Sal (batch no. 17111321) was obtained from Shanghai Green Valley Pharmaceutical Co., Ltd. DMEM was purchased from Gibco (Thermo Fisher Scientific, Inc.). FBS was purchased from Serana Europe GmbH. VEGF protein (cat. no. 100-20) was purchased from PeproTech, Inc. The rabbit monoclonal antibody against phosphorylated (p)-VEGFR2 (cat. no. 3770), rabbit monoclonal antibody against VEGFR2 (cat. no. 9698), mouse monoclonal antibody against β-actin (cat. no. 3700), rabbit monoclonal antibody against p-AKT (cat. no. 4060), rabbit monoclonal antibody against AKT (cat. no. 4696), rabbit anti-p38 MAPK monoclonal antibody (cat. no. 8690) and rabbit anti-p-p38 monoclonal antibody (cat. no. 4511) were purchased from Cell Signaling Technology, Inc. The mouse anti-F4/80 (C-7) monoclonal antibody (sc-377009) and rabbit anti-VEGF polyclonal antibody (sc-7269) were purchased from Santa Cruz Biotechnology, Inc. The rabbit anti-CD31 polyclonal antibody (GB12063) was purchased from Wuhan Servicebio Technology Co., Ltd. Horseradish peroxidase (HRP)-conjugated goat anti-mouse (A0216) and goat anti-rabbit (A0208) secondary antibodies were purchased from Beyotime Institute of Biotechnology.

All animal protocols and procedures were approved by the Shanghai University of Traditional Chinese Medicine Institutional Animal Care and Use Committee (grant no. PZSHUTCM200320004). All experiments were performed in accordance with the guidelines described in the Regulations for the Administration of Affairs Concerning Experimental Animals of China enacted in 1988. A total of 97 healthy male Sprague-Dawley rats (8 weeks old; weight, 300±20 g) were purchased from Shanghai Sino-British SIPPR/BK Lab Animal Co., Ltd. The rats were individually caged in a climate-controlled room (20-26˚C, relative humidity of 40-70%) housed under a 12-h dark/light cycle, and allowed free access to water and food. The animals were fasted without water deprivation for 12 h before the tMCAO procedure was performed.

Human umbilical vein endothelial cells (HUVECs; American Type Culture Collection HUV-EC-C cell line, cat. no. CRL-1730) were obtained from the Cancer Research Institute of Central South University. The murine RAW264.7 cell line was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Both types of cells were cultured in DMEM containing 10% FBS at 37˚C in a humidified atmosphere comprising 5% CO₂ in an incubator.

RAW264.7 cells (~3x10⁶ cells/ml) were seeded into cell culture dishes (60x15 mm) and incubated for 24 h. The cells were then incubated in medium without or with Sal (10 µM) at 37˚C. After culturing for 24 h, the medium was collected and centrifuged (3,500 x g) to remove cell debris. The following solutions were then collected: i) s-control, comprising the supernatant of RAW264.7 cells; and ii) s-Sal, comprising the supernatant of Sal (10 µM)-treated RAW264.7 cells.

In total, the following five groups were designated for the treatment of HUVECs: i) Control group, untreated HUVECs; ii) s-control group, where the HUVECs were treated with supernatant of RAW264.7 cells; iii) Sal group, where the HUVECs were treated with 10 µM Sal; iv) s-Sal group, where the HUVECs were treated with supernatant of Sal (10 µM)-treated RAW264.7 cells; and v) VEGF group, where the HUVECs were treated with 10 ng/ml VEGF as a positive control.

Cell viability was evaluated using the MTT assay (Sigma-Aldrich; Merck KGaA). HUVECs (2.5x10³ cells/well) were seeded into 96-well culture plates and incubated for 24 h. Subsequently, 100 µl complete medium containing Sal (10 µM), s-control or s-Sal was added. VEGF (10 ng/ml) was added as the positive control. After culturing for 24 h, 20 µl MTT (5 mg/ml) was then added to each well for an additional 4 h, prior to the addition of 150 µl DMSO to dissolve the formazan. The absorbance (optical density value) at 490 nm was detected.

HUVECs (5x10⁵ cells/well) were seeded into six-well culture plates to form a 90% confluent cell monolayer. The monolayer was scratched vertically along the center of each well with a 200-µl pipette tip and rinsed carefully with PBS three times to remove the cell debris. The HUVECs were incubated for 24 h with Sal (10 µM), s-control, s-Sal or VEGF (10 ng/ml; positive control) in the absence of FBS. In total, three randomly selected views along the wound line in each well were photographed using an inverted light microscope after incubation for 0 and 24 h. ImageJ software 1.50i (National Institutes of Health) was used to analyze the image migration distance and calculate the migration rate (MR). The MR was calculated according to the formula below: MR (%)=(1-24 h scratch distance/0 h scratch distance) x100.

A 50-µl volume of Matrigel (BD Biosciences) was pipetted into each well of a pre-chilled 96-well plate and allowed to solidify at 37˚C for 30 min. Subsequently, HUVECs (5x10⁴ cells/well) and 100 µl culture medium without or with Sal (10 µM), s-control, s-Sal or VEGF (10 ng/ml) were placed into each well and incubated for another 3 h. Tube formation was then observed and photographic images were captured using a Nikon light microscope (Nikon Corporation) and tube formation ability was measured by determining the number of master junctions with ImageJ software 1.50i (National Institutes of Health).

Cell cycle was detected using flow cytometry. HUVECs (3x10⁵ per well) were cultured in 60-mm culture plates for 24 h and then incubated with either medium alone or medium containing Sal (10 µM), s-control, s-Sal or VEGF (10 ng/ml) at 37˚C for 24 h. The cell cycle analysis was performed using a Cell Cycle Assay Kit-PI/RNase Staining (cat. no. C543) according to the manufacturer's protocol (Dojindo Molecular Technologies, Inc.). The cell cycle was detected via flow cytometry (CytoFLEX; Beckman coulter, Inc.) and analysis was used ModFit software LT5 (Verity Software House).

RAW264.7 cells (1x10⁶ per well) were cultured in 60-mm culture plates for 24 h and then incubated without or with varying concentrations of Sal (5, 10, 50, 100 µM) at 37˚C for 3 h. The culture medium was then removed, and total mRNA was extracted from the cells using RNAiso Plus (cat. no. 9108; Takara Bio, Inc.) according to the manufacturer's protocol. Reverse transcription was performed with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (cat. no. RR047A; Takara Bio, Inc.), and qPCR was performed using TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (cat. no. RR420A; Takara Bio, Inc.) both according to the manufacturer's instructions. The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 30 sec, denaturation (40 cycles) at 95˚C for 5 sec, and annealing/extending (40 cycles) at 60˚C for 30 sec; melt curve at 95˚C for 15 sec, at 60˚C for 1 min and at 95˚C for 15 sec. β-actin was used as a reference in the experiment. Relative mRNA expression was normalized to β-actin levels and analyzed with the 2^-∆∆Cq method (28). The mouse source primers were as follows: β-actin, forward: 5'-GTCCCTCACCCTCCCAAAAG-3' and reverse: 5'-GCTGCCTCAACACCTCAACCC-3'. Vegf, forward: 5'-TAGAGTACATCTTCAAGCCGTC-3', reverse: 5'-CTTTCTTTGGTCTGCATTCACA-3'. β-actin was used as the reference gene.

RAW264.7 cells (2x10⁶ per well) were cultured in 60-mm culture plates for 24 h and then incubated without or with 10 µM Sal at 37˚C for 24 h. The medium was collected and centrifuged at 3,500 x g at room temperature for 3 min to remove cell debris. The content of VEGF in the medium was determined using a Mouse VEGF ELISA Kit (cat. no. EMC103.96; Neobioscience Technology Co., Ltd.) according to the manufacturer's protocol.

Brain tissue and cell lysates were prepared using NP40 lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using the Enhanced BCA Protein Kit (Beyotime Institute of Biotechnology). An equal amount of each sample (30 µg) was separated by 10% SDS-PAGE and then transferred onto PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were washed with TBS-10% Tween 20 three times for 10 min each and then incubated with VEGF (1:1,000 dilution), VEGFR-2 (1:1,000 dilution), p-VEGFR-2 (1:1,000 dilution), AKT (1:1,000 dilution), p-AKT (1:1,000 dilution), p38 (1:1,000 dilution) or p-p38 (1:1,000 dilution) antibodies at 4˚C overnight. The membranes were then washed and incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The proteins were subsequently detected using a Clarity™ Western ECL Substrate kit (Bio-Rad Laboratories, Inc.). The densitometry analysis used AzureSpot software 2.0.062 (Azure Biosystems, Inc.).

The tMCAO rat model was established using a modified suture occlusion method. Rats were anesthetized with isoflurane (3% for induction and 2.5% for maintenance) before an incision was made in the neck of the rats precisely at the median position. The left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were carefully isolated. The CCA and ECA were then ligated, before a microaneurysm clip was placed around the ICA. A hole was cut in the CCA and the blunted tip of a nylon suture was inserted through the hole into the ICA until mild resistance was felt. The suture was left in place for 2 h and then withdrawn to allow reperfusion. In the sham-operated group, the rats were anesthetized, but only CCA and ECA ligation was performed.

Neurological functions were evaluated with the Longa scale (29) on the third day after MCAO/reperfusion. The neurological deficits were assessed using the following five-point scale: i) 0 points, no deficit; ii) 1 point, forelimb weakness, flexion of and inability to straighten the contralateral forelimb; iii) 2 points, circling to the affected side; iv) 3 points, inability to bear weight on the affected side and tilting to the contralateral side while walking; and v) 4 points, no spontaneous locomotor activity or loss of consciousness. Animals with scores of 0 or 4 points were excluded from the study.

Rats were randomly assigned into the following groups: i) Sham group (n=13), in which sham-operated rats were injected with normal saline by intraperitoneal injection; ii) infarct group (n=28), in which tMCAO model rats were injected with normal saline 2 h after reperfusion; iii) Sal groups, in which tMCAO rats were intraperitoneally injected with 5 (n=5), 10 (n=5), 20 (n=28) or 40 mg/kg (n=5) Sal 2 h after reperfusion; and iv) ED group (n=13), in which tMCAO model rats were treated with 30 mg/kg edaravone (Zhejiang Shengtong Biotechnology Co., Ltd.) 2 h after reperfusion as a positive control. The treatment was administered for 3 days with a frequency of once a day. To observe the effects of Sal (20 mg/kg) at different time periods, brain tissues were collected after 0, 1 and 3 days from mice in the infarct and Sal groups.

Rats from the different groups were anesthetized with 2% sodium pentobarbital (30 mg/kg) and then the brain tissues were rapidly collected and cut into 2-mm slices. The brain slices were immersed in 2% TTC (Sigma-Aldrich; Merck KGaA) solution at 37˚C for 15 min, after which they were soaked and fixed in 4% paraformaldehyde solution at room temperature for 15 min. The ischemic area of each brain slice was photographed and analyzed using ImageJ software 1.50i. The infarct area is the area of viable brain tissue in the right hemisphere minus the area of viable brain tissue in the left hemisphere. The infarct volume was calculated according to the following formula:

where h=2 mm, which represents the distance between each section and S represents the infarct area (mm²) in each brain section.

At the end of the experiment, the rats were anesthetized with 2% sodium pentobarbital (30 mg/kg) and transcardially perfused with 150 ml PBS. The brain tissues were isolated and fixed in 4% paraformaldehyde solution at 4˚C overnight. The cerebral hemispheres were then cut in the coronal plane, stained with H&E at room temperature for 12 min and examined by light microscopy.

Immunohistochemical analysis was performed to detect the expression of VEGF and VEGFR-2. After 3 days, rats were sacrificed by deep anesthesia with 2% sodium pentobarbital (30 mg/kg) followed by transcardial perfusion with PBS, before the brain tissues were fixed with 4% paraformaldehyde at room temperature for 24 h. The 5-µm paraffinized brain sections were dewaxed and dehydrated in xylene and ethanol solutions, followed by antigen retrieval. Briefly, to deparaffinize and rehydrate, the sections were incubated in 3 changes of xylene for 10 min each and then dehydrate in 2 changes of pure ethanol for 5 min, followed by dehydration in gradient ethanol (85 and 75% ethanol) for 5 min each. The sections were then washed in distilled water. For antigen retrieval, the slides were immersed in EDTA antigen retrieval buffer (Wuhan Servicebio Technology Co., Ltd.) and maintained in boiling water for 35 min before being air cooled. The slides were then washed three times with PBS (pH 7.4) in a rocker device, for 5 min each, and blocked with 10% goat serum (Beyotime Institute of Biotechnology) at room temperature for 15 min. All sections were then incubated with anti-VEGF (1:100 dilution) and anti-VEGFR-2 (1:100 dilution) antibodies at 4˚C overnight. The sections were then washed in PBS three times prior to incubation with HRP-conjugated goat anti-rabbit IgG secondary antibodies for 10 min at room temperature. Next, the sections were visualized using a DAB kit and counterstained with hematoxylin at room temperature for 3 min. Using a light microscope, an investigator (FYW) blinded to the experimental groups randomly selected three separate tissue sections for each rat. The staining was analyzed using ImageJ analysis software 1.50i.

Paraffin-embedded brain sections were cut to a thickness of 5 µm in the coronal plane, deparaffinized using xylene, rehydrated with a descending ethanol gradient and washed in distilled water, prior to blocking with 10% goat serum (Beyotime Institute of Biotechnology) at room temperature for 1 h. The sections were then incubated with the primary antibodies targeting the following proteins overnight at 4˚C: VEGF (1:100 dilution) and F4/80 (a marker of macrophage cells; 1:350 dilution), or VEGFR-2 (1:100 dilution) and CD31 (a marker of vascular endothelial cells; 1:200 dilution). The sections were then washed in PBS and incubated with the appropriate secondary antibodies, namely anti-mouse IgG Alexa Fluor^® 488 Conjugate (cat. no. 4408; Cell Signaling Technology, Inc.) and Cy™3-conjugated Affinipure Donkey anti-rabbit IgG (cat no. 152679; Jackson ImmunoResearch Laboratories, Inc.) for 2 h at room temperature in the dark. The sections were finally mounted on coverslips with a drop of DAPI solution (Sigma-Aldrich; Merck KGaA). The stained cells were observed using a confocal microscope (A1; Nikon Corporation). The staining was analyzed using ImageJ analysis software 1.50i.

The neurological score data are presented as the median and interquartile range and were analyzed using the Kruskal-Wallis test followed by Dunn's post-hoc tests. All other data obtained are presented as the mean ± SD and were analyzed statistically using one-way or two-way analysis of variance followed by Bonferroni's multiple-comparisons tests. Statistical analysis was performed using GraphPad 8.0 software (GraphPad Software; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Firstly, the effect of Sal on VEGF expression and production in macrophages at the mRNA and protein levels, respectively, was assessed. The results showed that treatment with ≥10 µM Sal significantly promoted the expression of Vegf mRNA in RAW264.7 cells, and 10 µM Sal increased the secretion of VEGF protein into the supernatant of the cells (Fig. 1). The viability of HUVECs following various treatments was then evaluated using MTT assays. As shown in Fig. 2A, compared with that in the control group, s-control, s-Sal and VEGF treatment all significantly increased the proliferation of HUVECs, whilst treatment with Sal alone did not affect the viability of HUVECs. This suggests that Sal was unable to promote the proliferation of HUVECs directly. However, s-Sal significantly increased the proliferation of HUVECs compared with that in the s-control group, suggesting that Sal indirectly promoted the proliferation of HUVECs through its effect on macrophages. In addition, the cell cycle of the HUVECs was detected by flow cytometry, whereby s-Sal was found to significantly promote the number of HUVECs in the S phase compared with that in the control and Sal groups (Fig. 2B and C).

The migration of HUVECs was next analyzed using a wound healing assay. The s-control, s-Sal and VEGF groups all showed increased degrees of cell migration to the wounded area after 24 h of incubation compared with that in the control group (Fig. 2D and E). In addition, the migration rate in the s-Sal group was significantly higher compared with that in the Sal and s-control groups.

To examine the effect of Sal on the tubule formation of HUVECs, a tube formation assay was performed using Matrigel. Images of canaliculus formation are presented in Fig. 2G, and tube formation is expressed as the number of master junctions in Fig. 2F. No significant difference was detected between the Sal and control groups. However, s-Sal treatments significantly enhanced the extent of tube formation of the HUVECs compared with that in the s-control group. Together, these results suggest that Sal promoted endothelial cell tube formation via its effect on macrophages.

To investigate the effect of Sal on macrophage-mediated angiogenesis and the VEGF signaling pathway in vitro, western blotting was performed. Following VEGF binding to VEGFR2, the proliferation and migration of endothelial cells and their maturation into vessels are typically activated (7). Therefore, VEGFR2 expression levels and activation were assessed by western blotting analysis (Fig. 3A). s-Sal significantly promoted VEGFR2 phosphorylation at Tyr1175 compared with that in the control, s-control and Sal groups, which indicates that s-Sal induced the activation of this receptor (Fig. 3B). In addition, this form of VEGFR2 activation was found to be associated with the activation of AKT and p38 MAPK signaling downstream (Fig. 3A, C and D), as s-Sal significantly promoted the phosphorylation of AKT compared with that in the control and Sal groups and phosphorylation of p38 proteins compared with that in the control, s-control and Sal groups in the HUVECs. The activation of AKT and p38 MAPK is essential for cellular responses during angiogenesis (7).

To investigate the possible effects of Sal in the rat tMCAO model, tMCAO model rats were treated with Sal for 3 days and then the neurological deficit scores of the rats were determined. The scores are shown in Fig. 4A. The neurological deficit scores of the infarct group were observed to be significantly increased compared with those in the sham group. A significant reduction in neurological deficit scores of the 10, 20 and 40 mg/kg Sal groups and the ED group was observed compared with that in the infarct group, indicating improved neurological recovery following Sal or ED treatment.

The infarct volume was next determined by TTC staining. The infarct volume was found to be significantly increased in the infarct group compared with the sham group. Rats subjected to cerebral ischemia and treated with various doses of Sal exhibited a significantly smaller infarct volume compared with that in the infarct group (Fig. 4B and C). The neurological deficit scores and infarct volume after treatment with various concentrations of Sal indicated that 20 mg/kg was the most effective concentration, rendering 20 mg/kg as the dose selected for subsequent experiments. The results at the 0-, 1- and 3-day time points for the infarct and 20 mg/kg Sal group indicated that the infarct volume increased and nerve function deteriorated in the infarct group. By contrast, the rats in the Sal-administered group exhibited improvements with reductions in the extent of infarction and nerve function impairment (Fig. 4D-F).

To further investigate the protective effect of Sal against brain I/R injury, morphological changes in the brain tissues were observed by H&E staining after 3 days of treatment with Sal or ED. As shown in Fig. 5A, the characteristic histopathological features in the infarct group were nuclear atrophy, cytoplasmic eosinophilia and cellular edema. The 20 mg/kg Sal treatment group exhibited reduced histopathological abnormalities compared with those in the infarct group.

The effect of Sal on the VEGF signaling pathway was investigated in vivo. Immunohistochemical staining (Fig. 5B and C) was used to detect VEGF protein (Fig. 5B and D) and VEGFR-2 protein expression (Fig. 5C and E). The immunohistochemical staining intensity of VEGF and VEGFR-2 in the Sal group was found to be significantly higher compared with that in the infarct group.

To investigate whether Sal mediates angiogenesis via the VEGF signaling pathway in vivo, immunofluorescence analyses were performed. After 3 days of Sal treatment, F4/80 and VEGF double immunofluorescence staining and CD31 and VEGFR-2 double immunofluorescence staining were used to detect the cellular location and expression levels of VEGF and VEGFR-2 proteins in the brain tissue (Fig. 6). The results showed that VEGF protein was localized at sites of macrophage accumulation, where it tended to be highly expressed. In addition, VEGFR2 protein was found to colocalize with endothelial cells, and the expression of VEGFR2 in the Sal group was significantly increased compared with that in the infarct group, with higher microvessel density in the former.

To evaluate the efficacy of Sal in upregulating VEGF signaling activity in vivo, western blot analysis was performed on tissues in the peri-infarct area. As shown in the western blots in Fig. 7A, VEGF and VEGFR2 protein expression and VEGFR2 phosphorylation in the infarct group were lower compared with those in the sham group. Additionally, the protein expression of VEGF (Fig. 7B) and VEGFR2 (Fig. 7C) along with VEGFR-2 phosphorylation (Fig. 7D) were upregulated in the Sal group compared with those in the infarct group. The phosphorylation levels of AKT and p38, which lie downstream of the VEGF/VEGFR2 signaling pathway, were also significantly increased by the administration of Sal (Fig. 7A, E and F). These results suggest that Sal protected rats against acute cerebral ischemia by promoting angiogenesis in the brain. Specifically, the mechanism of its pro-angiogenic activity appears to be the promotion of VEGF signaling and in turn the downstream AKT and p38 signaling pathways.