Microglia were isolated from primary mixed glial cell cultures prepared from newborn C57BL/6J mice (Guangdong Medical Lab Animal Center, China) on day 10 by shaking the flasks overnight at 300 rpm on a rotary shaker at 37°C. Purified microglia were resuspended and cultured in complete medium containing 1% microglia growth supplement (ScienCell, USA) for 2–3 days. The purity of the cultures was almost 100%, as determined by immunostaining with an anti-Iba1 antibody (Abcam, USA). The protocols for the animal experiments were approved by the Animal Experiment Committee of Guangzhou Medical University. Mouse EOC 2 microglial cells were obtained from the American Type Culture Collection (http://www.ATCC.org) and were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco-BRL) containing 10% fetal bovine serum (FBS; Gibco-BRL).

The cells were seeded in 6-well plates at 1 × 10⁵ cells/ml/well 1 day before the transfection. The following day the transfection was performed when the cells reached ~70% confluence. The final concentration of IL-17A or HMGB1 siRNA (Santa Cruz) was 100 nM. The transfection was conducted with the X-tremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's instructions. The transfection medium was replaced 4–6 h after the transfection. After siRNA transfection, Tenovin-6 (10 μM; SantaCruz, USA) and MK-2206 (5 μM; Selleck, China) were added or not.

After transfection for 24 h, the cells were replaced medium with glucose-free Earle's balanced salt solution, and then placed in an oxygen deprived incubator (95% N₂/3% CO₂/2% O₂) at 37°C for 2 h. The control cells were incubated in Earle's balanced salt solution with 10 mM glucose under normal conditions (95% air/5% CO₂) for the same period of time. The cells were moved to normal conditions to terminate the OGD and start reperfusion. The protein concentrations of IL-17A (eBioscience, USA) and HMGB1 (SHINO-TEST, Japan) in the culture supernatants were determined by an ELISA kit following the manufacturer's protocol.

The growth-inhibitory effect was measured using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. Microglial cells (6 × 10⁴ cells/well) were seeded in a 96-well tissue culture microplate for 24 h, and then, the cells were exposed to the different treatments. After that, 25 μl of the MTT solution (5 mg/ml in phosphate buffered saline) was added into each well and was incubated for 4 h at 37°C. The metabolized MTT product was dissolved in DMSO and was quantified by measuring the optical density at 570 nm on a microplate reader (Dynex Technologies, USA).

Cell proliferation was estimated in 96-well plates using a colorimetric immunoassay, based on the measurement of BrdU incorporation during DNA synthesis (BrdU ELISA kit, Roche Diagnostics, Germany). The cells were labeled with BrdU (10 mM 5-bromo-2′-deoxyuridine) for 3 h at 37°C. Cells were fixed, and incubated with peroxidase-conjugated anti-BrdU antibody for 90 min at room temperature. Then the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine was added, and BrdU incorporation was quantitated by differences in absorbance at wavelength 370 minus 492 nm. Cell proliferation was expressed as the mean percentage of the control values (set at 100%).

There are certain characteristics of apoptotic cells that can be identified and used to detect apoptotic cells in an otherwise healthy population of cells. Technically the easiest characteristic to detect is loss of DNA from permeabilized cells due to DNA fragmentation. When cells are permeabilized, for example by 70% ethanol, the fragmented 182 bp DNA multimers leak out of the cell. The result is a population of cells with a reduced DNA content. If the cells are then stained with a DNA intercalating dye like propidium iodide, then a DNA profile representing cells in G1, S-phase, and G2M will be observed with apoptotic cells being represented by a sub G0/G1 population seen to the left of the G0/G1 peak. Microglial cells were washed with PBS and fixed in 75% ethanol overnight at –20°C. The fixed cells were stained with PI (1.21 mg/ml Tris, 700 U/ml RNase, 50.1 μg/ml PI, pH 8.0) for 4 h in the dark. The red PI fluorescence was measured with a Coulter Epics XL flow cytometer (Coulter, Germany) using CXP software. In each sample, 10,000 events were measure for the apoptosis detection and cell cycle analysis. The data were analyzed with the Expo32 analysis tool. A DNA histogram represented the proportion of cells in the G0/G1, S, and G2/M phases. The apoptotic cells with a hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern.

The activities of caspase-3, -8, -9, and PARP in the lysates of the microglial cells were monitored by a fluorometric method. The cell lysates and specific substrates were placed in 96-well plates and were then incubated at 37°C for 2 h. The enzymatic activities were determined by measuring the fluorescence intensity in a fluorescence microplate reader (MD) with the excitation and emission wavelengths at 380 and 440 nm, respectively.

For the protein extraction, 2D-gel electrophoresis, image analysis, protein identification, tryptic digestion, and MALDI-TOF mass spectrometry, please see the Supplementary Methods (Data Sheet 1). The differentially expressed proteins identified by 2D-gel electrophoresis and MALDI-TOF/TOF-MS were aggregated and analyzed using the UNIPROT database and the DAVID data analysis software and were classified into GO (gene ontology) categories (http://www.geneontology.org/) according to their biological functions and the physiological processes in the body.

The cells were collected, washed twice with PBS, and lysed in RIPA buffer containing protease inhibitors. After determining the protein concentration with the BCA Protein Assay Reagent Kit (Pierce), equal amounts of protein were separated on 8% SDS-PAGE, electrically transferred to a PVDF membrane and were blocked with 5% skim milk. The membranes were incubated with IL-17A (1:800; CST, USA), HMGB1 (1:800; CST, USA), Cleaved Caspase-3 (1:800; CST, USA), Osteopontin (1:600; Abcam, USA), p53/p-p53 (1:1,000; CST, USA), Akt/p-Akt (1:1,000; CST, USA), and β-actin (1:2,000; Sigma, USA) antibodies respectively, overnight at 4°C. After washing, the membranes were then incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Southern Biotech) at room temperature for 1 h. The membranes were finally incubated with a West Femto chemiluminescence substrate (Pierce), and the images were visualized and recorded.

The intracellular reactive oxygen species (ROS) levels were determined by a fluorometric assay (DCF-DA assay). Briefly, the microglial cells were seeded in 96-well microplates at 6 × 10⁴ cells/well for 24 h, and then, the cells were incubated with the different treatments for different periods of time. After incubation, the treated cells were incubated with 10 μM DCFH-DA at 37°C for 15 min. The medium was aspirated, and the cells were washed with PBS twice. Before the measurement, 100 μl of PBS was added into each well and the ROS was immediately measured. The generation of ROS was determined by the fluorescence intensity with the excitation and emission wavelengths set at 485 and 525 nm, respectively. The change in the intracellular ROS levels of each group was determined by calculating the ΔF = (F − F0)/F0, where F represents the fluorescence read at each time point, and F0 is the control fluorescence.

TUNEL-DAPI co-staining assay was used to analyze the apoptotic cells based on the In situ cell Death Detection kit (Roche). Briefly, 4% formaldehyde was used for fixation at 4°C for 25 min, and then, the cells was washed by PBS. Next, 0.2% Triton X-100 was then added into cells for a 5 min incubation. Then, the cells were mixed with 100 μl of Equilibration Buffer at room temperature for 10 min. The cells were washed with Saline Sodium Citrate (SSC) for 15 min after an incubation with 50 μl of the TUNEL reaction mixture, containing the nucleotide mixture and terminal deoxynucleotidyl transferase (TdT), for 60 min at 37°C. Moreover, the cells were incubated with 0.3% H₂O₂ for 10 min and a Streptavidin working solution for 30 min at room temperature. The cells were incubated with 0.5 μg/ml of DAPI in a humidified chamber in the dark for 5 min at room temperature. Eventually, a fluorescence microscope (Nikon Eclipse 80i) was used to examine and take photos of the stained cells.

The experiments were carried out at least in triplicate, and the results were expressed as the mean ± SD. All variances are homogeneous and samples follow a normal distribution, so the differences between the two groups were analyzed by a two-tailed Student's t-test, while those between three or more groups were analyzed by a one-way analysis of variance (ANOVA). Differences with P < 0.05 (*) were considered statistically significant.

The IL-17 family includes six homologous members, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F (Wang et al., 2009; Zhu and Qian, 2012), in which IL-17A and IL-17F receive more attention. IL-17A plays a more important role in autoimmune diseases than IL-17F (Ishigame et al., 2009), and is a cytokine with a strong pro-inflammatory action. In addition, IL-17A is a major factor that causes ischemia-reperfusion tissue injury (Li et al., 2005; Xue et al., 2011), and it causes inflammatory injury to local tissues and apoptosis through multiple pathways. However, the underlying mechanisms remain unclear. Therefore, the current study mainly focused on IL-17A. A model of OGD was established to simulate cerebral ischemia-reperfusion in vitro, and a significant up-regulation of IL-17A expression was found during OGD/reperfusion, while the addition of siRNA caused a reduction in IL-17A expression by using the expression of beta-actin in cells as internal reference (Figure 1). Using ELISA to detect IL-17A expression in the culture supernatants of EOC 2 and mouse primary microglia, the results were similar to those of the western blot (Figures 1D,E). Therefore, we concluded that the up-expression of IL-17A might play a critical role in microglial cells.

In the model of OGD, a decline in the proliferation and a significant elevation in apoptosis was observed in the microglial cells. When IL-17A expression was down-regulated, the number of proliferative microglial cells significantly increased, and the number of apoptotic cells reduced remarkably (Figure 2). The detection of apoptosis-related parameters showed that the activity of Caspase-3, Caspase-8, Caspase-9, and poly ADP ribose polymerase (PARP) was remarkably elevated in the microglial cells in the OGD model, while the down-regulation of IL-17A expression inhibited the activity of Caspase-3, Caspase-8, Caspase-9, and PARP, and the alteration of Caspase-3, Caspase-9, and PARP was most notable (Figure 3). Caspase-3, Caspase-9, and PARP are important factors associated with apoptosis, and IL-17A may exhibit a pro-apoptotic action via Caspase-3, Caspase-9, and PARP. Therefore, IL-17A is considered to play a critical role in microglial cells.

To investigate the mechanism of IL-17A in microglial cells, we used mass spectrometry to identify the protein expression profile regulated by IL-17A. Microglial cells were assigned into three groups, including the control group, the OGD group and the OGD + IL-17A siRNA group. Total protein was isolated from the microglial cells in the different groups, and the protein was separated using 2D gel electrophoresis. Then, each SDS-PAGE gel was scanned using a UMAX image scanner. As shown in Figure 4A, the altered expression of some proteins was seen in the OGD and OGD + IL-17A siRNA group. In addition, the proteomic profiles captured from 9 gels were analyzed using imaging analysis software. In the results of Mass spectrometric analysis, the gel points altered in OGD group and recovered in OGD + IL-17A siRNA group were selected for the subsequent identification. The proteins extracted from the spots with an average two-fold or greater changes (P < 0.05) were identified with MALDI-TOF/TOF-MS. After an in-gel tryptic digestion, MS analysis, an online identification of the peptide MS database using Mascot, and blasting the NCBI protein sequence database, the differentially expressed proteins were finally identified according to their Mascot scores, the molecular weight shown on the gel, and the isoelectric point information. As shown in Table 1 and Table S1, according to their function, these differentially expressed proteins could be classified into categories, such as signal transduction, cell proliferation, apoptosis, and oxidative stress-related proteins, as well as proteins with unknown functions.

Figure 4B, Tables S2, S3 show the GO classification (including CC, MF, BP) of the MS-identified proteins. These differential proteins are involved in processes, such as oxidative stress, cell metabolism, biological regulation, etc. For signal transduction pathways, part of the proteins are mainly involve in the apoptosis, p53 signaling pathway, the PI3K/Akt signaling pathway, the Toll-like receptor signaling pathway, etc. (Tables S4, S5). Caspase-3 is a key protease of apoptosis. HMGB1 plays a significant role in cerebral IRI. Osteopontin (OPN) is a key factor in the central nervous system repair and extracellular matrix remodeling after injury. Thus, the proteins Caspase-3, HMGB1, and Osteopontin were selected for validation. The results of the Western blot analysis showed that the protein expression of Caspase-3, HMGB1, and Osteopontin in the EOC 2 cells were consistent with the results of the MS (Figure 5). Therefore, in the microglial cells, IL-17A alters the expression of proteins related to signaling transduction, cell cycle, apoptosis, inflammation, and thereby regulates the activities of many signaling pathways to exert its anti-proliferation and pro-apoptotic effects.

Some important downstream pathways were selected for validation, including the p53 and PI3K/Akt signaling pathways. An increase was observed in the phosphorylation level of p53 in the OGD model, while the down-regulation of IL-17A expression caused a significant reduction in the phosphorylation level of p53 (Figures 6A,C). In addition, the phosphorylation level of Akt significantly decreased in the OGD model, while the down-regulation of IL-17A expression resulted in a restoration of the phosphorylation level of Akt (Figures 6B,C). In the OGD model, when an IL-17A siRNA transfection, Tenovin-6 (p53 activator), or MK-2206 (Akt inhibitor) was added, and then, the caspase-3 activity and apoptosis were detected. The results showed that Tenovin-6 or MK-2206 reversed the inhibitory effect of IL-17A siRNA on the caspase-3 activity (Figure 6D) and increased the apoptosis of EOC 2 cells and mouse primary microglia (Figure 7). This indicates that IL-17A regulated the occurrence of injury in the OGD model through the p53 and PI3K/Akt pathways. These findings demonstrate the role of IL-17A in cerebral IRI by affecting a series of important signaling transduction pathways and the expression of some important factors.

Of the factors that IL-17A regulates, HMGB1 is reported to promote the development of cerebral IRI (Kim et al., 2008; Yang et al., 2010; Zhang et al., 2014). In addition, HMGB1 promotes IL-17A expression in T cells (He et al., 2012; Shi et al., 2012; Zhu et al., 2013). It is therefore hypothesized that HMGB1 and IL-17A may be regulated mutually during cerebral IRI, thereby aggravating the injury. Our findings showed an elevated expression of both HMGB1 and IL-17A in the model of OGD (Figure 8), and the down-regulation of HMGB1 expression caused a reduced IL-17A expression (Figure 8C), while the down-regulation of IL-17A expression resulted in a reduction of HMGB1 expression (Figures 8A,B,D,E). The results demonstrate a synergistic effect between HMGB1 and IL-17A through mutual regulation. We further assessed the effect of HMGB1 on Caspase-3 activity, and our findings showed that down-regulation of HMGB1 expression reduced Caspase-3 activity in the OGD model, indicating that HMGB1 has a pro-apoptotic action (Figure 8F).

A large amount of ROS is produced during cerebral ischemia and reperfusion (Kahles and Brandes, 2012), and mass spectrometry demonstrates that IL-17A correlates with oxidative stress. It is therefore speculated that there may be a relationship between ROS and IL-17A. In the model of OGD, the amount of ROS was found to almost double in the microglial cells, and the production of ROS almost recovered to the level in the control group following the addition of the ROS inhibitor N-acetyl-L-cysteine (NAC) (Figure 9A). In addition, an obvious down-regulation was seen in the expression of IL-17A and HMGB1, and notably, IL-17A and the up-regulation of IL-17A expression induced by OGD/reperfusion were completely blocked (Figures 9B,C). The results demonstrated that the production of a large amount of ROS may promote IL-17A and HMGB1 expression, and the down-regulation of IL-17A or HMGB1 expression caused a reduction in the production of ROS (Figure 9D), which, however, still appeared as an increasing trend relative to the control group. It is therefore considered that ROS may be located upstream of HMGB1 and IL-17A. Furthermore, IL-17A siRNA, NAC, and HMGB1 siRNA increased the microglial cell proliferation in the in vitro model of OGD, but p53 activator Tenovin-6 or Akt inhibitor MK-2206 blocked the above effects (Figure 9E). So, ROS, IL-17A, and HMGB1 all can regulate the microglial cell proliferation through p53 and PI3K/Akt pathways.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.