Cell culture and treatment
Rat pheochromocytoma PC-12 cells and human embryonic kidney 293 (HEK293) cells, displaying a semi-differentiated phenotype with neuronal projections, were used as model neuronal cells. An RPMI1640 medium (Sigma-Aldrich, USA) supplemented with penicillin (100 U/mL, Sigma-Aldrich), streptomycin (100 µg/ml, Sigma-Aldrich) and 10% FBS (Scientifix Pty Ltd, French) at 37 ℃ in a humidified 95% air/5% CO2 incubator was used as cell culture medium. This medium was changed daily.
Evaluation of cytotoxicity of HL-008 in vitro
Cell viability was determined by measuring the formazan produced by the reduction of MTT (No. M5665-5G, Sigma), which shows the mitochondrial activity of living cells. Cells were seeded at 1 × 104 cells per well in a 96-well plate and incubated for 24 h to allow adherence. A 1 × PBS (pH = 7.4) was first used to dissolve the HL-008 and then diluted to the final stock concentrations prior to addition to the cells. Cells were treated with the serially diluted HL-008 solution at different concentrations and then incubated for 48 h at 37 ℃. Following this, the cell culture medium was replaced by 100 µL of 0.5 mg/mL MTT and incubated for 4 h. Finally, 80 µL of 20% SDS in 0.02 M HCl was added, and the plate was incubated for 18 h. The absorbance of the plate was measured at 570 nm using a microplate reader.
Evaluation of the effect of HL-008 on cytotoxicity induced by hydrogen peroxide
PC-12 cells were seeded at 1 × 104 cells per well and incubated for 24 h to allow adherence. The cells were incubated with HL-008 for 15 min prior to the addition of H2O2 at different concentrations (100 µM, 150 µM and 200 µM) and then incubated for 24 h at 37 ℃. Following this, the medium was replaced with 0.5 mg/mL of MTT and incubated for 4 h at 37 ℃. Finally, 80 µL of 20% SDS in 0.02 M HCl was added, and the plate was incubated for 18 h. The absorbance of the plate was measured at 570 nm using a microplate reader.
All male SD rats (200–300 g) used in this study were purchased from SPF Biotechnology Co., Ltd. (Beijing) and bred in a certified animal facility. The temperature in the feeding room was 24–26 ℃; humidity was 37–51%; air change was performed 10–20 times/h, and the day-night alternation time was 12 h/12 h.
All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (IRM, CAMS; permit number: 2017053). The animals were cared for in accordance with the guidelines of the National Animal Welfare Law of China. Meanwhile, all the studies were carried out in compliance with the Animals in Research: Reporting in vivo Experiments (ARRIVE) guidelines.
Evaluation of the BBB permeability of HL-008
The number of SD male rats used was 27. They were divided into nice groups; three rats in each group. After a single intraperitoneal injection of 200 mg/kg of HL-008, whole blood and brain were collected at 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 6, and 8 h after administration. A volume of 200 µL of whole blood was collected in a blood collecting vessel containing EDTA, placed on ice, and separated at 4000 rpm for 20 min. The separated plasma was placed in a 1.5 ml EP tube. After euthanizing the rats, the brain was quickly removed. 50 µL plasma or brain tissue sample was added to 96 well plate, mixed thoroughly with 250 µL 35% trichloroacetic acid, and centrifugated at low temperature for 20 minutes. Approximately 150 µL of the supernatant was transferred to new 96-well plate and mixed with 150 µL of ultrapure water. Finally, 10 µL of the sample was obtained to enter into the LC-MSMS system (API4000 Q-Trap).
Establishment of an animal model of AIS
The AIS model was established by MCAO in the rats. The body temperature of the anesthetized rats was maintained at 37 ± 0.5 ℃. With a median cervical incision, the distal end of the external carotid artery was ligated with a 6 − 0 silk thread, 4 mm from the common carotid artery bifurcation. Another 6 − 0 silk thread was inserted into the external carotid artery, and a living knot was made near the common carotid artery bifurcation. A head-treated 0.33 mm diameter nylon wire was then inserted into the middle cerebral artery. The insertion depth of the nylon wire was approximately 16 ± 1 mm at the bifurcation of the common carotid artery. After ischemia, at 90 min, the thread bolt was removed, the proximal end of the external artery with 6 − 0 silk thread was ligated, and the neck wound was sutured with 3 − 0 silk thread. The rats were then placed on a heating pad.
Evaluation the protective effect of HL-008 on the MCAO model
A total of 25 male SD rats (200–250 g) were divided into five groups (n = 5 each), including a control group, ischemia reperfusion (I/R) group, HL-008 100 mg/kg group, HL-008 50 mg/kg group, and HL-008 25 mg/kg group. During reperfusion, HL-008 was administered intravenously. After 24 h of reperfusion, the neurological function score and cerebral infarction area were evaluated.
Assessment of neurological deficits and the infarct area
The neurological behaviors were evaluated based on Longa and Bederson's 5-point scoring system [24–26]; 0, observed behavioral deficits; 1, unable to fully extend the opposite forepaw; 2, failure to turn to the opposite side; 3, falling to the opposite side; 4, no spontaneous walking. The higher the score, the more serious the neurological impairment of the animal.
For assessment of infarct volume, the brain tissue of rats was obtained and cut into 2 mm thick slices along the coronal plane after neurological scoring. Half of the brain slices were placed into 2% TTC dye solution at intervals. After incubation at 37 ℃ for 10 min, the slices were turned regularly for uniform staining. The normal brain tissue was bright red and the infarcted area was pale in color. The infarct area was measured and analyzed by Image Pro Plus 6.0 software.
Comparison of efficacy between HL-008 and edaravone
A total of 20 male SD rats (200–250 g) were divided into four groups (n = 5 each), including a control group, ischemia reperfusion (I/R) group, HL-008 100 mg/kg group and edaravone 3 mg/kg group. During reperfusion, HL-008 and edaravone were administered intravenously. After 24 h of reperfusion, the neurological function score and cerebral infarction area were evaluated.
Investigation of the HL-008 mechanism
A total of 15 male SD rats (200–250 g) were divided into three groups (n = 5 each), including a control group, ischemia reperfusion (I/R) group, and HL-008 100 mg/kg group. During reperfusion, HL-008 and edaravone were administered intravenously. After 24 of reperfusion, the rats were killed and the brain tissue was removed for further study.
Measurement of ROS levels in the brain
Brain tissue weighing 0.1 g was fully homogenized with 1 mL of extracting solution on ice and centrifuged at 10000 g and 4 ℃. The supernatant was obtained and placed on ice for testing. The ROS content in the brain tissue was analyzed using an ROS detection kit (No. E004,Nanjing Jiancheng Bioengineering Institute, China). The procedure was conducted according to manufacturer's instructions.
Evaluation of malondialdehyde (MDA) in the brain
Brain tissue weighing 0.1 g was fully homogenized with 1 mL of extracting solution on ice and centrifuged at 8000 g, at 4 ℃, for 20 min. The supernatant was obtained and placed on ice for testing. The MDA content in brain tissue was analyzed using an MDA detection kit (No. X-W-A401, Sino Best Biological Technology Co., Ltd., China), according to the manufacturer's instructions.
Evaluation of antioxidant activity
Brain tissue weighing 0.1 g was fully homogenized with 1 mL of extracting solution on ice and centrifuged at 10000 g, at 4 ℃ for 20 min. The supernatant was obtained and placed on ice for testing. The activities of SOD (No. YX-W-A500, Sino Best Biological Technology Co., Ltd., China), CAT (YX-W-A501, Sino Best Biological Technology Co., Ltd., China), and GSH-Px (A005, Nanjing Jiancheng Bioengineering Institute, China) in rat brain tissues were tested using commercial kits, according to the manufacturer's instructions.
Hematoxylin and eosin (HE) staining
The brain tissue samples fixed with 4% paraformaldehyde, were embedded in paraffin, and cut into 5 µm sections using a paraffin slicer. After slicing, the slices were slightly dried in air and baked overnight in an oven at 60 ℃. After the slices were successfully obtained, they were dewaxed with xylene and absolute ethanol to water, then stained with HE (71014460; Shanghai Jingke Chemical Technology Co., Ltd.) and analyzed using the Pannoramic SCAN (3D HISTECH). The nucleus was dyed blue and the cytoplasm dyed red. Based on the staining results in each group, the morphological changes were analyzed.
Analysis of TUNEL staining
The paraffin section was dewaxed with xylene and absolute ethanol to water. A concentration of 20 µg/ml protease K without DNase was added at 20–37 ℃ for 15–30 min. After washing thrice with PBST, each slice was added to 50 µL of TUNEL reaction solution (No. 11684795910, Roche) and incubated in a constant temperature incubator at 37 ℃ in the dark for 60 min. Each slice was washed thrice with PBST and combined with 100 µL 4',6-Diamidino-2-phenylindole (DAPI, 1:1000 PBS pH = 7.4), away from light for 10 min at room temperature. Finally, PBST (pH = 7.4) was used to wash the slices thrice, and a fluorescence quenching agent was used to seal the slices for microscopic inspection (Pannoramic SCAN, 3D HISTECH). DAPI was stained with blue fluorescence in the nucleus and green fluorescence in the positive part.
The fixed tissue sections were dewaxed to water; washed with PBS thrice for 3 min each; blocked by 0.3% hydrogen peroxide; incubated at room temperature for 20 min; washed by PBS again thrice for 3 min each, using 0.01 M citric acid (pH 6.0) buffer solution for antigen repair. After natural cooling, they were washed with PBS (pH = 7.4) thrice, for 3 min each, and dripped and sealed with normal goat serum at 37 ℃ for 30 min. The serum was poured out, and 1:100 diluted TNF-α (No. Ab167161, abcam), IL-1β (No. Ab8348, abcam) and 1:300 diluted GFAP (No. Ab68428, abcam) antibody were added and incubated overnight at 4 ℃. They were then washed thrice with PBS, and a 1:200 diluted fluorescent coupling antibody was added and incubated for 1 h in the dark at room temperature. The nucleus was then dyed with DAPI, washed thrice with PBS, sealed with 50% buffer glycerin, and observed using fluorescence microscopy during which photographs were obtained.
The tissue was washed with cold PBS (pH = 7.4) twice or thrice, cut into small pieces, and homogenated. Add 10 times the tissue volume of lytic fluid (add protease inhibitor a few minutes before use) to the ice for complete homogenization. The supernatant was collected by centrifugation of 12000 g for 5 min, resulting in the total protein solution. The protein concentration was measured using the BCA method, and the protein was separated using SDS-PAGE electrophoresis. After electrophoresis, the blocked membrane was incubated using the first antibody, including Nrf2 (No. AF0639, Affinity), HO-1(No. AF5393, Affinity), Bax (No. AF0120, Affinity), Caspase-3 (No. AF6311, Affinity), P-p38(No. AF4001, Affinity), p-NF-κB (No. AF2006, Affinity), Bcl-2(No. AF6193, Affinity), p-ERK (AF1015, Affinity), p-JNK (AF3318, Affinity), P-p53(AF3075, Affinity), NOX4 (DF6924, Affinity), and GAPDH (ab 68428, Abcom). Following this, the second antibody was incubated. The chemiluminescent substrate was dripped onto the membrane and incubated in a dark room for approximately 5 min. The luminous substrate was discarded, the film was placed between X-ray films of equal size, and exposed for approximately 1–3 min. Then an X-ray film was taken out every other minute. Finally, the X-ray film was developed for 5 minutes, fixed for 5 minutes, and washed for more than 10 min and dried.
The tissue samples were ground into powder with liquid nitrogen, and 6 mL of protein extract I (40 g trichloroacetic acid + 400 mL protein extract II, -20 ℃, 1 h) was added, and centrifuged at 4 ℃, 12000 rpm for 20 min. Following this, 6 mL of protein extract II (400 mL acetone + 280 µL β- mercaptoethanol, -20 ℃, 1 h) was added to the precipitate, and centrifuged at 4 ℃ 12000 rpm for 20 min. The precipitate was vacuumed dry to obtain the crude protein powder. Then, a certain amount of dry powder was added to 400 µL of the SDT (8 M urea, 100 mM Tris-Hcl, 1 mM PMSF, PH = 8.5) cracking solution for dissolution, and placed at room temperature for 1 h. Centrifugation at 9000 rpm at 4 ℃ for 10 min was performed to obtain the protein sample. The protein concentration was determined using the Bradford method and identified by SDS-PAGE. The total protein was hydrolyzed with trypsin in an incubator set at 37 ℃ for 12–16 h. The hydrolysate was washed with 50 mM of ammonium bicarbonate, acidified with trifluoroacetic acid (TFA), vibrated, centrifuged to remove sodium deoxycholate (SDC), and desalinated with the C18 column. The desalted samples were lyophilized in a freeze-drying apparatus, then re-dissolved in 0.1% formic acid after lyophilization, and detected using a mass spectrometer (Q-active mass spectrometer, Thermo Fisher). The retrieved proteomic database was from the UniProt, Rattus norvegicus (rat) protein, with a 29944 protein count. Maxquant (version 184.108.40.206) and Protein Discoverer (edition 1.4) were used for database retrieval and relative quantification.