Cell culture and experimental treatments

THP-1 cells were purchased from Procell (Wuhan, China). THP-1 monocytes (1.0 × 105 cells/mL) were added dropwise into six-well plates with complete medium containing 100 ng/ml phorbol-12-myristate-13-acetate (PMA; Sigma Chemical Co. St. Louis, MO, USA) and incubated for 48 h. THP-1 macrophages were treated with 1 µM nicotine for 24 h. In addition, drugs and inhibitors, such as n-acetyl cysteine (NAC, 1 mM), 3-TYP (50 µM), VX-765 (10 µM) and Z-DEVD-FMK (100 µM), were administered before MT (30 mM).

Establishment of the in vivo AS model

This study was approved by the Laboratory Animal Ethics Committee of Tianjin Chest Hospital on January 5, 2023 (registration number: TJCH-2003-001). All animal experiments were performed according to procedures approved by the Laboratory Animal Ethics Committee of Tianjin Chest Hospital and complied with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and regulations.

Ten-week-old male ApoE−/− mice (weighing 25.5–29.5 g) were divided into six groups of 10 mice each: the standard diet group (NCD), the standard diet group treated with nicotine (NCD + nicotine), the standard diet group treated with nicotine and MT (NCD + nicotine + MT), the high-fat diet (0.15% cholesterol and 21% fat) (Huafukang, Beijing, China) group (HFD), the high-fat diet group treated with nicotine and MT (HFD + nicotine + MT), and the high-fat diet group treated with nicotine (HFD + nicotine). Each mouse was given an average of 8 g of feed and 12 mL of water daily. The mice in the HFD group were maintained on a high-fat diet for 12 weeks to establish the AS mouse model. The NCD + nicotine and HFD + nicotine groups were also treated for 12 weeks to observe the formation of atherosclerotic plaques. Nicotine was dissolved in distilled water at a concentration of 0.1 mg/mL and was given in drinking water for 12 weeks. Dissolve MT in distilled water to a 1 mg/mL concentration, and the solvent will not affect subsequent experiments. According to the literature [9], 10 mg/kg/day daily was intragastric administration for 12 weeks.

Cell viability assay

THP-1 macrophages were exposed to nicotine/MT. Then, 100 µL of medium containing 10% CCK-8 (Yeason, Shanghai, China) was added to each well. After incubation for 1 h in the dark, the absorption at a wavelength of 450 nm was measured using a microplate reader (Varian Australia Pty Ltd, Australia).

Enzyme-linked immunosorbent assay (ELISA)

The supernatant of THP-1 macrophages was collected to detect soluble IL-18, IL-1β, and 4-HNE; THP-1 macrophage lysate was collected to detect soluble Caspase-1 and Caspase-3; and mouse serum was collected to detect IL-18, IL-1β, and testosterone levels by using ELISA kits (Elabscience Biotechnology Co. Ltd., Wuhan, China).

Immunofluorescence analysis

THP-1 macrophages were fixed with 4% paraformaldehyde for 30 min and permeabilized with 1% Triton X-100 for 20 min. The macrophages were blocked with 3% BSA and incubated with NLRP3, ASC and IL-18 antibodies overnight at 4 °C. Finally, the macrophages were incubated with fluorescence-labelled secondary antibodies at 37 ℃ for 1 h in the dark. DAPI staining was used to counterstain the nucleus. Fluorescence intensities were quantified with a laser scanning confocal microscope (Nikon, Japan).

Reactive oxygen species (ROS) analysis

Treated cells were incubated with the fluorescent dye 2′-7′-dichlorofluorescein diacetate (DCFH-DA)/MitoSOX Red Mitochondrial Superoxide Indicator for 30 min at 37 °C in the dark. The cells were analyzed by fluorescence spectrophotometry (Nikon, Japan) or flow cytometry (Agilent, America).

Western blot (WB) analysis

The membranes were blocked with 5% nonskimmed dry milk containing 0.05% Tween 20 in Tris-buffered saline for 1.5 h. The membrane was probed with primary antibodies against NLRP3 (proteintech), ASC (Omnimabs), pro-caspase-1/caspase-1 (Abcam), IL-18 (proteintech) and IL-1β (proteintech), GAPDH (proteintech), FOXO3α (proteintech), SIRT3 (proteintech), mnSOD (proteintech), 4-HNE (Abcam), Catalase (proteintech), Caspase-3 (proteintech), GSDMD (proteintech), GSDME (proteintech), all diluted at 1:1000 in 5%BAS-TBST buffer 4 °C overnight. This was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; proteintech) for 1.5 h. The immunoblots were visualized by chemiluminescence using an imaging system (Bio-Rad, America). Protein bands were quantified using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Due to the need to display the results of proteins with different molecular weights on the same PVDF membrane, we performed imprinting cleavage before antibody hybridization. To ensure the rigor of the experiment, we cut the blots of the exact result graph of the same grouping on a PVDF membrane. However, the exposure time of different bands is inconsistent due to differences in antibodies. Therefore, we adopted a separate color development method (simultaneous color development may lead to underexposure/overexposure of some blots).

Quantitative PCR (q-RT‒PCR)

The UNIQ-10 Columnar Total RNA Extraction Kit (Sangon Biotech, Shanghai, China) was used for RNA extraction, and reverse transcription was performed using the RT Easy II First Strand cDNA Synthesis Kit (FORGENE, Sichuan, China). Then, 1 µL of cDNA was amplified using Real-Time PCR Easy (SYBR Green I) (FORGENE, Sichuan, China) on an ABI 7900HT Sequence Detection System (ABI Applied Biosystems, Foster City, CA, USA). Table 1 shows the primer sequences.

Transfection of small interfering RNAs (siRNAs)

Five microlitres of siRNA (20 µM) and 8 µL of Hieff TransTM siRNA (Yeason, Shanghai, China) were mixed with 200 µL of RPMI 1640 medium. The mixture was added to individual THP-1 macrophage cultures in a six-well plate containing fresh medium and incubated for 48 h. The caspase-1 siRNA duplexes S1 (sense: CCUGUGAUGUGGAGGAAAUTT; and anti sense: AUUUCCUCCACAUCAXAGGTT), S2 (sense: UGG AAGACUCAUUGAACAUTT, anti sense: AUGUUCA AUGAGUCUUCCATT), S3 (sense: GAAGACUCAU UGAACAUAUTT, anti sense: AUAUGUUCAAU GAGUCUUCTT) were used. The FOXO3α siRNA duplexes S1 (sense: GCUGUCUCCAUGGACAAUATT, anti-sense: UAUUGUCCAUGGAGACAGCTT), S2 (sense: GCUCACUUCGGACUCACUUTT, anti-sense: AAGUGAGUCCGAAGUGAGCTT), and S3 (sense: CCUCAUCUCCACACAGAAUTT; and anti-sense: AUUCUGUGUGGAGAUGAGGTT) were used.

Lentiviral transduction

THP-1 macrophages were inoculated, and the virus was added according to the previously determined multiplicity of infection (MOI). The cells were randomly divided into blank control and overexpression groups. The blank control group was cultured with ordinary medium, while the overexpression group was transfected with the FOXO3α-overexpressing lentivirus. After 48 h of transfection, complete medium was added to the cells in the overexpression group to establish a stably transfected cell line.

Immunoprecipitation (co-IP)

THP-1 macrophages were lysed, and the IP lysate and protein samples were mixed with caspase-1 and caspase-3 antibodies and protein A/G-agarose beads (Absin, Shanghai, China) overnight at 4 ℃ with gentle shaking. Subsequent steps were performed according to the co-IP kit instructions. Negative controls using nonimmune IgG antibodies were used under the same experimental conditions to ensure the antibodies’ specificity for immunoprecipitation.

Immunohistology of AS lesions

The heart’s aortic root and basal portion were fixed in 4% paraformaldehyde, followed by OCT compound embedding (Sakura, USA), before being cut into 6 μm-thick sections. Atherosclerotic lesions of the aortic root were observed by using HE staining. Oil red O staining was performed according to the manufacturer’s instructions to examine lipid deposition. Briefly, the frozen sections were soaked in reagent I for 10 min, followed by a 20-second wash using distilled water at 37 °C. Then, reagent II was added to the sections and incubated for 5 min, followed by a 60 s wash and microscopic examination. In addition, Oil red O staining, HE staining, Masson’s staining, and immunohistochemistry were performed on the aortic root. Images were captured using a confocal laser scanning microscope (NanoZoomerS210, Japan).

Hoechst assay

Cells were fixed, washed twice with PBS and stained with Hoechst staining solution according to the manufacturer’s instructions (Beyotime Biotechnology, Jiangsu, China). Stained nuclei were observed under a fluorescence microscope (Nikon, Tokyo, Japan).

Transmission electron microscopy (TEM)

After treatments, the cells were harvested by centrifugation and processed for TEM analysis on a JEM-1220 device (JEOL, Tokyo, Japan).

Statistical analysis

All experiments were repeated at least three times independently. The data were analysed using one-way analysis of variance (ANOVA) and Student’s t test. The results are presented as the mean ± standard deviation (SD). A P value < 0.05 was considered statistically significant.