2.1 Materials and Reagents
The 32 amino-acid long Elabela 32 peptide (ELA32, QRPVNLTMRRKLRKHNCLQRRCMPLH SRVPFP) with purity more than 98% was synthesized by GenScript (Piscataway, NJ). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, penicillin-streptomycin solution were purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was obtained from Biolog industries (BI, Israel). Dihydroethidium (DHE), Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and 2,3,5-triphenyltetrazolium chloride (TTC) were obtained from Sigma (St. Louis, MO, USA). JC-1 mitochondrial membrane potential detection kit was obtained from Beyotime (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Kaiji Biotechnology (Hangzhou, China). Cell Counting Kit (CCK8) assay kit (ZETA Life Inc., CA, USA). The kits for the detection of ROS, Malondialdehyde (MDA), 8-Oxo-dG, protein carbonyl, Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GSH-Px), and Glutathione (GSH) were obtained from Jiancheng Bioengineering Institute (Nanjing, China). MitoSOX Red was obtained from Molecular Probes (Eugene, OR). The primary antibodies for NQO-1, HO-1, P-PGC-1α, PGC-1α, SIRT3, AMPK, P-AMPK, Nrf2, Akt, P-Akt, P-GSK-3β, GSK-3β, APLNR, cleaved-caspase 3, cleaved-caspase 9, Bax, Bcl-2, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA). All other reagents were commercial obtained from Chinese suppliers.
2.2 Animal and middle cerebral artery occlusion (MCAO) model
Male Sprague-Dawley rats (6 weeks age, 200-230g) were obtained from the Experimental Animal Center of the Fourth Military Medical University. Rats were kept at the environment with 22-25℃, 45-50% humidity and 12-h light/dark cycle, and free to water and standard cages. The in vivo experiments were designed based on the STAIR-criteria and randomization, dose-response assessment, blinding and extensive physiological monitoring were performed strictly. Protocols were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996.
Rats were divided into sham, model, ELA 32 (30 ng/kg), ELA 32 (60 ng/kg), and ELA 32 (120 ng/kg), randomly, and 15 rats in every group. MCAO model was induced by intraluminal filament method as the direction of previous studies. Briefly, after anesthetized with chloral hydrate (400 mg/kg, ip), the middle cerebral artery was ligated using a 3-0 nylon suture. Laser Doppler flowmetry was used to detect the blood flow, and a blood-flow drop to 80% was considered as successful blockage. After ischemia for 1.5h, the intraluminal suture was withdrawn for reperfusion. ELA 32 was dissolved in saline and intracerebral ventricle injected (3.5 mm depth, ± 1.5 mm mediolateral) 15 min before reperfusion using a stereotaxic frame (Alctt Biotech, Shanghai, China). ELA 32 was given once a day for 3 days.
2.3 Neurological deficit evaluation
Neurological deficit scores were always used to evaluate the neurological function. In this study, a modified scoring method developed from Longa was used. The grade was scored as 0-5: 0 (there was no deficits observed), 1 (rats were failure to extend left forepaw fully), 2 (rats circled to the left), 3 (rats fall to the left), 4 (rats had no spontaneous walking with a depressed level of consciousness) and 5 (dead). The evaluation was performed by two another observers blinding to the study.
2.4 Infarct size measurement
The whole brains were collected and cut into 5 slices with 2 mm thickness. Slices were incubated with 2% TTC solution at 37℃ for 20 min, and fixed in 4% paraformaldehyde. Slices were photographed by a camera and images were analyzed. Infarct size was expressed as percentage of the contralateral hemisphere.
2.5 Cerebral edema measurement
The brain water content was used to evaluate the edema extent. After sacrificed, brains were quickly removed and weighed to get the wet weight (WW). Then, brains were dried at 110℃ for 24h to get the dry weight (DW). The formula for calculating water content (%): (WW - DW)/WW × 100.
2.6 Cell culture
HT22 cell, a mouse hippocampal neuron cell line, was purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM medium which contains 10% FBS, and antibiotics in 5% CO2 at 37℃. The culture medium was changed every other day and subcultured when cell growth to 90% confluence.
2.7 OGD/R model and drug treatments
HT22 cells were seeded into 96 or 6 well plates at appropriate concentration and pretreated with ELA 32 (0.5, 1 and 1.5 μM) for 6h. For OGD, medium was changed with the Earle’s balanced salt solution which containing 116 mmol/L NaCl, 5.4 mmol/L KCl, 0.8 mmol/L MgSO4, l mmol/L NaH2PO4, 0.9 mmol/L CaCl2, and 10 mg/L phenol red, and incubated in a hypoxia chamber (Thermo Scientific, USA) pre-gassed with N2/CO2 (95%/5%) gas mixture 37℃ for 3 h. After 3 h challenge, culture medium were replaced with the normal medium containing ELA 32, and cultured for another 6h to imitate the reperfusion process. Cells in control group were treated with normal medium in normal condition. Cells in model group were treated with medium without Elabela 32 when reperfusion.
2.8 Cell viability determination
Cells were seeded into 96 well plates and treated as above, and then the supernatant was replaced by normal medium containing Cell Counting Kit (CCK8). After 4h incubation at 37℃, the optical density was assessed at 450nm using a (Bio-Rad Laboratory, Hercules, CA). The results were showed as the fold of control.
2.9 Apoptosis rate determination
After different treatments, the apoptosis rate of HT22 cells were measured using an Annexin V-FITC/PI apoptosis detection kit according to the instruction. The apoptosis was measured by using flow cytometer analysis (BD FACS Aria II).
2.10 ROS measurement
ROS levels in brain tissues were measured by 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and the fluorescence intensity was measured by a spectrofluorometer (Shimadzu Corp., Japan). The results were showed as the fold of sham group. O2⋅- levels in mitochondria of HT22 cells were measured by MitoSOX Red. H2O2 levels in cells were measured by DCFH-DA. O2⋅- levels in cells were measured by DHE. A laser confocal microscope (Nikon, Japan) was used to acquire images.
2.11 Mitochondrial membrane potential (MMP) measurement
The MMP was measured by tetrechloro-tetraethylbenzimidazol carbocyanine iodide (JC-1) as the direction of manufacturer. Briefly, after cells treated with different treatments, JC-1 at a final concentration of 10 μg/ml was added into the cell cultures, and incubated at 37 ℃ for 30min. Cells were washed by PBS and the fluorescence was observed by a laser confocal microscope (Nikon, Japan).
2.12 ATP level measurement
Tissues or cells were lysed sufficiently and centrifuged at 12000g, 4℃ for 10min. The supernatant was mixed with ATP working dilution as 1:1, after incubation, the fluorescence intensity was measured by a spectrofluorometer. The ATP levels were expressed as the fold of control.
2.13 Mitochondrial enzyme activities measurement
Mitochondria were isolated and purified by differential centrifugations. The mitochondria-located enzymes complex I, succinate dehydrogenase (SDH) and complex V were measured by commercial kits as the direction of manufacturer.
2.14 Biochemical analysis
Brain tissues and HT22 cells were lysed by RIPA buffer, and the supernatant was collected for the determination of biochemical values. Levels of MDA, GSH, LDH, CAT, 8-Oxo-dG, protein carbonyl and GSH-Px were measured by commercial kits as the direction of manufacturer.
2.15 siRNA transfection
AMPK, SIRT3, APLNR, Akt, and Nrf2 specific short interfering RNA (siRNA) were designed and chemically synthesized by Shanghai Genechem Company. HT22 cells were transfected by siRNA molecules using the Lipofectamine RNAi MAX reagent as the manufacturer’s direction (Life Technologies, CA, USA). After transfection for 48h, the transfection efficiency was measured by Western blotting. Tranfected cells were treated with ELA 32 and OGD/R for the further studies.
2.16 Western blotting
After different treatments, brain tissues and HT22 cells were collected, washed by cold PBS and lysed by RIPA buffer with protease inhibitor cocktail. Protein concentrations in samples were measured by BCA protein assay kit. Proteins were separated by 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane. After blocked by 5% nonfat dry milk solution, membranes were incubated at 4 ℃ overnight with the corresponding primary anti-bodies: NQO-1 (1:1000), HO-1 (1:1000), PGC-1α (1:1000), P-PGC-1α (1:1000), SIRT3 (1:8000), AMPK (1:1000), P-AMPK (1:1000), Nrf2 (1:8000), Akt (1:1500), P-Akt (1:1500), P-GSK-3β (1:1000), GSK-3β (1:1000), APLNR (1:800), cleaved-caspase 3 (1:1000), cleaved-caspase 9 (1:1000), Bax (1:1000), Bcl-2 (1:1000), and GAPDH (1:1500). After incubated with secondary antibodies, membranes were visualized by an enhanced chemiluminescent substrate (Thermo, USA). The densities were scanned and quantified using image-analysis systems (Bio-Rad, USA). The results were showed as the fold of control.
2.17 Acetylation assays
PGC-1α acetylation level was measured by immunoprecipitation (IP) followed by Western blotting using antiacetylated lysine antibodies.
2.18 Statistical analysis
Experimental data were collected from triplicate parallel experiments unless otherwise indicated. Results were analyzed by GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA) and expressed as mean ± SD. Statistical analyses were performed using One-way ANOVA followed by the Tukey test and P<0.05 was considered to be statistically significant.