Drugs and reagents
Cornin (purity > 99.0%, molecular formula: C17H24O10, molecular weight: 388.37) was obtained from SenBeiJia Biological Technology Co., Ltd. (Nanjing, China).
Antibodies against mTOR (Lot No: 2972S, 10), phospho-mTOR (Lot No: 2971S, 26), Akt (Lot No: 9272S, 28), phospho-Akt (Lot No: 13038S, 5), Bax (Lot No: 14796S, 2), and Bcl-2 (Lot No: 3498 T,3) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against microtubule-associated proteins 1A/1B light chain 3B (LC3B) (ab48394, Lot No: GR3199111-5), cleaved caspase-3 (ab13585, Lot No: GR274345-1), Beclin-1 (ab62557, Lot No: GR3205839-5), LY294002 (PI3K inhibitor) (ab146593, Lot No: APN08061-6–12-S), p62 (ab56416, Lot No: GR3234289-1), and GFAP (ab7260, Lot No: GR3249186-1) were purchased from Abcam (Cambridge, UK). mTOR siRNA (sc-35409, Lot No: G0518) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Alexa Fluor 488-labelled anti-rabbit antibody (Abcam, UK, ab150077, Lot No: GR3245102-1) was used for immunofluorescence. Tetrazolium chloride (TTC) and NeuN (MAB377, Lot No: 2919676) were obtained from Sigma (Shanghai, PR China), whereas Evans blue was bought from Urchem (Shanghai, PR China).
Male Sprague Dawley rats were obtained from Shandong Luye Pharmaceutical Company (Shandong, PR China, Permit Number: SCXK 2018 0003). The animals were housed with the following conditions: 22 °C ± 2 °C, 50% ± 10% relative humidity, 12 h light/dark cycle, with food and water ad libitum. The Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, USA), revised in 1996, was followed when conducting all animal studies. The studies were also carried out in compliance with the ARRIVE guidelines and the animal welfare compliance Guide for the Care and Use of Laboratory Animals (8thed., 2011). All experiments and procedures were conducted following the Animal Care Guidelines of the Animal Experiment Committee of Binzhou Medical University (China; authorization number BYLY 2015–74).
Induction of cerebral ischemia by ischemia/reperfusion procedure
After acclimatization for one week, rats within weight of 270–300 g were injected intramuscularly with ketamine 100 mg/kg and xylazine 10 mg/kg to achieve anesthesia. The rectal temperature was kept constant throughout the surgical procedure, and the rectal temperature was recorded and maintained at 37 °C. Middle cerebral artery occlusion (MCAO) was performed as described elsewhere . In brief, occlusion of the left common carotid artery was performed, and then the branches of the external carotid artery were isolated and divided. The internal carotid artery was traced rostrally, and the 4–0 filament (a diameter of 0.25 mm), which had a tip diameter of 0.34 mm, was used to form a globular stopper and inserted into the internal carotid artery, and the filament was then pushed forward until resistance was observed. The filament was removed after 90 min.
One hundred rats were divided into into five groups (Group I ~ V, half animals for Neurological Severity Score (mNSS) and infarct volume detection, the other animals for BBB detection, western blotting and immunohistochemical staining detection) with 20 rats per group: I) non-ischemia/reperfusion (IR) (sham) group; II) I/R group (treated with saline only); III) 2.5 mg/kg cornin group; IV) 5 mg/kg cornin group; and V) cornin 10 mg/kg group. After reperfusion for 15 min, cornin were intravenously injected (i.v.) at various dosages via the tail vein. After 24 h of reperfusion, rats neurological deficit scores, as well as the blood–brain barrier (BBB) permeability were performed [19, 20]. The rats were placed in a clear glass box of approximately 10 L with a CO2 perfusion rate of 2 L per minute for euthanized. The brains were collected for brain infarct volume determination after transcardial perfusion with phosphate-buffered saline (PBS), the brains corresponding to the ischemic core, peri-infarct cortex rat brain tissues were collected and cutted into two pieces, one piece stored at -80 °C and utilized in western blotting, the other piece was fixed with postfixed with 4% paraformaldehyde in PBS overnight at 4 °C for subsequent immunohistochemical staining in the second group.
For the mechanism study, 50 rats were divided into 5 groups : VI) non-ischemia/reperfusion (IR) (sham) group; VII) I/R group (treated with saline only); VIII) 10 mg/kg cornin group; IX) 10 mg/kg cornin combined with LY294002 group; and X) LY294002 group. After 24 h of reperfusion, the rats were euthanized with carbon dioxide (CO2). The brains corresponding to the peri-infarct cortex were collected and utilized for Western blot analysis.
Assessment of neurological deficit scores
Neurological deficit scores were assessed at 24 h after reperfusion using the flowing method . Briefly, neurological function was graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18). The mNSS is a composite of sensory, reflex, and balance tests. In the severity scores of injury, 1 score point is awarded for the inability to perform the test or for the lack of a tested reflex. Thus, a higher score corresponds to a more severe injury.
Evans blue extravasation and measurement of infarct volume
At 24 h post-reperfusion, rats were sacrificed using deep anesthesia, and the brains were immediately collected. Five coronal sections of the brain with 2.0-mm thickness were prepared and incubated in a 2% 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) solution at 37 °C for 20 min. Infarct brain tissues appear white, while normal brain tissues appear red. The sections were digitized, and the infarct areas were measured using Image Pro Plus software to calculate the percentage of cerebral infarct areas .
At 24 h post reperfusion, 0.1 ml of 4% Evans blue mixed in 0.9% saline was administered intravenously. A quantitative assay using Evans blue was performed to determine blood–brain barrier (BBB) leakage based on a method described previously .
Western blot analysis in vivo
Extraction of total protein from the ischemic penumbra in the cerebral cortex was performed in the experiment. The samples were homogenized with lysis buffer containing a protein inhibitor, the supernatant was obtained, and the amount of protein in the supernatant was determined using the BCA assay. Approximately 50 μg tissue protein samples were separated by SDS-PAGE and then analyzed by western blotting using specific antibodies against p-mTOR, mTOR, Bcl-2, Bax, p-Akt, Akt, Beclin-1, GFAP, p62, LC3B, cleaved caspase-3, and GAPDH at 4˚C overnight. A Gel Doc2000 system (Bio-Rad, USA) was used for scanning and quantification of strip optical density. The data were normalized to GAPDH.
PI3K inhibitor LY294002 intervention experiment
Rats were injected intramuscularly with ketamine 100 mg/kg (i.m.) and xylazine 10 mg/kg (i.m.) to achieve anesthesia. The CI/R rats were injected with LY294002 via the lateral ventricle approximately 30 min prior to reperfusion. LY294002 was dissolved in dimethyl sulfoxide (DMSO) immediately before the operation to obtain a final concentration of 10 mM, and 10 μL of LY294002 solution was injected into the rat’s lateral ventricle using a brain stereotaxic device (Reward, Shenzhen, China) . The stereotactic intracerebroventricular (ICV) injection site was selected from the following areas of the bregma: anterior and posterior, 0.8 mm; lateral, 1.5 mm; and depth, 3.5 mm.
The ischemic penumbra in the cerebral cortex was fixed in 4% paraformaldehyde for 48 h, then dehydrated, embedded in paraffin, and sectioned at 4-μm thickness. The histological sections were placed on glass slides coated with polylysine, deparaffinized in xylene, and rehydrated across an alcohol gradient, then stained by immunohistochemistry. The expressions of neuronal nuclei (NeuN), GFAP, LC3-II, and Beclin-1 were detected. Positive expression was assessed using an Image-Pro Plus analysis system.
Human glioma U87 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. The U87 cells were cultured in dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics consisting of 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C and 5% CO2. The cells were subcultured upon reaching 80% confluency.
In vitro oxygen glucose deprivation/reperfusion model
To simulate in vitro glucose and oxygen deprivation, the U87 cells were subjected to a hypoxic environment for three hours. An in vitro oxygen glucose deprivation/reperfusion (OGD/R) model was established as in previous study . Before hypoxia, pretreatment of cells was performed using various cornin concentrations (10, 30, 100, 300 and 1000 nM) for 48 h. A negative control was prepared using a normal culture using DMEM with 1% FBS in 5% CO2 air and 20% O2, whereas a hypoxia solution culture was employed as the control.
Cell viability assays
The U87 cells were kept in the hypoxic solution with or without cornin for three hours, and then cell viability was evaluated using an cell counting Kit-8 (CCK-8) assay. Cells at a density of 1 × 105 cells/well were seeded into 96-well plates. The CCK-8 solution (10 μL/well) was placed into each well and further cultured at 37˚C for 4 h. Absorbance at 450 nm was then assessed with a Molecular Devices SpectraMax M5 instrument (Sunnyvale, CA, USA).
Western blot analysis in vitro
The U87 cells were cultured for 48 h, then PI3K inhibitor LY294002 intervention was performed, and mTOR siRNA was added for transfection. Treated cells were followed by washing three times with ice-cold phosphate buffered sline (PBS) and lysing using RIPA lysis buffer (Beyotime, PR China) containing 1 mM PMSF (Sigma-Aldrich). Similar amounts of cell protein (30 μg) were separated by SDS-PAGE, and then analyzed by western blotting using specific antibodies against p-mTOR, mTOR, Beclin-1, p62, LC3B, p-Akt, and Akt, with β-actin as reference, at 4˚C overnight. The strips were scanned, and optical densities were quantified using a Gel Doc2000 (Bio-Rad, USA). Data normalization was performed using β-actin.
Determination of apoptosis
An Annexin-V FITC apoptosis detection kit was employed to identify apoptotic cells. First, the cells were collected, washed, and then cultured in the dark in the presence of Annexin-V FITC, as well as propidium iodide, at 4 °C for 30 min, followed by analysis using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) .
The distribution and expression of LC3B were detected by immunofluorescence in U87 cells. After cell treatment, imaging was conducted under a fluorescence microscope (Olympus IX73, Japan).
All experiments were performed in triplicate. The data from the experiments were expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Differences with P < 0.05 were considered statistically significant.