Patients and tissue samples
Before the study, written informed consents were obtained from the patients, and this study was granted approval of the Ethics Committee of The First Hospital of Hebei Medical University (Ethical approval: 20190506). All participates were over 16 years of age. The study protocols were also based on the ethical principles of the Declaration of Helsinki for medical research involving human subjects. 80 patients with IA participated in this study. IA tissue samples were obtained during the surgery, and superficial temporal artery (STA) vascular tissue samples were collected as controls. All samples were fixed in 10% formaldehyde (Yuanmu Biotech, Shanghai, China) and embedded in paraffin.
VSMC isolation and cell culture
Sprague-Dawley rats (male, 8 weeks old, 300-320 g) were purchased from Hunan SJA Laboratory Animal CO., LTD (Hunan, China). Animal assays were carried out under the guideline of the Ethics Committee of The First Hospital of Hebei Medical University (Ethical approval: 2019-DWLL0506A). The experiment procedures were consistent with guidelines on the Use and Care of Laboratory Animals for Biomedical Research published by National Institutes of Health. Rat brain VSMCs were separated from the Circle of Willis. The brain vessels were collected under sterile conditions followed by phosphate buffer solution (PBS; Absin Biotechnology, Shanghai, China) washing. After washing, excess fat and fascia were removed. Subsequently, the vessels were placed in enzyme solution containing 0.2% collagenase II and 1 mg/ml soybean trypsin inhibitor for 20 min at 37℃. For VSMCs isolation, the blood vessels were cut off along the long axis and the intima was tore off. Next, the vessels were cut into pieces (1 mm3) and 0.125% trypsin was added. After incubation for 15 min at 37℃, DMEM (Sunncell Biotech, Hubei, Wuhan, China) supplemented with 10% fetal bovine serum (FBS; Excell Biotechnology, Shanghai, China) was added. Then the digested VSMCs were collected by centrifugation and the supernatant was removed. The collected cells were cultured in DMEM supplemented with 10% FBS, 100U/ml penicillin, and 100 mg/ml streptomycin at 37℃ with 5% CO2 in a humidified condition. VSMCs at passages 3 to 8 were used for subsequent experiments.
Cell treatment and CCK-8 assays
The first step of the study was to find out the optimal concentration of hydrogen peroxide (H2O2) and DEX by conducting CCK-8 assays were. The VSMCs were first exposed to different concentrations (0, 0.1, 1, 10 μM) of DEX for 120 min. The treated cells seeded in the 96-well plates (5×103 cells/well) until 80% confluence. Then 10 μl of CCK-8 reagent (Yeasen Biotechnology, Shanghai, China) was added into the culture medium. Another 2 h was given the cells to incubate at 37℃. Absorbance at 450 nm was measured using a microscope (Bio-Tek, Winooski, VT, USA). After confirmed the optimal concentration of DEX, the cells were exposed to different concentrations (0.2, 0.5, 1, 10 mM/ml) of H2O2 for 12 h, and then CCK-8 assays were conducted as described above. The optimal concentration of DEX and H2O2 was 1 μM and 1 mM/ml, respectively. Then the VSMCs were divided into the control group (DMEM), H2O2 group (1 mM/ml H2O2), H2O2 + NS group (1 mM/ml H2O2 + normal saline), and H2O2 + DEX group (1 mM/ml H2O2 + 1 μM DEX).
Intracellular ROS measurement
The VSMCs cells were seeded into 6-well plates (1×105 cells/well) to measure the intracellular ROS levels. After treatment as described above, PBS washing was conducted. The cells were then incubated at 37℃ with 10 μM DCFH-DA (Beyotime, Shanghai, China) for 1 h. Next, the VSMCs were washed three times using the serum-free DMEM and suspended in PBS. A fluorescence microscope (Leica Biosystems, Shanghai, China) was used to visualize the fluorescence intensity of DCF at 488 nm (excitation) and 525 nm (emission). A flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the fluorescence.
Wound healing assays
The cells were seeded into 24-well plates (5×104 cells/well) and incubated at 37℃ with 5% CO2 for 24 h. Until the cells reached 90% confluence, a pipette tip (10 μl) was used to make scratches on the cells. The cells were then washed using PBS and subsequently cultured at 37℃ with 5% CO2 for 24 h. The wound width was imaged using an AxioCam camera (Carl Meditec, Jena, Germany) at 0 and 24 h. ImageJ software was used to evaluate the remaining area .
The cells were plated in transwell inserts with 8-μM pore size in serum free DMEM. DMEM with 10% PBS was added to the lower chamber. After 12 h, the cells on the upper side of the insert were removed and the migrated cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with crystal violet. Number of migrated cells were measured using an inverted microscope (Leica). Six fields were randomly selected, and the average was calculated .
The proteins were collected using RIPA lysis buffer (Sigma-Aldrich) containing protease inhibitor. Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (PVDF). After blocking with 5% skimmed milk, the membranes were incubated overnight with primary antibodies against SM22α (ab14106, 1 μg/ml; Abcam), αSMA (ab7817, 0.341 μg/ml; Abcam), α2AR (ab85570, 1 μg/ml; Abcam), p-GSK-3β (ab107166, 1 μg/ml; Abcam), GSK-3β (ab93926, 1:1000; Abcam), MKP-1 (ab138265, 1:1000; Abcam), NRF2 (ab92946, 1:1000; Abcam) and GAPDH (ab8245, 1:1000; Abcam) at 4℃. Then the membranes were washed three times. After that, the membranes were incubated with secondary antibodies for 2 h at room temperature. After being probed with enhanced chemiluminescence (Yeasen Biotechnology), the proteins were visualized using an image analysis system (Tanon, Shanghai, China). The protein intensity was evaluated by ImageJ software.
IA and STA tissue samples were stored at -80℃ and placed in pre-cooled lysis buffer (Sigma-Aldrich) after stirring with an electric homogenate machine. After centrifugation at 1200 rpm for 30 min, the supernatant was collected. TRIzol (Sigma-Aldrich) was used for total RNA isolation from lysed tissues and VSMCs. Reverse transcription into cDNA was done using the Reverse Transcription Kit (Sigma-Aldrich). RT-qPCR was performed on the ABI 7300 system (Applied Biosystems, Foster City, UA, USA). The relative level was calculated using 2-△△Ct , and expressed as ratio of GAPDH. The primer sequences were listed in Table 1.
Measurement of TNF-α, IL-6 and MCP-1 levels
Cellular supernatants were collected after 1000g centrifugation for 5 min. Interleukin-1β, IL-6 and tumor necrosis factor (TNF-α) ELISA kits (Enzyme-linked Biotechnology, Shanghai, China) were applied to measure the levels of inflammatory cytokines according to the kit instructions.
The paraffin-embedded IA and STA tissue sections were cut into 5 μM and then placed on poly-L-lysine coated slides. For immunohistochemistry, the slides were incubated with primary antibody against α2AR (ab85570, 1 μg/ml; Abcam) overnight at 4℃. After washing, the slides were incubated with secondary antibody. The slides were visualized using a DAB plus chromogen (Thermo Fisher Scientific). Under 400 × magnification, the pictures were taken in 5 random fields.
Each experiment was performed three times. Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad, Sam Diego, CA, USA). Data were described as the mean ± standard deviation. Student’s t test and one way analysis of variance followed by Tukey’s post hoc test were used to compare differences. p < 0.05 was considered statistically significant.