Effects of β‑hydroxybutyrate on restoring autophagic flux and mitochondrial function in macrophages exposed to oxidized low‑density lipoprotein

Introduction

Cardiovascular diseases (CVDs) are a leading cause of morbidity and mortality worldwide, accounting for an estimated 19.7 million deaths annually, representing ~32% of all global deaths (1). Among the risk factors for CVDs, oxidized low-density lipoprotein (ox-LDL) is a key driver of atherosclerosis, a disease characterized by the accumulation of fatty deposits in arterial walls (2,3). Previous studies have highlighted the detrimental effects of ox-LDL on macrophage function, especially its ability to promote inflammation and cell senescence (4,5). Moreover, the fact that ox-LDL contributes to atherosclerosis by inducing oxidative stress and inflammatory responses is well-established (6,7).

Autophagy is a key cellular process that degrades and recycles damaged organelles and proteins, thereby maintaining cell homeostasis (8). In macrophages, autophagy is particularly important because it regulates inflammation and lipid metabolism (9,10). Impaired autophagic activity is associated with pathophysiological conditions, including CVD and neurodegeneration (11). Macrophage autophagy promotes the catabolism of cytoplasmic lipid droplets, maintains cell lipid homeostasis and plays an important role in anti-atherosclerotic processes (12). Moreover, mitochondrial dysfunction, typically a consequence of oxidative stress, exacerbates these conditions (13). The interaction between autophagy and mitochondrial function is complex and understanding this interplay is essential for developing effective therapeutic interventions.

β-hydroxybutyrate (BHB), a ketone body generated during fatty acid metabolism, possesses anti-inflammatory properties and may protect cells from oxidative stress-induced damage (14). BHB also promotes autophagosome formation, suggesting that it may be used therapeutically in metabolic disorders (15). The STAT signaling pathway is a critical component of cellular communication, primarily involved in regulating the transcription of target genes that are essential for cellular function (16). The clinical significance of the relationship between STAT signaling and autophagy is becoming increasingly evident (17,18). In cancer, for example, the activation of the JAK/STAT pathway leads to enhanced autophagic activity, which may contribute to tumor cell survival and resistance to therapy (19). Conversely, inhibiting the JAK/STAT pathway is associated with the promotion of autophagy and tumor cell death, indicating that targeting this signaling pathway could be a viable strategy for cancer treatment (20). The activation of the IL-12/STAT4 signaling pathway enhances autophagic activity, thereby contributing to the clearance of Mycobacterium tuberculosis in infected macrophages (21). Autophagy-related protein 5 can modulate immune responses by enhancing the production of CCL2, which influences STAT4 activation in CD4+ T helper 1 cells during malaria infections (22). When macrophages are exposed to ox-LDL, STAT4 is phosphorylated and translocates to the nucleus, where it regulates the transcription of genes involved in inflammation (23). This leads to the production of pro-inflammatory cytokines such as IFN-γ and TNF-α, which are essential for the activation and recruitment of immune cells to the site of inflammation (24).

The molecular mechanisms by which BHB regulates autophagy and the role of STAT4 in this process remain incompletely understood. Further investigations are needed to delineate these pathways and assess their therapeutic relevance for autophagy-targeted interventions in lipid-related diseases. The present study aimed to examine the effects of BHB on autophagy and mitochondrial function in THP-1 macrophage exposed to ox-LDL and determine how BHB may restore cellular functions that are impaired by ox-LDL, with particular emphasis on the signaling molecule STAT4. The present findings could inform the development of novel therapeutic strategies for metabolic disorders and CVD.

Materials and methods

Reagents

(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.

Cell culture and treatment

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.

Western blot analysis

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.

Autophagy detection

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.

ΔΨm assessment

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).

Senescence-associated β-Gal (SA-β-Gal) staining

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.

Fluorescent ox-LDL uptake assay

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 extraction and reverse transcription-quantitative PCR (RT-qPCR)

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′.

STAT4 overexpression via Lipofectamine-mediated transfection

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.

STAT4 silencing

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.

Statistical analysis

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.

Results

ox-LDL induces impaired autophagic flux and decreased ΔΨm in THP-1 cells

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.

Figure 1. ox-LDL impairs autophagic flux and reduces the mitochondrial membrane potential. (A) Western blot analysis of (B) p62 and LC3 in THP-1 cells treated with ox-LDL at various doses for 24 h. (C) Quantification of LC3II/LC3-I ratios. (D) Confocal microscopy image of autophagosome puncta in ox-LDL-treated cells stained with dansylcadaverine. Nuclei were visualized by counterstaining with DAPI (magnification, ×60). (E) JC-1 staining of THP-1 cells. Fluorescence was recorded using 488 nm excitation (magnification, ×60). Relative fluorescence intensity of (F) autophagosome puncta and (G) JC-1 aggregates. *P<0.05, **P<0.01. ox-LDL, oxidized low-density lipoprotein.

BHB restores impaired autophagic flux and ΔΨm in ox-LDL-treated THP-1 cells

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.

Figure 2. BHB restores impaired autophagic flux and mitochondrial membrane potential. (A) Western blot analysis of (B) p62 and LC3 in THP-1 cells treated with ox-LDL and BHB for 24 h. (C) LC3II/LC3-I ratios. (D) Confocal microscopy image of autophagosome puncta in cells treated with ox-LDL and BHB. Nuclei were counterstained with DAPI (magnification, ×60). (E) JC-1 staining of THP-1 cells visualized by fluorescence confocal microscopy. Fluorescence was recorded using 488 nm excitation (magnification, ×60). Relative fluorescence intensity of (F) autophagosome puncta and (G) JC-1 aggregates in cells treated with ox-LDL and BHB. *P<0.05, **P<0.01, ***P<0.001. ox-LDL, oxidized low-density lipoprotein; BHB, β-hydroxybutyrate; ns, not significant.

BHB decrease ox-LDL accumulation and β-Gal activity in THP-1 cells

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.

Figure 3. BHB decreases ox-LDL accumulation and β-Gal activity. (A) Representative fluorescence microscopy of (B) Dil-ox-LDL uptake in THP-1 cells. Scale bar, 100 µm. (C) Representative (D) SA-β-Gal staining. Scale bar, 50 µm. *P<0.05, **P<0.01. BHB, β-hydroxybutyrate; Dil-ox-LDL, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-oxidized low-density lipoprotein; SA-β-Gal, senescence-associated β-galactosidase.

BHB downregulates STAT4 expression in ox-LDL-treated THP-1 cells

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.

Figure 4. BHB downregulates STAT4 expression in ox-LDL-treated THP-1 cells. (A) Western blot analysis of (B) STAT4 expression in THP-1 cells treated with ox-LDL and BHB. *P<0.05. BHB, β-hydroxybutyrate; ox-LDL, oxidized low-density lipoprotein.

STAT4 overexpression impairs BHB-mediated restoration of autophagic flux

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.

Figure 5. STAT4 overexpression impairs the ability of BHB to restore autophagic flux. (A) Western blot analysis of (B) STAT4 and (C) p62 in STAT4-overexpressing THP-1 cells treated with ox-LDL and BHB. (D) Confocal microscopy image of autophagosome puncta in STAT4-overexpressing THP-1 cells following ox-LDL and BHB treatment. Nuclei were visualized by counterstaining with DAPI (magnification, ×60). (E) JC-1 staining of THP-1 cells imaged by fluorescence confocal microscopy. Fluorescence was recorded using 488 nm excitation (magnification, ×60). Relative fluorescence intensity of (F) autophagosome puncta and (G) JC-1 aggregates in STAT4-overexpressing THP-1 cells after ox-LDL and BHB treatment. The group without 50 mg/l ox-LDL, 3 mM BHB and PCDNA3.1-STAT4 treatment was transfected with the empty vector. *P<0.05, **P<0.01. BHB, β-hydroxybutyrate; ox-LDL, oxidized low-density lipoprotein; ns, not significant.

STAT4 silencing prevents Ox-LDL-induced impairment of autophagic flux and Ox-LDL-induced reduction of the ΔΨm in THP-1 cells

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.

Figure 6. STAT4 silencing prevents ox-LDL-induced impairment of autophagic flux and mitochondrial dysfunction. (A) Western blot analysis of (B) STAT4 and (C) p62 in STAT4-silencing THP-1 cells treated with ox-LDL and BHB. (D) Confocal microscopy image of autophagosome puncta in siRNA-STAT4 THP-1 cells after treatment. Nuclei were visualized by counterstaining with DAPI (magnification, ×60). (E) JC-1 staining of THP-1 cells visualized by fluorescence confocal microscopy. Fluorescence was recorded using 488 nm excitation (magnification, ×60). Relative fluorescence intensity of (F) autophagosome puncta and (G) JC-1 aggregates in siRNA-STAT4 THP-1 cells after treatment. The group without 50 mg/l ox-LDL and siRNA-STAT4 treatment was transfected with negative control siRNA. *P<0.05, **P<0.01. ox-LDL, oxidized low-density lipoprotein; BHB, β-hydroxybutyrate; si/siRNA, small interfering; ns, not significant.

Discussion

The pathophysiology of CVD, particularly atherosclerosis, is influenced by the accumulation of ox-LDL, which serves a pivotal role in macrophage dysfunction and foam cell formation (27). This accumulation initiates a cascade of inflammatory responses and promotes cell senescence, contributing to disease progression and enhancing susceptibility to other types of metabolic disorder, such as glucose metabolism disorders (28,29).

The present study examined the impact of BHB on autophagy and mitochondrial function in THP-1 macrophages exposed to ox-LDL. ox-LDL impairs autophagic flux and compromises mitochondrial integrity, resulting in elevated cell stress and inflammation (30). Here, BHB significantly restored autophagic function and enhanced ΔΨm in these cells, implying a potential therapeutic benefit of BHB in mitigating the deleterious effects of ox-LDL. Moreover, modulation of the STAT4 signaling pathway is a key mechanism by which BHB exerts its restorative effects, underscoring the importance of targeting autophagy to improve macrophage function in atherosclerosis (31).

Recent studies have demonstrated that ox-LDL-induced impairment of autophagy flux and mitochondrial dysfunction in THP-1 macrophages serve critical roles in the progression of atherosclerosis (32,33). Gu et al (34) showed that ox-LDL impairs autophagy flux in THP-1 cells, whereas treatment with nicotinate-curcumin rescues the impaired autophagic flux by significantly increasing LC3-II levels, autolysosome numbers and p62 degradation in ox-LDL-treated THP-1 cells, similar to the present study. The present study examined the expression of the key selective autophagy protein p62. Accumulation of p62 protein typically suggests impairment or inhibition of autophagic flux (35). Changes in p62 alongside LC3-II levels reflect autophagic activity (36). The present study demonstrated an increase in LC3-II levels along with a decrease in p62, indicating overall activation of autophagic flux (37,38).

In human umbilical vein endothelial cells (HUVECs) treated with ox-LDL, silencing of syntaxin 17, a key regulator of autophagosome-lysosome fusion, exacerbates autophagic flux impairment and inflammatory responses (39). Here, ox-LDL induced decreased ΔΨm in THP-1 macrophages, a phenomenon increasingly associated with oxidative stress and inflammatory signaling (40,41). Erianin reverses ox-LDL-induced decreased ΔΨm in HUVECs by activating the Nrf2 pathway, which enhances antioxidant defenses (42). These findings align with the present observations, suggesting that decreased ΔΨm may stem from impaired electron transport chain activity and ROS accumulation.

BHB, a primary ketone body, serves a key role in regulating macrophage function and foam cell formation. A recent study has demonstrated that BHB suppresses pro-inflammatory M1 macrophage polarization by inducing histone β-hydroxybutyrylation (Kbhb) of STAT1 at lysine 679, thereby inhibiting its phosphorylation and inflammatory cytokine production (such as IL-1β and TNF-α) (43). Conversely, BHB promotes anti-inflammatory M2 polarization via STAT6-dependent pathways, enhancing the secretion of IL-10 and arginase-1 (Arg-1), which facilitates tissue repair and decreases lipid accumulation in macrophages (44). In atherosclerosis models, BHB decreases ox-LDL uptake by downregulating scavenger receptors (such as CD36) and upregulating cholesterol efflux transporters (ATP-binding cassette protein A1 and ATP-binding cassette transporter G1), thereby attenuating foam cell formation (45,46). These immunometabolic shifts underscore the dual role of BGB in mitigating inflammation and lipid overload, positioning it as a promising therapeutic candidate for vascular pathology.

BHB directly modulates STAT transcription factors through epigenetic and post-translational mechanisms (47). Bai et al (43) identified that BHB induces Kbhb modification of STAT1 at lysine 679, which impairs its phosphorylation and nuclear translocation, thereby inhibiting lipopolysaccharide-driven M1 polarization. Conversely, BHB activates STAT6 phosphorylation via IL-4 receptor α signaling, driving M2-associated gene expression. In endothelial cells, BHB enhances STAT6-mediated Nrf2 activation, thereby boosting antioxidant defenses and decreasing oxidative stress (48). This STAT-centric regulation underpins the ability of BHB to rebalance macrophage phenotypes by suppressing STAT1/NF-κB-driven inflammation and amplifying STAT6-mediated resolution pathways. These mechanisms may contribute to the decreased plaque instability observed in atherosclerotic models treated with BHB or ketogenic diets (49,50).

STAT signaling intersects with mitochondrial homeostasis and autophagy, which are key for macrophage lipid metabolism (51). STAT1 hyperactivation promotes mitochondrial ROS overproduction and membrane depolarization, thereby exacerbating oxidative damage and impairing mitophagy (52). By contrast, STAT6 activation by BHB upregulates Nrf2, which enhances mitochondrial biogenesis and stabilizes membrane potential via peroxisome proliferator-activated receptor γ coactivator 1-α (48,53). Furthermore, STAT6-dependent induction of Arg-1 supports autophagy flux through mTOR inhibition, facilitating the clearance of ox-lipids (44). In BHB-treated macrophages, the suppression of STAT1 coupled with the activation of STAT6 preserves mitochondrial integrity and promotes autophagy-mediated cholesterol efflux, thereby decreasing foam cell transformation (54). This crosstalk highlights STATs as a link between metabolic reprogramming and cell quality control.

Mitochondrial dysfunction is increasingly recognized as a central driver of vascular injury (55,56). The NADH dehydrogenase (ubiquinone) Fe-S protein 4 (NDUFS4)-sirtuin 5 (SIRT5)-dual specificity phosphatase 1 (DUSP1) axis regulates mitochondrial unfolded protein response (mtUPR) and energy metabolism, thereby protecting against coronary microvascular injury during ischemia-reperfusion (57). Similarly, targeting the SIRT5/DUSP1 pathway is a promising strategy for mitochondrial repair in doxorubicin-induced cardiotoxicity (58). In the present study, BHB restored autophagic activity and ΔΨm, which may involve analogous pathways, as STAT4 modulation may intersect with these mitochondrial regulatory networks.

The interplay between mitochondria and the endoplasmic reticulum (ER) has emerged as a key determinant of cell stress responses (59). Astragaloside IV attenuates mechanical stress-induced cardiac remodeling through the Piezo1-voltage-dependent anion channel 1 axis, which regulates ER UPR (60). Tanshinone IIA modulates METTL3- and SIRT5-mediated UPR pathways to govern mitochondria-ER crosstalk in coronary microvascular injury (61). The potential involvement of ER-mitochondria coupling in ox-LDL-challenged macrophages warrants further investigation.

Mitophagy and mitochondrial dynamics are key nodes in mitochondrial quality control networks. The nuclear receptor subfamily 4 group A member 1-mitochondrial fission factor-FUN14 domain containing 1 axis regulates mitochondrial fragmentation and mitophagy in myocardial ischemia-reperfusion injury (62). In atherosclerosis, targeting of the transmembrane BAX inhibitor motif containing 6/NDUFS4 pathway restores mitophagy and suppresses necroptosis (63). The present study demonstrated that BHB enhancement autophagic function may extend to mitophagy regulation, potentially via STAT4-dependent mechanisms. In addition, the DNA-dependent protein kinase catalytic subunit-yes-associated protein 1-ferroptosis axis is implicated in diabetic cardiomyopathy (64), suggesting broader crosstalk between DNA damage responses and organelle homeostasis.

The present study had certain limitations that warrant consideration. A key limitation of this study is the assessment of autophagy primarily through steady-state levels of LC3-II and p62 without the use of lysosomal inhibitors to directly measure autophagic flux. Future investigations should establish the dynamics of the autophagic process and include standard pharmacological approaches using bafilomycin A1 or chloroquine to trace LC3-II and p62 turnover. The present study used monodansylcadaverine-based staining, which does not distinguish between autophagosomes and autolysosomes and does not give a definitive assessment of autophagic flux. Furthermore, employing more advanced techniques such as live-cell imaging with tandem fluorescent-tagged LC3 [red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3] is required to confirm whether the present changes represent an upregulation of functional autophagy or a potential impairment at the fusion/degradation stage. This method allows precise discrimination between autophagosomes (GFP+/RFP+) and autolysosomes (GFP-/RFP+), providing a direct and quantitative measure of autophagic flux and maturation independently of lysosomal inhibitors (65). The present findings were derived from in vitro experiments using THP-1 macrophage cell lines, which may not fully replicate the complex physiological and pathological contexts of human atherosclerosis. The modulation of STAT4 underscores the complexity of the signaling pathways involved. Specifically, the present data do not demonstrate whether STAT4 acts upstream or downstream of mitochondrial functional changes. To identify the mechanistic interplay between STAT4 signaling and autophagy, subsequent experiments should assess levels of phosphorylated STAT4 and STAT4 nuclear localization to validate the activation status and subcellular distribution of STAT4 in response to BHB.

In conclusion, the present study demonstrated the detrimental effects of ox-LDL on autophagic flux and mitochondrial function in THP-1 macrophages, while highlighting the potential of BHB to restore these processes. The present findings contribute to understanding of the cell mechanisms underlying lipid metabolism and inflammation and suggest BHB may be a promising therapeutic candidate for the treatment of atherosclerosis. Future studies should investigate the translational potential of these findings in vivo to validate the clinical applicability of BHB in cardiomyocyte energy metabolism and CVD.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by the Joint Construction Project of the Henan Provincial Medical Science and Technology Research Program (grant no. LHGJ20230035), Henan Natural Science Foundation (grant no. 222300420355) from the Henan Provincial Department of Education and the Henan Province Science and Technology Research Program Project (grant no. SBGJ202103016) from the Henan Provincial Health Commission.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

WL designed the study and wrote the manuscript. MH performed the experiments. WL and HW analyzed the data. WL and HW confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References