Fetal bovine serum (FBS) was purchased from Biological Industries (Beit-Haemek, Israel). Dulbecco’s Modified Eagle’s Medium (DMEM) and phosphate buffer saline (PBS) were purchased from Hyclone Laboratories Inc (Logan, Utah, USA). Rabbit polyclonal antibody against NF-κB p65, matrix metalloprotein 9 (MMP-9) and rat polyclonal antibody against proliferation cell nuclear antigen (PCNA) were purchased from Proteintech Group Inc. (Wuhan, China). Secondary antibodies were purchased from CWBio (Beijing, China). Cell counting kit-8 and mitochondrial membrane potential detection kit was purchased from Solarbio Science & Technology (Beijing, China). N-acety-L-cysteine (NAC) 2’, 7’-dichlorofluorescindiacetate (DCFH-DA) was purchased from Beyotime Biotechnology (Shanghai, China). Collagenase-II was purchased from Biosharp (Nanshan, Guangdong, China). Trans-well plates were purchased from Millipore (Bedford, MA, USA).
Male Sprague-Dawley rats (5-8weeks) were purchased from HFK Bioscience Company (Beijing, China). All procedures were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Henan University of Science and Technology, which is in compliance with the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines on animal research.
- Cell Culture
VSMCs were prepared from the thoracic aorta of Sprague-Dawley rats. Whole thoracic aorta was isolated from sacrificed rats. The thoracic aorta was cut open longitudinally and the endothelial cells were removed with a sterile Elbow tweezer scraping back and forth twice. The vascular adventitia was carefully stripped with ophthalmic tweezers, and then was rinsed with PBS twice. The aortic tissue was cut into small pieces (1 mm2). The pieces of aortic tissue were digested with collagenase (125 U) for 2h (shaking once every 30 min) at 37°C in a humidified 5% CO2 air atmosphere. When a great mass of cells slipt, five folds compelet medium added into tube and centrifuged at 300 g for 5 min; At last, the cells were plated in T25. The cells were grown to confluence in DMEM with 5.5 mM glucose, 10% FBS, at 37°C in a humidified 5% CO2. Cells were grown to 80% confluence and cells were used up to 8th (Ren, et al., 2007).
- Cell Treatment Protocol
VSMCs were divided into four groups. Control group (NG): DMEM medium with 5.5 mM D-glucose; high glucose group (HG): DMEM medium with 25 mM D-glucose; mannitol group (MG): DMEM medium with 5.5 mM D-glucose and 19.5 mM mannitol; yixintongmai group (HG+Y): DMEM medium with 25 mM D-glucose and 360 μg/ml yixintongmai.
- CCK-8 Assay
VSMCs proliferation was assayed with CCK-8 cell viability kit (Solarbio, Beijing, China). Firstly, VSMCs were randomly seeded at the density of 5×103 cells/well and cultured in a 96-well plate. After cells reached 20-30% confluence, VSMCs were starved for 24 h with free serum DMEM medium, and then incubated for 24 h to stimulate cell proliferation in the different conditions as mentioned above. Subsequently, CCK-8 reagent (10 μL) was added and cells were further incubated for 2 h at 37oC. The absorbance wavelength was read at 450nm using spectrophotometric plate reader (Jeong, et al., 2011).
- Western Blot
Total protein was extracted using an extraction kit (Solarbio, Beijing, China) according to the manufacturer’s protocols, and the protein concentration was determined by a BCA protein assay kit (Solarbio, Beijing, China). Protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (40 mg per lane, 80 V, 2 h); then transferred to polyvinylidene fluoride membranes (90 V, 120 min). After blocking with 5% non-fat dried milk in Tris-buffered saline with Tween-20 (PBST) for 1 h, the membranes were separately incubated with the appropriate primary antibody, β-actin (1:5000), PCNA (1:3000), NF-κB P65 (1:3000), MMP-9 (1:3000). They were then incubated with horseradish peroxidase-conjugated secondary antibody (Bioss, Beijing, China) and visualized using the ECL plus detection kit (Pierce Protein Research Products, Rockford, IL, USA). Protein expression was quantified by densitometry using Image J (Bio-Rad, Hercules, CA, USA) with β-actin as an internal loading control.
- Wound Healing Assay
Wound healing assay was used to evaluate the migration of VSMCs. VSMCs (4×104 cells/well) were seeded and cultured in a six-well plate. After VSMCs grown to 80% confluence, VSMCs were incubated in serum-deprivation media for 24 h, and then a sterile plastic 1 ml micropipette tip was used to create a 1 mm scratch wound. Cells were continually cultured in the different culture media as mentioned above. The scratched region was photographed immediately and at 48h after scratching, and the migrating distance was thus calculated using image pro plus 6.0 (Shi, et al., 2017).
- Trans-well Assay
VSMCs migration was analyzed by Trans-well assay. The cells were cultured in the different culture media as mentioned above. After 24 h, cells were trypsinized with 0.25% (v/v) trypsin and re-suspended in the serum-free DMEM. Cell were counted and seeded in the upper chamber of each Trans-well at the concentration of 1×105 cells in 0.2 ml serum-free DMEM. 0.8 ml of DMEM supplemented with 20% FBS (Huang, et al., 2013) was added to the lower chamber of each Trans-well. Chambers were incubated for 12 h at 37°C with 5% CO2. Cells that migrated to the underside of the Trans-well filter were fixed with 4% formaldehyde (w/v) for 20 min at room temperature and then stained with DAPI (1:10) for 10 min. The staining was examined by fluorescence microscopy at 200× magnification. The numbers of cells were calculated using image pro plus 6.0.
- Mitochondrial Membrane Potential Assay
VSMCs (4×105 cells/well) were seeded in 6-well plates. After VSMCs grown to 30% confluence, VSMCs were incubated in serum-deprivation media for 24h. And then the cells were cultured in the different culture media as mentioned above. Subsequently, mitochondrial membrane potential was analyzed by mitochondrial membrane potential assay kit with JC-1 according to manufacturer’s instructions (Solarbio, Beijing, China).
- Measurements of Intracellular ROS
Intracellular ROS was detected using the oxidant-sensitive probe DCFH-DA. VSMCs were seeded on six-well plates. After starving with serum-free DMEM medium for 24 h, the cells cultured in the different culture media as mentioned above for 48 h. And then cells were washed twice with PBS and were incubated with 5 μmol/L of DCFH-DA for 30 min. The relative DCF fluorescence intensity was detected by fluorescent microscopy (Nikon, Tokyo, Japan). The examination wavelength was 488 nm and the emission wavelength was 530 nm respectively.
- Statistics analysis
All statistical analysis of the data was performed using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA). The results are presented as the means ± standard deviation (SD) of at least three independent experiments. One-way analysis of variance was used for statistical analysis of the data. In all cases P values less than 0.05 were considered significant.