Chemical Materials
Pentobarbital sodium was safeguarded in Harbin Medical Pharmacological Laboratory (Harbin, China). BSA and rhTrx-1 were purchased from R&D System (St. Paul, Minnesota, USA). Penicillin/streptomycin solution, Hoechst and 2% TTC dyeing solution were purchased from Solarbio (Beijing, China). ROS and Necrostatin-1 was purchased from Sigma (St. Louis, MO, USA). Annexin V-PE/7AAD assay was purchased from BD Biosciences (San Jose, CA, USA). All the ELISA kits were purchased from Cusabio (Wuhan, China). Dapi dyeing solution and JC-1 kit were purchased from Beyotime (Shanghai, Beijing). DMEM, FBS amd MEM/EBSS were purchased from Hyclone (Logan, Utah, USA). The primary antibodies used were: anti-RIPK1, anti-RIPK3, anti-MLKL, anti-pMLKL, anti-CCL2 and anti-Iba-1 (Abcam, Cambridge, MA, USA); another anti-Iba-1 (Wako, Japan); anti-NLRP3, anti-ASC, anti-caspase-1, anti-caspase-3 and anti-β-actin (ABclonal, Wuhan, China); anti-CD206 and anti-MMP-9 (R&D System, St. Paul, Minnesota, USA); anti-CD16 (Bioss, Beijing, China). The second antibodies used were: Alexa Fluor 800-conjugated Goat-anti rabbit (LI-COR, Lincoln, NE, USA), Alexa Fluor 555-conjugated Donkey-anti Rabbit, Alexa Fluor 488-conjugated Donkey-anti Rabbit, Alexa Fluor 488-conjugated Donkey-anti Goat (Abcam, Cambridge, MA, USA).
MCAO Procedure And Drug Administration
C57 BL/6 male mice received 60 min of MCAO as previous described with some modifications [24]. Briefly, thirty-eight mice were randomly divided into three groups: Sham, MCAO and MCAO + rhTrx-1 group. All mice were anesthetized with pentobarbital sodium before inducing MCAO. During the operation, mice body temperature was kept at 37℃. For MCAO procedure, common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were exposed and separated. After clipping the common carotid artery, ligation was performed at the ECA and a small incision was made. Through the incision, thread embolism was slowly inserted into ICA. After 60 min occlusion, thread embolism was pulled out and the reperfusion was carried out. Mice in Sham group received the same procedure, but did not receive the embolus. The mice in MCAO + rhTrx-1 group received 10 mg/kg rhTrx-1 by tail vein injection after reperfusion. As control, mice in MCAO group were injected with equal volume of 0.9% sterile saline. All mice were sacrificed 24 h after reperfusion for further analysis.
Infarct Volume Assessment
TTC staining was executed to evaluate the size of cerebral infarction. In brief, the brain was taken to make coronal brain sections after the mice were sacrificed. The slices were placed in 2% TTC staining solution and immersed for 10 min at 37℃ avoiding meeting up. After staining, the slices were fixed in 4% PFA overnight, and pictured the next day. Each infarct area was measured by using Image J software.
Neurobehavioral Testing
To assess the degree of sensory and motor damage in each group of mice, Bederson score and corner test were carried out based on previous studies [25, 26]. In order to evaluate Bederson score, scores were recorded according to the physical signs of the mice in the tail suspension state as followed: 0 points, normal; 1 point, the contralateral forelimb could not be fully extended; 2 points, the resistance to thrust of the contralateral forelimb decreased; 3 points, turning to the contralateral side of the lesion. For evaluation corner test, mice were placed in the depth of 30-degree angle, and the direction of turning was observed. Normal mice turned randomly and with equal probability toward both sides, whereas mice turned toward the lesion side after cerebral ischemia.
OGD Procedure And Treatment
BV-2 cell line were cultured in complete medium, and incubated in 37℃ incubator containing 5% CO2. The complete medium was composed of DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin solution. For the OGD program, after replacing the cell supernatant with EBSS solution, the cells were transferred to three gas incubators (37℃, containing 95% N2 and 5% CO2) and cultured for 1, 2, or 4 h, respectively. Cells in the Nec-1 group were treated with Nec-1 (20 µM) after re-oxygenation and maintained for 6 h, 12 h, 24 h. Cells in the rhTrx-1 group were incubated with 5, 10 or 25 µg/ml rhTrx-1 (dissolved in sterile PBS) for 24 h after reoxygenation.
Flow Cytometry
To detect apoptotic rate and intracellular ROS expression level, Annexin V-PE/7AAD kit and DCFH-DA solution were used according to the manufacturer's instructions. Briefly, to analyze the extent of apoptosis, cells were harvested and double-stained with Annexin V-PE and 7AAD for 15 min at room temperature in the dark. To detect intracellular ROS levels, cells were harvested and stained with DCFH-DA solution for 20 min in the dark at 37℃. Immediately after the above staining, the cells were detected and analyzed using a Beckman CytoFLEX flow cytometer (CA, USA).
Western Blot Analysis
The protein levels were detected by western blot. In a brief, cells or brain tissues were lysed in precooled RIPA buffer and centrifuged to collect the supernatant for protein extraction. Samples were loaded onto SDS-PAGE and then transferred onto nitrocellulose membranes. Membranes were blocked with 5% BSA or 5% non-fat dry milk and finally incubated with primary antibodies (anti-RIPK1, anti-RIPK3, anti-MLKL, anti-pMLKL, anti-CCL2, anti-MMP-9, anti-NLRP3, anti-ASC, anti-caspase-1, anti-caspase-3 and anti-β-actin) at 4℃ overnight. The second day membranes were incubated with Alexa Fluor 800-conjugated Goat-anti rabbit antibody at room temperature. Protein bands were imaged and analyzed with the Odyssey system (LI-COR Biosciences, Lincoln NE, USA).
Transmission Electron Microscope (TEM)
After collecting the cell precipitation, glutaraldehyde was added slowly for fixation. Ultrathin cell sections (100 nm thick) prepared using ultramicrotome were mounted on a copper grid, and then stained with uranyl acetate and lead citrate. Observed the sections at an accelerating voltage of 80 kV on a transmission electron microscope (Hitachi H-7100, Hitachinaka, Japan) by single blind.
Immunofluorescence Staining
Briefly, for in vitro test, cells were fixed with 4% paraformaldehyde (PFA) for 15 min and then washed with PBS for 3 times and then incubated with first antibodies. After incubated with the anti-CD206 or anti-CD16 primary antibody respectively at 4℃ overnight, cells were incubated with the secondary antibodies at room temperature for 1 h, and were added Hoechst finally. For in vivo test, frozen slices were only washed with PBS for 3 times and then blocked with 5% BSA at room temperature for 1 h. The anti-RIPK1, anti-CD206, anti-CD16 and Iba-1 primary antibody were incubated respectively. The Alexa Fluor 555-conjugated Donkey-anti Rabbit, Alexa Fluor 488-conjugated Donkey-anti Rabbit, Alexa Fluor 488-conjugated Donkey-anti Goat second antibodies were used finally. The slices and cells were imaged with fluorescence microscopy (ZEISS, Jena, Germany). The whole scan were imaged with Leica image scope (Leica, Heidelberg, Germany) .
ROS Detection
Before collecting cells, DCFH-DA was diluted with serum-free DMEM at a dilution ratio of 1:1000. To detect the intracellular generation of ROS in vitro, treated cells were incubated with diluted DCFH-DA solution for 20 min in a 37℃ incubator protected from light. After washed three times with serum-free cell culture medium, cells were immediately acquired under a fluorescence microscope (ZEISS) and analyzed by using Image J software.
Mitochondrial Membrane Potential (MMP) Assay
JC-1 staining kit was used to measure the MMP in vitro according to instructions. Briefly, JC-1 buffer solution and working solution was prepared after the cells subjected to 4 h OGD and 24 h reoxygenation. The appropriate volume of JC-1 working solution was added to fully cover the cells instead of cell culture medium, and then cultured the cells in 37℃ incubator for 20 min. After washing 3 times with JC-1 buffer solution, cells were examined immediately under ZEISS fluorescence microscope.
ELISA
To detect the inflammatory factor levels, cell supernatant or brain tissues were collected and ELISA were performed according to the instructions. Briefly, 50 µL sample and 100 µL antibody being tested were added to the reaction wells respectively and then placed the wells-plate in a 37℃ incubator and incubated for 60 min in the dark. After incubation with termination solution, the OD value was measured at 450 to measure the expression levels of TNF-α, IL-1β and TGF-β.
Molecular Docking
The structures of rhTrx-1 and RIPK1 were processed and optimized using Accelrys Discovery Studio 2016 platform (San Diego, CA, USA), and protein docking was performed and calculated in ZDOCK module. Poses with the best scores were selected and then further energy optimization was performed using the RDock program. The interaction between rhTrx-1 and RIPK1 was analyzed using the Analyze Protein Interface module. Finally, Pymol (DeLano Scientific. Palo Alto, CA, USA) was used for mapping.
Statistical Analysis
GraphPad Prism (Version 6.0c) software was used for statistics. Student's T test and One-factor Analysis of Variance (One-Way ANOVA) were used to evaluate the statistical significance. The results were presented using mean standard deviation. P < 0.05 was considered statistically significant.