1. Animals and reagents
Sprague-Dawley (SD) rats (8 weeks) were purchased from Zhejiang Laboratory Animal Center, 12% yellow rice wine and alcohol were obtained from Shaoxing Yellow Rice Wine Group and 12% red wine was supplied by Languedoc-Roussillon of France; HFN (human fibronectin). Vascular endothelial growth factor (VEGF) and in vitro angiogenesis kit were purchased from Chemicon company. Endothelial growth medium-2 (EGM-2) was procured from Cambrex company; 0.25% trypsin-EDTA and fetal bovine serum were acquired from Gibco company; rat lymphocyte separation medium was purchased from Tianjin Haoyang Biological Manufacture Co., Ltd. FITC-UEA-I (fluorescein isothiocyanate-conjugated lectin Ulex europaeus agglutinin-1) was purchased from Sigma. The acetylated (ac) LDL (acLDL-DiL) was obtained from Molecular Probe. The anti-rat vWF-FITC antibody was obtained from Abcam company, the anti ratCD31-APC antibody was from eBioscience company and the rabbit anti-rat CD133 antibody was acquired from Proteintech. MTT was purchased from Sino-American Biotechnology Co., Ltd (imported and repackaged) and 5 µm transwell was purchased from Corning company. The other analytical reagents were obtained locally within China.
2. Methods
2.1 Isolation and culture of EPCs
SD rats were euthanized by cervical dislocation. Bilateral femur and tibia were removed under sterile conditions and cultured with D-Hanks’ solution after cutting the epiphysis at both ends of femur and tibia to expose the marrow cavity. A 5 mL injector was used to absorb the D-Hanks’ solution (including fetal bovine serum with 10% volume fraction) and flush the marrow cavity. After centrifugation at 1,000 rpm for 5 min, the cell pellets were collected and the D-Hanks’ solution was added. Cell pellets were gently blown and beaten until the 5 mL D-Hanks’ solution was thoroughly mixed with the cells. A 15 mL centrifuge was used to slowly overlay solution on the upper layer of an equal amount of lymphocyte separation medium, and centrifuged at 2,000 r/min for 20 min. The mononuclear cells in the buffy coat were collected and washed with the D-Hanks’ solution twice (centrifuged at 1,000 r/min for 5 min). The supernatant was discarded, and the complete medium EGM-2 (including fetal bovine serum with 10% volume fraction and 10 µg/L of endothelial growth factor) was added to suspend the cells again(Ziebart et al., 2016). The cells were planted in the culture plate coated with HFN at a density of 1×109/L, and incubated for 24 h at 37°C in a CO2 incubator under saturated humidity and 5% volume fraction. The non-adherent cells were transferred to the newly coated wells. The complete medium was cultured for another 7 days, and the adherent cells were used for the experiment.
2.2 Staining and identification of cells
The cells were cultured for 10 h with acLDL-DiL (2.4 mg/L) at 37°C, fixed with 4% paraformaldehyde for 10 min and cleaned with D-Hanks’ solution. FITC-UEA-I (10 mg/L) was added to the above samples, and further cultured for 1 h at 37°C. A multi-wavelength laser confocal microscope was used for observation. Cells with red fluorescence were positive acLDL-DiL cells, cells with green fluorescence were positive FITC-UEA-I and double-positive (yellow) acLDL-DiL and FITC-UEA-I cells represented differentiating EPCs(Qin et al., 2018).
2.3 Flow cytometry of cell surface antigen
Major surface antigens of late EPCs include CD31, vWF and CD133(Morrone et al., 2021). A 0.25% trypsin was used to digest, collect and count adherent cells, and 5×105 adherent cells were combined with CD31, vWF and CD133 in turn, cultured for 30 min in a 4°C water bath. After washing twice with D-Hanks’ solution, the cells were suspended in 300 µL of D-Hanks’ solution and subjected to flow cytometry.
2.4 Test groups
The role of Hcy on the activity of EPCs was determined by dividing the samples into five groups using a series of Hcy concentration gradients: 0 µmol/L, 100 µmol/L, 200 µmol/L, 300 µmol/L and 400 µmol/L, and cultured with EPCs for 24 h. EPCs were cultured with 300 µmol/L Hcy for 12, 24 and 48 h. MTT was used to test the experimental concentrations selected for cellular activity. The effect of yellow rice wine and red wine on EPCs function was tested in five groups including a control group, a Hcy group (300 µmol/L Hcy), a Hcy yellow rice wine group (300 µmol/L Hcy and 0.4% alcohol), a Hcy red wine group (300 µmol/L Hcy and 0.4% alcohol) and a Hcy alcohol group (300 µmol/L Hcy and 0.4% alcohol). Samples were collected after 24-h culture, MTTand transwell assays were peformed, and apoptosis and in vitro angiogenesis of EPCs were determined. Combined with the preliminary results, each wine concentration (0%, 0.2%, 0.4%, 0.8% and 1.6%) was mixed with 300 µmol/L Hcy to co-culture EPCs for 24 h. MTT was used to test the impact of wine on cellular activity and the optimal concentration was determined.
2.5 Test of cell migration
The cells were counted and transferred to a six-well plate at the rate of 2 mL per well. The culture medium was added to each well to obtain a final alcohol concentration of 0.4% in each group. After culturing for 2 days, the cells in each group were collected, counted and suspended again without serum medium to obtain 2.5×105/mL cell suspension. A 24-well transwell plate was supplemented with 600 µL complete medium in the lower well and inoculate with 200 µL suspension in the upper well. The cells were cultured for 6 h in a 37°C incubator. The transwell was transferred into 0.1% crystal violet solution and cultured for 6 h at room temperature. The crystal violet solution remaining in the upper well was wiped off, and the migrating cells in the lower well were visualized with a 200X microscope. Seven stochastic fields were counted randomly.
2.6 Cell proliferation
Cells were counted as above, cultured for two days in a 37°C incubator at 5% CO2 and saturated humidity. After addition of 10 µL CCK-8 to each well and cultured for 1 h, the absorbance was tested using a microplate reader at 450 nm and 630 nm.
2.7 Angiogenesis
After 12 days, the cultured EPCs were digested with 0.25% trypsin and counted. The cells (1×105/ml ) were inoculated in a 6-well plate at the rate of 2 mL per well, After adherence of cells to the wall, the medium was replaced with the prepared medium (final alcohol concentration of each group was 0.4%) and cultured for 2 days in a 37°C incubator with 5% CO2 and saturated humidity. The cells in each group were collected and counted. The cells were inoculated in a 96-well plate with properly-laid Matrigel (performed in triplicate for each group) with a cell count of 3×105/mL, and cultured for 16 h in a 37°C incubator with 5% CO2 and saturated humidity. A 100X microscope was used to observe five random stochastic fields and the areas of angiogenesis were counted.
2.8 Apoptosis
The cells were collected and counted as above, using 0.25% trypsin to digest the cells. Cell pellets were collected after centrifugation and washed with PBS. According to the manufacturer’s instructions, the cells were suspended again with 500 µL binding buffer, followed by addition of Annexin V 5 µL and PI 10 µL, respectively. The cells were mixed and cultured at room temperature for 5 min and finally tested for apoptosis(Chen et al., 2007).
2.9 Testing for NOS using Western blot
The cells in each group were collected and 100 µL lysate containing PMSF (1 mM) was added to each group for 30 min of lysis at 4oC. Cell lysis was carried out by shaking the centrifuge tube. After lysis, the cells were centrifuged at 4oC for 5 min at a speed of 11,000 rpm. The supernatant after centrifugation was transferred to a 1.5 mL centrifuge tube and stored under − 80oC. The BCA method was used to test the protein concentration, and 1L transfer buffer was prepared and cooled to 4oC. The PVDF membrane was immersed in methanol for about 30 s, soaked in the transfer buffer for 10 min, and finally suspended in electrotransfer buffer with filter paper and gel. A constant current of 200 mA was used for the electrotransfer at a speed of about 1 min/kd according to the molecular weight of the transferred protein. The PVDF membrane was soaked in the liquid containing 5% nonfat dry milk or bovine serum albumin, vortexed gently, and sealed at room temperature overnight. The sealed PVDF membrane was removed, immersed in 1×TBST buffer, and washed slowly on the shaker for 5 min. It was transferred to a small bag containing the primary antibody and cultured for 2 h at room temperature. PVDF membrane was transferred to the glass dish containing the secondary antibody, which was cultured for 1 h at room temperature on the shaker. The PVDF membrane was immersed in the 1×TBST buffer solution and washed for 15 min each time slowly three times, on the shaker. Liquids A and B were taken in moderate and equal amounts from the ECL kit and mixed, and added to the surface of the membrane. The membrane was transferred into the gel imaging analyzer for exposure to photosensitive chemicals. The optical density of each strip was analyzed using the software Image J(Guo et al., 2021).
2.10 Production of p-eNOS
The protein concentration of p-eNOS was tested as above.
2.11 NO synthesis
The EPCs cultured for 15 days were digested with 0.25% trypsin and counted. The cells were inoculated into the 6-well plate in aliquots of 2 mL each containing 1×105 cells/mL. The samples were divided into 5 groups: normal (CK), Hcy (300 µM Hcy), yellow rice wine (300 µM Hcy and yellow rice wine), red wine (300 µM Hcy and red wine) and ethanol groups (300 µM Hcy and ethanol). The cells were cultured for 2 d in a 37°C incubator with 5% CO2 and saturated humidity. The cell culture supernatant (3,000 rpm for 10 min) was collected and stored at -80°C. The supernatant and NO test kit were removed and balanced at room temperature for 30 min. The reagents were prepared and the test was conducted according to the manufacturer’s instructions (Aicher et al., 2003, Thum et al., 2005).
3. Statistical analysis
All data were expressed as mean ± standard deviation (x̄ ± s). The statistical software SPSS18.0 was used for analysis, the t-test was used to compare the two groups, and one-way analysis of variance was used to compare multiple groups. P < 0.05 indicates statistical significance of the differences.