Construction of AD mouse model
All animal experiments were approved by The Institutional Animal Care and Use Committee of First Affiliated Hospital of Gannan Medical University. Eighteen C57BL/6 male mice (3 weeks old, weighing 10–12 g) were obtained from SPF Beijing Biotechnology Co., Ltd. (Beijing, China). To construct an animal model of AD, mice were given 0.1% β-aminopropionitrile (BAPN; Sigma-Aldrich, St. Louis, Missouri, USA) in drinking water for four weeks and the body weights were measured and recorded weekly. Angiotensin II (Ang II; 1,000 ng/kg/min; Sigma-Aldrich) was then administered subcutaneously using an Alzet osmotic minipump (model 2004, Durect Corp; Cupertino, CA, USA)17. Mice in the control group were administered normal water and then received osmotic minipumps filled with normal saline (0.9% sodium chloride). After 72 h, the mice were deeply anesthetized and euthanized by an intraperitoneal injection of sodium pentobarbital (60 mg/kg). The thoracic aorta of the mice was harvested to observe the formation of AD and perform a pathological examination. In cases of premature death, the mice were promptly dissected and blood accumulation in the thoracic and abdominal cavities was detected to determine whether the aortic coarctation had ruptured. Tissue and blood samples were collected from mice for further analysis. The LILRB4 knockdown model was constructed using adeno-associated virus serotype 2 (AAV2; ViGene Biosciences, Shandong, China), which has broad tropism, allowing it to infect many cell types and tissues, especially the liver. Therefore, AAV2 is a versatile tool in gene therapy and biomedical research18,19,20. All mice were randomly divided into three groups (six per group): control (untreated AD mice), AD + AAV-NC (negative control group), and AD + AAV-shLILRB4 (LILRB4 knockdown group). Mice in the AD + AAV-shLILRB4 group were injected with recombinant AAV-shLILRB4 adenovirus at a titer of 1 × 1011 vg/mL in the tail vein. In the AD + AAV-NC group, mice were injected with the same amount of AAV-shNC adenovirus in the same way.
Cell culture and processing
Human aortic smooth muscle cells (HASMCs) were acquired from icellbioscience biotechnology Co. Ltd. (Shanghai, China). Cells were cultured in SMC medium (B310-500, Sigma-Aldrich) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific, New York, USA), 1% smooth muscle cell growth supplement (311-GS, Sigma-Aldrich), and 100 U/mL penicillin and 100 μg/mL streptomycin in 37 °C with 5% CO2. Platelet-derived growth factor BB (PDGF-BB) is an essential mitogen that promotes the phenotypic switching and migration of VSMCs and significantly contributes to AD21. To simulate AD in vitro, cells were exposed to PDGF-BB (20 ng/mL; P3201, Sigma-Aldrich) for 12 h. In addition, HASMCs were pre-treated with AG490 (10 μM, Santa Cruz Biotechnology, Santa Cruz, California, USA) for 1 h to inhibit the JAK/STAT signaling pathway.
Cell transfection
LILRB4 expression in HASMCs was suppressed using siRNAs targeting LILRB4 (si-LILRB4). A non-targeting siRNA (si-NC) was used as a control (Supplementary Table 1 for primer sequences). The cells were seeded into a six-well plate at a density of 1 × 105 cells per well. Once the cell density reached 30–50%, transfection was performed. A total of 20 pmol si-NC or si-LILRB4 was introduced into HASMCs using Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer’s guidelines.
Hematoxylin–eosin (HE) staining
At the end of the experiment, the aorta was rapidly removed and cleaned with saline. The entire aorta was fixed in 4% paraformaldehyde for 24 h. Further, aorta tissues were paraffin-embedded and sliced into 4 µm slices. The slices were stained with hematoxylin, rinsed under a running water stream, soaked in 1% hydrochloric acid ethanol, dehydrated, and immersed in an eosin solution. After staining, the sections were sealed with neutral gum and observed under a light microscope (OLYMPUS, Tokyo, Japan).
Biochemical analysis
Serum samples were collected to assess the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in liver tissues; blood urea nitrogen (BUN) and creatinine (Cr) in kidney tissues; superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels in liver and kidney tissue using a biochemical autoanalyzer (Fuji Medical System, Tokyo, Japan).
Enzyme-linked immunosorbent assay (ELISA)
Tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and interferon-gamma (IFN-γ) levels were measured in serum of mice using ELISA kits (Esebio Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instructions.
Western blot analysis
Protease inhibitor-supplied radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China) was used to lyse mouse aortic tissue and HASMCs. Protein samples were separated using 10% SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membranes (Roche, Basel, Switzerland). The PVDF membranes were coated with 5% defatted milk powder for 2 h. Then PVDF membranes were incubated with primary antibodies (dilution: 1:2000) overnight at 4 °C. The blots were then incubated with the corresponding secondary antibodies for 1 h at room temperature. Protein blots were visualized using ECL luminescent reagent (Amersham, Little Chalfont, UK) and analyzed using the ImageJ software. The primary antibodies in this study included anti-LILRB4 (ab229747, Abcam, Cambridge, UK), anti-α-SMA (ab5694, Abcam), anti-SM22α (ab14106, Abcam), anti-S100A4 (ab197896, Abcam), anti-LOX (ab174316, Abcam), anti-MMP2 (ab250476, Abcam), anti-MMP9 (ab283575, Abcam), anti-NLRP3 (ab263899, Abcam), anti-GSDMD-N (ab215203, Abcam), anti-caspase-1 (D7F10, Cell Signaling, Massachusetts, USA), and anti-β-actin (ab5694, Abcam).
Cell counting kit-8 (CCK8)
Cells (1 × 104) were seeded in a 96-well plate and cell viability was assessed. Cells were treated with 10 µL of CCK-8 reagent (Solarbio) at predetermined intervals (0, 24, 48, and 72 h) and then cultivated for 2 h. The absorbance was measured at 450 nm using a microplate reader (DALB, Shanghai, China) to quantify cell viability.
Wound healing assay
The cells were plated in six-well plates at a density of 1 × 104/well. A straight-line scratch was created at a cell concentration of 80% or higher using a pipette tip. Subsequently, cells were grown in serum-free SMCM medium. Images were captured at 0 and 24 h and ImageJ software was used to analyze cell migration.
Apoptosis and cycle analysis
Apoptosis in HASMCs was measured using FITC-labeled Annexin V and propidium iodide (PI), according to the manufacturer’s guidelines. Briefly, a total of 100 µL of cell suspension and 5 µL of FITC-labeled Annexin V were mixed and incubated for 5 min at room temperature in the dark. Subsequently, 5 µL of PI solution was introduced, followed by 400 µL of PBS. Apoptosis was analyzed using flow cytometry. For cell cycle analysis, HASMCs were stained with PI for 30 min at room temperature after being fixed with 70% ethanol for an overnight at 4 °C. The G0-G1, S, and G2-M phase cell percentages were determined by flow cytometry (Becton, Dickinson and Company, New Jersey, USA).
Signaling pathway screening
AD-related genes were screened using GeneCards (https://genealacart.genecards.org/). Genes co-expressed with LILRB4 were identified using the COXPRESdb database (https://coxpresdb.jp/). The Venn tool (https://bioinformatics.psb.ugent.be/webtools/Venn/) was used to identify shared genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the shared genes was performed using the DAVID database (https://david.ncifcrf.gov/).
Statistical analysis
Three independent experiments were conducted. GraphPad Prism software (version 8.0) was used for the data analysis. The findings are reported in the form of the mean ± standard deviation (SD) of the measures. Statistical differences between two groups were evaluated using a t-test. For multiple group comparisons, ANOVA was used and Tukey’s multiple comparison test was performed. Statistical significance was set at p < 0.05.
Ethical approval
The experiments conformed to the Guide for the Care and Use of Laboratory Animals. Animal study has been approved by the Animal Ethics Committee of First Affiliated Hospital of Gannan Medical University (LLSC2023-153). All methods are reported in accordance with ARRIVE guidelines.