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miR-155-5p in Extracellular Vesicles Derived from Choroid Plexus Epithelial Cells Promotes Autophagy and Inflammation to Aggravate Ischemic Brain Injury in Mice

Ischemic stroke is a common disease of the central nervous system, and ischemic brain injury (IBI) is its main manifestation. Recently, extracellular vesicles (EVs) have been strongly related to the diagnosis and treatment of IBI. However, the underlying mechanism of their effects remains enigmatic. In the present study, we aimed to study how miR-155-5p plays a role in choroid plexus epithelial (CPE) cell-derived EVs in IBI pathology. We found that miR-155-5p expression was enriched in CPE cell-derived EVs, which were subsequently internalized by neurons, enabling the delivery of miR-155-5p into neurons. An inducible oxygen and glucose deprivation and reoxygenation (OGD/R) cell model was developed to mimic ischemic neuronal injury in vitro. miR-155-5p overexpression led to reduced neuron viability, promoted apoptosis, elevated autophagic proteins' expression, and activated NLR family pyrin domain-containing 3- (NLRP3-) related inflammasomes, thereby aggravating OGD-induced neuronal injury. A dual-luciferase reporter assay exhibited that miR-155-5p could inhibit the Ras homolog enriched in brain (Rheb) expression, a mechanism critical for miR-155-5p-mediated neuronal injury. Furthermore, a mouse IBI model was developed using the transient middle cerebral artery occlusion (tMCAO) method. Animal experiments verified that miR-155p delivery via CPE cell-derived EVs aggravated IBI by suppressing Rheb expression. In conclusion, miR-155-5p in CPE-derived EVs can aggravate IBI pathology by suppressing Rheb expression and promoting NLRP3-mediated inflammasomes, suggesting its role as a potential therapeutic target in IBI.


Introduction
Stroke is primarily classified into hemorrhagic and ischemic subtypes. Although less fatal, ischemia accounts for 60-80% of all types of stroke globally, significantly impacting human health and quality of life [1]. Ischemic stroke has been correlated with high morbidity rates, physical disability, mortality, and recurrence [2]. Pathologically, ischemia is characterized by cerebral thrombosis or embolism, leading to hypoxia and glucose deficiency. Importantly, sudden depletion of oxygen and glucose most likely induces neuroinflammation and cell death as well as secondary injury to the brain during cerebral ischemia/reperfusion (I/R) and hypoperfusion [3]. A growing body of evidence has revealed the involvement of multimodal pathophysiologies in the cerebral I/R process, resulting in diverse outcomes, such as energy depletion, oxidative stress, excitotoxicity, ion imbalance, and altered gene expression pattern-mediated brain damage and motor/cognitive dysfunctions [4]. Ischemic brain injury (IBI) is unique in that once ischemia begins, irreparable brain damage can occur within minutes to hours [5]. It has been observed that disruption of the blood-brain barrier (BBB) integrity is one of the frequently occurring I/ R-induced brain encephalopathy [6]. With the recent advancements in stroke management and rehabilitation, remote ischemic preconditioning (RIPC) therapy has been proven safe and effective in suppressing recurring IS events [7]. However, the main obstacle to IBI treatment remains the efficient and targeted delivery of therapeutics to ischemic foci [8]. Recently, accumulating evidence has pointed out that extracellular vesicles (EVs) possess the potential to treat I/R-induced brain injury in premature infants [9].
EVs are nanosized vesicular structures with lipid bilayer membranes that are secreted into the extracellular space by most cell types under certain pathophysiological conditions [10]. EVs, released by both prokaryotes and eukaryotes, serve as the key mediator of intra-and intercellular communications through physical interactions with the target cell type [11]. Specifically, choroid plexus epithelial (CPE) cell-derived EVs have been identified as novel factors in the signaling communications across the BBB during peripheral inflammation. Additionally, CPE-derived EVs can secrete proinflammatory microRNAs (miRNAs) in the presence of recipient brain parenchymal cells [12]. In particular, miRNA-155 (miR-155) expression has been linked to the promotion of autophagic and neuroinflammatory responses in the IBI [13,14]. Furthermore, an increased level of matured miR-155-5p in relation to ischemic injury has been validated in the rodent model of middle cerebral artery occlusion (MCAO) as well as in the cell model of induced oxygen and glucose deprivation and subsequent reoxygenation (OGD/R) [15,16]. Through the bioinformatics analysis of miRNA expression datasets in combination with luciferase reporter assay for miR-155 expression, we found that both miR-155-5p and GTP-binding Ras homolog enriched in brain (Rheb) protein were enriched following the IBI. Rheb has been shown to interact with a myriad of signaling molecules, playing an essential role in regulating apoptosis and autophagy [17,18]. Rheb also functions as the inducer of mammalian target of rapamycin (mTOR) signaling. It has been found that Rheb/mTOR signaling interaction can inhibit both autophagic and apoptotic responses in neuronal cells, reflecting the induction of underlying neuroprotective mechanism [19]. Stroke pathology-induced miR-155 upregulation mechanistically disrupts the Rheb and mTOR pathway interaction promoting autophagy and inflammasome activation [20]. Among the mTOR complexes, namely, mTORC1 and mTORC2, mTORC1 regulates the translation of its downstream effectors by phosphorylation-mediated posttranslational modifications of 4E-binding protein 1 (4E-BP1), in addition to p70 ribosomal S6 protein kinases (S6Ks) like initiation factors, that modulate cellular proliferation and maturation processes [21].
Rheb expression has been linked to the inhibition of inflammasome activation, particularly the NOD-like receptor family protein pyrin domain-containing 3-(NLRP3-) related inflammasome [21]. The NLRP3 inflammasome signaling modulates cell damage mechanisms by assembling NLRP3 and oligomerized ASC and activates the caspase1 signaling axis in response to microbial infection and other stressors [22]. Suppression of NLRP3 inflammasome activation alleviates IBI pathology [23]. Therefore, we aimed to investigate the mechanism of EV-miR-155-5p-mediated regulation of the Rheb/mTORC1 signaling axis in IBI pathology.

Materials and Methods
2.1. Ethical Approval. The study protocols for animal experiments were approved by the Animals Ethics Committee of the Affiliated Hospital of Guizhou Medical University.

Choroid Plexus Epithelial (CPE) Cell
Culture. Mouse primary CPE cells were cultured as described elsewhere [12]. Briefly, 500 mg/kg bodyweight of tribromoethanol was intraperitoneally injected in C57BL/6J mice, aged 2-9 days (provided by Liaoning Changsheng Biotechnology Co. Ltd., Chinese Academy of Sciences, Liaoning, China), for whole body anesthesia, followed by cervical dislocation prior to harvesting the brains. The choroid plexus (CP) was separated under an anatomical microscope. Next, the CP was incubated with streptomycin protease (isolated from Streptomyces griseus; Sigma-Aldrich, USA) for 5-7 min for enzymatic hydrolysis, then excess enzyme was removed by washing 2 times with HBSS (Hank's balanced salt solution) medium to terminate the digestion and remove residual enzyme. Cells were then grown in the conditioned medium (CM; DMEM-F12+10% EV-depleted serum) for 48 h on a laminin-coated plate or transwell system. To eliminate the fibroblast cells from the culture, cytosine arabinoside (Ara-C) treatment was initiated after 48 h of culture. Cells were maintained in a humidified chamber at 37°C with 5% CO 2 .
2.3. Primary Neuron Culture. Primary cortical neurons were isolated from the cerebral cortex of C57 mouse embryos aged E16 to E18 days. Briefly, cortical neurons (7 × 10 5 cells/well) were seeded on the poly-D-lysine (Sigma-Aldrich) coated 6-well plate in DMEM medium, which was replaced with B27-supplemented neurobasal medium (Gibco, USA) 4 h postseeding, and was maintained in a moisturized condition with 5% CO 2 supply for 7-10 days at 37°C, before the experiments.
2.4. Construction of an OGD/R Cell Model. CPE cells and primary neurons were washed 2 times with glucose-free DMEM basal medium prior to seeding them for culture in DMEM with 10% FBS in a low-oxygen incubator supplied with 94% N 2 , 5% CO 2 , and 1% O 2 and then treated with OGD. The culture was removed from the incubator after 45 min for the replacement of the OGD induction medium with the maintenance medium, and the OGD-induction solution was changed with the maintenance medium. Following that, cells were transferred to a conventional cell culture-grade incubator allowing the cells to recover for 24 h for subsequent analyses [24].

Evaluation of EVs.
The OGD/R-CPE cell-derived exosomes were isolated in high purity from the supernatant culture medium. Stepwise, after removing the OGD/R-CPE cell culture medium, cells were cleaned on-plate for 2 times with DPBS, and the exosome-free medium (ultracentrifugation at 100,000 × g for 16 h at 4°C) was added to the cells to culture for 48 h, prior to collecting the supernatant. The supernatant was then sequentially centrifuged at was at 2,000 × g for 30 min and 100,00 × g for 30 min and washed for one time in PBS at 100,000 × g for 70 min at 4°C. At the final step, exosomes were resuspended for downstream characterizations.
After precipitation, EVs were quickly cross-linked by 2.5% glutaraldehyde (GTA) at 4°C. Then, EVs were subjected to gradient alcohol dehydration steps and finally immersed in epoxy resin. EVs were observed under transmission electron 2 Oxidative Medicine and Cellular Longevity microscopy (TEM) (JEOL 1230, Japan) by staining the finely sectioned slices with lead citrate-uranyl acetate solutions. The remaining portions of the purified EVs were resuspended in PBS buffer to reach the concentration of 10 6 -10 9 particles/ mL for injection into the NanoSight analyzer (ZetaView PMX 110, Germany) using a 1 mL syringe. EV-specific markers, like calnexin, CD63, CD81, and Hsp70, were subsequently analyzed by immunoblotting. When the neurons were becoming 30-50% confluent, the nutrient solution was aspirated from the culture. Next, 1 mL/well of a complete nutrient solution containing a 10fold volume of diluted virus (dilution factor was between 10 -3 and 10 -7 ) was simultaneously added with polybrene (H8761; Solarbio), and these treated neurons were main-tained under standard culture condition. The next day, the viral particle-loaded medium was replaced with 2 mL of complete medium for another ON culture. The GFP expression was observed after 5 days with a fluorescence microscope. >95% of cells were found to be GFP-positive. Then, stable positive cells were selected by treating with 0.5 μg/ mL of puromycin.
2.9. Cell Transfection. CPE cells and neurons were plated separately in 6-well plates at 1 × 10 5 cells/mL density, one day pretransfection. After reaching between 50% and 70% confluency, successfully modeled CPE cells or neurons were treated with 100 nM of mimic-NC, miR-155-5p mimic, inhibitor-NC, or miR-155-5p inhibitor (GenePharma, China) each by Lipofectamine-2000 (11668027; Thermo Fisher, USA). After modeling, the neurons were immediately transduced with overexpressing-(oe-) NC or oe-NLRP3 adenoviral particles. Cells were selected by G418 (600 mg/ L) after 24 h, and the solution was renewed every 3 days. Twelve days postselection, 46% of transduced cells survived. Subsequently, the culture was expanded under a 300 mg/L maintenance concentration to obtain a stably transfected cell line.
2.11. Cell Counting Kit-8 (CCK-8). Cells (5 × 10 4 cells/mL) were resuspended in DMEM containing 10% FBS. Next, 100 μL of media with cells was put in each well in a 96-well plate and incubated for 24 h, 48 h, and 72 h. After incubation, the supernatant was removed, followed by the addition of 10 μL of CCK-8 solution (WH1199; Shanghai Weiao, China) to every well, and kept for 2 h at 37°C. The absorbance was measured by a microplate reader Multiskan FC (51119080; Thermo Fisher) [24]. Triplicated wells were measured for each group to calculate the mean value.

TUNEL Staining.
We performed the TUNEL assay to determine whether EV treatment reduced the apoptosis rate of cortical neurons. The cells were treated with 4% PFA. Following PBS washing, a TUNEL assay was performed using fluorescein-coupled probes, as directed in the protocol. Cells were then counterstained with DAPI (1 : 1000; Beyotime, China) for 10 min at room temperature to evaluate nuclear morphology. Each slide was washed, and images were captured by a fluorescence microscope. To assess whether the EV treatment reduced the neuronal apoptosis rate, we performed the TUNEL assay. We calculated the populations of TUNEL+NeuN and NeuN-only positive cells in 5 distinct brain regions. In this in vitro study, we calculated the 3 Oxidative Medicine and Cellular Longevity number of TUNEL+DAPI double-positive cells and only DAPI-positive cells in the similar 5 brain regions in each slide for statistical analysis. The ratio of injured to uninjured cells was calculated by dividing the count of DAPI/TUNEL double-positive cells to only DAPI-positive cells.

Construction of a Transient Middle Cerebral Artery
Occlusion (tMCAO) Mouse Model. All adult male C57BL/6J mice were 8-10 weeks old (provided by Liaoning Changsheng Biotechnology Co. Ltd., China). At all stages of the study, the researchers were unaware of the experimental conditions. Mice were randomly assigned to different treatment groups: (1) sham group: mice that underwent sham surgery; (2) model group: no intervention; (3) model intervention group: NC EV-agomir, EV-miR-155a-5p agomir, NC antagomir, EV-miR-155a-5p antagomir, oe-NC, oe-Rheb, oe-Rheb+EV-agomir-NC, and oe-Rheb+EV-miR-155a-5p agomir-treated groups. They were given ad libitum access to fine-grained feed and water and provided with natural light in a 12 h light/dark cycle. After anesthetizing mice with 30-70% oxygen/nitrous oxide combined with 1.5-2% isoflurane, a 6-0 nylon suture (silicone-coated) was guided through the external to the internal carotid artery and then to the MCA. The efficiency of the occlusion model was determined from the surface cerebral blood flow (CBF) rate by a laser Doppler flowmeter (Moor Instruments, UK) until 10% of the baseline CBF was reached. One hour after successful occlusion, the suture was taken out to allow reperfusion, and immediately after this, EVs were administered through the tail vein at 100 μg per day dose for 3 days [25].

Neurobehavioral Tests.
Neurobehavioral examinations were conducted before and after 3 days of the establishment of the tMCAO model by investigators unaware of the exper-imental design. The neurological assessment was based on the modified neurological severity score (mNSS) system, which combines reflex, motor, and balance test scores [26]. The severity score ranges from 0 to 14, where 0 indicates normal, and increasingly higher scores indicate increasing injury severity [27].
2.16. Brain Weight. Three days after tMCAO surgery, mice were euthanized, and the brains were collected without perfusion. After dissecting out the cerebellum and brainstem portions from the forebrain, the remaining whole brain was cut through the midline and weighed using a precision balance (sensitivity, 0.001 g). Then, the mass ratio of the ipsilateral (right) to the contralateral (left) hemisphere was calculated.
2.17. Triphenyltetrazolium Chloride (TTC) Treatment. The whole brains were harvested without perfusion from the EVtreated tMCAO mice after proper euthanasia. The brain was sliced into 2 mm sections, incubated in TTC solution (2%) (Sigma-Aldrich) in the dark for 10 min, and fixed with 10% PFA. The cerebral infarction volumes were analyzed with Ima-geJ (NIH, USA). The percentage of infarcts was determined using the following formula: ðtotal area of the contralateral hemisphere − no infarction area of the ipsilateral hemisphereÞ /ðtotal area of the contralateral hemisphere × 2Þ.
2.18. Enzyme-Linked Immunosorbent Assay (ELISA). ELISA kits (69-21178 and 69-21183; MSKBIO, China) were used to determine the levels of IL-18 and IL-1β in the mouse brain tissue lysates. Mouse brain tissues in sterile PBS were ground, the mixture was centrifuged at 10000 rpm for 10 min, and the supernatant was collected for detection. The absorbance (A) values of the wells were measured at 450 nm within 3 min of preparation using an all-purpose enzyme marker (Synergy 2, BioTek, Winooski, VT, USA).

Real-Time Quantitative-Polymerase Chain Reaction
(RT-qPCR). EV-containing miRNAs were purified using the SeraMir exosome RNA purification kit (System Biosciences, USA). Total cellular RNA was extracted by the TRIzol method (15596026; Invitrogen). Complementary DNA (cDNA) was prepared from miRNA by miScript Reverse Transcription kit (Qiagen GmbH, Germany). U6 served as the internal control in the stem-loop RT-qPCR assay (Gen-ePharma, China).
A PrimeScript™ RT reagent kit (TaKaRa) was employed for cDNA synthesis from 1 μg of total RNA. Next, a

Oxidative Medicine and Cellular Longevity
StepOnePlus™ RT-PCR System (Invitrogen) was utilized for quantitative mRNA expression analysis of Rheb, NLRP3, and transthyretin (TTR) genes. The primer sequences are listed in Table 1. β-Actin served as an internal reference. SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa) was used for cDNA amplification. The 2 -ΔΔCt method was used to calculate the relative mRNA or miRNA expressions.
2.20. Western Blot Analysis. Cells or tissue lysates were prepared by lysing the sample in lysis buffer (1 mL) with protease inhibitor (P0013J; Beyotime) for 45 min on ice, followed by spinning at 4000 × g for 30 min at 4°C. The protein concentration of each supernatant sample was measured using the BCA kit (PC0020; Solarbio). The 20 μg protein sample from each group was resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Next, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (66485; Pall Corp, USA). The membrane was then blocked with 5% nonfat skim milk for 2 h at RT. The The viability of neurons exposed to OGD/R after coculture was determined using a CCK-8 kit. (g) The number of TUNEL + neurons exposed to OGD/R after coculture was determined by TUNEL staining. * p < 0:05 vs. the brain group or control group. # p < 0:05 vs. neurons treated with OGD/R+PBS (n = 3).

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Oxidative Medicine and Cellular Longevity  2.21. Statistical Analysis. SPSS 21.0 software (IBM, USA) was used for the statistical analyses. The data were expressed as means ± standard deviation (SD). An unpaired t test was conducted for comparisons of data between two groups. For multiple group comparisons, a one-way analysis of variance (ANOVA) was performed, followed by Tukey's post hoc test. For comparisons of data between groups at different time points, two-way ANOVA was performed, followed by Bonferroni's test. Statistical significance was established at p < 0:05.

CPE Cell-Derived EVs Promote Neuronal Injury.
To investigate the regulatory mechanisms of CPE cell-derived EVs in IBI, we established a mouse primary CPE cell model. Next, the CPE cells were subjected to an OGD/R environment, and EVs were subsequently isolated from the normal and OGD/R culture. We observed the typical cup-shaped EV morphology under TEM. Membranous structures were visualized around the vesicles with the central low electron density region (Figure 1(a)). The NanoSight tracking analysis revealed that the EVs had a diameter between 40 and 160 nm exhibiting irregular Brownian motion, and the number of CPE cell-derived EVs was increased when exposed to OGD/R (Figure 1(b)). Western blot analysis indicated that CPE-derived EVs could express the relevant marker proteins CD81, CD63, and Hsp70 but not the calnexin (Figure 1(c)), suggesting that the ischemic environment stimulated the production of more EVs in CPE cells. Therefore, subsequent experiments were conducted using EVs isolated from OGD/ R-treated CPE cells.
To verify whether CPE-derived EVs could enter neurons and affect their functions, we first constructed an OGD/R model with neurons. Subsequently, PKH26-labeled EVs were cocultured with the neuron for 6 h, and CPE-derived EVs were found to be absorbed by neurons (Figure 1(d)). Next, to examine the effect of these EVs on neurons, we detected the formation of autophagosomes in neurons and measured the expression of related proteins (Figure 1(e)), assessed cell viability (Figure 1(f)), and determined the apoptosis rate (Figure 1(g)). The number of autophagosomes was significantly increased, and LC3 II/I, Beclin-1, and LAMP-1 expressions were notably elevated, but P62 expression was strikingly downregulated (Figure 1(e)), with decreased cell viability (Figure 1(f)), and increased the number of TUNEL-positive (Figure 1(g)) neurons exposed to OGD/R compared with normal controls. Additionally, neurons cotreated with OGD/R+EV showed an increased number of autophagosomes; elevated Beclin-1, LC3 II/I, and LAMP-1 protein expression; decreased P62 protein expression (Figure 1(e)); decreased cell viability (Figure 1(f)); and an increased number of TUNEL-positive cells (Figure 1(g)) compared with neurons cotreated with OGD/R+PBS. These results suggest that CPE-derived EVs can promote neuronal autophagy and apoptosis.

CPE Cell-Derived EVs
Aggravate IBI through miR-155-5p. Next, a tMCAO mouse model was constructed and administered with miR-155-5p mimic or inhibitor treated CPEderived EVs. Compared with the mice receiving sham treatment, the miR-155-5p level was significantly increased (Figure 3(a)), the mass ratio of the ipsilateral (right) hemisphere to the opposite (left) hemisphere of the brain was significantly reduced (Figure 3(b)), the mNSS was increased (Figure 3(c)), the cerebral infarction rate was significantly increased (Figure 3(d)), and the levels of LC3 II/I, Beclin-1, and LAMP-1 were increased considerably, while P62 expression was significantly reduced (Figure 3(e)) in the mice that had undergone the tMCAO operation, suggesting the successful establishment of the tMCAO model. Compared with mice treated with EV+mimic-NC, mice treated with EV+miR-155-5p mimic exhibited a similar trend. However, compared with mice cotreated with EV+inhibitor-NC, mice cotreated with EV+miR-155-5p inhibitor exhibited opposite results. These results indicated that CPE-derived EVs promoted autophagic activity, activated inflammatory factors, and aggravated IBI through miR-155-5p expression in the tMCAO mouse model.

miR-155-5p in CPE Cell-Derived EVs Can Target and
Inhibit Rheb to Enhance Neuronal Injury. We analyzed the miRDB, TargetScan, and RNA22 databases to predict the

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Oxidative Medicine and Cellular Longevity  Figure 5: Rheb activates the NLRP3 inflammasome to enhance neuronal autophagy and apoptosis. (a) Rheb and NLRP3 were coexpressed as determined using the MEM tool. (b) NLRP3 expression was estimated by RT-qPCR. (c) NLRP3 expression in the OGD/R-exposed cell model was measured by RT-qPCR. (d) oe-NLRP3 neuron transfection efficiency was quantitated by RT-qPCR. (e) Rheb and NLRP3 expression in oe-Rheb+oe-NC cotransfected neurons exposed to OGD/R conditions as measured by RT-qPCR. (f, g) The secretion levels of IL-18 (f) and IL-1β (g) from neurons cotransfected with oe-Rheb+oe-NC and exposed to OGD/R conditions were measured by ELISA. (h) The activity of neurons was determined using a CCK-8 kit. (i) Expressions of autophagy-linked proteins (Beclin-1, LC3 II/I, LAMP-1, and P62) and NLRP3 inflammasome activation-related proteins as measured by western blot analysis. (j) Protein expressions of total caspase3 and cleaved-caspase3 in neurons were determined by western blot analysis. (k) The number of TUNEL + neurons was determined by TUNEL staining. * p < 0:05 vs. control or oe-NC-transfected neurons. # p < 0:05 vs. oe-Rheb+oe-NC-cotransfected neurons (n = 3).

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Oxidative Medicine and Cellular Longevity

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Oxidative Medicine and Cellular Longevity targets of miR-155-5p and found that Rheb was the only gene regulated by miR-155-5p identified in each of these datasets (Figure 4(a)). As shown in Figure 4(b), the binding sites of miR-155-5p and Rheb in humans, rats, and mice were identified using TargetScan. Additionally, the Rheb/ mTORC1 axis exerts inhibitory effects on autophagy and inflammasome activation following stroke [20]. Therefore, we hypothesized that miR-155-5p might regulate neuronal function via the Rheb/mTORC1 axis. The luciferase assay showed that the bioluminescence intensity in neurons cotreated with the miR-155-5p mimic and Rheb-3′ UTR-WT was significantly reduced, as compared with that of mimic-NC plus Rheb-3′ UTR-WT cotransfected cells, suggesting that miR-155-5p could bind to the Rheb gene specifically (Figure 4(c)). We examined whether miR-155-5p delivered to neurons by CPE-derived EVs could inhibit Rheb/mTORC1 activity and thereby might affect neuronal function. We observed that the miR-155-5p level was drastically elevated, whereas Rheb expression was decreased in oe-Rheb+EV-and miR-155-5p mimic-cocultured neurons compared with that in oe-Rheb+EV-and mimic-NC cotreated cells (Figure 4(d)). Therefore, increased phosphorylations of mTOR, S6K, and 4EBP1 were observed in neurons cotreated with oe-Rheb+PBS compared with neurons cotreated with oe-Rheb+EV-and miR-155-5pcotreated mimic (Figure 4(e)).
3.6. miR-155-5p in EVs Derived from CPE Cells Suppresses Rheb Expression to Aggravate IBI. To further understand the mechanism of CPE-derived EVs on IBI in tMCAO mice, we treated mice with oe-Rheb+EV with mimic-NC or oe-Rheb+EV with miR-155-5p mimic. RT-qPCR indicated an increase of the miR-155-5p expression with a decreased Rheb expression in the tMCAO model compared with the corresponding expression levels in sham-treated mice. Interestingly, miR-155-5p expression remained unchanged, whereas Rheb expression was elevated in oe-Rheb-infected mice compared with the corresponding expression levels in tMCAO and oe-NC-infected mice, but this trend was reversed by EV and miR-155-5p mimic cotreatment, which also led to increased miR-155-5p expression (Figures 6(a) and 6(b)). Compared with that in sham-treated mice, the mass ratio of the ipsilateral (right) to the contralateral (left) hemisphere in the mouse model of tMCAO was reduced ( Figure 6(c)), and the mNSS (Figure 6(d)) and cerebral infarction rates were increased ( Figure 6(e)). The expression was significantly reduced, and NLRP3 expression was sharply increased (Figure 6(f)). The levels of proinflammatory factors IL-18 and IL-1β in the mouse brain tissue lysates were remarkably increased (Figures 6(g) and 6(h)). The protein expressions of Beclin-1, LC3II/I, and LAMP-1 in the brain tissues of these mice were significantly increased, and P62 expression was dramatically reduced (Figure 6(i)).
Compared with tMCAO-and oe-NC-infected mice, the mass ratio of the ipsilateral (right) hemisphere to the contralateral (left) brain hemisphere in the oe-Rheb-infected mice was significantly increased, while the mNSS and cerebral infarction rates were significantly reduced. The expression was increased, and NLRP3 expression was significantly reduced. The IL-18 and IL-1β concentrations in the brain samples were strikingly decreased. The LC3II/I, Beclin-1, and LAMP-1 expressions in the brain tissues were significantly reduced, but P62 expression was notably increased, the effect of which was rescued by EV and miR-155-5p mimic cotreatment. Notably, miR-155-5p in CPE cell-derived EVs suppressed Rheb to aggravate IBI.  Figure 7: Schematics of potential mechanisms involved in the EV-delivered miR-155-5p and IBI. CPE-derived EV-derived miR-155-5p promoted inflammation-and/or autophagy-related protein expression to promote inflammation and autophagy of neurons through the Rheb/mTORC1-NLRP3 axis in a hypoxic environment.

Discussion
Stroke is characterized as a cerebrovascular disease, leading to high disability and mortality rates, especially among the elderly population [29,30]. Increasing evidence has highlighted that EVs are critically involved in the ischemic pathomechanism, and EVs derived from different cell types can induce neuroprotection and neurorestorative effects by modulating gene, protein, and miRNA expressions in their target cell and tissue types [31]. However, to date, studies have focused only on the potential of EVs in treating ischemic stroke. Previous studies have revealed that LPS-stimulated primary CPE cell-derived EVs can enter the brain parenchyma to inhibit the miRNA targets and inflammatory gene upregulation, resulting in aggravated brain injury [12]. Studies on the mechanisms involved in brain injury after cerebral ischemia have mainly investigated neuronal responses after ischemic stroke. No study has revealed the molecular signal mediating interactions between CPE cells and neurons to exacerbate a neuronal injury. Identifying the molecular mechanism by which CPE cells contribute to neuronal injury would facilitate the development of new strategies to treat ischemic stroke. Initially, we found that CPE cell-derived EVs promoted cell inflammation, as evidenced by increased IL-18 and IL-1β levels; enhanced autophagy characterized by increased LC3II/I, Beclin-1, and LAMP-1 levels; reduced P62 levels; decreased cell viability; and elevated apoptosis rate. Similar findings have been previously reported; for example, the addition of EVs increased the expression of Beclin-1 and LC3B II/I in mice after focal brain irradiation [32]. Mesenchymal stem cell-derived EVs can inhibit the proliferation/migration by the ERK pathway activation in RSC96 cells, promoting apoptosis [33]. IL-18 acts as the proinflammatory cytokine inducing tissue damage, inflammation, and apoptosis, and increased IL-18 levels are associated with myocardial injury after ischemia or infarction [34]. Our experiments revealed that CPEderived EVs could deliver miR-155-5p to aggravate neuronal injury. Therefore, CPE cell-derived EVs constituted a novel mechanism of communication between the peripheral blood under inflammatory conditions and the brain, with systemic inflammation resulting in an increase in EVs and associated proinflammatory miRNAs, including miR-155 [12]. It has been observed that the miR-155-5p level is increased in EVs present in the cerebrospinal fluid [35]. Additionally, a previous study showed that miR-155-5p restoration could promote autophagy, while miR-155-5p inhibition could decrease P62 expression [36].
Subsequently, our study findings suggested that miR-155-5p expression could impact the IBI through Rheb signaling. Reportedly, miR-155 binds the 3′ UTR of Rheb and inhibits its expression [20]. The same report suggests that reduced Rheb expression downregulates mTORC1 expression, which is associated with considerable cerebral infarction volumes and cell apoptosis during ischemic stroke [20,37]. Consistently, we found that CPE cell-derived EVs contained miR-155-5p that promoted neuronal injury by targeted inhibition of Rheb expression. Earlier, it was shown that Rheb activation promotes the differentiation of neurons [38]. Rheb also inhibits NLRP3 inflammasome activation [21]. Additionally, NLRP3 is overexpressed in cells exposed to OGD/R conditions, whereas NLRP3 inhibitor treatment decreases NLRP3 expression in ischemic stroke [39]. Furthermore, studies have revealed that inhibition of NLRP3 inflammasome activation can attenuate hypoxic IBI in newborn male rats [23]. NLRP3 inhibitors clearly have unique anti-inflammatory effects, protecting the injured brain after traumatic brain injury [40].

Conclusion
In summary, CPE cell-derived EVs containing miR-155-5p can aggravate IBI by suppressing the Rheb/mTORC1 expression and activating the NLRP3-mediated inflammasome, highlighting the miR-155-5p expression as the potential therapeutic target in IBI (Figure 7). Progressive steps to elucidate the mechanisms of IBI and prevent its incidence are urgently needed in the future.

Data Availability
The datasets generated/analyzed in preparing the current study are available.

Ethical Approval
The animal study protocol was approved by the Ethics Committee of the Affiliated Hospital of Guizhou Medical University.