Four adult male Sprague-Dawley rats weighing 300–350 g were sacrificed by decapitation in each treatment group. A total of 12 rats were used in the present study, purchased from the Animal Center of Chinese Academy of Sciences (Shanghai, China). All animals were housed at a constant temperature of 22°C, under a 12-h light/dark cycle (lights switched on at 6:00 a.m.) with free access to food and water. All rats were placed under general anesthesia prior to fixation-perfusion and euthanasia procedures. All procedures were approved by the Institutional Animal Care Committee of the Zhangjiagang Hospital of Traditional Chinese Medicine (Suzhou, China and were performed in accordance with the guidelines of the National Institutes of Health on the care and use of animals. Whole brains were dissected under sterile conditions and immediately placed in a culture dish with Dulbecco's modified Eagles medium supplemented with Gibco^® nutrient mixture F-12 (DMEM/F12), 100,000 U/l penicillin and 100,000 U/l streptomycin (Thermo Fisher Scientific, Inc. Waltham, MA, USA). Basilar arteries were dissected out in a laminar flow hood and rinsed repeatedly with DMEM/F12 medium supplemented with 100,000 U/l penicillin and 100,000 U/l streptomycin. (Thermo Fisher Scientific, Inc.). Following excision of the adventitial layer, the vessels were sectioned into small segments of length ~0.2 mm. The tissue was digested in 1 ml 0.1% collagenase I solution (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at 37°C with 5% CO₂ for 30 min until the tissue appeared swollen. In order to digest the tissue further, 1 ml 0.125% trypsin solution (Gibco; Thermo Fisher Scientific, Inc.) was added for an additional 10 min. The dispersed cells were collected and transferred to a centrifuge tube, and DMEM/F12 medium supplemented with 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) was added to terminate digestion. The cell suspension was subsequently centrifuged at 200 × g for 5 min, and the supernatant discarded. The cells were resuspended in DMEM/F12 medium with 20% FBS, seeded into a 60 mm culture dish and incubated at 37°C with 5% CO₂. The medium was changed every 3 days. After 10 days, the culture had reached 80–90% confluence, following trypsin digestion the third passage was used for further experiments.

Hemolysate was prepared using a freeze-thaw method previously described (10), although with several modifications, to lyse red blood cells. A heparinized sterile syringe was used to collect 1 ml blood from the rat tail artery. The blood was transferred to a sterile centrifuge tube, and then centrifuged at 2,500 × g for 15 min at 4°C. The serum was discarded, and the red blood cells were resuspended in sterile distilled water. The cells were frozen at −20°C for 20 min and then immediately transferred to a 39°C water bath. After the cells had completely thawed, they were centrifuged at 12,000 × g for 30 min at 4°C. The supernatant containing the hemolysate was collected. Hemolysate concentration was calculated as follows: Concentration = (hemolysate mass - distilled water mass) / hemolysate volume. All procedures were performed under sterile conditions.

Certain of these experiments included 2-[(4-phenoxyphenylsulfonyl)methyl]-thiirane (SB-3CT), a selective MMP-9 inhibitor purchased from Sigma-Aldrich; Merck Millipore. As shown in Fig. 1A, the cells were randomly assigned into three groups: i) The control group, where cerebrovascular smooth muscle cells were cultured under normal conditions; ii) the hemolysate treatment group, where the cells were treated with 1 mg/ml hemolysate for 24 h; and iii) the SB-3CT treatment group, where cells were pretreated with 1 µM SB-3CT for 6 h, and subsequently treated with 1 mg/ml hemolysate for a further 24 h.

Following treatment, the culture medium was discarded and cells were rinsed three times with phosphate-buffered saline (PBS). Cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) was added and the cells were scraped off the dishes and maintained on ice. Cell lysates were placed on ice for 30 min prior to centrifugation at 12,000 × g for 10 min at 4°C. Protein concentration was determined using the bicinchoninic acid assay (BCA) method. The cell lysates were transferred to a 96-well plate, and 200 µl BCA working solution was added to each well. After a 30 min incubation at 37°C, the absorbance at 562 nm was determined using a microplate reader. A standard curve was generated and used to determine the concentrations of the samples. The samples were then separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gels), and transferred to a nitrocellulose membrane. The membranes were blocked with 1X PBS-Tween 20 (PBST) solution with 5% bovine serum albumin (BSA; Beyotime Institute of Biotechnology) for 1 h at room temperature. The membranes were then incubated with primary antibodies against MMP-9 (cat. no. ab119906), type IV collagen (cat. no. ab19808), type V collagen (cat. no. ab114072) and β-actin (cat. no. ab8227; all at a dilution of 1:1,000, and obtained from Abcam, Cambridge, MA, USA) overnight at 4°C. The membranes were rinsed three times with PBST (5 min per rinse) and then incubated with a horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab6789; ab 6721) at 1:5,000 dilution for 2 h at room temperature. Finally, the membranes were rinsed with PBST three times (5 min per rinse) and visualized using Pierce ECL Western Blotting substrate (cat. no. 32106; Thermo Fisher Scientific, Inc.).

Cover slides were placed in 12-well plates, coated with 0.1% poly-lysine (Sigma-Aldrich, Merck Millipore) overnight and rinsed three times with sterile distilled water. Cells were seeded into 12-well plates at a density of 1.0×10⁵ cells/well, cultured until they reached 70% confluence, and then treated with hemolysate and SB-3CT, as described above. Following the treatment, the medium was removed and the cells were rinsed three times with PBS and fixed in 4% paraformaldehyde for 20 min. The cells were rinsed with PBS and blocked with PBS with 5% BSA for 30 min at room temperature. Following blocking, the cells were incubated with a 1:1,000 dilution of primary antibody against α-smooth muscle actin (α-SMA; a marker for cerebrovascular smooth muscle cells; cat. no. ab7817; Abcam), MMP-9, type IV and V collagen (Abcam) overnight at 4°C. The cells were then washed three times with PBS and incubated with fluorescent, labeled secondary antibodies (cat nos. ab150077; ab150115; Abcam) at room temperature for 30 min. Following three additional washes using PBS, the cell nuclei were stained using 4′,6-diamidino-2-phenylindole. The slides were then sealed and observed under a BX50/BX-FLA/DP70 fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Cell contraction assay kits were purchased from Cell Biolabs, Inc. (San Diego, CA, USA) and used according to the manufacturer's protocol. Following treatment (Fig. 1), cells were trypsinized and suspended, rinsed three times with PBS, centrifuged at 1,500 × g for 3 min and resuspended in phenol red-free DMEM (Thermo Fisher Scientific, Inc.) at a final concentration of 5.0×106 cells/ml. Rat tail collagen solution (Gibco; Thermo Fisher Scientific, Inc.) was diluted to a final concentration of 3 mg/ml with 0.3 ml phenol red-free DMEM (pH 7.3–7.4). Collagen solution, cell suspension and FBS were then combined at a ratio of 8:1:1 to yield a final concentration of 5.0×105 cells/ml. Aliquots (1 ml/well) of the collagen-cell mixture were added to a 24-well plate and the plates were incubated for 30 min at 37°C with 5% CO2 to promote polymerization. Following the solidification of the gel, the edges of the gel attached to the wells were scored using pipette tips and an appropriate quantity of phenol red-free DMEM was added. Gels were incubated for an additional 24 h prior to image capture. Cell contractility was calculated using the following formula: Contraction index = (well area - gel area) / well area × 100%.

The western blotting and immunofluorescent staining were analyzed using ImageJ version 1.46 software (National Institutes of Health, Bethesda, MD, USA. The mean grayscale value of the control group was normalized to 1, and the ratios of the grayscale values of the experimental groups to the control group were calculated and analyzed. Statistical analysis was performed using GraphPad Prism version 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± standard error. One-way analysis of variance and Fisher's least significant difference test was used for pairwise comparison of groups. P<0.05 was considered to indicate a statistically significant difference.

Immunofluorescence staining was performed to determine the phenotype of the adherent cells. As presented in Fig. 1B, the rat cerebrovascular smooth muscle cells had a polygonal or long, spindle-like morphology and expanded into a monolayer when observed under a microscope. It was demonstrated that the majority of isolated cells stained positively for α-SMA, a specific marker of cerebrovascular smooth muscle cells. This indicated that the cerebrovascular smooth muscle cells had been successfully isolated (Fig. 1B).

As presented in Fig. 2, the contractility of cerebrovascular smooth muscle cells was determined using a collagen gel contraction assay. Significant gel contraction was observed in the hemolysate treatment group when compared with the control group (P<0.01; Fig 2B). However, pretreatment with SB-3CT significantly inhibited the hemolysate-induced gel contraction when compared with the hemolysate treatment group (P<0.01; Fig. 2B).

Western blot analysis was used to demonstrate that hemolysate stimulation significantly increased MMP-9 protein expression levels (P<0.01; Fig. 3) and reduced collagen IV and V protein expression levels (P<0.01; Fig. 3B) when the hemolysate treatment group was compared with the control. However, pretreatment of cerebrovascular smooth muscle cells with SB-3CT significantly reduced the expression levels of MMP-9 when compared with the hemolysate treatment group (P<0.01; Fig. 3B). Conversely, collagen IV and V protein expression levels were significantly greater in the SB-3CT pretreatment group compared with the hemolysate group (P<0.01; Fig. 3B).

Immunofluorescence staining also demonstrated that hemolysate treatment significantly increased the protein expression levels of MMP-9 (P<0.01; Fig. 4) and reduced the protein expression levels of collagens IV and V (P<0.01; Fig. 4) in cerebrovascular smooth muscle cells compared with the control group. However, pretreatment with SB-3CT significantly reduced the MMP-9 expression levels (P<0.01; Fig. 4D). Collagen IV and V protein expression levels were significantly increased in the SB-3CT pretreatment group compared with the hemolysate group (P<0.01; Fig. 4).