Male Sprague-Dawley (SD) rats (250~300g) were purchased from the Laboratory Animal Center, Xiangya School of Medicine, Central South University, China. Food was withheld from the animals for 24h before the experiments, but they are free access to tap water. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH Publication, 8th edition, 2011) and the ARRIVE guidelines (Animal Research: Reporting In-Vivo Experiments). The animal protocols were approved by the Institutional Animal Care and Use Committee of Central South University.
2.2 Ischemic stroke rat model
A rat model of ischemic stroke was established by middle cerebral artery occlusion (MACO) as we described previously . Briefly, the rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and then the left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were surgically isolated. A nylon suture with a rounded tip (0.40 mm diameter) was inserted into internal carotid artery (ICA) through a tiny incision in ECA and gently advanced 18~20 mm past the carotid bifurcation to occlude the origin of middle cerebral artery (MCA). After 2h of ischemia, the nylon suture was withdrawn to allow reperfusion. The sham-operated rats were subjected to the same procedure except no nylon suture was inserted.
2.3 Protocols for the experiments in animals
The first set of animal experiments was designed to examine the expression of Pellino3 after stroke. The rats were randomly divided into 3 groups (n = 12 per group): (1) the control group, no treatment for rats; (2) the sham group, rats were subjected to surgical procedures but with no ischemic insult; and (3) the stroke group, the rats were subjected to 2 h-ischemia plus 24 h-reperfusion.
In the second set of experiments, the effects of caspofungin on Pellino3 expression as well as necroptosis were explored. The animals were divided into 5 groups (n=12 per group): (1) the control group; (2) the sham group; (3) the stroke group; (4) and (5) the caspofungin (low dose, L) group and the caspofungin (high dose, H) group: the rat received caspofungin (Selleckchem, USA) at 2 mg/kg and 6 mg/kg (dissolved in saline, i.m.) at 1h after the ischemia, respectively.
After 24h-reperfusion, neurological deficit score was assessed first, and then the brain tissues of 6 rats from each group were saved for infarct volume measurement, while the brain tissues of the remaining 6 rats from each group were saved for morphological, biochemical and molecular analysis.
2.4 Protocols for the experiments in cells
The well-differentiated PC12 cells (Chinese Academy of Sciences, Shanghai, China) were seeded at a density of 1 × 104 cells/cm2 and cultured in DMEM with 10% FBS. According to the instruction provided by the cell supplier, the differentiation of PC12 cells was induced by nerve growth factor. The cells were maintained at 37°C in 95% air/5% CO2 in a humidified incubator. Under such conditions, cells grew well and exhibited spindle-shaped cell morphology similar to neuronal cells, which was consistent with morphological characteristics of well-differentiated PC12 cells. The cells were kept in the serum-free DMEM for 16h before the experiments.
To verify the anti-necroptosis effect of caspofungin via upregulation of pellino3 in the stroke rat brain, the PC12 cells were cultured in a medium without glucose under hypoxic condition (N2/CO2, 95:5), which is widely used to mimic the ischemic stroke in vitro . The cells were divided into 4 groups (6 individual experiments per group): (1) The control group, cells were cultured under normoxic condition; (2) The hypoxia group, cells were cultured for 8h-hypoxia plus 24h-reoxygenation; (3) The hypoxia plus pellino3 overexpression group, cells transfected with Adenovirus-mediated pellino3 gene and subjected to hypoxia; and (4) The hypoxia plus negative control group, cells transfected with Adenovirus-mediated GFP and subjected to hypoxia.
At the end of the experiments, cells and culture mediums were collected for flow cytometry, LDH release and molecular analysis.
2.5 Assessment of neurological function and infarct volume
The neurological behavioral tests were carried out after 24h reperfusion by an investigator blinded to the experimental groups using five-point neurological deficit score (0 = no deficit, 1 = failure to extend the left forepaw, 2 = decreased grip strength of left forepaw, 3 = circling to the left by pulling the tail, 4 = spontaneous circling).
After neurological behavioral tests, the rats were killed under anesthesia, and the brains were rapidly removed and sliced into 2-mm thick coronal sections with the aid of a brain matrix. The sections were stained with 1% triphenyltetrazolium chloride (TTC) for 15 min at 37 °C and scanned into a computer. The images of brain sections with TTC-staining were analyzed with the imaging software (Image J, NIH, USA). The absence or presence of infarction was determined by TTC staining.
The infarct volume (in mm3) of each section was calculated as infarct area (in mm2) multiplied by the section thickness (2 mm). The total infarct volume of each brain was equal to the summation of the infarct volume of all sections. To eliminate the effect of edema on the accuracy of infarct volume assay, the final infarct volume was corrected by following equation: corrected infarct volume=total infarct volume× (left hemisphere volume/right hemisphere volume). Here, left hemisphere refers to no-ischemic hemisphere of brain while right hemisphere refers to ischemic contralateral side.
2.6 Hematoxylin-eosin (HE) staining
HE staining was performed to evaluate the morphological changes in brain. Briefly, the brain tissues were fixed in 4 % paraformaldehyde and embedded in paraffin, and then they were cut into 5-μm sections. The slices underwent hematoxylin and eosin staining for 20 and 2 min, respectively. After HE staining, the results were imaged by microscope (Nikon Eclipse 80i, Japan) to evaluate the morphological changes among the experimental groups.
2.7 Overexpression of Pellino 3
The Adenoviral constructs carrying rat pellino 3 gene (ADV1 ×Flag-Pellino3) (1×1010 pfu/mL) was purchased from GenePharma (Shanghai, China). PC12 cells were seeded in 6-well plates at 1 ×106 per well and transduced at approximately 70~80% confluence with the adenoviral vectors at MOI 8 according to a standard protocol for 24h. Afterward, the media were exchanged for fresh DMEM media to proceed the following experiments. The specificity or efficiency of Pellino 3 overexpression was evaluated by Western blot. The negative control cells were transfected with adenoviral constructs carrying GFP (Ad-GFP) (1×1010pfu/mL).
2.8 Lactate dehydrogenase release and flow cytometry analysis
Culture medium was collected for analysis of lactate dehydrogenase (LDH) release (an indicator of cellular necrosis) by using a colorimetric assay kit (Beyotime, Jiangsu, China) following the protocol provided by the manufacturer. Released LDH was determined by a coupled enzymatic reaction that resulted in the conversion of a tetrazolium salt into a red color formazan by diaphorase. Briefly, 120 µL of culture medium was mixed with 60 μL of LDH work solution, then incubated at 25~30°C for 30 min. The absorbance was measured at 490 nm. The percentage of LDH release was calculated by a formula provided by the protocol.
Flow cytometry was performed to further evaluate the type of cell death. In brief, the PC12 cells were collected and centrifuged at 1000 rpm for 5 min. After washing 2 times with PBS, 200 μL of FITC-conjugated Annexin V (195 μL AnnexinV-FITC Binding Solution + 5 μL AnnexinV-FITC) were added to the cells and incubated at room temperature for 15 min in the dark. Ten μL of propidium iodide (PI) were added before flow cytometry analysis. PI+ /Annexin V+ cells were considered as necroptotic cells.
2.9 Western blot analysis
Brain tissues or PC12 cells were lysed in ice-cold lysis buffer containing proteinase inhibitor to obtain the proteins following a regular method. The protein concentration in homogenate was measured by BCA Protein Assay kit (Beyotime, Jiangsu, China). Protein samples (20~40μg) were separated on 8~10 % SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, USA). The membranes were then blocked in Tris-buffered saline containing Tween-20 (TBST) with 5% skim milk powder for 1 h at room temperature and incubated with primary antibodies against Pellino 3 (Santa Cruz, California, USA), RIPK1 (Boster, Wuhan, China), RIPK3 (Biovision, CA, USA), MLKL (Abcam, Cambridge, UK), p-RIPK1 (CST, Boston, USA), p-RIPK3 (Abcam, Cambridge, UK), p-MLKL (Abcam, Cambridge, UK) or β-actin (Beyotime, Jiangsu, China) overnight at 4 °C. The PVDF membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime, Jiangsu, China) for 1 h at room temperature. Protein signals were detected by Luminata Creseendo Western HRP substrate through Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, PA). Quantitative analysis of protein bands was conducted using Image J software. β-actin served as the internal control. Arbitrary optical density units of the targeting protein were normalized against control and expressed as fold change.
2.10 Co-immunoprecipitation (Co-IP) assay
Co-IP was performed to evaluate the RIPK1 ubiquitination and the interaction between RIPK1 and RIPK3 following a protocol provided by the kit supplier (Absin, Shanghai, China). Briefly, the brain tissues or PC12 cells were lysed in lysed in IP buffer supplemented with protease inhibitors. The lysates were immunoprecipitated with the anti-RIPK1 antibody (Proteintech, Wuhan, China) or rabbit IgG (Beyotime, Jiangsu, China) overnight at 4°C, and then incubated with Protein A/G Agarose beads for another 4h at 4°C. Agarose beads were washed with ice-cold washing buffer (supplied by the kit) followed by elution of bound proteins. Precipitated proteins were determined by Western blot with anti-Ub (CST, Boston, USA), anti-K48 linked UB (CST, Boston, USA), anti-K63 linked UB (CST, Boston, USA), or anti-RIPK3 (Biovision, CA, USA) antibody. Rat IgG was used as negative control. The samples in the input group only proceeded Western blot and served as positive controls.
2.11 Statistical analysis
Prism GraphPad 6.0 was used for statistical analysis. The data were presented as mean ± SEM. Differences in measured values among multiple groups were analyzed by one-way ANOVA followed by Tukey test for comparing multiple groups. The neurological deficit scores were analyzed by Kruscal-Wallis H and Wilcoxon tests. Differences were considered significant when P < 0.05.