Activation of CRHR1 contributes to cerebral endothelial barrier impairment via cPLA2 phosphorylation in experimental ischemic stroke

Journal Pre-proof Activation of CRHR1 contributes to cerebral endothelial barrier impairment via cPLA2 phosphorylation in experimental ischemic stroke

Changchun Cao, Jun Zhou, Xiaoli Wu, Yuanyuan Qian, Yali Hong, Junyu Mu, Lai Jin, Chao Zhu, Shengnan Li PII:

S0898-6568(19)30263-3

DOI:

https://doi.org/10.1016/j.cellsig.2019.109467

Reference:

CLS 109467

To appear in:

Cellular Signalling

Received date:

23 August 2019

Revised date:

6 November 2019

Accepted date:

8 November 2019

Please cite this article as: C. Cao, J. Zhou, X. Wu, et al., Activation of CRHR1 contributes to cerebral endothelial barrier impairment via cPLA2 phosphorylation in experimental ischemic stroke, Cellular Signalling(2018), https://doi.org/10.1016/j.cellsig.2019.109467

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© 2018 Published by Elsevier.

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Activation of CRHR1 contributes to cerebral endothelial barrier impairment via cPLA2 phosphorylation in experimental ischemic stroke Changchun Caoa,b, Jun Zhoua, Xiaoli Wub, Yuanyuan Qiana, Yali Honga, Junyu Mua, Lai Jina, Chao Zhua, Shengnan Lia,* a

Department of Pharmacology, Nanjing Medical University, 101 Longmian Avenue,

Nanjing, Jiangsu, China

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b

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Department of Pharmacy, The Affiliated Huaian NO.1 People's Hospital of Nanjing

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Medical University, 1 Huanghe West Road, Huaian, Jiangsu, China

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Original Research

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Corresponding author: Shengnan Li

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Tel: +86-25-86869403;

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E-mail address: [email protected];

Fax: +86-25-86869321.

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Abstract The activation of corticotrophin-releasing hormone receptor (CRHR) 1 is implicated in neuronal injury in experimental stroke. However, little is known about the relationship between CRHR1 activation and brain endothelial barrier impairment after ischemia and reperfusion (I/R). Recently we have demonstrated that the activation of extracellular

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signal-regulated kinase (Erk) 1/2 as well as p38 is required for hydrogen peroxide

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(H2 O 2 )-increased cytosolic phospholipase A2 (cPLA2 ) phosphorylation in bEnd3 cells. Using

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this in vitro ischemic- like model, we found that both blockade and interference of CRHR1

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inhibited H2 O2 -enhancd p38, Erk1/2 and cPLA2 phosphorylation and in turn suppressed

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monolayer hyperpermeability and ZO-1 redistribution. Then using the transient middle cerebral artery occlusion (tMCAO) mouse model, we revealed that CRHR1 antagonist

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NBI27914 pretreatment attenuated cPLA2 phosphorylation, Evans blue dye (EBD)

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extravasation, tight junction disruption and mitochondrial cytochrome c release. CRHR1

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interference also inhibited cortical vascular hyperpermeability. Furthermore, NBI27914 administration attenuated neurovascular injury. After 30 minutes MCAO with 7 days reperfusion CRHR1 interference alleviated hippocampal blood-brain barrier (BBB) leakage and improved spatial cognitive dysfunction. Thus, our study demonstrates that during ischemic stroke the activation of endothelial CRHR1 contributes to BBB impairment via cPLA2 phosphorylation. Keywords corticotrophin-releasing hormone receptor; reperfusion; endothelial permeability; oxidative stress; phosphorylation; junction 2

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1. Introduction Currently, the clinical effective therapy for acute ischemic stroke is to establish early reperfusion by timely recanalization [1]. However, cerebral I/R can lead to life-threatening vasogenic edema and hemorrhagic transformation [1, 2], arising from increased reactive oxygen species (ROS) generation [3] and irreversible BBB disruption [4-6]. As ischemia

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impairs not only neuronal functions but also vascular barrier properties [7, 8], protecting

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endothelial integrity is necessary despite potential neuroprotection [9, 10]. In neurovascular

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unit (NVU), the microvascular endothelial cells (ECs) creating BBB by complex and

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continuous tight junctions (TJs) [11, 12] play a vital role in ROS production following

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reperfusion [13]. Moreover, TJ restricts the paracellular diffusional pathway which is integral to the barrier function [14], and I/R initiates rapid pathological changes in brain ECs that

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eventually promote paracellular hyperpermeability [15]. Therefore, endothelial junction

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impairment is responsible for BBB breakdown, and the detailed understanding of which is

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important for vascular protection.

The CRH belongs to the mammalian central stress hormone [16] and regulates various responses by binding to CRHR1, which is a G protein-coupled receptor expressed in several brain regions including cerebral cortex and hippocampus [17]. After permanent focal cerebral ischemia (FCI) in rats, the selective CRHR1 antagonists attenuate brain damage and swelling [18]. Additionally, mice lacking CRHR1 also show attenuated neuronal injury after cerebral ischemic reperfusion [19]. Notably, CRHR1 is present in mouse cerebral microvessels [20] and FCI increases CRH vascular immunoreactivity on the cortical surface [21]. The endothelium- neuron interaction promotes healthy brain function [22]. Nevertheless, the 3

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association of endothelial CRHR1 with neurovascular injury in ischemic stroke is elusive. Oxidative stress disrupts BBB in vivo through the p38/cPLA2 pathway after I/R [6]. On the other hand, cPLA2 amplifies ROS formation and mitogen-activated protein kinase (MAPK) phosphorylation shortly after I/R [23]. Recently we have explored that cPLA2 activation modulates the membrane-permeant and stable ROS H2 O 2 -triggered TJ alteration and

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paracellular hyperpermeability via p38 and Erk1/2 in mouse brain microvascular EC (BMEC)

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line, bEnd3 [24]. More intriguingly, the urocortin-enhanced cPLA2 expression and

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phosphorylation in vascular ECs involves CRHR1 [25]. In light of these studies, we

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hypothesized that CRHR1 may regulate cerebrovascular endothelial permeability by

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activating the key enzyme cPLA2 in I/R injury. Thus, in this study, we evaluated the potential of CRHR1 in the modulation of endothelial barrier integrity following I/R and its possible

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2. Materials and methods

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cellular mechanism.

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2.1 Animals

All experiments were approved by the Animal Experiment Committee of the Nanjing Medical University. In accordance with the guidelines of the Nanjing Medical University’s Regulations of Animal Experiments, all mice provided by the Model Animal Research Center of Nanjing University (Nanjing, China) were raised in a pathogen-free barrier facility with a regulated 12-hour light/dark cycle with water and food ad libitum for 7 days before experiment to adapt to the circumstances. A total of 229 adult male C57BL/6 mice (8-10 weeks) were used. 2.2 Cell culture and chemical 4

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Immortalized murine BMECs bEnd3 (American Type Culture Collection), passage 25-35, were grown as a monolayer in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 ºC in a humidified atmosphere of 5% CO 2 and 95% air, as described previously [10, 24, 26]. NBI27914 was purchased from TOCRIS, and CRH was synthesized by ChinaPeptides

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(Shanghai, China).

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2.3 Lentiviral constructs and short hairpin RNA (shRNA) for CRHR1

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CRHR1 shRNA (shCRHR1) and negative control shRNA (shNC) lentivirus vectors with a

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green fluorescent protein (GFP) tag were obtained from GENECHEM (Shanghai, China). To

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generate the shCRHR1, a target sequence was designed against mouse CRHR1: TCCTGGTCCTGCTGATCAATT. According to the manufacturer ’s instructions, bEnd3 cells

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were incubated with lentivirus for 24 hours and then the medium was changed. After

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incubation for an additional 72 hours, the knockdown efficiency at the protein level was

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assessed by western blot analysis. 2.4 Transwell assay

Transendothelial permeability was measured as previously described [24, 27]. Briefly, bEnd3 cells seeded on 24-well hanging inserts (0.4 µm pore size, Millipore Millicell) in 24-well dishes were serum-starved for 4 hours and treated with vehicle (DSMO, Sigma-Aldrich) or NBI27914 for 2 hours prior H2 O2 (2 mM) stimulation [24, 28] for 1 hour. Immediately after H2 O2 addition 5 µL of FITC-dextran (molecular weight 40 000, Sigma-Aldrich, final concentration 1 mg/mL) was added to the inserts. The fluorescence intensity of FITC-dextran in the samples taken from the lower dishes after H2 O2 treatment 5

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was measured in duplicate per condition at 492/520 nm excitation /emission wavelengths. 2.5 Immunofluorescence staining As previously reported [24, 29], the confluent cells were incubated with anti-ZO-1 (21773-1-AP, 1:100, Proteintech) primary antibody at 4°C overnight, followed by incubation with the Alexa Fluor 594-conjugated secondary antibody (SA00006-4, 1:100, Proteintech).

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The nuclei were stained with DAPI (KeyGEN BioTECH, Nanjing, China). Images were

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captured with a confocal laser-scanning microscope (LSM710, Carl Zeiss).

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2.6 Transient MCAO and treatment

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The tMCAO was performed following previously described methods with minor

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modification [10]. Briefly, surgery was performed by a dissecting surgical microscope. Body temperature was maintained constant (37±0.5 ºC) with a heating pad and lamp. Under chloral

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hydrate anesthesia (300 mg/kg, i.p.), a nylon monofilament (0.104 mm) with a

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silicone-coated head end (0.22±0.02 mm) (RWD Life Science, Shenzhen, China) was gently

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inserted from the right external carotid artery stump to the internal carotid artery and stopped at the origin of the MCA. The distance from the bifurcation of internal/external carotid artery to MCA was 10±0.5 mm. Successful occlusion was verified by monitoring regional cerebral blood flow with laser Doppler flowmetry (Moor Instruments). After 90 minutes occlusion, the filament was withdrawn to allow reperfusion. In the sham-operated mice, the filament was inserted along the internal carotid artery and then immediately withdrawn. To conduct the short-term ischemia with long-term reperfusion, the MCA was occluded for 30 minutes [30]. Animals were randomly assigned to the treatment groups. Vehicle or NBI27914 (20 mg/kg) [18] was injected intraperitoneally 30 minutes before the onset of reperfusion. 6

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2.7 Neurological score evaluation Neurological scoring was determined by an investigator blinded to the experimental groups at 24 hours after reperfusion, according to a previously described 5-point scale (0, normal; 1, forelimb flexion; 2, circling to the contralateral side but normal posture at rest; 3, leaning to contralateral side at rest; and 4, no spontaneous movement) [10].

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2.8 Determination of infarct and edema ratios

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As described previously [10, 24, 31], mice were euthanized and brains were harvested

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rapidly after neurological score assessment. Seven coronal sections of the brain (1 mm

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thickness) were incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC,

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Sigma-Aldrich) in phosphate buffer solution (PBS) for 30 minutes at 37 ºC. Scanned images were used to calculate infarct and edema ratios by image analysis software (Image J, NIH) .

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2.9 EBD extravasation assay

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Two hours before the animals were euthanized, 2% EBD (Sigma-Aldrich) solution (4

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mL/kg) in normal saline was administered intravenously. The animals were anesthetized and perfused through the left ventricle with ice-cold normal saline to remove intravascular dye. Brains were harvested, sliced and scanned. The hemispheres or hippocampi dissected over ice were weighted and homogenized by sonication in 50% trichloroacetic acid solution, then centrifuged at 20 000 g for 20 minutes. Then the supernatant was diluted 4-fold with ethanol. The extravascular EBD amount was calculated by measuring the fluorescence intensity (620/680 nm excitation/emission) of the supernatant [10, 24]. 2.10 Stereotactic injection For all surgical procedures, mice were anesthetized. As previously described with minor 7

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modification [31-33], each mouse was subjected to shNC or shCRHR1 lentivirus stereotaxic injection in the right hemisphere (i.e., ipsilateral to the MCAO). The coordinates for the cortex were as follows: point 1, 1.5 mm anterior to the bregma, 2.5 mm lateral, 2 mm deep; point 2, 0.5 mm posterior to the bregma, 3 mm lateral, 2 mm deep; point 3, 2 mm posterior to the bregma, 3 mm lateral, 2 mm deep. And the coordinates used for hippocampal injectio n were as follows: 2 mm posterior to the bregma, 2.5 mm lateral, 2.3 mm deep. 1 μL of

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lentivirus suspension containing 1×109 TU/mL was injected in each point at a rate of 0.2

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μL/minute. The needle was withdrawn over a course of 15 minutes. Mice were allowed 7

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days of recovery and then subjected to histology or tMCAO as described above.

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2.11 Histology

Mice were anesthetized and perfused transcardially as previously described [24, 32]. The

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brains were post-fixed in 4% paraformaldehyde (PFA) for 24 hours and then transferred to a

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30% sucrose solution in PBS for 2 days. 20μm thick coronal sections sliced on a freezing

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microtome (Leica CM1950) were incubated with DAPI for 10 minutes. Endo genous viral expression of fluorophores was imaged on a fluorescence microscope (OLYMPUS IX70). 2.12 Morris water maze (MWM) The whole procedure was performed in a circular stainless steel tank painted white on the interior (122 cm in diameter, Jiliang Neuroscience Inc.), as described previously with a few adjustments [30, 34], divided into three parts: visible platform training, hidden platform training and probe test. Four cardinal points around the edge of the pool were demarcated as N, E, S, and W, which divided the pool into four quadrants. The animal facing the tank wall is released into the water at water- level. The training paradigm for the visible platform version 8

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consisted of 4 trials (1minute maximum; 15 minutes interval) on the first day. The circular platform (10 cm in diameter) submerged 0.5 cm below the surface had a ping-pong ball mounted 12 cm above the platform on a plastic rod. Both platform location and start position varied for each trial [34]. Following visible platform training, the training paradigm for the hidden platform version consisted of 4 trials (1minute maximum; 15 minutes interval) per

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day for 5 consecutive days. The platform just submerged was located in the SW quadrant and

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start positions were semi-randomly defined [34]. Distal cues were provided on the walls

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around the pool. When an animal failed to find the platform within the allotted time, it was

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picked up and placed on the platform for 15 seconds. One 30-second probe trial starting from

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NE with the platform removed was administered 24 hours after the completion of hidden version on day 7 [34]. The swim paths were recorded and analyzed by a video tracking

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system (EthoVision XT). The data of the mice which were floating or jumping off the

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platform was excluded.

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2.13 Isolation of mitochondria

Mitochondria were isolated from the cortical tissues using a tissue mitochondria isolation kit (C3606, Beyotime, China) according to the previously described method [35]. In brief, freshly collected ischemic brain tissues were weighed, rinsed with ice-cold PBS, and homogenized 10 times with mitochondrial isolating regent A on the ice. The homogenate was centrifuged at 600 g, 4 ℃ for 5 minutes and the supernatant was centrifuged at 11,000 g, 4 ℃ for 10 minutes. Then, the mitochondrial pellet was collected. Mitochondrial protein concentration was determined by using the Bradford protein assay. 2.14 Western blot analysis 9

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As reported previously [23, 24], protein extracts from cultured cells and brain tissues were prepared and analyzed by western blot. Primary antibodies were anti-phospho-p38 (Thr180/Tyr182) (#4511), anti-p38 (#9212), anti-phospho-cPLA2 (Ser505) (#2831), anti-CD31 (#77699) (all 1:1000, Cell Signaling Technology), anti-Claudin-5 (ab15106), anti-CRHR1 (ab77686), anti-VE-cadherin (ab205336) (all 1:1000, Abcam), anti-cPLA2

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(sc-454), anti-phospho- Erk1/2 (Thr202/Tyr204) (sc-136521), anti- Erk1/2 (sc-514302) (all

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1:500, Santa Cruz Biotechnology), anti-COXⅣ (11242-1-AP), anti-CRHR2 (25267-1-AP),

anti- ZO-1

(21773-1-AP)

(all

1:1000,

Proteintech).

Appropriate

horseradish

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and

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anti-Cytochrome C (10993-1-AP), anti-Occludin (13409-1-AP), anti-PSD95 (20665-1-AP)

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peroxidase- linked secondary antibodies (1:10000, Proteintech) were used for detection by enhanced chemiluminescence (Bio-Rad). To control cell sample loading and protein transfer,

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the PVDF membranes (Roche) were stripped and reprobed with Tubulin-beta antibody

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(10094-1-AP, 1:5000, Proteintech). For brain tissue sample, GAPDH antibody (60004-1-Ig,

control.

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1:5000, Proteintech) was used. For mitochondrial protein, COXⅣ is used as a loading

2.15 Statistical analysis

All values reported are mean ± SEM. For comparison between 2 groups, Statistical differences were evaluated by the unpaired Student t test. For comparison among multiple groups, statistical differences were calculated by one-way ANOVA followed by Newman-Keuls test. The neurological scores were analyzed with a nonparametric test (Mann-Whitney U-test). The criterion for statistical significance was set at P＜0.05. 3. Results 10

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3.1 CRHR1 inactivation inhibited H2 O2 -increased cPLA2 phosphorylation and monolayer permeability in bEnd3 cells Our recent study have shown that the activation of Erk1/2 as well as p38 accounts for H2 O 2-enhanced cPLA2 phosphorylation and brain endothelial permeability in vitro [24]. To address whether CRHR1 antagonist inhibits this oxidative stress-triggered endothelial

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signaling, bEnd3 cells were pretreated with different concentrations of NBI27914 and then

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exposed to H2 O2 . Western blot analysis showed that H2 O2 -induced concurrent increase in

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Erk1/2 and cPLA2 phosphorylation was inhibited dose-dependently by NBI27914 from 5 μM

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up to a maximum with 20 μM, whereas the increased p38 phosphorylation was significantly

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suppressed at 10 and 20 μM (Fig. 1A, Fig. S1). Moreover, H2 O2 incubation increased the phosphorylation of p38, Erk1/2 and cPLA2 in a dose-dependent manner, which was slightly

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suppressed by 5 μM NBI27914 pretreatment (F ig. S2). We then measured the permeability of

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high molecular weight FITC-dextran across bEnd3 cell monolayer by the transwell assay.

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H2 O 2-enhanced endothelial monolayer permeability (P＜0.01) was not decreased after 10 μM NBI27914 pretreatment (Fig. 1B). As shown in Fig. 6C, NBI27914 at 20 μM markedly attenuated H2 O2 -increased endothelial permeability (P ＜ 0.01), similar to the effect of sphingosine-1-phosphate receptor-2 (S1PR2) antagonist JTE013 at 1 μM (P＜0.01). Thus, we selected the optimal concentration 20 μM for subsequent experiments. Besides, concomitant treatment of two receptor antagonists slightly augmented the decrease in H 2 O2-induced endothelial hyperpermeability (P＜0.001, Fig. 1C). Furthermore, CRHR1 agonist CRH alone dose-dependently

increased

endothelial

monolayer

permeability

(Fig.

1D)

and

phosphorylation of p38, Erk1/2 and cPLA2 (Fig. 1E). In H2 O 2-stimulated cells, CRH at 250 11

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nM

that substantially

enhanced

endothelial permeability

(P ＜ 0.001) abrogated

NBI27914-attenuated phosphorylation of p38, Erk1/2 and cPLA2 (Fig. 1F). To further address the critical role of CRHR1 signaling in H2 O2-increased monolayer permeability, we applied the RNA interference approach and observed that the shCRHR1 lentivirus effectively decreased CRHR1 protein expression (0.39±0.04, P＜0.01) without

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affecting CRHR2 expression in bEnd3 cells compared with shNC (Fig. 2A-2C). As shown in

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Fig. 2D-2G, H2 O2 stimulation significantly increased the phosphorylation levels of p38 (P＜

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0.001), Erk1/2 (P ＜ 0.001) and cPLA2 (P ＜ 0.01) in shNC-infected cells, which was

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suppressed by shCRHR1 infection. Although the basal paracellular permeability in

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shCRHR1- infected cells increased slightly, the H2 O2-increased monolayer permeability was remarkably reduced when compared with shNC-treated cells (P＜0.05, Fig. 2H).

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3.2 CRHR1 inactivation inhibited H2 O2 -induced TJ disruption in bEnd3 cells

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In response to H2 O2 , the cPLA2 activation alters ZO-1 junctional localization in bEnd3

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cells [24]. Thus, we explored the effect of CRHR1 activation on TJ structure under oxidative stress. The immunofluorescence staining exhibited a continuous distribution of ZO-1 between neighboring cells in the absence of H2 O2 , while a more discontinuous pattern with loss of junctional localization was observed after H2 O 2 stimulation (Fig. 3A). The pretreatment with NBI27914, meanwhile, maintained the continuous ZO-1 distribution along cell junctions in the presence of H2 O2 and did not affect the ZO-1 localization alone. Next, we further investigated the effect of CRHR1 knockdown on the junctional localization of ZO-1 after H2 O2 stimulation. As shown in Fig. 3B, we observed a continuous distribution of ZO-1 at cell-cell contacts in both shNC-infected and shCRHR1- infected 12

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bEnd3 cells. And in the presence of H2 O2 , shCRHR1 infection retained the continuous pattern of ZO-1 staining instead of the zigzag pattern observed in shNC-infected cells. 3.3 CRHR1 antagonist alleviated cPLA2 phosphorylation after stroke To address the involvement of cPLA2 in mediating the BBB disruption via CRHR1 in the tMCAO model, we detected the protein phosphorylation in ischemic brain (Fig. 4A). While

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inhibiting p38 phosphorylation slightly (Fig. 4B), NBI27914 administration attenuated

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Erk1/2 phosphorylation significantly (P＜0.01, Fig. 4C) in ipsilateral hemispheres compared

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to contralateral hemispheres after 90 minutes MCAO with 6 hours reperfusion. Intriguingly, a

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slight increase in cPLA2 phosphorylation in the ipsilateral hemispheres in vehicle-treated

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mice was observed, which was suppressed substantially in NBI27914-treated mice (P＜0.001, Fig. 4D).

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3.4 CRHR1 antagonist protected the BBB from disruption after stroke

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We firstly evaluated the in vivo effect of CRHR1 inactivation on BBB disruption in the

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early stage of stroke using the tMCAO mouse model and EBD extravasation assay (Fig. 5A). After 90 minutes MCAO with 6 hours reperfusion, EBD extravasation ratio increased remarkably in vehicle-treated mouse brain compared with sham animals (P＜0.001, Fig. 5B), which was ameliorated by NBI27914 administration (P＜0.05, Fig. 5B). To further investigate whether CRHR1 activation provokes cerebral EC junction disruption, we detected the total protein levels in ischemic brain. As shown in Fig. 5C, the significant decrease in protein levels of ZO-1 (P＜0.01, Fig. S3A), CD31 (P＜0.05, Fig. S3B), occludin (P ＜ 0.05, Fig. S3E) and claudin-5 (P ＜ 0.01, Fig. S3G) was observed in ipsilateral hemispheres at 6 hours after reperfusion compared to contralateral hemispheres, which was 13

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reversed by NBI27914 treatment (P＜0.05). Meanwhile, the selective CRHR1 antagonist did not mitigate the decrease in protein levels of VE-cadherin (Fig. S3C) and PSD95 (Fig. S3D) in the ipsilateral hemispheres. Interestingly, there was no significant change in the CRHR1 protein level between contralateral and ipsilateral hemispheres (Fig. S3F). The release of cytochrome c from mitochondria into cytosol is considered as a death signal

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that triggers the cascade of apoptosis [35]. In vehicle-treated group, the level of cytochrome c

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in mitochondria was reduced in ipsilateral hemispheres compa red to that in contralateral

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hemispheres at 6 hours after reperfusion (P＜0.05, Fig. 5D and E). However, there was no

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significant difference in mitochondrial cytochrome c level between ipsilateral and

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contralateral hemispheres after NBI27914 treatment (Fig. 5E). 3.5 CRHR1 knockdown alleviated BBB disruption in the cortex after stroke

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To assess the direct contribution of CRHR1 activation to BBB damage after I/R, mice

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received unilateral cortical injections of either shNC or shCRHR1 lentivirus. One week post

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injection, we observed GFP expression (Fig. 6A) and reduced CRHR1 expression (0.50±0.05, P＜0.05, Fig. 6B and C) in the cortices of mice. Then we conducted tMCAO in the right hemisphere and measured EBD leakage (Fig. 6D). After 90 minutes MCAO with 6 hours reperfusion, the shCRHR1-treated mice showed dramatically decreased EBD extravasation in the ipsilateral hemispheres compared to the shNC-treated mice (P＜0.05, Fig. 6E). 3.6 CRHR1 antagonist attenuated brain edema, infarction volume and neurological deficit following stroke Next, we explored whether CRHR1 activation exacerbates the neurovascular responses to I/R injury. Mice were subjected to tMCAO and the infarct areas were analyzed via TTC 14

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staining (Fig. 7A). As compared with the vehicle-treated mice, the NBI27914-treated mice showed a dramatic decrease in both total cerebral edema ratio (P＜0.01, Fig. 7B) and infarct ratio (P＜0.05, Fig. 7C) at 24 hours after reperfusion. Besides, the administration of CRHR1 antagonist resulted in a significant improvement of the neurological scores at 24 hours after reperfusion (P＜0.05, Fig. 7D).

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3.7 CRHR1 knockdown alleviated BBB disruption in the hippocampus and spatial learning

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and memory impairment after short-term ischemia with long-term reperfusion

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The short-term ischemia with long-term reperfusion results in oxidative stress and BBB

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breakdown in the hippocampal region and impairs spatial cognitive function [30]. To examine

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the effect of CRHR1 inactivation on hippocampal BBB breakdown, mice were unilaterally injected with either shNC or shCRHR1 lentivirus. As shown in Fig. 8A, GFP expression was

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visualized in the hippocampal dentate gyrus 7 days after injection. We detected decreased

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CRHR1 protein expression in the hippocampi of shCRHR1-treated mice (0.72±0.07, P＜0.05,

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Fig. 8B, C). Then mice were subjected to 30 minutes MCAO. Compared with shNC, the EBD extravasation was attenuated in shCRHR1-treated brain after 7 days reperfusion (P＜0.05, Fig. 8D, E).

To further understand the effect of CRHR1 inactivation on spatial learning and memory impairment associated with stroke, we subjected the mice to MWM test after 30 minutes MCAO with 7 days reperfusion. During visible platform training the escape latencies of shCRHR1- infused mice were gradually decreased and there was no significant difference compared to the performance of shNC-infused mice (Fig. 9A). In hidden platform training, both shNC and shCRHR1- infused mice showed a trend of learning. However, mice infused 15

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with shCRHR1 lentivirus spent less time finding the hidden platform underneath the water than mice infused with shNC lentivirus as illustrated by the escape latency value (Fig. 9B). In the probe trail, although there was no difference regarding the time spent in target quadrant between the two groups (Fig. 9C), the shCRHR1 treatment increased platform- site crossings compared with shNC (Fig. 9D).

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4. Discussion

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In the present study, we revealed that inactivation of CRHR1 counteracts H2 O2-stimulated

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paracellular hyperpermeability and TJ protein localization alteration by inhibiting p38,

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Erk1/2 and cPLA2 phosphorylation in bEnd3 cells, an in vitro model of endothelium at the

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BBB [36]. Congruously, pharmacological inactivation of CRHR1 prevented tMCAO- induced cPLA2 phosphorylation, BBB damage and TJ disruption. Genetic inactivation of CRHR1 in

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vivo after I/R attenuated cortical and hippocampal BBB leakage, as well as ameliorating

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spatial learning and memory impairment. Our findings indicate that endothelial CRHR1

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activation has important implications for ischemic neurovascular injury. The binding of CRHR1 with its endogenous ligand CRH regulates the MAPK signal pathway [37]. Moreover, the CRHR1 is essential for CRH- induced Erk1/2 activation in the CA3 and CA1 hippocampal subfields and basolateral complex of the amygdala [38], implicating this receptor as a regulator of CRH-dependent MAPK activation. Importantly, our recent study has unravelled the distinct role of another G protein-coupled receptor S1PR2 in regulating oxidative stress-enhanced paracellular permeability of BMECs via p38 and Erk1/2-activated cPLA2 [24]. Hence, we assumed that MAPK-activated cPLA2 as a common pathway mediates the regulatory effect of CRHR1 on cerebrovascular endothelial integrity 16

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under oxidative stress. Similar to the inhibitory effect of S1PR2 blockade [24], CRHR1 blockade inhibited the exogenous H2 O2-induced concomitant increase in p38, Erk1/2 and cPLA2 phosphorylation in bEnd3 cells. Consistent with our hypothesis, 20 μM NBI27914 identical to S1PR2 antagonist JTE013 mitigated paracellular hyperpermeability in H2 O 2-stimulated bEnd3 cells. Additionally, blockade of both CRHR1 and S1PR2 together

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exhibited a slight additive effect, suggesting that these two different receptors modulated

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endothelial paracellular permeability in oxidative injury via p38 and Erk1/2-activated cPLA2 .

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CRH secreted by ECs in both autocrine and paracrine manners regulates vascular

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endothelial homeostasis [39], and CRHR1 transduces this peptide-stimulated intracellular

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ROS elevation [40]. Consistently, our in vitro results showed that CRH directly inducing hyperpermeability and intracellular signaling activation restored H2 O2-triggered concomitant

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phosphorylation in the presence of NBI27914, suggesting that CRHR1 activation intensifies

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the membrane-permeable ROS-impaired cerebral endothelial integrity. Nevertheless, whether

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CRHR1 activation facilitates the release of CRH in brain ECs needs further investigation. Similar with S1PR2 interference [24], lentivirus- mediated CRHR1 inactivation protected bEnd3 cells from oxidative stress. Although cPLA2 resides in the cytosol [41], it can translocate to cellular membranes minutes after reperfusion [23]. The translocation of cPLA2 controls the transport of transmembrane junction proteins, contributing to the maintenance of endothelial TJs [42]. Moreover, the cytoplasmic scaffolding proteins which link the junctional molecules claudin and occludin via cingulin to the cytoskeleton regulates the effectiveness of TJs, the key feature of the BBB [14]. Recently we have also found that the subcellular localization of 17

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endogenous scaffolding protein ZO-1 was no longer continuous and obvious at the margin of bEnd3 cells after H2 O2 stimulation [24]. Here we revealed that CRHR1 inactivtaion maintained the continuous ZO-1 distribution after H2 O2 exposure. These morphologic results, similar with S1PR2 inactivtaion [24], suggesting that ROS may affect the TJ structure via CRHR1-MAPK-cPLA2 activation.

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Once the normal blood flow is interrupted by ischemic insults, cerebral microcirculation

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disturbance leads to local accumulation of CRH [21]. The above in vitro results prompted us

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to explore whether CRHR1 signaling is necessary for endothelial barrier disruption in stroke

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in vivo. Herein, we found that NBI27914 administration attenuated cPLA2 phosphorylation

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and cerebral vascular EBD extravasation after 90 minutes MCAO with 6 hours reperfusion, supporting the close relation between CRHR1-cPLA2 activation and early BBB disruption.

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Subsequently, we found that CRHR1 knockdown decreased early BBB damage in ischemic

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cortex, in line with the in vivo observations using NBI27914.

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At the early stage of reperfusion, we also observed NBI27914 treatment significantly mitigated the decreased expression of TJ protein claudin-5, occludin, and ZO-1 as well as CD31 in ipsilateral hemispheres, but did not relieve the reduced expression of AJ protein VE-cadherin and synaptic protein PSD95. Given that there is a surge in ROS production following tMCAO [43], these findings indicate that cerebrovascular ECs as well as neuronal cells are vulnerable to oxidative stress. Adherens junctions (AJs) holding the cerebral ECs together promotes the formation of TJs, whereas disruption of AJs compromises endothelial barrier [14]. CD31 which is localized in the EC contacts outside of TJ also modulates the barrier stabilization [12, 44]. Compared to TJ, the relatively loose AJ structure may facilitate 18

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the irreversible decrease of VE-cadherin expression after tMCAO. Although CRHR1 antagonist protected the TJ proteins against I/R insult, the molecular mechanism remains to be clarified. In addition, mitochondria essential for ROS generation are considered as the targets of oxidative stress [45]. We provided evidence that blockade of CRHR1 prevented cytochrome c escaping from mitochondria, which partly accounts for the reduction of

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cerebral I/R-induced damage.

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S1PR2 signal induces brain endothelial cell activation by binding endothelium-derived or

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serum-derived S1P, participating in neurovascular responses to I/R injury [10]. In common

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with S1PR2 signaling, we revealed that NBI27914 administration alleviated neuronal

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damages, cerebral edema and neurological deficit in transient FCI in mice, suggesting that CRHR1 activation aggravates neurovascular I/R injury.

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Indeed, complete occlusion of cerebral blood flow causes rapid damage within minutes in

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vulnerable areas [8]. The 30 minutes MCAO with 7 days reperfusion leads to increased

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oxidative stress and severe BBB breakdown in the hippocampus, associated with impaired spatial learning and memory [30]. We found that CRHR1 knockdown alleviated BBB breakdown in the ipsilateral hippocampus after this short-term ischemia with long-term reperfusion, suggesting that CRHR1 activation accounts for the I/R-induced oxidative damage and endothelial barrier impairment. We also found that after long-term reperfusion shCRHR1-treated mice performed better than shNC-treated mice in MWM, as reflected by the markedly shorter escape latencies in hidden platform training and increased target crossings in probe test, indicating that CRHR1 inactivation improves spatial cognitive dysfunction. However, the endothelial-specific CRHR1-deficient mice would be required to 19

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elucidate the precise role of CRHR1 activation in ischemic microvessels. 5. Conclusions In summary, we uncover a novel link between CRHR1 signaling and brain endothelial barrier impairment in stroke. I/R-induced oxidative stress activates the cerebrovascular endothelial CRHR1, which mediates TJ alteration via p38 and Erk1/2-dependent cPLA2

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phosphorylation, promoting BBB disruption and exacerbating neurovascular injury (Fig. 10).

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Thus, CRHR1 inactivation can be considered as an alternative strategy for acute

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cerebrovascular protection, and we hope this work will encourage further studies on

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endothelial CRHR1 as a relevant target for clinical treatment of ischemic stroke.

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Acknowledgements

This work was supported by grants from the Natural Science foundation of China

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Author Contributions

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(No.81773724 & 81573424) and Huaian city science and technology program (HAB201925).

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SNL and CCC conceived the project and designed the study. CCC, JZ, XLW, YYQ and JYM performed the experiments and analyzed the data. YLH and LJ contributed to lentivirus experiments. CZ contributed to transwell assay experiments. XLW contributed to Morris water maze experiments. CCC and SNL wrote the manuscript and prepared the figures. All authors reviewed the manuscript. Conflicts of interest The authors declare that no competing interests exist. References [1] J.L. Saver, Improving reperfusion therapy for acute ischaemic stroke, Journal of thrombosis and haemostasis : JTH 9 Suppl 1 (2011) 333-43. 20

Journal Pre-proof [2] C.A. Molina, J. Alvarez-Sabin, Recanalization and reperfusion therapies for acute ischemic stroke, Cerebrovascular diseases 27 Suppl 1 (2009) 162-7. [3] C. Lehner, R. Gehwolf, H. Tempfer, I. Krizbai, B. Hennig, H.C. Bauer, H. Bauer, Oxidative stress and blood-brain barrier dysfunction under particular consideration of matrix metalloproteinases, Antioxidants & redox signaling 15(5) (2011) 1305-23. [4] J.R. Piro, G.L. Suidan, J. Quan, Y. Pi, S.M. O'Neill, M. Ilardi, N. Pozdnyakov, T.A. Lanz, H. Xi, R.D. Bell, T.A. Samad, Inhibition of 2-AG hydrolysis differentially regulates blood brain barrier permeability after injury, Journal of neuroinflammation 15(1) (2018) 142. [5] C. Betzen, R. White, C.M. Zehendner, E. Pietrowski, B. Bender, H.J. Luhmann, C.R. Kuhlmann, Oxidative stress upregulates the NMDA receptor on cerebrovascular endothelium, Free radical biology & medicine 47(8 ) (2009) 1212-20. [6] C. Nito, H. Kamada, H. Endo, K. Niizuma, D.J. Myer, P.H. Chan, Role of the p38 mitogen -activated protein

f

kinase/cytosolic phospholipase A2 signaling pathway in blood-brain barrier disruption after focal cerebral

oo

ischemia and reperfusi on, J Cereb Blood Flow Metab 28(10) (2008) 1686-96.

[7] M. Krueger, I. Bechmann, K. Immig, A. Reichenbach, W. Hartig, D. Michalski, Blood -brain barrier breakdown

pr

involves four distinct stages of vascular damage in various models of experimental focal cereb ral ischemia, J Cereb Blood Flow Metab 35(2) (2015) 292-303.

[8] L. Zhu, L. Wang, F. Ju, Y. Ran, C. Wang, S. Zhang, Transient global cerebral ischemia induces rapid and

e-

sustained reorganization of synaptic structures, J Cereb Blood Flow Metab 37(8) (2017) 2756-2767. [9] C. Bouleti, T. Mathivet, B. Coqueran, J.M. Serfaty, M. Lesage, E. Berland, C. Ardidie-Robouant, G.

Pr

Kauffenstein, D. Henrion, B. Lapergue, M. Mazighi, C. Duyckaerts, G. Thurston, D.M. Valenzuela, A.J. Murphy, G.D. Yancopoulos, C. Monnot, I. Margaill, S. Germain, Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke, European heart journal 34(47) (2013) 3657 -68.

al

[10] G.S. Kim, L. Yang, G. Zhang, H. Zhao, M. Selim, L.D. McCullough, M.J. Kluk, T. Sanchez, Critical role of sphingosine-1-phosphate receptor-2 in the disruption of cerebrovascular integrity in experimental stroke,

rn

Nature communications 6 (2015) 7893.

[11] B. Engelhardt, S. Liebner, Novel insights into the development and maintenance of the blood-brain barrier,

Jo u

Cell and tissue research 355(3) (2014) 687-99. [12] B.V. Zlokovic, The blood-brain barrier in health and chronic neurodegenerative disorders, Neuron 57(2) (2008) 178-201.

[13] P.A. Fraser, The role of free radical generation in increasing cerebrovascular permeability, Free radical biology & medicine 51(5) (2011) 967-77. [14] N.J. Abbott, A.A. Patabendige, D.E. Dolman, S.R. Yusof, D.J. Begley, Structure and function of the blood-brain barrier, Neurobiology of disease 37(1) (2010) 13-25. [15] Y. Shi, L. Zhang, H. Pu, L. Mao, X. Hu, X. Jiang, N. Xu, R.A. Stetler, F. Zhang, X. Liu, R.K. Leak, R.F. Keep, X. Ji, J. Chen, Rapid endothelial cytoskeletal reorganization enables early blood-brain barrier disruption and long-term ischaemic reperfusion brain injury, Nature communications 7 (2016) 10523. [16] P. Barra de la Tremblaye, H. Plamondon, Alterations in the corticotropin -releasing hormone (CRH) neurocircuitry: Insights into post stroke functional impairments, Frontiers in neuroendocrinology 42 (2016) 53-75. [17] Y. Sztainberg, Y. Kuperman, N. Justice, A. Chen, An anxiolytic role for CRF receptor type 1 in the globus pallidus, The Journal of neuroscience : the official journal of the Society for Neuroscience 31(48) (2011) 17416-24. [18] K.B. Mackay, H. Bozigian, D.E. Grigoriadis, S.A. Loddick, G. Verge, A.C. Foster, Neuroprotective effects of the 21

Journal Pre-proof CRF1 antagonist R121920 after permanent focal ischemia in the rat, J Cereb Blood Flow Metab 21(10) (2001) 1208-14. [19] S.L. Stevens, T.E. Shaw, E. Dykhuizen, N.S. Lessov, J.K. Hill, W. Wurst, M.P. Stenzel -Poore, Reduced cerebral injury in CRH-R1 deficient mice after focal ischemia: a potential link to microglia and atrocytes that express CRH-R1, J Cereb Blood Flow Metab 23(10) (2003) 1151-9. [20] H. Tu, A.J. Kastin, W. Pan, Corticotropin-releasing hormone receptor (CRHR)1 and CRHR2 are both trafficking and signaling receptors for urocortin, Molecular endocrinology 21(3) (2007) 700 -11. [21] M. De Michele, G. Sette, D.T. Chalmers, D. Dewar, D. Toni, G. Sancesario, J. McCulloch, Focal cerebral ischaemia induces corticotropin releasing factor (CRF) vascular immunoreactivity in rat occluded hemisphere, Regulatory peptides 143(1-3) (2007) 69-75. [22] K.W. Wu, L.L. Lv, Y. Lei, C. Qian, F.Y. Sun, Endothelial cells promote excitatory synaptogenesis and improve ischemia-induced motor deficits in neonatal mice, Neurobiology of disease 121 (2019) 230 -239.

f

[23] K. Kishimoto, R.C. Li, J. Zhang, J.A. Klaus, K.K. Kibler, S. Dore, R.C. Koehler, A. Sapir stein, Cytosolic

oo

phospholipase A2 alpha amplifies early cyclooxygenase-2 expression, oxidative stress and MAP kinase phosphorylation after cerebral ischemia in mice, Journal of neuroinflammation 7 (2010) 42.

pr

[24] C. Cao, L. Dai, J. Mu, X. Wang, Y. Hong, C. Zhu, L. Jin, S. Li, S1PR2 antagonist alleviates oxidative stress-enhanced brain endothelial permeability by attenuating p38 and Erk1/2 -dependent cPLA2 phosphorylation, Cellular signalling 53 (2019) 151-161.

e-

[25] R. Wan, Y. Liu, L. Li, C. Zhu, L. Jin, S. Li, Urocortin increased endothelial ICAM1 by cPLA2-dependent NF-kappaB and PKA pathways in HUVECs, Journal of molecular endocrinology 52(1) (2014) 43 -53.

Pr

[26] J.A. Shin, J.C. Yoon, M. Kim, E.M. Park, Activation of classical estrogen receptor subtypes reduces tight junction disruption of brain endothelial cells under ischemia/reperfusion injury, Free radical biology & medicine 92 (2016) 78-89.

al

[27] B.Y. Jin, A.J. Lin, D.E. Golan, T. Michel, MARCKS protein mediates hydrogen peroxide regulation of 109(37) (2012) 14864-9.

rn

endothelial permeability, Proceedings of the National Academy of Sciences of the United States of America [28] K. Takeuchi, Y. Morizane, C. Kamami -Levy, J. Suzuki, M. Kayama, W. Cai, J.W. Miller, D.G. Vavvas,

Jo u

AMP-dependent kinase inhibits oxidative stress-induced caveolin-1 phosphorylation and endocytosis by suppressing the dissociation between c-Abl and Prdx1 proteins in endothelial cells, The Journal of biological chemistry 288(28) (2013) 20581-91.

[29] S. Fischer, M. Wiesnet, D. Renz, W. Schaper, H2O2 induces paracellular permeability of porcine brain-derived microvascular endothelial cells by activation of the p44/42 MAP kinase pathway, European journal of cell biology 84(7) (2005) 687-97. [30] X. Zhang, P.K. Yeung, G.M. Mc Alonan, S.S. Chung, S.K. Chung, Transgenic mice over-expressing endothelial endothelin-1 show cognitive deficit with blood-brain barrier breakdown after transient ischemia with long-term reperfusion, Neurobiology of learning and memory 101 (2013) 46 -54. [31] L. Zhou, F. Li, H.B. Xu, C.X. Luo, H.Y. Wu, M.M. Zhu, W. Lu, X. Ji, Q.G. Zhou, D.Y. Zhu, Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95, Nature medicine 16(12) (2010) 1439-43. [32] J.C. Jimenez, K. Su, A.R. Goldberg, V.M. Luna , J.S. Biane, G. Ordek, P. Zhou, S.K. Ong, M.A. Wright, L. Zweifel, L. Paninski, R. Hen, M.A. Kheirbek, Anxiety Cells in a Hippocampal -Hypothalamic Circuit, Neuron 97(3) (2018) 670-683 e6. [33] K. Kanninen, R. Heikkinen, T. Malm, T. Rolova, S. Kuhmonen, H. Leinonen, S. Yla-Herttuala, H. Tanila, A.L. Levonen, M. Koistinaho, J. Koistinaho, Intrahippocampal injection of a lentiviral vector expressing Nrf2 22

Journal Pre-proof improves spatial learning in a mouse model of Alzheimer's disease, Proceedings of the National Academy of Sciences of the United States of America 106(38) (2009) 16505 -10. [34] C.V. Vorhees, M.T. Williams, Morris water maze: procedures for assessing spatial and related forms of learning and memory, Nature protocols 1(2) (2006) 848-58. [35] W. Yang, X. Chen, J. Pan, H. Ge, K. Yin, Z. Wu, X. Li, D. Sha, Y. Xu, Malibatol A protects against brain injury through reversing mitochondrial dysfunction in experimental stroke, Neurochemistry international 80 (2015) 33-40. [36] B. Xu, Y. Zhang, X.F. Du, J. Li, H.X. Zi, J.W. Bu, Y. Yan, H. Han, J.L. Du, Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity, Cell research 27(7) (2017) 882 -897. [37] D. Kovalovsky, D. Refojo, A.C. Liberman, D. Hochbaum, M.P. Per eda, O.A. Coso, G.K. Stalla, F. Holsboer, E. Arzt, Activation and induction of NUR77/NURR1 in corticotrophs by CRH/cAMP: involvement of calcium, protein kinase A, and MAPK pathways, Molecular endocrinology 16(7) (2002) 1638 -51.

f

[38] D. Refojo, C. Echenique, M.B. Muller, J.M. Reul, J.M. Deussing, W. Wurst, I. Sillaber, M. Paez-Pereda, F.

oo

Holsboer, E. Arzt, Corticotropin-releasing hormone activates ERK1/2 MAPK in specific brain areas, Proceedings of the National Academy of Sciences of the United States of America 102(17) (2005) 6183 -8.

pr

[39] T. Simoncini, R. Apa, F.M. Reis, F. Miceli, M. Stomati, L. Driul, A. Lanzone, A.R. Genazzani, F. Petraglia, Human umbilical vein endothelial cells: a new source and potential target for corticotropin -releasing factor, The Journal of clinical endocrinology and metabolism 84(8) (1999) 2802-6.

e-

[40] S. Gougoura, P. Liakos, G.N. Koukoulis, Effect of CRH on NO bioavailability, ROS production and antioxidant defense systems in endothelial EAhy926 cells, Free radical research 44(7) (2010) 803 -12.

Pr

[41] J.D. Clark, L.L. Lin, R.W. Kriz, C.S. Ramesha, L.A. Sultzman, A.Y. Lin, N. Milona, J.L. Knopf, A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP, Cell 65(6) (1991) 1043-51.

al

[42] E. Regan-Klapisz, V. Krouwer, M. Langelaar-Makkinje, L. Nallan, M. Gelb, H. Gerritsen, A.J. Verkleij, J.A. Post, Golgi-associated cPLA2alpha regulates endothelial cell -cell junction integrity by controlling the trafficking of

rn

transmembrane junction proteins, Molecular biology of the cell 20(19) (2009) 4225-34. [43] O. Peters, T. Back, U. Lindauer, C. Busch, D. Megow, J. Dreier, U. Dirnagl, Increased formation of reactive

Jo u

oxygen species after permanent and reversible middle cerebral artery occlusion in the rat, J Cereb Blood Fl ow Metab 18(2) (1998) 196-205.

[44] J.R. Privratsky, P.J. Newman, PECAM-1 : regulator of endothelial junctional integrity, Cell and tissue research 355(3) (2014) 607-19.

[45] T. Kalogeris, C.P. Baines, M. Krenz, R.J. Korthuis, Cell biology of ischemia/reperfusion injury, International review of cell and molecular biology 298 (2012) 229-317.

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Figure Legends Fig. 1. CRHR1 antagonist inhibited H2 O2 -increased phosphorylation of p38, Erk1/2 and cPLA2 and cerebral endothelial permeability in vitro. (A) The lysates of confluent bEnd3 cells pretreated with NBI27914 at the indicated concentrations for 2 hours and then exposed to H2 O 2 (2 mM) for 30 minutes were immunoblotted with antibodies indicated. A

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representative immunoblot is shown. (B-D) Endothelial permeability was measured by the

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FITC-dextran transwell assay. (B and C) After 4 hours of serum starvation, bEnd3 cells

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seeded in transwell inserts were subjected to vehicle, NBI27914 (10μM or 20 μM) or JTE013

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(1μM) as indicated for 2 hours before 2 mM H2 O2 stimulation for 1 hour. (D) The confluent

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bEnd3 cells were treated with CRH at the indicated concentrations for 2 hours. Fluorescence intensity was normalized to vehicle-treated cells. Data are expressed as mean ± SEM, ＊

P ＜ 0.05,

＊＊

P ＜ 0.01,

＊＊＊

P ＜ 0.001;

##

P ＜ 0.01,

###

P ＜ 0.001. (E) CRH

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n=4/group.

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stimulates phosphorylation of p38, Erk1/2 and cPLA2 . The lysates of confluent bEnd3 cells

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treated with CRH at the indicated concentrations for 1 hour were immunoblotted with antibodies indicated. (F) CRH counteracts NBI27914- inhibited phosphorylation of p38, Erk1/2 and cPLA2 in H2 O2 -treated bEnd3 cells. After 1 hour pretreatment with 20 μM NBI27914, the confluent bEnd3 cells were pretreated with CRH (250 nM) for 1 hour and then exposed to H2 O 2 (2 mM) for 30 minutes. Fig. 2. CRHR1 knockdown inhibited H2 O2 -increased cPLA2 phosphorylation and cerebral endothelial permeability in vitro. (A) GFP-positive cells indicated the successful infection of shNC or shCRHR1 lentivirus. Scale bar, 50 μm. (B) After 96 hours of infection, CRHR1 and CRHR2 protein expression was detected by western blot analysis. (C) Quantification of 24

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CRHR1 protein level in (B). n=3/group. (D) After 96 hours of infection and 4 hours of serum starvation, the lysates of lentivirus- infected bEnd3 cells exposed to H2 O 2 (2 mM) for 30 minutes were immunoblotted with antibodies indicated. (E-G) Quantification of p38, Erk1/2 and cPLA2 phosphorylation in (D). Data are obtained from at least 3 independent experiments. (H) Endothelial permeability of lentivirus- infected bEnd3 cells was measured by the

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FITC-dextran transwell assay. The lentivirus- infected bEnd3 cells were serum-starved for 4

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hours and then exposed to H2 O2 (2 mM) for 1 hour. The fluorescence intensity was

＊＊

P＜0.01,

＊＊＊

P＜0.001; # P＜0.05, ## P＜0.01, ### P＜0.001.

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n=4/group.

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normalized to vehicle-treated shNC-infected cells. Data are presented as mean ± SEM,

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Fig. 3. CRHR1 regulated H2 O2 -induced ZO-1 redistribution in vitro. (A and B) Confocal images of ZO-1-immunolabeled (red) and DAPI-stained (blue) bEnd3 cells. (A) After 4 hours

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of serum starvation, cells were treated with vehicle or 20 μM NBI27914 for 2 hours before 2

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mM H2 O 2 stimulation for 1 hour. (B) After 96 hours of infection and 4 hours of serum

bar, 20 μm.

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starvation, the lentivirus- infected bEnd3 cells were exposed to H2 O2 (2 mM) for 1 hour. Scale

Fig. 4. CRHR1 antagonist alleviated cPLA2 phosphorylation after stroke. (A) The cortex lysates of vehicle or NBI27914-treated mice after 90 minutes MCAO with 6 hours reperfusion were immunoblotted with antibodies indicated (n=3/group). (B-D) Quantification of p38, Erk1/2 and cPLA2 phosphorylation in (A). I indicates ipsilateral hemisphere; C, contralateral hemisphere. Fig. 5. CRHR1 antagonist protected the BBB after I/R injury. (A) Cerebrovascular permeability was determined by EBD extravasation assay at 6 hours after reperfusion (7.5 25

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hours after the onset of MCAO). Brain coronal slices of representative animals. Blue area showed extravasated EBD. (B) Quantification of extravasc ular EBD (n=8/group). Ipsilateral/contralateral (IL/CL) ratios are plotted. Scale bar, 5 mm. Data are mean ± SEM. P ＜ 0.01,

＊ ＊＊

＊＊

P＜ 0.001; #P ＜ 0.05. The levels of total protein (C) and mitochondrial

cytochrome c (D) in the cortices of vehicle or NBI27914-treated mice after 6 hours of

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reperfusion were analyzed by western blot. (E) Quantification of cytochrome c protein level

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in mitochondria in (D). Representative images of the indicated protein are shown (n=3/group).

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I indicates ipsilateral hemisphere; C, contralateral hemisphere.

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Fig. 6. CRHR1 knockdown alleviated BBB breakdown in the cortex after I/R injury. (A)

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The representative fluorescence images of the cortices infected with shCRHR1 lentivirus vectors expressed GFP (green) and stained with DAPI (blue). Sca le bar, 50 μm. (B) CRHR1

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expression in the cortex of mice was assessed by western blot analysis after 7 days of

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infection. Representative images of the indicated protein are shown (n=3/group). (C)

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Quantification of CRHR1 in (B). (D) BBB permeability was studied using EBD extravasation after 90 minutes MCAO with 6 hours reperfusion. (E) Quantification of extravascular EBD (n=8/group). Ipsilateral/contralateral (IL/CL) ratios are plotted. Scale bar, 5mm. Data are mean ± SEM.

＊

P＜0.05.

Fig. 7. CRHR1 antagonist decreased cerebral edema, neuronal injury and neurological deficits after stroke. (A) Representative images of TTC staining of seven, 1- mm-thick brain coronal slices at 24 hours after reperfusion. Edema (B) and infarct (C) ratios were calculated by image analysis and reported as a ratio of the contralateral hemisphere. (D) Neurological scores were evaluated at 24 hours after reperfusion. Scale bar, 5 mm. Data are mean ± SEM, 26

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n=8/group.

＊

P＜0.05,

＊＊

P＜0.01.

Fig. 8. CRHR1 knockdown alleviated BBB breakdown in the hippocampus after short-term ischemia with long-term reperfusion. (A) The fluorescence images of hippocampal region from brain sections of mice injected with shCRHR1 lentivirus stained with DAPI 1 week after injection. GFP is green; DAPI is blue. Scale bar, 50 μm. (B) The CRHR1 protein

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expression was knocked down in hippocampus of mice infected with shCRHR1 lentivirus

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(n=3/group). (C) Quantification of CRHR1 protein level in (B). (D) The BBB integrity was

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determined by EBD extravasation assay after 30 minutes MCAO with 7 days reperfusion. (E)

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Quantification of hippocampal extravascular EBD (n=8/group). Ipsilateral/contralateral ＊

P＜0.05.

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(IL/CL) ratios are plotted. Scale bar, 5mm. Data are represented as mean ± SEM.

Fig. 9. CRHR1 knockdown ameliorated spatial cognitive impairment after short-term

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ischemia with long-term reperfusion. Escape latency in visible platform training (A) and

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hidden platform training (B), percent time spent in the target quadrant (C) and platform-site

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crossings (D) in probe test were measured in MWM task after 30 min MCAO with 7 days ＊

reperfusion. Data are mean ± SEM, n=10. P＜0.05,

＊＊

P＜0.01.

Fig. 10. Schematic model of the effect of CRHR1 activation on disruption of cerebrovascular integrity after tMCAO. Fig. S1. Western blot analysis from vehicle-treated and NBI27914-treated bEnd3 cells. (A-C) Quantification of p38, Erk1/2 and cPLA2 phosphorylation in Fig. 1A. Data are obtained from at least 3 independent experiments and represented as mean ± SEM. 0.01,

＊＊＊

＊＊

P＜

P＜0.001 versus vehicle. # P＜0.05, ## P＜0.01, ### P＜0.001 versus vehicle + H2 O2 .

Fig. S2. The effect of 5 μM NBI27914 on H2 O2 -enhanced p38, Erk1/2 and cPLA 2 27

Journal Pre-proof phosphorylation in bEnd3 cells. The lysates of confluent bEnd3 cells pretreated with NBI27914 (5 μM) for 2 hours and then exposed to H2 O2 at the concentrations indicated for 30 minutes were immunoblotted with antibodies indicated. Fig. S3. Western blot analysis from vehicle-treated and NBI27914-treated mice at 7.5 hours after the onset of MCAO. (A-G) Quantification of the indicated protein in Fig. 5C after

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normalization to GAPDH. I indicates ipsilateral hemisphere; C, contralateral hemisphere.

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P＜0.01; # P＜0.05; NS, non-significant.

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＊＊

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0.05,

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Data expressed as mean ± SEM are obtained from at least 3 independent experiments.

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＊

P＜

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Highlights CRHR1 inactivation suppresses H2 O2-increased cPLA2 phosphorylation and paracellular permeability. CRHR1 inactivation suppresses H2 O2 -induced tight junction alteration. CRHR1 antagonist suppresses cPLA2 phosphorylation after reperfusion.

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CRHR1 inactivation protects BBB integrity against reperfusion injury.

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CRHR1 interference improved spatial cognitive dysfunction after short-term ischemia with

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long-term reperfusion.

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