Animal experiments
Twelve ApoE−/− female mice (8 weeks, 18–21 g) and 3 C57BL/6 female mice (8 weeks, 18–21 g) were purchased from Beijing Vital River Experimental Animal Technology Co, Ltd. (Beijing, China). All mice maintained a normal diet and circadian rhythm for one week. Then, the ApoE−/− female mice were fed with high-fat diet (21% fat and 0.15% cholesterol). The C57BL/6 female mice were fed for normal diet for 12 weeks, and at the last 4 weeks the mice were randomly assigned into 4 groups (3 in each group): MOD group (high-fat diet treatment), T10 group (high-fat diet treatment + 10 mg/kg Tan injection each day), T30 (high-fat diet treatment + 30 mg/kg Tan injection each day), T90 (high-fat diet treatment + 90 mg/kg Tan injection each day). Tan solution was purchased form Sigma-Aldrich (Shanghai, China). The C57BL/6 female mice with normal diet were as CON group.
Then the mice were intraperitoneally anaesthetized with 75 mg/kg pentobarbital sodium (Sigma-Aldrich, Shanghai, China). The blood was collected by the 1.0-1.5-mm glass capillary (immersed in 1% heparin solution) through eyeball insertion. The blood was added with ethylene diamine tetra acetic acid solution (Thermo Fisher Scientific, Rockford, IL, USA) immediately to avoid clotting, and allowed to stand still in an Eppendorf tube (Sangon Bio., Shanghai, China) for 1 h at room temperature. After that, the blood was centrifugated at 4℃ at 1200 g for 10 min with the supernatant removed, and then store at − 80℃ for subsequent analysis. The mice were injected with pentobarbital sodium at 75 mg/ kg intraperitoneally and moistened with gauze. After anesthesia, the skin of the anterior chest was cut to expose abdominal cavity and chest. A total of 500 mL phosphate buffer saline (PBS) solution (Thermo Fisher Scientific, Rockford, IL, USA) was pumped into the left ventricle using a DL10-14 peristaltic pump (LEAD FLUID, Baoding, Hebei, China) at 4℃ until the outflow fluid was clear. The perfusion was lasted for 4 h from a relatively slow speed to a higher speed until the death of animals, which was confirmed by observing the lack of heartbeat, respiratory arrest, and nerve reflex. After the perfusion, the lungs were cut off. Under the microscope (LIOOS600T, Trilobite, Beijing, China), the aortic trees were separated, the outer membrane fat was removed and the whole aortic tree was separated from the spine and fixed in 4% paraformaldehyde for subsequent experiments.
Hematoxylin-eosin (HE) staining
The cross-section tissues of the aorta of the experimental mice were fixed, embedded in paraffin, sliced at 4 µm, dewaxed in xylene for 5 min, soaked in alcohol of gradient concentration for 2 min respectively. After washed out alcohol, the sections were soaked in hematoxylin dye solution (Leagene, Anhui, China) for 12 min followed by floated color washing, soaked in 1% hydrochloric acid alcohol for 10 s, washed with running water for 40 min, and then immersed into eosin solution (Leagene, Anhui, China) for 4 min followed by rinsing. Subsequently, the sections were dehydrated in gradient alcohol, then transferred into xylene I and xylene II for transparency, and then sealed with gum and observed under a microscope.
Plasma lipid determination
Total cholesterol (TC), high-density lipoprotein (HDL), low density lipoprotein (LDL) and triglyceride (TG) were measured by automatic biochemical analyzer (RX-2000, Tarrytown, NY, USA). TC and TG were detected by enzyme method, and LDL and HDL were detected by turbidimetry.
Enzyme-linked immunosorbent assay (ELISA)
The contents of necrosis factor (TNF)-α and interleukin (IL)-6 were determined by Enzyme Immunoassay kits (Enzo Life Sciences, Shanghai, China). The specific antibody globulin was diluted to 10 ug /mL, which was added to the plate (0.3 mL/well) and incubated at 4℃ overnight. The cells were incubated with 0.2 mL diluted plasma at 37℃ for 1 h, 0.2 mL diluted enzyme labeled antibody solution at 37℃ for 1 h and 0.2 mL substrate at room temperature for 30 min. Finally, 0.05 mL H2SO4 was added to each well to terminate the reaction. The enzyme marker (OPD = 492 nm) was applied to determine optical density (OD) value.
Microarray analysis
Total RNA (0.5 µg) was extracted from mouse aortic plasma. cDNA was synthetized by GeneChip Invitro Tran-scription Express Kit (902789, Thermo Fisher Scientific, Rockford, IL, USA). Then, cDNA fragments were hybridized with miRCURY LNA Array v.16.0 (Axon GenePix, Sunnyvale, CA, USA). The microarray was washed and scanned by GeneChipTM Scanner 3000 7G system (000213, Thermo Fisher Scientific, Rockford, IL, USA).
RNA isolation and quantitation
Total RNA from cardiovascular tissues and THP-1‐derived macrophages was isolated by TRIzol. Reverse transcription and amplification of the same amount of RNA into the complementary DNA (cDNA) were conducted according to the manufacturer's instructions PrimeScript™RT Master Mix Kit (Takara, Tokyo, Japan). Gene expression was quantified by SYBR®Premix Ex Taq™II kit (Takara, Tokyo, Japan). The primers involved in this study were as follows:
miR-23b: Forward, 5′-CAGGCAAGATGCTGTTGCA-3′; Reverse, 5′-GCGAGCACAGAATTAATACGACTC-3′;
Tribbles 1 (TRIB1): Forward, 5′-ACGTTGGATGTAGAAGTCCCCTTCCCTTAG-3′; Reverse, 5′-ACGTTGGATGGAACAAGGACTTTCGTCCTC-3′.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed on Mx3005P quantitative PCR system (Stratagene, Shanghai, China) according to the instructions: 95℃ for 10 min, 40 cycles of 95℃ for 15 s, 55℃ for 20 s and 72 °C for 30 s. β-actin was used as an internal control. All samples were analyzed in triplicate. The relative expression levels of RNA were calculated using 2−ΔΔCt method.
Cell culture and treatment
Human monocytes THP-1 cells were obtained from the cultured specimens (American Tissue Culture Collection, Manassas, VA) and grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37℃ and an atmosphere of < 5% CO2. THP-1 cells were differentiated into macrophages by incubating with 150 ng/mL phorbol 12-myristate 13-acetate for 48 h. After washed by PBS, the cells were observed under a microscope (LIOOS600T, Trilobite, Beijing, China). THP-1-derived macrophages were stimulated with10 µmol/L Tan for 1 h at 37℃ and 5% CO2. Finally, the macrophages were washed by PBS after Tan absorption and used in further experiments.
Twenty-four h before transfection, the differentiated macrophages were cultured in antibiotics-free RPMI-1640 with 10% PBS and without and placed in a 24-well plate. Before transfection, the culture medium was changed to Opti-MEM I reducing serum medium (Grand Island, NY, USA). Using Lipofectamine 2000 (Invitrogen, Carlsbad, USA), the miR-23b mimic, empty plasmid, or small interfering RNA-targeting TRIB1 (si-TRIB1) were transfected to the macrophages for 6 h. The cell transfection efficiency was observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Transwell assay
After adding Matrigel (BD Biosciences, Franklin lakes, NJ, USA) to the apical chamber for 30 min under specific pathogen free condition, 30 µL RPMI-1640 medium was added to the apical chamber and placed in CO2 incubator for standby. The cells were digested, centrifuged, resuspended without serum and diluted to 5 × 105 cells/mL. then, 500 µL RPMI 1640 medium containing 10% FBS was added into the basolateral chamber of Transwell while 200 µL cell suspension was added into the upper chamber, and then the Transwell plate was cultured in 37℃, 5% CO2 incubator for 48 h. The chamber was taken out, and the culture medium was washed out with PBS. After that, the cells were stained with crystal violet for 10 min with the floating color and suspended cells washed off. Finally, the invasive cells were observed under the microscope (LIOOS600T, Trilobite, Beijing, China) and observed and the number of cells was counted.
Cell adhesion assay
Ten µg/mL of fibronectin (Abcam Inc., Cambridge, MA, USA) at 70 µL/well was injected into the 96-well plates and incubated overnight at 4℃. After 3 times of PBS cleaning, the cells were sealed with 1% bovine serum albumin (Sigma Aldrich, Shanghai, China) at 37℃ for 1 h. After PBS cleaning 3 times, the cells at the logarithmic growth phase were digested, suspended in the serum-free medium. The cell concentration was adjusted at 5 × 104 cells/mL and 100 µL/well. Each group was established three wells. The cells were incubated at 37℃ for 1 h, the suspension was washed off by PBS. The cells were fixed by formaldehyde and stained by crystal violet followed by PBS washing. Under the microscope, 5 visual fields were selected for photographing and counting. The average value was taken.
Dual luciferase reporter gene assay
The 3’UTR binding sequences of miR-23b were predicted by online prediction software Starbase (http://starbase.sysu.edu.cn/). The 3’UTR of wild type (WT) and mutant (MUT) of miR-23b was synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and inserted into pMIR-REPORTTM (Thermo Fisher Scientific Inc., Waltham, MA, USA) luciferase reporter vector [15]. Using the Lipofectamine 3000 transfection Kit (Invitrogen, Carlsbad, CA, USA), WT/MUT plasmids and miR-23b were co-transfected into THP-1 cells. After 24 h, the cells were lysed. Luciferase activity intensity (intensity = RLU1/RLU2, RLU1 is the luciferase activity of firefly, RLU1 is the luciferase activity of renilla) was detected by dual luciferase reporter assay system (Promega Corporation, WI, USA) [15].
Immunohistochemistry
After dewaxing and hydration, the sections of mouse aorta were infiltrated with sealing permeable liquid (Solarbio, Beijing, China) for 30 min. The sections were added into sodium citrate buffer solution (50 ×, Solarbio, Beijing, China) and heat for 6 min × 4 times, and dropped with 5% sheep serum (Solarbio, Beijing, China) and allowed to stand still for 30 min at room temperature. The serum was removed and the cells were incubated with the primary antibodies against nuclear factor kappa B (NF-κB) (ab16502) and Toll-like receptor 4 (TLR4) (1:500, ab13556) at room temperature for 1 h and 4℃ overnight. Then, the cells were washed by PBS and incubated with the secondary antibody (1:50000, ab205718) at 37℃ for 30 min. subsequently, the cells were incubated with streptavidin peroxidase (Solarbio, Beijing, China) at 37℃ for 30 min. finally, the cells were added with Diaminobenzidine (20×, Solarbio, Beijing, China) for 5 min followed by 20-s hematoxylin staining. The cells were dehydrated, sealed in gum and observed under a microscope.
Statistical analysis
All statistical analyses were performed using SPSS 21.0 (IBM Corp. Armonk, NY, USA). Data were in normal distribution according to Kolmogorov-Smirnov method and described as mean ± standard deviation. Differences between two groups were analyzed by t test. Differences among multiple groups were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA. Tukey’s multiple comparisons test was used for the pairwise comparison after ANOVA analysis. p was obtained by two-tailed test and p < 0.05 was considered statistically significant.