Animal preparation
Male Sprague-Dawley rats (200-230g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China, No.1103241911033018). Rats were maintained in an air-conditioned room (temperature: 21±2°C) under a 12h day-night cycle with free access to food and water, and were acclimated for 3 days prior to the experiment. Animal handling procedures were performed in accordance with the guide of the Ethics Committee of Xi Yuan Hospital of China Academy of Chinese Medical Sciences (Protocol No. 2019XLC015-2). And all animal housing, care, feeding, and experimental procedures were in compliance with the National Guidelines for Animal Protection.
Establishment of cerebral infarction model in rats with permanent middle cerebral artery occlusion (pMCAO)
Rats were anesthetized by an intraperitoneal injection with 1% pentobarbital sodium (80 mg/kg). Under anesthesia, the right common carotid, the right external carotid, and the right internal carotid were separated and were carefully exposed. The right external carotid and the right common carotid were ligated with a suture silk. Thereafter, a 3-0 monofilament nylon suture with a rounded tip diameter 0.32mm (Item#2432A1, Beijing Sunbio Biotech Co Ltd) was introduced into the bifurcation of the right common carotid and then was intracranially advanced for approximately 18 mm to block the blood flow of the right middle cerebral artery. During this procedure, the body temperature was maintained at 37°C with a warm pad. For the sham-operated group, only skin incisions were performed under anesthesia.
Drug treatments in rats
SLT was provided by the ShenWei Pharmaceutical Corporation (Heibei, China). SLT was soluted in saline, and was injected into the duodenum immediately after the right middle cerebral artery was blocked. The doses of SLT were set as 16.5 mg/kg (SLT-L) and 33 mg/kg (SLT-H). Rats in the sham group and model group were injected with saline in the same way and at the same time point.
Measurement of neurological deficits
The neurological function deficit scores of rats were blindly evaluated 24h after MCAO. A 5 point scale was used as follows: 0, no neurological deficits; 1, failure to fully extend the left forelimb; 2, decreased resistance to a lateral push toward the right side and failure to fully extend the left forepaw; 3, circling to the left side; and 4, inability to walk spontaneously and lack of response to stimulation [24].
Assessment of infarct volume
After neurological function tests, the rats were killed by decapitation, and their brains were taken out and were sectioned into slices of 2 mm thickness; the slices were incubated in a 2% 2,3,5-Triphenyltetrazolium chloride (TTC) solution at 37°C for 15 minutes. TTC stained the noninfarcted region with a deep red pigment, while the infarcted brain areas were stained with white [24]. Stained sections were photographed, and the images were analyzed to calculate the infarct volumes by using an Image Pro-Plus 6.0 analysis system (Media Cybernetics, Rockville, MD, USA).
Brain tissue fixations, embeddings, and sections and HE stainings
24h after MCAO, the rats were killed by decapitation, and the brains were then rapidly taken out and were fixed in 4% paraformaldehyde for 7 days. The brains were then embedded in paraffin and were sectioned into slices of 7 µm. The sections were stained with HE and were observed with an Olympus BX51 microscope.
Evaluation of blood water content of the brain
24h after MCAO, rats were sacrificed by decapitation, and their brains were taken out. The wet brains were weighed and were dried at 60°C for 3 days, and then the weights of the dry brains were measured. The brain water content = (1-dry weight/wet weight)×100%.
Immunohistochemistry examinations
The brain sections were deparaffinized, rehydrated, and blocked. Next, the sections were incubated overnight at 4°C with anti-claudin-1 antibody (1:500, ab15098, abcam), anti-occludin antibody (1:500, 27260-1-AP, proteintech), and anti-heme oxygenase-1 (HO-1) antibody (1:500, 66743-1-Ig, proteintech), respectively. The sections were rinsed with PBS for three times, then were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG at 37°C for 20 min, and were colorized with DAB. Lastly, hematoxylin restaining was performed. The sections were observed under an Olympus BX51 microscope.
Western blot assay
The nuclear and cytosol protein extractions for Nrf2 and total protein extractions for Nrf2, HO-1, occludin, and claudin 1 from the samples (in in vivo experiments, were cerebral cortex; in in vitro experiment, were hCMEC/D3 cells) were performed by using the nuclear-cytosol extraction kit (Applygen Technologies Inc., Beijing) or the total protein extraction kit (Solarbio Science & Technology Co. Ltd Beijing). Equal amounts of protein (50 µg) were loaded into 10% or 12.5% SDS-PAGE gels, and then were subjected to electrophoresis, lastly were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, and then were incubated overnight at 4 °C with anti-claudin-1 antibody (1:1000), anti-occludin antibody (1:1000), anti-Nrf2 antibody (1:1000), anti-HO-1 antibody (1:1000), anti-GAPDH antibody (1:1000,60004-1-Ig, proteintech), anti-β-actin antibody (1:1000, 60008-1-Ig, proteintech) or anti-histone H3 antibody (17168-1-AP, proteintech). The membranes were incubated with HRP-conjugated secondary antibodies for 1.5h at room temperature. The protein bands were enlightened with an enhanced chemiluminescence kit, and their brightness were quantified by using Image LabTM Software.
Determination of the activities of superoxide dismutase (SOD) and the contents of glutathione (GSH)
All samples (brains and cells) were made into homogenates by an ultrasounic cell disrupter at 0 °C. SOD activities and GSH contents in the homogenates were analyzed with the merchant kits (Institute of Biological Engineering of Nanjing Jiancheng, Nanjing, China).
Cell culture
Human brain microvascular endothelial cell line (hCMEC/D3, iCellBioscience, Inc. Shanghai, China) were cultured at 37℃ with 5% CO2 in Endothelial Cell Medium (ECM, PriMed-iCell-0016, China) supplemented with 5% fetal bovine serum, 1% ECGS, 100 U·mL-1 penicillin and 100 μg·mL-1 streptomycin, and were passaged with 0.25% trypsin.
Oxygen-glucose deprivation and reoxygenation (OGD/R) model and SLT treatments
For oxygen–glucose deprivation (OGD), hCMEC/D3 cells were incubated in a glucose-free Dulbecco's Modified Eagle's Medium (DMEM, Gibco, USA) in a customer-made chamber filled with 95% N2/5% CO2 for 4h at 37 °C. After OGD, for reoxygenation, the incubation media was replaced with normal ECM and the cells were cultured under normal atmosphere with 5% CO2.
The mock group was incubated in normal DMEM with 5% CO2 for 4h at 37 °C, and was then incubated with ECM.
SLT was soluted with DMSO, and the solutions were added into the incubation medium with the volume ratio of 1:1000 at the beginnings of OGD and reoxygenation. The mock group and the OGD/R model group were treated with DMSO in the same way at the same time points.
Cell viability
The cells were incubated with CCK8-ECM solution (1:10) at 37°C for 2h. The absorbance at 450 nm was measured with a microplate reader. Then the cell viabilities were obtained through normalization to the average absorbance of the mock (normal control) group.
Immunofluorescence assay
Paraformaldehyde-fixed hCMEC/D3 cells were incubated with anti-Nrf2 antibody or anti-HO-1 antibody (both 1:200), followed by the incubation with secondary antibodies, i.e. Fluorescein-Conjugated AffiniPure Goat anti-Rabbit IgG (1:100, Yuabio, china), and were stained with DAPI finally. Cells were observed and imaged with an Olympus IX81 live cell station.
RNA interference by siNrf2 and drug treatment
Transfections in HCMEC/D3 cells were conducted with Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA). High purity control siRNAs (negative control siRNA and GAPDH siRNA) and Nrf2 siRNAs were obtained from JTSBIO (Wuhan, China). The Nrf2 siRNA sequences were: Nrf2 siRNA-1, forward, CCCUGAAAGCACAGCAGAATT, and reverse, UUCUGCUGUGCUUUCAG- GGTT; Nrf2 siRNA-2, forward, CCAGAACACUCAGUGGAAUTT, and reverse, AUUCCACUGAGUGUUCUGGTT; Nrf2 siRNA-3, forward, GCCUGUAAGU- CCUGGUCAUTT, and reverse, AUGACCAGGACUUACAGGCTT. Negative control (NC) siRNA sequences were: forward, UUCUCCGAACGUGUCACGUTT, and reverse, ACGUGACACGUUCGGAGAATT. GAPDH sequences were: forward, UGACCUCAACUACAUGGUUTT, and reverse, AACCAUGUAGUUGAGGU- CATT.
Paracellular Permeability Measurement
Cell monolayer integrity was assessed by diffusion of fluorescein isothiocyanate (FITC)-dextran (4kDa, Lot:64878, MCE, USA) as previously described [13]. After OGD/R, 400 μL FITC-dextran (0.5 mg/mL) solutions were added to the upper chamber of 12-well transwell culture plate inserts (A190059, Millicell, Germany), on the bottom of which the hCMEC/D3 cell confluents grew. Inserts were placed in 12-well culture plates containing 1000 μL of DMEM/F12 media (serum-free and without phenol red) per well. Then the cells were incubated at 37℃ for 60 min in the dark. Inserts were removed and the solutions in the wells were collected and transferred into a back-96-well plate. The fluorescence intensities were measured by using the excitation and emission wavelengths as 490 and 520 nm, respectively, and were converted to the concentrations of FITC-dextran with the calibration curve.
Statistical analysis
The data are expressed as Mean ± SEM and were statistically analyzed by using the Statistical Product and Service Solutions (SPSS) 16.0 software; data comparisons between two groups were conducted with t-test, that among multiple groups were conducted with one-way or two-way analysis of variance (ANOVA). The statistical significance level was set to 0.05, i.e. differences were deemed statistically significant as P < 0.05.