This study was approved by the Ethics Committee of Hunan Provincial People's Hospital (Changsha, China), and all of the experiments performed on animals were performed in compliance with the principles of experimental animal ethics. A total of 48 Sprague-Dawley male rats at the age of 8 weeks, weighing 300–350 g, were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The rats were maintained under controlled conditions (22±2°C; 55% humidity) with a 12 h light/dark cycle, and free access to food and fresh water. The permanent middle cerebral artery occlusion (MCAO) model was established in accordance with the Longa et al study (22). Briefly, the rats in the MCAO group were anesthetized via an intraperitoneal injection of 10% chloral hydrate (300 mg/kg intraperitoneal injection). Subsequently, the right common carotid artery was exposed through a 2 cm midline incision in the neck. To occlude the middle cerebral artery, a 4-0 nylon suture with a silicone tip was inserted into the internal carotid artery until mild resistance was felt. At 6, 12, 24 and 72 h time intervals following MCAO, the rats were sacrificed via an intraperitoneal injection of pentobarbital (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Rats in the sham group were anesthetized and underwent surgery without MCAO. The neurological function of the rats was then tested 2 h post-MCAO in accordance with the Longa et al study (22), and rats with scores of between one and three were held for further experiments. Human TSLP recombinant protein was sourced from Abnova (Taipei, Taiwan). The MCAO rats (n=6/group) were then given either 10 µg of recombinant TSLP or phosphate-buffered saline (PBS) intraperitoneally for 24 h. Subsequently, the rats were sacrificed and cerebral infarct areas were collected.

HUVECs (American Type Culture Collection, Manassas, VA, USA) were cultured in a RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml streptomycin/penicillin at 37°C. Cells cultured under normal conditions were maintained in a humidified atmosphere of 95% air and 5% CO₂. Cells in the OGD condition were cultured in the RPMI-1640 without glucose, and maintained in a humidified atmosphere of 94% N₂, 1% O₂ and 5% CO₂ for 2 h. Following this, the OGD-treated cells were cultured in the RPMI-1640 with 5.5 mmol/l glucose under normoxic conditions for reoxygenation for 24 h. The cells subjected to OGD were treated with TSLP at a concentration of 20 ng/ml. LY294002 (50 µM; Cell Signaling Technology, Inc., Danvers, MA, USA) was administered in order to suppress the PI3K/AKT pathway.

Tissues of the cerebral infarct area and HUVEC cell supernatants were collected, and the concentration of TSLP was then determined by ELISA assay according to the manufacturer's instructions (DTSLP0, R&D Systems, Inc., Minneapolis, MN, USA).

Total proteins were prepared from the cerebral infarct area and HUVEC cells using a Total Protein Extraction kit (Thermo Fisher Scientific, Inc.), and protein concentrations were then determined using a Bicinchoninic Acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Equal masses of protein samples (50 µg) were subsequently separated on a 10% SDS-PAGE gel and electrophoretically transferred to nitrocellulose membranes. Following blocking with Tris-buffered saline 0.1% Tween (TBST) containing 5% non-fat milk for 2 h at room temperature, the membranes were then incubated with the primary antibodies at 4°C overnight. The primary antibodies used in these analyses included anti-TSLPR (1:500; sc-517429, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), vascular endothelial growth factor A (VEGFA; 1:800; sc-507, Santa Cruz Biotechnology, Inc.), angiopoietin 2 (Ang-2; 1:400; sc-74402, Santa Cruz Biotechnology, Inc.), phosphorylated AKT (p-AKT; Ser 473 1:400; 12694, Cell Signaling Technology, Inc.), AKT (1:800; 2920, Cell Signaling Technology, Inc.) and GAPDH (1:1,000; sc-47724, Santa Cruz Biotechnology, Inc.). Following washing with TBST, the membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; sc-2005, Santa Cruz Biotechnology, Inc.) at 37°C for 2 h. The target bands were then developed using the super ECL reagent (Thermo Fisher Scientific, Inc.). The density of bands was analyzed using Image-Pro Plus, version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The MTT Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). According to the manufacturer's instructions, 2×10³ cells were plated into the 96-well plates, and treated with 20 ng/ml TSLP. The cells were allowed to grow for 12, 24, 48 and 72 h time intervals, and then 10 µl MTT solution (5 mg/ml) was added into each well. Following incubation at 37°C for 4 h, 10 µl formazan solution was added into each well and incubated at 37°C for a further 4 h in order to dissolve the formazan crystals. The absorbance at 570 nm was then determined using a microplate reader (Multiskan Spectrum; Thermo Fishers Scientific, Inc.).

A 6-well Transwell system (8 µm; Corning Incorporated, Corning, NY, USA) was used in the present study to determine cell migration. Following washing with PBS, the cells were suspended in RPMI-1640 cell medium without serum at a density of 5×10⁴ cells/ml in the upper chambers, and 2 ml of cell suspension was then added to the Transwell plates. RPMI-1640 cell medium with 10% fetal calf serum (1 ml) was then added to the lower chambers. The plates were incubated at 37°C for 24 h, and the upper chamber was then fixed in 95% ethanol at room temperature for 15 min and stained with 10% hematoxylin for 15 min at room temperature. The number of migrated cells was revealed using an Eclipse TS100 microscope (magnification ×400; Nikon Corporation, Tokyo, Japan).

The BD Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA) was thawed overnight on ice at 4°C, and was subsequently added to pre-chilled 24-well plates and incubated at 37°C for 1 h for solidification. The cells were then digested at a density of 4×10⁵ cells/ml, and 50 µl of the cell suspension was then added to each well. The plates were then incubated at 37°C, and formation of tube structure was observed 8 h later using a Leica DFC345 FX microscope. Tube lengths for each group were then measured using ImageJ software, version 1.45 (GraphPad Software, Inc., La Jolla, CA, USA), in 10 randomly selected fields.

All statistical analyses were performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA), and the data are expressed as the mean ± standard deviation. Comparisons between two groups were performed using the Student's t test, and one-way analysis of variance followed by Fisher's Least Significant Difference test was used to compare the statistically significant differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

The rat MCAO model was constructed and the expression of TSLP and TSLPR was then examined using ELISA and western blot analyses at 6, 12, 24 and 72 h time intervals following MCAO. As presented in Fig. 1, compared with the sham at 6 h, the expression levels of both TSLP and TSLPR were significantly increased at 12, 24 and 72 h time intervals following MCAO.

In order to investigate whether TSLP affects angiogenesis following MCAO, the rats in the MCAO group were injected with TSLP (10 µg), and the expression levels of VEGFA and Ang-2 in the cerebral infarct area were determined by western blot analysis. It was revealed that compared with the sham, the expression levels of both VEGFA and Ang-2 were significantly upregulated following MCAO. VEGFA and Ang-2 expression levels were further increased in the MCAO rats injected with TSLP (Fig. 2).

In order to investigate whether or not the PI3K/AKT pathway could be activated by TSLP following MCAO, p-AKT was investigated using western blot. It was subsequently revealed that the level of p-AKT was significantly increased in the MCAO group compared with the control group. Additionally, 10 µg TSLP injection caused further activation of the PI3K/AKT signaling pathway in the MCAO rats (Fig. 3).

In order to create the in vitro MCAO model, the HUVECs were exposed to OGD, and the expression levels of both TSLP and TSLPR were subsequently examined using ELISA and western blot analyses, respectively. As demonstrated in Fig. 4, the expression levels of TSLP and TSLPR in OGD-treated HUVECs, in comparison with the cells cultured under normal conditions, were significantly increased.

The HUVECs subjected to OGD were treated with TSLP, and then cell viability, cell migration and tube formation were examined in order to determine the effect of TSLP on the angiogenic capacity of OGD-treated HUVECs. It was observed that cell viability was significantly increased following TSLP treatment (Fig. 5A). Furthermore, in the TSLP group, the number of migrated cells was significantly increased in comparison with the control (Fig. 5B). Additionally, TSLP treatment led to an increase in the tube lengths of OGD-treated HUVECs compared with the control treatment (Fig. 5C).

The level of p-AKT was examined in OGD-treated HUVECs in order to determine whether or not the PI3K/AKT pathway is involved in mediating the effect of TSLP on angiogenesis in vitro. As revealed in Fig. 6, the level of p-AKT was significantly increased in OGD-treated HUVECs following TSLP treatment compared with the control group.

LY294002, a PI3K inhibitor, was used to determine whether the PI3K/AKT signaling pathway mediates the effect of TSLP on in vitro angiogenesis of OGD-treated HUVECs. As demonstrated by western blot analysis, LY294002 inhibited the expression of p-AKT induced by TSLP in OGD-treated HUVECs (Fig. 7A).

Additionally, cell viability was investigated using MTT. It was subsequently revealed that the effect of TSLP on cell proliferation was reversed by the addition of LY294002 (Fig. 7B). In addition, TSLP-induced cell migration was suppressed by LY294002 (Fig. 7C). Tube lengths were also assessed using the tube formation assay. As demonstrated by Fig. 7D, the effect of TSLP on tube formation of OGD-treated HUVECs was attenuated by the addition of LY294002.