High-fatty diet (HFD) model and Rb1 administration
All the animal experiments were performed following the accepted principles for laboratory animal use and care as found in for example the European Community guidelines with the approval of the Ethics Committee of the First Affiliated Hospital of Shandong First Medical University. Six-week-old male Sprague Dawley (SD) rats (180-200 g) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and housed individually under 12-h light/dark cycle with ad libitum to water. To assess the effects of Rb1 on endothelium injures, 12 rats were randomly divided into four groups (three for each group): Control group, rats fed with a standard chow diet (24% protein, 66% carbohydrates, and 10% fat) for ten weeks; HFD group, rats fed with a HFD (20% protein, 20% carbohydrates, and 60% fat) for ten weeks; HFD + L group, rats subjected to HFD model and the diet containing low dose of Rb1 (20 mg/kg body weight) (Victory, Sichuan, China); HFD + H group, rats subjected to HFD model and the diet containing low dose of Rb1 (40 mg/kg body weight).
Detection of hemodynamics parameters
Upon completion of the experiments, the changes in hemodynamics parameters of model rats, including interventricular septal thickness (IVS), left ventricular posterior wall thickness (LVPW), aortic diameter (AO), left ventricular internal diameter (LV), and right atrium diameter (RV) were measured using the algorithms of ultrasound system using Philips iE33 system (Philips Ultrasound, Bothell, WA). Then rats were sacrificed with i.p. injection of overdose (200 mg/kg body weight) pentobarbital sodium, and the abdominal aorta tissues and blood samples were collected for subsequent detections.
Detection of blood lipids and cytokines
The blood levels of lipid species, including total cholesterol (TC), high-density lipoprotein (HDL), low density lipoprotein (LDL), and triglyceride were measured following the previously described method (23). The blood levels of cytokines, including IL-6 (H007, Nanjing Jiancheng Bioengineering Institute, China), IL-1β (H002, Nanjing Jiancheng Bioengineering Institute, China), and TNF-α (H052, Nanjing Jiancheng Bioengineering Institute, China), were measured using corresponding ELISA kits following the procedures in the manufacturers’ instructions.
Detection of miR-21-5p/KIRT1 axis activity
Total RNA was extracted using Trizol method and cDNA templates were achieved from RNA using M-MLV according to the manufacturer’s instruction. The relative expression levels of different genes (miR‐21-5p, forward: 5′‐CGGCGGTAGCTTATCAGACTGA‐3′, reverse: 5′‐ATCCAGTGCAGGGTCCGAGG‐3′; U6, forward: 5′‐AGAGAAGATTAGCATGGCCCCTG‐3′, reverse: 5′‐ATCCAGTGCAGGGTCCGAGG‐3′) were detected using ExicyclerTM 96 (BIONEER, South Korea) and calculated according to the formula of 2-△△ct.
Total protein was extracted using the RIPA lysis buffer and was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were firstly incubated with primary antibodies, including KIRT1 (1:500) (ab196025, Abcam, USA) and GAPDH (1:1000) (ab8245, Abcam, USA), at 4˚C overnight and then with secondary IgG-HRP antibodies at 37˚C for 45 min. Protein blots were developed using Beyo ECL Plus reagent (P0018, Beyotime Biotechnology, China) and the images were scanned in the Gel Imaging System (WD-9413B, Liuyi Factory, China). The relative expression levels of the different proteins were calculated based on the data of integrated optical density measured with Gel-Pro-Analyzer (Media Cybernetics, USA).
Cell culture and treatments
HUVECs were purchased from Wuhan Procell Life Science and Technology and cultured in endothelial cell culture medium (Ham’s F-12K) supplemented with 10% fetal bovine serum (FBS) in an atmosphere consisting of 5% CO2 at 37°C. To detect the protective effects of Rb1 on HUVECs against ED, the cells were divided into four groups: Control group, healthy HUVECs cultured in a density of 2 × 105 cells/mL. OxLDL group, HUVECs (2 × 105 cells/mL) incubated with oxLDL (Guangzhou Yiyuan Biotechnologies, Guangzhou, China) for 24 h. OxLDL + Rb1 group, HUVECs (2 × 105 cells/mL) incubated with oxLDL (Guangzhou Yiyuan Biotechnologies, Guangzhou, China) and 30 μM Rb1 for for 24 h.
Detection of cell apoptosis
Apoptotic rate in HUVECs was detected using the apoptosis detection kit (KGA106, KeyGEN BioTECH, China): briefly, 5 μl Annexin V was incubated with cells for 10 min at room temperature. Then the cells were resuspended with 1×Binding buffer and added with 5 μl Propidium Iodide (PI). The total apoptotic rate was analyzed using a FACScan flow cytometer (Accuri C6, BD, USA), which was equal to the sum of the late apoptotic rate and the early apoptotic rate.
Modulation of miR-21-5p level
To determine the role miR-21-5p in the protective effects of Rb1 on HUVECs, HUVECs were further transfected with miR-21-5p using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) 24 h before oxLDL and Rb1 (30 μM) treatments. Then the effects of miR-21-5p overexpression on cell apoptosis and inflammation were detected.
Dual luciferase assay
Wild type (WT) and mutant type (MUT) of KRIT1 3’UTR were inserted into pmirGLO plasmid (between NheI and Sal I) to form luciferase vector. Mimics of miR-21-5p (5’-UAGCUUAUCAGACUGAUGUUGA-3’) and negative control (NC) mimics (5’-UUCUCCGAACGUGUCACGUTT-3’) were used to detect the bind of miR-21-5p to KIRT1 3’UTR. Transfections int HUVECs were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Luciferase activity of firefly was detected with Luciferase Activity Detection Kit (E1910, Promega, USA) using a Chemiluminescence Apparatus (Lumat LB 9507, Berthold, German).
Statistical analysis
Data were expressed in the format of mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with post-hoc comparisons using Tukey method was performed using Graphpad Prism version 6.0 (GraphPad Software, Inc., San Diego, CA) with a significant level of 0.05 (two-tailed).