The murine macrophage cell line ANA.1 (National Infrastructure of Cell Line Resource, 3101MOUGNM 2) was generously provided by Professor Yuexia Liao (Yangzhou University). Cells were cultured at 37 °C with 5% CO₂ in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (ExCell Bio, China) and 1% penicillin-streptomycin (Gibco, USA). For M1 polarization, cells were first treated with Antro and then simultaneously stimulated with 1 μg/ml LPS from Escherichia coli O55:B5 (Sigma, USA, catalog number L2880) and 20 ng/ml IFN-γ (Proteintech, USA) for 6 h. The medium was aspirated following stimulation, and cells were washed thoroughly three times with warm PBS to remove residual LPS. Fresh complete medium containing the same concentration of Antro was then added, and both cells and supernatants were harvested for the further analysis after 6 h treatment.

Antro used in this study was extracted and purified from the fermented products of A. camphorata by Wuxi AppTec Co., Ltd. The culture conditions for A. camphorata and the extraction protocol for Antro followed a previously established method (22). Following extraction and purification, the purity of Antro was determined by High Performance Liquid Chromatography (HPLC), and its structure was verified by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) analyses.

The protocol of primary peritoneal exudate macrophages (PEMs) have been described previously (23). Briefly, murine PEMs were isolated from ICR mice (6 weeks) 3 days after intraperitoneal injection of 4% thioglycolate (Sigma, Germany). Cells were harvested by peritoneal lavage with phosphate-buffered saline (PBS, Gibco, USA), lysed with red blood cell lysis buffer (CWBIO, China), and cultured in DMEM medium (supplemented with 10% FBS and 1% penicillin-streptomycin). Macrophage purity was determined to be 94.6%. The culture conditions and handling procedures for PEMs are identical to those for established cell lines.

Cell viability was monitored using CCK8 kit (Dojindo, Japan) according to the protocol provide by manufacturers. Briefly, M0 or M1 macrophages (1 × 10⁵ cells/well) were seeded in 96-well plates and treated with varying concentrations of Antro (extracted by WuXi AppTec, China). After treatment, 10 μl of CCK-8 solution was added to each well, followed by incubation at 37 °C for 1 h. Absorbance was then measured at 450 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, USA).

Cell supernatant was harvested and centrifuged at 1,000 g for 5 min, and the levels of pro-inflammatory cytokines were measured using ELISA (CSB-E08054m, Cusabio, China) according to the manufacturer's instruction.

Total RNA was isolated with RNA Purification Kit (EZ Bioscience, USA) and reverse transcribed into cDNA using Color Reverse Transcription Kit (EZ Bioscience, USA). qPCR was then performed using SYBR Green qPCR Master Mix (EZ Bioscience, USA) on QuantStudio 6 Pro system (Thermo Fisher Scientific, USA). Primer sequences used in this study are listed in Table 1. Data were normalized to β-actin as the reference gene and analyzed using the 2^−ΔΔCT method.

Cells were lysed using RIPA Lysis Buffer (Beyotime, China), and protein concentration was quantified with the enhanced BCA Protein Assay Kit (Beyotime, China) prior to separation via SDS-PAGE. Resolved proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with Tris-Tween-buffered saline buffer (TBST) and incubated with primary antibodies at 4 °C overnight. Following TBST washes, membranes were probed with HRP-conjugated secondary antibodies at room temperature for 90 min. Chemiluminescent substrate was applied, and signals were captured by X-ray film exposure. After film development, band intensities were quantified using Quantity One software (v4.6.6).

Genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen, China), and global DNA methylation levels were quantified from purified DNA samples using the MethylFlash Global DNA Methylation (5-mC) ELISA Kit (EpigenTek, USA) according to the manufacturer's protocol.

DNA (3 μg) was diluted to 50 μl with sterile double-distilled water and treated with NaOH to a final concentration of 0.2 M, followed by incubation at 42 °C for 30 min. Subsequently, 30 μl of 10 mM hydroquinone, 520 μl of 3 M sodium bisulfite (pH 5.0), and 200 μl of paraffin oil were added. The mixture was incubated at 50 °C for 16 h in the dark. Bisulfite-converted DNA was purified by EZ-10 Column DNA Purification Kit (Sangon Biotech, China), and qPCR was performed with the Methylamp MS-qPCR Fast Kit (EpigenTek, USA). Primer sequences are listed in Table 2.

Nuclear proteins were extracted using the EpiQuik Nuclear Extraction Kit I (Epigentek USA). The extracted nuclear protein (10 μg) was assayed for DNA methyltransferase (DNMT) activity using the EpiQuik DNMT Activity/Inhibition ELISA Kit (Epigentek, USA) according to the manufacturer's protocol.

Molecular docking of Antro to the DNMT protein was performed using AutoDock Vina, with all water molecules excluded from the simulation. The resulting ligand-protein interactions were visualized and analyzed using a two-dimensional diagram generated with Discovery Studio 4.5.

Data were analyzed using GraphPad Prism (V9.5) and expressed as mean ± SD or mean ± SEM. Multi-group comparisons were evaluated by one-way ANOVA with Dunnett's test. For two-group comparisons, statistical tests were selected as follows: unpaired t-test for normally distributed data with equal variances; Welch's t-test for normally distributed data with unequal variances; and Mann-Whitney U-test for non-normally distributed data. The differences were considered significant at P < 0.05.

Given the unknown effects of Antro on macrophage activity, we employed a cell viability assay to evaluate its impact on primary elicited macrophages (PEMs; M0 and M1 phenotypes) and the ANA.1 cell line. Results indicated that Antro treatment significantly increased the cell viability of peritoneal M0 macrophages (P < 0.05) but had no significant effect on peritoneal M1 macrophages (Figure 1B). Furthermore, Antro did not significantly alter ANA.1 cell viability (Figure 1C). Collectively, these data demonstrate that Antro did not suppress the cell viability of tested macrophages.

Based on cell viability results, a concentration of 1 μg/ml Antro was selected for subsequent experiments. To further assess the impact of Antro on macrophage inflammatory responses, we next measured characteristic M1 macrophage cytokines (IL-1β and TNF-α) for 12h after Antro treatment. Results showed that Antro significantly reduced Il1b gene expression and IL-1β protein production in primary macrophages (P < 0.05) but did not affect Tnfa expression or TNF-α production (Figures 2A, B), and the similar results was also found in ANA.1 cell line (Figure 2C). Together, these findings demonstrate that Antro selectively inhibits IL-1β production in M1-polarized macrophages without altering TNF-α production.

IL-1β production is regulated by signaling pathways including NF-κB, mammalian target of rapamycin (mTOR), and inflammasome pathways (24–26). To investigate the mechanism by which Antro inhibits IL-1β production in M1 macrophages, we analyzed the expression of those key signaling components. Antro significantly reduced TLR4 and Myd88 protein expression and decreased phosphorylation ratios of IκB (p-IκB/IκB) and p65 (p-p65/p65; P < 0.05, Figure 3A). Correspondingly, Tlr4, Myd88, and Trif mRNA levels were also downregulated (P < 0.05, Figure 3B). In addition to NF-κB pathway, Antro reduced p-mTOR/mTOR and p-S6K/S6K ratios and NLRP3 and Caspase1 protein expression (P < 0.05, Figure 3C), and downregulated the mRNA expression of Nlrp3 and Caspase1 (P < 0.05, Figure 3D). These results demonstrate that Antro inhibits IL-1β production through suppression of NF-κB signaling pathway.

As NF-κB functions upstream of mTOR and inflammasome pathways, we speculated that Antro reduces IL-1β production in M1 macrophages primarily through suppressing the mRNA expression of Tlr4 (the most upstream initiator). To verify this, we employed a genetic approach to silence Tlr4 expression by siRNA (Figures 4A, B). Tlr4 silencing abrogated the inhibitory effects of Antro on Il1b expression and IL-1β production in M1 macrophages (P < 0.05, Figure 4C). Furthermore, it eliminated the suppression of Antro on the p-p65/p65 ratio and protein levels of Caspase1 and IL-1β (P < 0.05, Figure 4D). To further validate the critical role of Tlr4 in Antro-induced IL-1β reduction, we constructed and transfected the Tlr4 overexpression plasmids into M1 macrophages (Figure 4E). As expected, Tlr4 overexpression rescued the inhibitory effects of Antro on Il1b expression and IL-1β production in M1 macrophages (Figure 4F). Collectively, these results demonstrated that Antro inhibits IL-1β production in M1 macrophages through downregulating the mRNA expression of Tlr4.

Previous studies have demonstrated that Antroquinonol D, a ubiquinone derivative isolated from A. camphorata, functions as a DNA methyltransferase regulator (27). Given the structural similarity between Antroquinonol D and Antro, we speculated Antro might regulate DNA methylation to modulate Tlr4 expression. Consequently, we measured global DNA methylation in M1 macrophages treated with Antro. The results showed that Antro significantly increased the percentage of 5-methylcytosine (5-mC; P < 0.05, Figure 5A), indicating enhanced global DNA methylation. To assess whether Antro increased the DNA methylation of Tlr4, we performed quantitative methylation specific PCR and observed increased DNA methylation of Tlr4 (P < 0.05, Figure 5B). Subsequently, we treated M1 macrophages with the DNA methyltransferase inhibitor 5-aza-cytidine hydrate (5-Aza), which abolished Antro-mediated suppression of Tlr4 and Il1b expression, as well as IL-1β production (P < 0.05, Figures 5C–E). Collectively, these results indicate that Antro suppresses Tlr4 expression through promoting the DNA methylation of Tlr4.

In the catalysis of DNMTs, DNA undergoes methylation to alter chromatin structure or DNA conformation through the transfer of methyl groups from S-adenosylmethionine (SAM), thereby regulating the gene expression (28, 29). To clarify the mechanism by which Antro regulates DNA methylation, we analyzed the impact of Antro on substrate level and enzyme activity. Antro did not alter cellular SAM levels (Figure 6A) but significantly increased DNMT activity (P < 0.05, Figure 6B), indicating that Antro regulates DNA methylation by enhancing DNMT activities. We next investigated how Antro affects DNA methyltransferase activity at the mRNA and protein expression level. Interestingly, the results showed that Antro decreased the mRNA expression of DNMT-encoding genes, including Dnmt1, Dnmt3a, and Dnmt3l (P < 0.05, Figure 6C). In addition, Antro reduced the protein expression of DNMT1, DNMT3A, and DNMT3L (P < 0.05, Figure 6D). These findings indicate that Antro increases DNMT activity independently of transcriptional or translational processes, likely through direct binding to DNMTs to enhance their enzymatic activity. To validate the potential binding interactions between Antro and DNMTs, we performed molecular docking analyses, which demonstrated strong binding affinities between Antro and five target DNMTs (Figure 6E). These results provide structural evidence for direct molecular interactions, supporting a mechanism where Antro boosts DNMT activity post-translationally.