Balloon injury model and treatment
The healthy male SD rats were provided by the Experimental Animal Center of Shanxi Medical University, weighing 250-300g. Standard feed and free drinking water. All animal experiments complied with the National Institutes of Health guide for the care and use of Laboratory animals. 2% sodium pentobarbital (50mg/kg) was used to inject intraperitoneally for anesthesia. Incising the skin and subcutaneous tissue along the anterior midline of the neck, and exposing the left common carotid artery and internal and external carotid arteries in the anterior cervical triangle. Then, the distal end of the external carotid artery was ligated; the blood flow at the proximal end of the common carotid artery was closed with a vascular clip. Incising a wedge-shaped inside the distal end of the external carotid artery, inserting a 1.5F balloon catheter from the external carotid artery to the beginning of the common carotid artery, and inflating the balloon to block the blood flow for 30 seconds, then slowly pulling the balloon back to the bifurcation of the internal and external carotid arteries for three times, and withdrawing the balloon. At last, the outside of the neck arteries was ligated, subcutaneous tissue and skin was sutured layer by layer.
The low-HRP (L-HRP) and high-HRP (H-HRP) groups were treated with 10 μg/kg and 100 μg/kg HRP (inhibitor of (P)RR, glbiochem, shanghai) respectively by tail vein injection. The positive control group was intragastric administration with 100 mg/kg Losartan (Los, antagonist of angiotensin I receptor, Sigma). All these treatments lasted for 17 consecutive days from 3 days before surgery.
Cell culture and treatment
Vascular smooth muscle cells (VSMCs) of rats, purchased from Procell (Wuhan, China), were cultured in Dulbecco's modified Eagle's (DMEM) medium (Gibco, USA) containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin, and 100 μg/ml of streptomycin under 5% CO2 at 37 °C. Cells were treated with different concentration of renin (Sigma-Aldrich, USA), 10-6 mol/L Los, 10-5 mol/L PD123319 (antagonist of angiotensin II receptor, Sigma), 10nmol/L platelet-derived growth factor-BB (PDGF-BB, PeproTech), or 10-5 mol/L diphenyleneiodonium chloride (DPI, inhibitor of NADPH, Sigma-Aldrich, USA). Renin, PDGF-BB, and DPI were added after cells were treated with Los and PD123319 for 30 min.
Construction of miRNA interference plasmid of (P)RR
Four pairs of miRNAs and negative control (NC) sequence were designed and synthesized according to the gene sequence (GenBank accession number: AB188298). Then, miRNAs and NC were all cloned into pcDNA™6.2-GW/EmGFP vector (Invitrogen, USA) to determine the most effective miRNA. Cells were transfected with miRNA-(P)RR using Lipofectamine™2000 reagent (Invitrogen, USA).
Cell counting kit-8 assay
Collecting the logarithmic phase VSMCs, and adjusting the cell suspension concentration to 1×104/well. Cells treated or transfected with different reagent were incubated with 5% CO2 at 37°C overnight until the cell monolayer covered the bottom of the 96-well plate. Then, cells in each well were added 10 μl CCK-8 (Solarbio, China) solution and continued to culture for another 4 h. Microplate reader was used to detect the optical density of each well at 450 nm wavelength.
Detection of cell cycle
Cells were seeded into 6-well plates at a density of 1×106/well and cultured overnight. Then, cells were treated or transfected with corresponding reagent and continue culturing for another 48 h at 37℃, 5% CO2 incubator. Collecting the cells after centrifugation, discarding the supernatant, washing the cells three times with pre-cooled PBS, adding pre-cooled 70% ethanol, and fixing the cells at 4°C overnight. Next, cells were collected, washed with PBS, and added into RNAase for incubation at 37℃, 30 min. Then, cells were stained with 50 μl PI and incubated at 4℃ for 30 min in dark. Lastly, cell cycle was detected by flow cytometry (FCM) and analyzed by ModFit software.
Measurement of MDA and SOD
VMSCs were seeded into plates and cultured overnight. Then cells were collected for experiments. The production of malondialdehyde (MDA) and antioxidant enzymes superoxide dismutase (SOD) in VMSCs were examined using the MDA assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and Superoxide Dismutase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively on the basis of the manufacturer's specification.
Measurement of ROS
Reactive Oxygen Species Assay Kit (Beyotime, China) was used in this experiment. Briefly, VMSCs were inoculated in a 6-well plate (1×104 cells/well), cultured overnight until the cells were fused to 70%-80%, and performed the experiment. The positive control group was treated with H2O2 at a final concentration of 100 μM/L for 1 h. At the time point, the culture medium was discarded and rinsed three times with PBS at room temperature. Then, cells were added the DCFH-DA fluorescent probe (diluted probe without serum DMEM) at the final concentration of 10 μM/L, incubated at 37°C in the dark for 20 min and rinsed three times with PBS for observing the intracellular fluorescence under a microscope. Collecting and transferring the cell suspension into a flow tube, adding 400 μl PBS, and measuring the fluorescence intensity with a flow cytometer to indirectly present the level of reactive oxygen species (ROS).
Immunofluorescence staining assay
VMSCs in a 12-well plate were treated with different reagents for 24 h. Then the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 15 min, and then washed with PBS for three times. 5% skimmed milk in PBS was then added to the desired wells for 30 min at 37°C. Then, the cells were washed and stained with primary antibody (P)RR (Sigma-Aldrich, USA) and alpha-smooth muscle actin (α-SMA) (Abcam, UK) at 4°C overnight. After six times washing, the cells were incubated with fluorescein isocyanate (FITC)-conjugated goat anti-mouse (KPL, USA) as secondary antibody at 37°C for 1 h. DAPI (Solarbio, China) was added for incubation in dark for 5min, and the excess DAPI was washed by PBS for 5 min. Finally, images were acquired using fluorescent microscope (Nikon, Tokyo, Japan) at 400× magnification.
H&E staining
Common carotid artery tissues from each group were fixed in 4% paraformaldehyde at room temperature, embedded in paraffin, sectioned at a thickness of 5 μm, and stained with hematoxylin and eosin (H&E). The vascular intimal hyperplasia and histopathological change were observed under the light microscope (Olympus, Tokyo, Japan) at 100× and 400× magnification and quantified by the intima/media area ratio (I/M).
Enzyme-linked immunosorbent assay (ELISA)
Arterial blood from mice and VMSCs in each group were collected, the levels of renin (Abcam, UK) and PDGF-BB (PeproTech, China) in mice and proinflammatory cytokines including tumor necrosis factor-α (TNF-α, Beyotime, China), interleukin-6 (IL-6, Abcam, UK), interleukin-1β (IL-1β, Beyotime, China), and inducible nitric oxide synthase (iNOS, Abcam, UK) were determined by ELISA based on the manufacturer’s instructions. The OD value of each well was immediately read at 450 nm.
Real time-PCR
Total RNAs from common carotid artery wall tissues or the VMSCs were isolated using TRIzol reagent according to the manufacturer's protocol (Invitrogen, Thermo Fisher Scientific). The expressions of NOX1, cyclin D1, proliferating cell nuclear antigen (PCNA) in tissues and (P)RR, NOX1, cyclin D1, PCNA in cells were determined using the SYBR Premix Ex TaqTM II Kit (Takara, Tokyo, Japan) on the basis of the manufacturer’s protocol. β-actin served as a control. The 2‑ΔΔCt method was used to quantify the relative expression levels of (P)RR, NOX1, cyclin D1, and PCNA.
Western blot assay
To confirm the expression levels of ERK1/2, p-ERK1/2, AKT, p-AKT, cyclin D1, PCNA, (P)RR, and NOX1 in common carotid artery wall tissues and VMSCs, HTR8/SVneo cells were lysed with RIPA buffer. Lysates were separated on 10% SDS-polyacrylamide gel and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Then, the membranes were blocked with 5% non-fat milk for 1 h at room temperature, and membranes were incubated with corresponding primary antibodies at 4ºC overnight following incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Protein expressions were detected with high sensitivity ECL chemiluminescence detection kit (Vazyme, China). The bands intensity was expressed as fold change by normalizing the data to the values of β-actin using ImageJ software.
Statistical analysis
Statistical analysis was performed by SPSS22.0 and GraphPad Prism 9 software. Data were showed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) or two-way ANOVA were used to compare the difference among multiple groups followed by post-hoc tests. p<0.05 was thought to be statistically significant.