Construction of an atherosclerosis model in type 2 diabetes mellitus mice

The present study used normal 8-week-old male C57BL/6 mice and mmu-miR-351−/− male C57BL/6 mice (mmu-miR-351 gene knockout mice were commissioned from Fudan University, Shanghai). They were divided into four groups: 1. WT group, wild-type C57BL/6, labeled WT. 2. miR-351−/− group: mmu-miR-351 knockout C57BL/6, labeled miR-351−/−. 3. HWT group: WT mice + high-fat diet. 4. H + miR-351−/− group: miR-351−/− group + high-fat diet. WT and miR-351−/− mice were the control groups, and HWT and H + miR-351−/− were the model groups. Each group contained 10 mice (each weighing 20 to 25 g). Referring to the Konstantin method used, mice in the HWT and H + miR-351−/− groups were used to construct a type 2 diabetes model (Belosludtsev et al. xxxx). The high-fat diet consisted of 20% lard + 2% cholesterol + 80% base feed (Maohua Bio, Shenyang, China). Atherosclerosis is induced by hyperlipidemia, and the whole induction process lasts for 6 months. At the end of the experiment, rats were killed by an overdose of 2% phenobarbital sodium. An eyeball was removed for blood collection, and the aortic arch and abdominal aorta were fixed in neutral formaldehyde solution. Part of the aorta was used for protein and gene detection.

This study was approved by the Shengjing Hospital of China Medical University Animal Care and Use Institutional Committee. The animals were euthanized with an overdose of phenobarbital when they rapidly lost 15–20% of their body weight or when they exhibited signs of eating disorders or organ infections.

Detection of blood biochemical indices

Peripheral blood was taken for testing between 8:00 am and 10:00 am after 6 months of high-lipid induction (n = 10). Triglycerides (TG, cat no. A110-1-1), low-density lipoprotein cholesterol (LDL-c, cat no. A113-1-1), high-density lipoprotein cholesterol (HDL-c, cat no. A112-1-1), nitric oxide (NO, cat no. A012-1-2), hypersensitivity C protein, hs-CRP (E024-1-1), and serum total cholesterol (TC, cat no. A111-1-1) were detected using kits from the Nanjing Jiancheng Institute of Bioengineering.

Detection of tissue biochemical indices

Fifty milligrams of aortic arch tissue was weighed, and 450 μL of PBS was added. Then, the tissue was homogenized by a tissue homogenizer to prepare a 5% tissue homogenate. The supernatant was centrifuged, and superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GSH-Px) were detected according to the kit instructions provided by Nanjing Jiancheng Institute of Bioengineering (n = 5).

Oil red O staining

After the mice were sacrificed, the thoracic aorta was removed and fixed in neutral formaldehyde solution at 4 °C for 24 h. The tissues were then immersed in Oil red O (60% isopropyl alcohol) for 1 h in PBS and stained for 15 min. The remaining oil red O dye was rinsed away with 60% isopropyl alcohol, and the specimens were washed with distilled water 3 times.

Pathological staining

Aortic arch and abdominal aorta tissue sections were taken from each group. After dewaxing with xylene and soaking in gradient concentrations of alcohol, hematoxylin–eosin staining and Masson staining were performed. Masson staining was performed following the instructions provided for the SolarBio kit (cat no. G1345). The slides were dehydrated and cleared with xylene, covered with a cover plate, sealed with neutral adhesive, and observed under an optical microscope (Leica, DM500). Oil red O staining of cell parts was be performed according to the staining method provided in the manual.

Quantitative real-time PCR

The gene expression levels of mmu-miR-351, endothelin 1 (EDN1), prostacyclin (PTGIS), angiotensin II (AGT), endothelial nitric oxide synthase (NOS3), cell adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1) were detected by real-time PCR. The TRIzol method was used to extract total RNA from mouse tissues or aortic endothelial cells. RNA was reverse-transcribed into cDNA using a reverse transcription kit (gDNA Purge, cat no. E047-01A, Novoprotein, JiangSu, China), and gene expression was detected according to the operating instructions of the NovoStart® SYBR qPCR SuperMix Plus kit (Novoprotein). Gapdh was used as an internal reference. For qPCR detection of miRNA, the relative expression level of the mmu-miR-351-5p gene was detected by the Guangzhou RiBo Biological miDETECT A Track miRNA qRT‒PCR Starter Kit. The U6 gene was used as the reference gene for miRNA qPCR detection. The relative gene expression levels were calculated by 2−ΔΔCt, and 3 replicates were performed for each sample. All the above qPCR tests were performed on an ABI 7500 system. Conventional amplification conditions included predenaturation at 95 °C for 5 min, denaturation at 95 ºC for 20 s; annealing at 60 °C for 20 s for 40 cycles, and extension at 72 °C for 10 min. The primer sequences are shown in Additional file 1: Supplementary materials 1-1.

Western blot

The following antibodies were used in this study: EDN1 (cat no. 12191-1-AP, Proteintech, USA), PTGIS (cat no. ER64571, HuaBio, Hangzhou, China), AGT (cat no. ET1705-6, HuaBio), NOS3 (cat no. A1548, ABclonal), ICAM1 (cat no. ER1910-98, HuaBio), VCAM1 (cat no. ET1601-18, HuaBio), and GAPDH (cat no. EM1101, HuaBio). Fifty milligrams of aorta or 5 × 106 cells were lysed with 200 μL RIPA protein lysis buffer. Specific methods can be found in the research of Zhang et al. (Zhang et al. 2021). ImageJ was used to quantify each protein band, and GAPDH was used as an internal reference gene for normalization.

For the cell experiment, the required cells were collected. After adding 100 μL of protein lysate, western blotting was performed according to animal experiments. The primary antibodies against ITGB3 (cat no. ET1702-41), Bcl-2 (cat no. ET1702-53), Bax (cat no. EM1203), active Caspase-3 (cat no. ET1602-47), and GAPDH were all purchased from Hua 'an Biotechnology Co., Ltd. Antibodies against PIK3R1 (cat no. 4249S), p-PIK3R1 (cat no. 4228S), Akt (cat no. 4691S), and p-Akt (cat no. 4060S) were purchased from Cell Signaling Technology. The experimental results were quantitatively analyzed by ImageJ software.

Construction of the atherosclerotic cell model

Mice were sacrificed by cervical dislocation, and cardiac aorta tissues were isolated from normal C57BL/6 J mice (aged 3–4 weeks). Primary culture of aortic endothelial cells (AECs) was carried out by referring to the method of Pedro et al. (Molina-Sánchez and Andrés 2015). All AEC cells used in the experiment were isolated from the 2nd generation to the 4th generation. Two to four generations of primary AEC were taken and treated with DMEM (containing 10% FBS + 1% penicillin/streptomycin) containing 50 μg/mL oxidized low-density lipoprotein (ox-LDL) and 30 mM glucose (HG) for 48 h to induce the diabetic atherosclerosis model (Wang and Bai 2021; Zhang et al. 2020), denoted as the model group. The Control group was left untreated. The cells were cultured in a constant temperature incubator with 5% CO2 at 37 ºC.

Primary identification of aortic endothelial cells

In cultured primary cells, immunofluorescence detected the expression levels of CD31 and CD34 markers in aortic endothelial cells. First, the primary cells were treated with silver, and after the cells were covered with cover glass, they were fixed with 4% paraformaldehyde. After PBS washing, 1% BSA was added and incubated at room temperature for 30 min. Rabbit anti-mouse CD31 (cat no. ER31219, Hua 'an) and CD34 (cat no. ER0802, Hua 'an) antibodies were added and incubated at 4 ºC overnight. After washing with PBS 3 times, red and green fluorescent-labeled secondary antibodies were added and incubated for 1 h at room temperature. DAPI was used to stain cells for 10 min with 1 μg/mL DAPI. After dropping the anti-fluorescence quenching agent, the tablets were sealed with nail polish. The results were photographed by a fluorescence microscope (Leica, DM500).

Construction of miR-351-silenced cell lines

The NCBI website (https://www.ncbi.nlm.nih.gov) was searched to obtain the sequence of mmu-miR-351 in mice, and the shRNA sequence was designed through the GPP Web Portal online website (https://portals.broadinstitute.org/gpp/public/), taking the first three higher-score sequences of mmu-miR-351-5p for the carrier construction. Two complementary single-stranded oligonucleotides containing the target sequence were chemically synthesized and annealed. Double-stranded oligonucleotides were inserted between the pLKO-CMV-GFP-puro plasmid AgeI and EcoRI restriction sites, and the linked plasmids were transformed into Escherichia coli DH-5α competent cells for plasmid amplification. Plasmids from positive colonies were verified by RT‒PCR and DNA sequencing.

The recombinant lentivirus was prepared by cotransfection. The lentiviral vectors △8.91 and pVSV-G (10:10:1) were transfected into 293 T cells (X-Tremegene HP DNA transfection reagent, Roche) using the cationic lipid complex method. The cell density was 1 × 106 cells/pore. The cells were divided into a control group (negative control lentiviral vector infection) and a mmu-miR-351 shRNA 1–3 group (lentiviral vector infection). AECs were seeded into 6-well plates at a density of 5 × 105 cells/well. The next day, cells were infected with viruses at the same titer containing 2 µg/mL polybrene, and after 72 h of infection, the medium was replaced with medium containing 2 μg/mL puromycin. Cell growth was maintained for 7–9 days. The medium was then replaced with medium without puromycin, and some cells from each group were used for qPCR detection of the mmu-miR-351 expression level to determine the silencing efficiency. The primer sequences are shown in Additional file 1: Supplementary materials 1-2.

Cell transfection

The siRNA sequence of ITGB3 was designed according to its CDS region and was commissioned to be synthesized by Guangzhou RiboBio. When the convergence degree of lentivirus-infected AECs was 70–90%, the ITGB3 siRNA (50 nM) was transfected with Lipofectamine RNAiMAX (Thermo Fisher Scientific), and the cells were incubated in a CO2 incubator at 37 °C for 48 h. The standard medium was replaced, and a portion of the cells was used for qPCR and western blot detection. The other portion of the cells was treated with 50 μg/mL ox-LDL for 48 h for subsequent experimental detection. The siRNA primer sequences are shown in Additional file 1: Supplementary materials 1-3.

Measurement of cell viability by MTT

The cells were inoculated into 96-well plates (Gibco-BRL) with 200 μL medium per well and cultured at 37 °C for 24 h. The plates were then treated with medium containing 50 μg/mL ox-LDL and 30 mM glucose for 48 h, and 10 μL MTT (5 mg/mL) was added to each well. The plates were then incubated at 37 °C for 4 h. The medium was removed, 150 μL DMSO (Sigma‒Aldrich) was added to each well, and the plate was shaken for 5 min. A microplate reader (Molecular Devices, USA) was used to perform absorbance measurements at 570 nm.

DAPI staining

MiR-351-silenced and normal AEC cells (1 × 105) were plated in 6-well plates, and 3 cover slides were placed at the bottom of each well plate. When the cells grew to 90% confluence, the medium was replaced with medium containing 50 μg/mL oxidized low-density lipoprotein (ox-LDL) and 30 mM glucose for 48 h. After removing the cover glasses, the cells were fixed with methanol at 4 °C 15 min, washed with PBS 3 times, and then incubated with 1 μg/mL DAPI drops for 10 min. After washing with PBS 3 times, the anti-fluorescence quenching agent was added dropwise, sealed with nail polish, and photographed. The results were obtained with a fluorescence microscope (Leica, DM500).

Apoptosis detection

Apoptosis was detected by Annexin V-FITC/PI double staining. Lentivirus-infected cells in each group were treated with 50 μg/mL ox-LDL and 30 mM glucose for 48 h by following the instructions of the Solarbio (Beijing, China) Annexin V-FITC/PI apoptosis detection kit (cat no. CA1020). The cell apoptosis rate was detected by an Attune N × T flow cytometer (ABI, USA). The experiment was repeated three times.

Oil red O staining

A total of 5 × 104 cells from each group were seeded and cultured in 24-well plates. After 24 h, the cells had adhered to the wall, and the convergence degree was approximately 70%. After 48 h, oil red O staining was performed according to the instructions of the oil red O staining kit (cat no. G1262, Solarbio). The cells were imaged with an inverted microscope (Leica).

Reactive oxygen species (ROS) detection

Superoxide anion formation was detected by dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma‒Aldrich). The 2nd and 3rd generations of myocardial microvascular endothelial cells were taken and inoculated on cover glasses. After cell adherence, the cells in each group were treated according to their pregroup. After treatment, each group was divided into two EP tubes with no less than 1 × 106 cells in each tube. DCFH-DA solution was added to the cell culture medium at a final concentration of 5 μmol/L and incubated for approximately 30 min. After the cell slides were removed and washed with PBS, one tube was taken from each group, and the ROS content was detected by flow cytometry to determine the degree of oxidative damage to the cells. The fluorescence intensity was analyzed to quantitatively determine the degree of oxidative damage. The excitation wavelength was 504 nm, and the emission wavelength was 529 nm.

Oxidative stress index detection

Lentivirus-infected cells were treated with high glucose and low-density lipoprotein for 48 h and then digested and collected with trypsin. SOD (A001-3–2), MDA (A003-1–2) and GSH-Px (A005-1–2) indices were detected. All kits were purchased from Nanjing Jiancheng Institute of Biological Engineering.

Fluorescence in situ hybridization

Guangzhou RiboBio biosynthesis of the miR-351-5p FISH probe was commissioned, FISH hybridization was conducted according to the FISH kit instructions, and the influence of miRNA-351 expression on AEC cells cultured in slides after treatment with ox-LDL and high-glucose medium was determined.

Bioinformatics analysis

Genes related to atherosclerosis and vascular disease lipid metabolism disorders were obtained from the GeneCard website. Target gene prediction of the mmu-miR-351-5p sequence was performed using miRDB (http://mirdb.org). The obtained disease-related genes were screened by intersection with mmu-miR-351-5p-interacting genes to determine the target genes.

Dual-luciferase reporter assay

Partial sequences of the ITGB3-3'UTR containing wild-type and mutated miR-351 binding sites were cloned into pIS0 luciferase vectors (Promega, Madison, WI, USA) to generate ITGB3-WT and ITGB3-MUT. pRL-TK was used as an internal reference plasmid. 293 T cells were transfected at 60–80% fusion degree. The miR-351 mimic and NC mimic (2.5 μL, final concentration of 100 nM) were purchased from Genewiz Company for synthesis. Lipofectamine 2000 was used for transfection. A Promega GloMax was used to measure the dual-luciferase reporting system (Promega). Relative luciferase activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity. The primer sequences involved are provided in Additional file 1: Supplementary materials 1-4.

Statistical methods

In the animal experiments, 3 samples were randomly selected from mice in each group for experimental detection unless otherwise indicated. Cytological tests were performed in triplicate. All data are represented as the mean ± standard deviation. Statistical significance between groups was assessed by a two-tailed unpaired Student’s t test. All results were calculated using GraphPad Prism Software (version Prism 8; GraphPad Software, Inc.). P < 0.05 was considered to indicate a statistically significant difference.