital of Central South University.All rats were bred in a speci c pathogen-free environment in 12-h light-dark cycle and fed with rodent diet and water.All rats were anaesthetized with inhaling iso urane (2%, CAS NO. 64181101, Lunan Pharmaceutical Co., LTD.Shandong, China) and sacri ced by cervical dislocation.The whole brain was removed after opening the cranial cavity.High-purity brain microvascular fragments were obtained, followed by homogenization and fractionation.Microvessels were subjected to incubation at 37 ℃ and 5% CO 2 in a tissue culture ask coated by collagen which contained FBS (10%), heparin (100 milligram per milliliter) and basic broblast growth factors (10 nanogram per milliliter).RBMVECs (the rat brain microvascular endothelial cells) were observed using microscopy, and factor VIII-related antigen expression was detected.RBMVECs were transferred into an anaerobic incubator con gured to an atmosphere of 5% CO 2 , 94% N 2 , 1% O 2 , 98% humidity and 37°C to induce oxygen and glucose deprivation (OGD).The cells were reperfused by returning them to the incubation medium containing different concentrations of glucose (5, 10, 30, 45 and 75 mM) for 24 h of culture.The RBMVECs cultured in a medium with 5 mM glucose served as the control.
All experiments were approved by Institutional Animal Care and Use committee in Central South University.The protocol for the use of rats followed the guidelines of the Care and Use of Laboratory animals.

Lactate dehydrogenase (LDH) and cell viability assay
Using a commercial CCK-8 (cell counting kit-8; CK04, provided by Dojindo, Tokyo, Japan), cell viability was assessed.Brie y, after the indicated experiments, a CCK-8 working solution was added to each well and incubated for two hours at thirty-seven centigrade degree.The absorbance values were determined at 450 nm using a microplate spectrophotometer (Bio-Tek, Winooski, VT, USA).A LDH detection kit (purchased from Roche, Basel, Switzerland) was used as previously described [29,30], and OD (optical density) values were recorded at 450 nm.Cell survival rates and the values of LDH release were standardized to the values in the control group and expressed as percentages.

Real-time quantitative polymerase chain reaction (PCR)
After the indicated experiments, the total RNA was extracted by using Trizol (provided by Invitrogen, Carlsbad, CA, USA) according to the product instructions.PCR was performed with speci c primers and a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA).The 2 -ΔΔ Ct method was used to calculate the relative levels of lncRNA-MEG3, p53, FTH1, GPX4 and ACSL4 [31].The primer sequences are shown in Table-1.

Iron concentration assay
After indicated treatments, RBMVECs were immediately homogenized in PBS (phosphate-buffered saline).The supernatant was collected after centrifugation.The iron concentration was measured by using an Iron Assay Kit (ab83366, Abcam, UK) according to the product instructions.Iron concentrations were expressed as increments compared with the values of the control group (100%).

Lipid reactive oxygen species (ROS) measurement
After experiments, RBMVECs were incubated with C11-BODIPY 581/591 (D3861, Invitrogen).Then, the transplanted RBMVECs were cultured with the reagent at a working concentration of 2.5 µM for 30 min in an incubator.Using ow cytometry (provided by Becton Dickinson FACS Canto TM, USA), cellular uorescence intensity was analyzed and cell images were acquired through a uorescence microscope (IX81; Olympus).Lipid ROS production was expressed as increments compared with the values of the control group (100%).

Assessment of MPO (myeloperoxidase) and GSH (glutathione) expressions
RBMVECs were homogenized and the supernatant was collected for the analysis of MPO and GSH expressions by using a MPO assay kit (A044, Jiancheng Bioengineering Institute, Nanjing, China) and a total GSH/oxidized GSH assay kit (A06, Jiancheng Bioengineering Institute, Nanjing, China), respectively.The ratio of GSH/GSSG was calculated.The levels of MPO and GSH were standardized to the values of the control group and were expressed as percentages.

Detection of cell apoptosis
The apoptosis of RBMVECs was detected using an Annexin V-FITC/PI apoptosis detection kit (Sigma-Aldrich Trading Co., Shanghai, China) following the manufacturer's instructions.Apoptotic cells were quanti ed using a FACSCalibur cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

ChIP (Chromatin immunoprecipitation) assay
Sonicated nuclear lysates were puri ed, and using a ChIP assay kit (P2078, Beyotime), immunoprecipitation was performed according to the method described in a previous study of the authors in this research [33].

Statistical analysis
All data were presented as mean ± SD.One-way analysis of variance was applied with a Bonferroni post hoc test or Student's t-test.Data were analyzed with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA).P< 0.05 indicated signi cant difference.

A mimetic model of diabetic brain ischemic reperfusion injury was successfully established in vitro
To induce diabetic brain ischemic reperfusion injury in vitro, RBMVECs were exposed to 6 h of in vitro ischemia induced by OGD in an anaerobic incubator, followed by subsequent reperfusion via returning the RBMVECs to normoxic conditions, in which the RBMVECs were cultured for 24 h in culture media containing different concentrations of glucose (5, 10, 30, 45 and 75 mM).To elevate the in vitro insult to RBMVECs, a survival rate of approximately 50-60% was considered appropriate.The data demonstrated that as compared to 5 mM glucose (served as control), the exposure to higher glucose concentrations (≥30 mM) during the reperfusion period caused a signi cant reduction in cell viability and a marked increase of LDH release in a dose-dependent manner after exposure to OGD (P<0.05,Fig. 1A and B).However, higher concentrations of glucose (45 or 75 mM) caused too excessive damage to RBMVECs (with a survival rate of less than 50%).Therefore, continuous incubation at a relatively high concentration of glucose (30 mM) was chosen for subsequent experiments.In addition, 25 mM of mannitol were added into the 5 mM glucose medium to produce the same osmotic pressure as that produced by 30 mM of glucose, and the medium was used to treat RBMVECs for 24 h.There was no change in cell viability and LDH leakage between 5 mM and mannitol groups (P>0.05,Fig. 1C and D), which suggested that the impact of osmolality on cell survival was minimal.Thus, these ndings indicated that an in vitro mimetic model of diabetic brain ischemic reperfusion injury was successfully established.

LncRNA-MEG3 mediated the cellular damage induced by exposure to OGD + high glucose
After 6 h of exposure to OGD-induced injury and subsequent reperfusion in normal (5 mM) or high glucose (30 mM) media for 24 h, the high glucose (HG) condition signi cantly induced the up-regulation of lncRNA-MEG3 expression (P<0.05,see Fig. 2A).Mannitol did not affect lncRNA-MEG3 expression (P>0.05,Fig. 2B).
si-MEG3 (lncRNA-MEG3 siRNA) was transfected into RBMVECs and the results showed that lncNA-MEG3 expression was markedly down-regulated in RBMVECs transfected with si-MEG3, which con rmed the successful transfection of RBMVECs with si-MEG3 (P<0.05,Fig. 2C).Next, to assess the effects of lncRNA-MEG3 knockdown on the survival of RBMVECs, cell viability and LDH leakage were detected under OGD + HG conditions following transfection with either si-MEG3 or NC-siRNA.The results indicated that lncRNA-MEG3 knockdown promoted cell survival and reduced the release of LDH in RBMVECs (P<0.05,Fig. 2D and E).Moreover, it was found that the exposure to moderately high glucose (30 mM) induced the apoptosis of RBMVECs after their exposure to the injury induced by OGD + hyperglycemic treatment.Similarly, lncRNA-MEG3 knockdown remarkably alleviated apoptosis triggered by OGD + HG exposure (P<0.05,Fig. 2F and G).
Inhibition of lncRNA-MEG3 conferred protection to RBMVECs against OGD + HG-induced ferroptosis Some pivotal proteins, such as FTH1 (ferritin heavy chain 1), ACSL4 (Acyl-CoA synthetase long-chain family member 4) and GPX4 (glutathione peroxidase 4), are considered as core regulators of ferroptosis.Therefore, the expression levels of these proteins were measured under OGD + HG conditions with or without si-MEG3 transfection.The ACSL4 (a positive regulator) level was notably increased, while the levels of GPX4 and FTH1 (two negative regulators) were reduced in RBMVECs exposed to OGD + HG (see Fig. 3A-D).Interestingly, the silencing of lncRNA-MEG3 by siRNA not only obviously inhibited ACSL4 expression, but also reversed the levels of FTH1 and GPX4.
Additionally, cellular iron concentration, generation of lipid ROS, the product of lipid peroxidation, such as MPO and GSH, and GSH/GSSG ratio were assayed after exposure to OGD + HG.Similar to the above results, OGD + HG signi cantly elevated intracellular iron concentration, lipid ROS generation and MPO content, while markedly reduced the level of GSH and the GSH/GSSG ratio.Similarly, the effects of OGD + hyperglycemic treatment on these ferroptosis-associated indictors were all abolished by si-MEG3 (P<0.05,Fig. 3E-I).
To gain more solid evidence, it was tested whether RBMVECs undergo ferroptosis in response to Erastin and RSL3, direct inhibitors of system XC -and GPX4, respectively.At rst, different concentrations of Erastin and RSL3 were used to induce the ferroptosis of RBMVECs to determine an optimum concentration.Depicted in Fig. 4A and B, Erastin suppressed cell viability and enhanced LDH leakage in a dose-dependent manner, and the optimum concentration was 5 μM.However, when 60, 120, and 180 μM of RSL3 were used, RSL3 triggered cytotoxicity in a dose-independent manner and the optimum concentration was 60 μM (P<0.05,Fig. 4C and D).The cytotoxicity induced by both Erastin and RSL3 was alleviated upon treatment with si-MEG (P<0.05,Fig. 4E-H).
Taken together, all these ndings proved that the inhibition of lncRNA-MEG3 neutralized the ferroptosis induced by OGD+ hyperglycemic treatment.
The lncRNA-MEG3-p53 axis mediated OGD + HG treatment-induced ferroptosis Previous studies have reported that lncRNA-MEG3 could stimulate p53-mediated ischemic neuronal death.In this present study, the results also demonstrated that lncRNA-MEG3 knockdown notably inhibited the elevation of p53 expression induced by OGD + hyperglycemic reperfusion injury, suggesting that p53 might act as a downstream target of lncRNA-MEG3 (P<0.05,Fig. 5A-C).To further validate whether lncRNA-MEG3 modulated OGD + HG-induced injury through targeting p53, rescue experiments were performed.Firstly, RBMVECs were transfected with a wild-type p53 encoded speci cally by plasmid DNA.Then, Western blotting indicated that p53 levels were dramatically increased by pcDNA-p53 in normal control cells (P<0.05,Fig. 5D and E).Secondly, it was shown that the transfection of pcDNA-p53 signi cantly restored the expression of p53 in si-MEG3-transfected RBMVECs exposed to OGD + HG stimulation (P<0.05, Figure 5F and G).Furthermore, the pcDNA-p53 treatment also reduced RBMVECs viability compared to that of RBMVECs transfected with si-MEG3 (P<0.05;Fig. 5H).Finally, LDH leakage was decreased by the transfection with si-MEG3, which was abolished by pcDNA-p53 transfection in RBMVECs treated by OGD + HG (P<0.05,see Fig. 5I).The results showed that the overexpression of p53 signi cantly abrogated the protective effect of si-MEG3.

P53 participated in OGD + HG-induced ferroptosis through modulating GPX4
In vitro gain of function and loss of function analyses of p53 were also employed to assess its effect on ferroptosis elicited by OGD+HG and the associated signaling pathway by using siRNAs.As summarized in Fig. 6A-B, p53 knockdown was validated on the protein level.
To further investigate the biological function of p53 in regulating OGD + HG-induced injury, the effect of p53 on OGD + HG reperfusion-induced ferroptosis was evaluated.It was found that the inhibition of p53 signi cantly improved cell survival, as evidenced by increased cell viability and reduced LDH release.Moreover, p53 knockdown not only reduced cellular iron concentrations, lipid ROS production and MPO levels, but also increased GSH contents and GSH/GSSG ratios.However, the overexpression of p53 exerted exactly opposite effects (P<0.05,Fig. 6C-H).
With a desire to better elucidate the underlying mechanisms of p53 in modulating the ferroptosis induced by OGD + hyperglycemic reperfusion injury, the impact of p53 overexpression or inhibition on the expression of FTH1, GPX4 and ACSL4 was assessed.The ndings revealed that neither the overexpression nor knockdown of p53 exerted any signi cant impact on the gene expression of FTH1 and ACSL4 (P>0.05,see Fig 7A and B).However, GPX4 gene expression was signi cantly down-regulated by p53 overexpression and remarkably up-regulated by p53 depletion in RBMVECs exposed to OGD + HGinduced injury (P<0.05,Fig. 7C).Meanwhile, it was also observed that the GPX4 protein expression was regulated by the overexpression or knockdown of p53 (P<0.05,Fig. 7D and E).Furthermore, the overexpression of p53 also reversed the impact of si-MEG3 on the protein expression of GPX4 (P<0.05,Fig. 7F and G).
Using a chromatin-anti-p53 immunoprecipitation assay, it was shown that p53 was structurally bound to GPX4 promoter.Upon OGD + HG insult, p53 interacted with GPX4 promoter to suppress its transcription.However, p53 depletion inhibited the role of p53 in a way similar to the results obtained from RBMVECs silenced by lncRNA-MEG3.In contrast, p53 overexpression rescued the interaction between p53 and GPX4 promoter in RBMVECs silenced by lncRNA-MEG3 (P<0.05,Fig. 7H and I).Collectively, these results indicated that lncRNA-MEG3 regulated OGD + hyperglycemic reperfusion-induced ferroptosis at least partially through p53-mediated GPX4 transcription.

Discussion
Perioperative stroke is featured by the acute development of cerebral circulation disorders caused by cerebral ischemia or embolism during the perioperative period, and the morbidity and mortality of perioperative stroke remain high.Perioperative stroke is also one of the common serious complications observed during or after surgery and anesthesia [1,2].Over the past decade, numerous risk factors, including diabetes mellitus (DM), have been well characterized for perioperative stroke [3,4].DM is a chronic, persistent, and severe metabolic health problem characterized by hyperglycemia.Individuals with DM have almost twice the risk of stroke compared to nondiabetics.Moreover, approximately 30% of perioperative stroke patients have DM [3,4].Unfortunately, so far, few data are available to clarify the possible molecular mechanisms of diabetic brain stroke.
Long non-coding RNAs are recognized as a unique class of non-coding RNAs with a length of above 200 bp.Recent studies have pointed to the essential role of lncRNAs in brain stroke [7,8,[16][17][18], and lncRNAs have been identi ed as pivotal biomarkers of diabetes mellitus [14,15].Maternally expressed gene 3 (MEG3), a newly discovered lncRNA, shows the strongest genetic susceptibility for neurovascular [16][17][18] and neurodegenerative disease [34,35].Recently, a growing body of evidence indicates that upregulated lncRNA-MEG3 expression is positively correlated with a poor prognosis of ischemic brain injury [16][17][18].Furthermore, previous studies reported that lncRNA-MEG3 is highly expressed in vascular endothelial cells [36].Nevertheless, up to now, few literatures have discussed the correlation between lncRNA-MEG3 and diabetic brain stroke.In this study, RBMVECs were exposed to OGD followed by reperfusion in a high glucose medium to simulate an in vitro diabetic brain stroke model.The results suggested that a relatively high concentration of glucose (30 mM) signi cantly reduced the survival of RBMVECs.Moreover, it was also found that lncRNA-MEG3 was robustly activated in RBMVECs, whereas the silencing of lncRNA-MEG3 expression using small interfering RNA (siRNA) resulted in prominent inhibition of cell death after OGD + hyperglycemic treatment and reperfusion, suggesting that lncRNA-MEG3 might accelerate the pathologic process of diabetic brain stroke.Thus, it is urgent to explore the mechanisms underlying the role of lncRNA-MEG3 in diabetic brain stroke.
As a new pathway of programmed cell death, ferroptosis is iron-dependent and activated by lipid peroxides.A study by Tuo et al. con rmed an elevated iron concentration in brain stroke injury and showed that ferroptotic inhibitors reduced the size of brain injury and improved neurological functions in a middle cerebral artery occlusion (MCAO) model [21], demonstrating the link between ferroptosis and ischemic brain injury.Since iron dysregulation and subsequent lipid peroxidation are indispensable for the induction of ferroptosis [19], iron concentrations, lipid ROS production, levels of MPO and GSH contents, and GSH/GSSG ratio were assayed in this study after OGD + hyperglycemic reperfusion.The results showed that OGD + HG led to signi cant cytoplasmic elevation of levels of iron, lipid ROS and MPO along with a remarkable reduction in the GSH content and GSH/GSSG ratio, implying ferroptosis.Furthermore, all events associated with ferroptosis were blocked by the treatment with si-MEG3.Moreover, to further verify the role of lncRNA-MEG3 in ferroptosis, Erastin and RSL3 were used to induce ferroptosis.The results showed a noticeably suppressive effect of si-MEG3 on the lethality of Erastin and RSL3.Additionally, several reports have described that FTH1, ACSL4, and GPX4 act as essential factors in ferroptosis regulation [37][38][39].Therefore, to obtain more evidence and further investigate the molecular mechanisms underlying the role of OGD + HG in ferroptosis regulation, the expression levels of above proteins were evaluated and the results showed that the expression of the positive ferroptosis regulator ACSL4 was enhanced, while the expression of two negative ferroptosis regulators (GPX4 and FTH1) was suppressed under OGD + HG conditions.Similar to the above results, the administration of si-MEG3 reversed the changes in the expression of these proteins in ferroptosis.Thus, these results suggested that the knockdown of lncRNA-MEG3 could inhibit ferroptosis following OGD + HG insult.
Additionally, it was found lncRNA-MEG3 was positively correlated with p53 expression in RBMVECs.The depletion of lncRNA-MEG3 caused the marked reduction of p53 expression.Notably, reintroduction of p53 reversed the protective effect of lncRNA-MEG3 inhibition against OGD + HG-induced injury.Furthermore, it was reported that lncRNA-MEG3 directly acted on p53 to regulate the expression of p53 and sensitize p53-mediated neuronal death in vivo and in vitro [16,17,24,25].Recently, Zhan and his colleagues also used an RNA-immunoprecipitation (RIP) assay to a rm that lncRNA-MEG3 could interact with p53 in RBMVECs [28].Hence, it seems plausible that p53 serves as a downstream target of lncRNA-MEG3 in brain stroke.P53 has been characterized as a positive regulator of ferroptosis associated with iron metabolism disorder and excessive lipid peroxidation in certain cancer cells [23,26].Moreover, the activation of p53 sensitizes cells to ferroptosis through transcriptional repression of TIGAR, GLS2 and SCO2 [40].Nevertheless, tumor cells expressing p53 mutations are more vulnerable to ferroptosis [41].Considering these facts, it is reasonable to speculate that the lncRNA-MEG3-p53 signaling pathway mediates ferroptosis in the model of this study.To validate this hypothesis, in vitro gain-and loss-offunction analyses of p53 were performed to estimate the effect of p53 on ferroptosis.Lipid peroxidation, down-regulated GPX4 expression and GSH depletion are crucial events in the ferroptosis assay.The data showed that the inhibition of p53 not only signi cantly alleviated lipid peroxidation and elevated iron and MPO contents upon exposure to OGD + hyperglycemic reperfusion, but also reserved the expression of GPX4 and GSH, whereas the overexpression of p53 exerted opposite effects.Although both overexpression and inhibition of p53 had no impact on FTH1 and ACSL4 expressions, it could still be concluded that p53 activation invariably caused ferroptosis in OGD + hyperglycemic reperfusion injury.
Ferroptosis can be activated by the treatment with RSL3 and direct inactivation of GPX4, which block the conversion of reduced GSH to oxidized GSH and lipid hydroperoxides/hydrogen peroxides to their end products, such as alcohol and water.Ferroptosis was induced by knockdown of GPX4 in a ROS-, MEK-, and iron-dependent manner, while RSL3 resistance was caused by GPX4 overexpression [42].Furthermore, cerebral neuronal loss was found in GPX4 knockout (neuron-speci c) mice, which was explained by induced ferroptosis in part [43].In this study, it was proven that p53 promoted GPX4 expression at the pre and post-transcriptional level.Mechanistically, the most pivotal issue to address is how p53 modulates GPX4 expression.SLC7A11 is a major component of System X C − and the latter is indispensable for importing cystine, which is used to synthesize antioxidant GSH [19,44].Previous studies have demonstrated that p53 directs the transcriptionally suppressed SLC7A11 expression [41,45,46], thereby suppressing system X C − activity to trigger ferroptosis.In a similar mechanism depicted in Fig. 7H, under OGD + HG conditions, p53 directly inhibits GPX4 transcription by binding to a speci c locus located on GPX4 promoter.Similarly, lncRNA-MEG3 knockdown blocked this interaction, while the reintroduction of p53 promoted this interaction in RBMVECs silenced by lncRNA-MEG3.Based on the ndings of this study, it can be conceived that diabetic cerebral ischemic reperfusion injury signi cantly up-regulated lncRNA-MEG3 expression, resulting the activation of its downstream target p53 and subsequently suppressing GPX4 expression, eventually leading to lipid peroxidation-triggered plasma membrane rupture and ferroptosis.

Conclusions
Taken together, we found that the knockdown of lncRNA-MEG3 protected RBMVECs against the injury induced by OGD + hyperglycemic treatment.Most importantly, the present study also provided novel insights that lncRNA-MEG3 served as a ferroptotic promoter to mediate diabetic brain ischemic damage by functionally activating p53 to inhibit GPX4 activity.Therefore, lncRNA-MEG3 promoted ferroptosis in RBMVECs induced by OGD + HG through the p53-GPX4 axis.Such information may help to develop a novel strategy for the therapeutic intervention of diabetic brain ischemia.