(R)-(−)-β-Hydroxybutyric acid sodium salt (cat. no. 298360) was purchased from Sigma-Aldrich (Merck KGaA). ox-LDL (cat. no. YB-002) and 1,1′-dioctadecyl--3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil)-ox-LDL (cat. no. YB-0010) were obtained from Guangzhou Yiyuan Biotech Co., Ltd. Phorbol 12-myristate 13-acetate (PMA; cat. no. S1819) and the JC-1 Mitochondrial Membrane Potential (ΔΨm) Assay kit (cat. no. C2003S) were obtained from Beyotime Biotechnology. FBS (cat. no. 11011-8611) was purchased from Beijing Solarbio Science & Technology Co., Ltd. The Senescence-Associated β-Galactosidase (SA-β-Gal) Stain Kit (cat. no. G1580), BSA (cat. no. IA0910), Autophagy Staining Detection kit (cat. no. G0170), DAPI Staining Solution (cat. no. C0065), High-Efficiency RIPA Cell/Tissue Lysis Buffer (cat. no. R0010) and trypsin (cat. no. T1300) were obtained from Beijing Solarbio Science & Technology Co., Ltd. Rabbit anti-rat p62 (cat. no. ab109012), anti-STAT4 (cat. no. ab28815) and anti-β-actin antibody (cat. no. ab8226) were obtained from Abcam. ECL Plus Ultra-Sensitive Detection Reagent (cat. no. P0018AM), PVDF membrane (cat. no. FFP28), HRP-labeled goat anti-mouse IgG (cat. no. A0216) and HRP-labeled goat anti-rabbit IgG (cat. no. A0208) were obtained from Beyotime Biotechnology. The PAGE Gel Rapid Preparation kit (cat. no. PG112) was obtained from Epizyme Biotech. High-glucose DMEM (cat. no. SH30022.01) was purchased from Cytiva. Lipofectamine 2000 (cat. no. 11668030) and Opti-MEM reduced-serum medium (cat. no. 31985070) were purchased from Thermo Fisher Scientific, Inc.

THP-1 cells were purchased from GenChem Inc. and maintained in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. All experiments were performed using cells between passages 3 and 10 at ~80% confluency.

For macrophage differentiation, 2×10⁵ THP-1 cells per well were seeded in 6-well plates and stimulated with 100 ng/ml PMA at 37°C for 24 h. Following PMA-induced monocyte-to-macrophage differentiation (confirmed by the transition to an adherent morphology), cells were washed twice with PBS. In the experiments involving both ox-LDL and BHB, the differentiated THP-1 macrophages were first incubated with 50 mg/l ox-LDL for 24 h at 37°C, and then incubated with BHB (1, 2 or 3 mM) for 24 h at 37°C.

A total of 2×10⁵ THP-1 cells per well seeded in 6-well plates were stimulated with 100 ng/ml PMA for 24 h at 37°C. The differentiated THP-1 macrophages were treated with either ox-LDL or BHB for an additional 24 h at 37°C. Cells were harvested and washed twice with ice-cold PBS. Protein extraction was performed using 100 µl high-efficiency RIPA lysis buffer supplemented with 1 µl PMSF by incubating on ice for 30 min. Lysates were centrifuged at 12,000 × g for 20 min at 4°C, mixed with 25 µl 5X loading buffer, denatured at 100°C for 10 min and immediately chilled on ice. Samples were stored at −20°C until analysis.

Protein concentration was determined using the BCA assay. Equal amounts of protein (20 µg/lane) were separated on 10% SDS-polyacrylamide gels and transferred onto PVDF membranes at 100 V. Membranes were blocked with 5% BSA for 2 h at room temperature, followed by incubation with primary antibodies (1:1,000) overnight at 4°C. HRP-conjugated goat anti-rabbit secondary antibody (1:5,000) or HRP-conjugated goat anti-mouse secondary antibody (1:5,000) was applied for 2 h at room temperature. Protein signals were detected using ECL reagents. Band intensities were quantified using ImageJ software (ImageJ 1.53t; National Institutes of Health) with β-actin as the loading control.

A total of 4×10⁴ THP-1 cells per well were seeded onto sterile glass coverslips placed in 24-well plates. Following stimulation with 100 ng/ml PMA at 37°C for 24 h, cells were washed twice with PBS. THP-1 cells in different experimental groups were treated as follows: In the experiment to determine the optimal concentration of ox-LDL, differentiated THP-1 macrophages were incubated with ox-LDL (0, 50 and 100 mg/l) at 37°C for 24 h; in experiments involving ox-LDL and BHB, differentiated THP-1 macrophages were first incubated with 50 mg/l ox-LDL at 37°C for 24 h, and then incubated with BHB (1, 2 or 3 mM) at 37°C for 24 h; and in experiments involving overexpression and inhibition of STAT4, after the overexpression and inhibition of STAT4, 50 mg/l ox-LDL was added (or not added) and cells were incubated at 37°C for 24 h, followed by the addition (or non-addition) of 3 mM BHB and incubation at 37°C for 24 h.

After the aforementioned treatment, the cells were processed with an Autophagy Staining Detection kit, according to the manufacturer's protocol. Briefly, cells were gently rinsed twice with wash buffer, then incubated with dansylcadaverine for 15–30 min at room temperature under light-protected conditions. Following two additional washes, nuclei were counterstained with DAPI (1 µg/ml) for 5–10 min at 25°C. Following final rinsing with wash buffer, the coverslips were mounted onto adhesive microscope slides using an anti-fade mounting medium (cat. no. S2100; Beijing Solarbio Science & Technology Co., Ltd.).

Autophagic vesicles were visualized with a laser-scanning confocal microscope (FV3000; Olympus Corporation). Excitation/emission settings were set to optimal dye parameters (488 nm excitation/530 nm emission) and for DAPI (405 nm excitation/461 nm emission). Images were acquired using sequential channel scanning to avoid fluorescence bleed-through.

A total of 4×10⁴ THP-1 cells per well grown on glass coverslips in 24-well plates. Following stimulation with 100 ng/ml PMA at 37°C for 24 h, cells were washed twice with PBS. Cell treatments and grouping were the same as described in the autophagy detection section.

After the aforementioned treatment, the cells were processed with the JC-1 Mitochondrial Membrane Potential (ΔΨm) Assay kit (cat. no. C2003S; Beyotime Biotechnology) according to the manufacturer's protocol. Briefly, a JC-1 dye working solution was prepared by diluting the stock solution 1:1 in complete culture medium (high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin). The cells were incubated with this mixture for 20 min at 37°C under light-protected conditions. After incubation, the coverslips were washed twice with JC-1 assay buffer, transferred to fresh culture medium and mounted face-down onto adhesive microscope slides using an anti-fade mounting medium. The ΔΨm assessment was performed by observing the aggregated (red) or monomeric (green) state of JC-1 in the cells with a laser-scanning confocal microscope (FV3000; Olympus Corporation). Fluorescence intensities were quantified using ImageJ software (ImageJ 1.53t; National Institutes of Health).

A total of 4×10⁴ THP-1 cells per well were seeded in 24-well plates. Following stimulation with 100 ng/ml PMA at 37°C for 24 h, cells were washed twice with PBS. Differentiated THP-1 macrophages were first incubated with 50 mg/l ox-LDL at 37°C for 24 h, and then incubated with BHB (0,1, 2 or 3 mM) at 37°C for 24 h. After the aforementioned treatment, the cells were washed twice with PBS and fixed with 4% formaldehyde for 15 min at room temperature. Following three PBS washes, the cells were incubated with 1 ml β-Gal staining solution [1 mg/ml X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl₂ in 40 mM citric-acid/sodium-phosphate buffer (pH 6.0)] under an airtight seal with paraffin film at 37°C in a non-CO₂ atmosphere for 24 h. After two additional PBS washes, SA-β-Gal-positive cells (blue staining) were examined under a fluorescence microscope. Semi-quantitative analysis of fluorescence intensity was performed using ImageJ software (version 1.54f; National Institutes of Health). In total, five random fields of view/well were imaged for statistical analysis.

A total of 4×10⁴ THP-1 cells per well were seeded in 24-well plates and differentiated with 100 ng/ml PMA for 24 h at 37°C. The resulting macrophages were incubated with 100 µg/ml DiI-ox-LDL together with BHB (0, 1, 2, 3 mM) for 24 h at 37°C, 5 % CO₂. After incubation, the cells were washed three times with PBS. Fluorescent micrographs were captured with an inverted fluorescence microscope. Intracellular DiI-ox-LDL accumulation was quantified with ImageJ software (version no. 1.53t, National Institutes of Health), by analyzing fluorescence intensity in three independent biological replicates.

Total RNA was isolated from THP-1 cells using an RNA isolation kit (cat. no. R0026; Beyotime Biotechnology) according to the manufacturer's instructions. Briefly, after adding the lysis buffer to the THP-1 cell culture plate, the cells were lysed and incubated at room temperature for 5 min. Subsequently, 200 µl chloroform was added, and the mixture was vigorously shaken for 15 sec, followed by incubation at room temperature for 3 min. The sample was then centrifuged at 13,000 × g for 10 min at 4°C. The supernatant was transferred to a new tube. An equal volume of ethanol was slowly added, followed by thorough mixing. The solution along with any precipitate was transferred into an adsorption column (CR3) and centrifuged at 13,000 × g for 30 sec at 4°C. Subsequently, 500 µl protein removal solution was added, followed by centrifugation at 13,000 × g for 30 sec at 4°C, and the flow-through was discarded. The CR3 column was placed into a collection tube, and 500 µl wash buffer was added. After incubation at room temperature for 2 min, the column was centrifuged at 13,000 × g for 30 sec at 4°C, and the flow-through was discarded. The column was transferred to a 2 ml collection tube and centrifuged at 13,000 × g for 2 min at 4°C to remove residual liquid. Finally, the CR3 column was placed into a new 1.5-ml microcentrifuge tube, and 40 µl RNase-free ddH₂O was added. After incubation at room temperature for 2 min, the tube was centrifuged at 13,000 × g for 2 min at 4°C. The extracted RNA was used for subsequent experiments.

Reverse transcription was performed using an RT-PCR kit (cat. no. RP1100; Beijing Solarbio Science & Technology Co., Ltd.). Briefly, in a total reaction volume of 14.5 µl, 2 µg of RNA and 2 µl of the specific primer Oligo(dT)₁₆ were added, and the volume was adjusted with RNase-free ddH₂O. The mixture was incubated at 70°C for 5 min and then rapidly cooled on ice for 2 min. After a brief centrifugation at 500 × g for 30 sec at 4°C to collect the reaction mixture, the following components were added: 4 µl 5X M-MLV Buffer, 1 µl dNTPs, 0.5 µl RNasin and 1 µl M-MLV reverse transcriptase. The reaction was incubated at 42°C for 60 min, followed by heating at 95°C for 5 min to terminate the reaction. The product was placed on ice for subsequent experiments.

qPCR was performed using 2X SYBRGreen PCR Mastermix (cat. no. SR1110; Beijing Solarbio Science & Technology Co., Ltd.). Briefly, the following components were added to a reaction tube: 12.5 µl of 2X SYBR Green PCR mix, 1 µl of primers (10 µM), 5 µl of template DNA and 5.5 µl of ddH₂O, to a final volume of 25 µl. The reaction was performed using an Applied Biosystems StepOnePlus real-time PCR system (Thermo Fisher Scientific, Inc.) under the following conditions: Initial denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 20 sec, annealing at 60°C for 25 sec and extension at 72°C for 30 sec (with fluorescence acquisition during the extension step). The cycle threshold (Ct) value was analyzed using StepOne software v2.3 (Thermo Fisher Scientific, Inc.), semilog amplification curves were evaluated using the comparative quantification method (2^−ΔΔCq) (25) and the gene expression levels were normalized to those of human β-actin. The primer pairs for qPCR were: STAT4 forward, 5′-CCTGACATTCCCAAAGACAAAGC-3′ and reverse, 5′-TCTCTCAACACCGCATACACAC-3′; and β-actin forward, 5′-GTGGACATCCGCAAAGAC-3′ and reverse, 5′-AAAGGGTGTAACGCAACTA-3′.

The PCDNA3.1-STAT4 (Homo sapiens, XM_054343566.1) expression plasmid was purchased from BGI Genomics and the empty PCDNA3.1 vector (BGI Genomics) was also transfected as a negative control. A total of 2×10⁵ THP-1 cells per well were seeded in 6-well plates at ~80 % confluency and differentiated with 100 ng/ml PMA for 24 h at 37°C. The medium was replaced with antibiotic-free complete DMEM (Cytiva). Lipofectamine 2000 (10 µl) was diluted in 250 µl Opti-MEM^® I Reduced-Serum Medium. Plasmid DNA (5 µg) was diluted in 250 µl Opti-MEM. Following a 5 min incubation at room temperature, the diluted DNA and Lipofectamine solutions were combined, vortexed gently and incubated for 15 min at room temperature. The resulting DNA-lipid complexes were added dropwise to the cells. Following 6 h of incubation at 37°C with 5 % CO₂, the transfection medium was replaced with fresh complete DMEM (Cytiva). The expression of STAT4 was validated by RT-qPCR. RT-qPCR was used to verify the overexpression efficiency of STAT4 before treating cells with ox-LDL or BHB. THP-1 cells were seeded into 6-well plates at a density of 2×10⁵ cells per well and stimulated with 100 ng/ml PMA at 37°C for 24 h. Following transfection according to the aforementioned procedure, the cells were collected for total RNA extraction. Subsequently, RT-qPCR was conducted to detect STAT4 mRNA levels in order to confirm the transfection efficiency.

The STAT4 gene-silencing kit (genOFF h-STAT4_2500A) was used according to the manufacturer's protocol (cat. no. SIGS0000957-4; Guangzhou RiboBio Co., Ltd.). The STAT4 siRNA target sequence was 5′-GCCTGACCATAGATTTGGA-3′ (the corresponding siRNA was provided by Guangzhou RiboBio Co., Ltd.), and the negative control siRNA (cat. no. SIGS0000957-4; Guangzhou RiboBio Co., Ltd.) was provided by Guangzhou RiboBio Co., Ltd. A total of 2×10⁵ THP-1 cells per well were seeded in 6-well plates. Following 24 h of stimulation with 100 ng/ml PMA at 37°C, the medium was replaced with antibiotic-free complete DMEM. The Ribo FECT™ CP buffer was diluted with PBS and the STAT4 siRNA and the negative control siRNA included in the STAT4 gene-silencing kit were briefly centrifuged (500 × g; 1 min) at 25°C, resuspended in 250 µl sterile de-ionized water, aliquoted and stored at −20°C. For the transfection mixture, 120 µl diluted Ribo FECT CP buffer, 10 µl 50 nM STAT4 siRNA and 12 µl Ribo FECT CP reagent were incubated at room temperature for 15 min and then added dropwise to the 6-well plates. Following 48 h of incubation at 37°C with 5% CO₂, the expression of STAT4 was validated by RT-qPCR. RT-qPCR was used to verify the silencing efficiency of STAT4 before treating cells with ox-LDL or BHB. THP-1 cells were seeded into 6-well plates at a density of 2×10⁵ cells per well and stimulated with 100 ng/ml PMA at 37°C for 24 h. Following transfection according to the aforementioned procedure, the cells were collected for total RNA extraction. Subsequently, RT-qPCR was conducted as aforementioned to detect STAT4 mRNA levels in order to confirm the transfection efficiency.

Data were analyzed using GraphPad Prism^® software (version 10.1.2; GraphPad Software, Inc.; Dotmatics). All experiments were independently repeated at least three times. All data are presented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The present study assessed the expression of p62, a selective substrate for autophagic degradation, in THP-1 cells following ox-LDL stimulation. Immunoblot analysis revealed that ox-LDL (50 and 100 mg/l) significantly increased p62 expression and decreased the ratio of LC3-II/LC3-I compared with untreated controls (Fig. 1A-C). To corroborate these findings, autophagosome formation was evaluated using confocal laser scanning microscopy. Cells treated with ox-LDL (100 mg/l) exhibited a marked reduction in autophagic puncta, consistent with the immunoblot data, indicating impaired autophagy (Fig. 1D and F). ΔΨm was measured using the JC-1 fluorescent probe. Red fluorescence, indicating high ΔΨm, was observed in control cells. By contrast, ox-LDL treatment (100 mg/l) led to significantly attenuated red fluorescence intensity and a concomitant increase in green fluorescence (indicating a reduction in ΔΨm; Fig. 1E and G). These results demonstrate that ox-LDL exposure induces a substantial decrease in ΔΨm in THP-1 cells. Collectively, these findings indicated mitochondrial dysfunction, reflecting a diminished capacity to maintain cell energy metabolism and homeostasis.

The present study assessed the effect of BHB on autophagic flux in THP-1 cells. p62 expression levels decreased significantly, while ratio of LC3-II/LC3-I increased markedly following BHB treatment, indicating that BHB effectively restored the impaired autophagic flux in THP-1 cells (Fig. 2A-C). Cells treated with both ox-LDL and BHB at 1 and 2 mM exhibited a higher expression of p62 compared with those exposed solely to ox-LDL, while a notable decrease in expression was observed at 3 mM BHB. Fluorescent staining confirmed this restoration: While 1 mM and 2 mM BHB reduced autophagic puncta formation and ΔΨm, 3 mM BHB markedly increased both autophagic puncta (Fig. 2D and F) and ΔΨm (Fig. 2E and G). Collectively, these results demonstrate that BHB enhances autophagic capacity and mitochondrial function in THP-1 macrophages by ameliorating ox-LDL-induced impairment of autophagic flux.

BHB co-treatment significantly decreased Dil-ox-LDL levels in THP-1 cells, indicating potent inhibition of ox-LDL accumulation in macrophages (Fig. 3A and B). As elevated β-Gal activity serves as a key marker of cell senescence (26), this was measured following 24 h of co-treatment with ox-LDL and BHB. BHB suppressed intracellular β-Gal activity in a dose-dependent manner (Fig. 3C and D), suggesting attenuation of cell aging processes.

ox-LDL significantly upregulated STAT4 protein expression in THP-1 cells (Fig. 4A and B). Conversely, BHB substantially decreased STAT4 levels in a dose-dependent manner.

The present study examined the role of STAT4 in mediating the rescue effect of BHB on ox-LDL-induced impairment of autophagic flux. Following overexpression of STAT4 (Fig. S1A), p62 levels increased significantly (Fig. 5A-C), whereas autophagic puncta decreased (Fig. 5D and F) and the ΔΨm declined (Fig. 5E and G). Following co-treatment with ox-LDL and BHB, STAT4-overexpressing cells exhibited no significant alterations in p62 expression, autophagosome count or ΔΨm. Collectively, these findings demonstrate that STAT4 overexpression abrogated BHB-mediated restoration of impaired autophagic flux and mitochondrial function.

siRNA transfection was performed to explore the role of STAT4 in autophagic recovery and ox-LDL uptake. Compared with cells transfected with scrambled RNA (Fig. S1B), STAT4 silencing significantly decreased p62 expression (Fig. 6A-C), increased autophagosome formation (Fig. 6D and F) and elevated ΔΨm (Fig. 6E and G). However, when ox-LDL was added following siRNA-STAT4 transfection in THP-1 cells, the results did not show the same pattern as before in non-siRNA-STAT4-transfected THP-1 cells [increased p62 expression levels, impaired autophagic flux (Fig. 1A-F) and decreased ΔΨm (Fig. 1E and G)]. As aforementioned, silencing of STAT4 markedly attenuated ox-LDL-induced impairment of autophagic flux and the accompanying decline in ΔΨm in THP-1 cells.