C57BL/6J mice were obtained from the Laboratory Animal Center of Nanfang Hospital, Southern Medical University (Medical Experimental Animal Number: SCXK-2016-0041). 1-MNA was sourced from Sigma-Aldrich (St. Louis, MO, USA). C57BL/6J mice with an IL-10 knockout (IL-10^–/–) background were purchased from Cyagen Biosciences (Suzhou, China). The IL-10^–/– mice were created by crossbreeding IL-10^–/– mice. 1-MNA was administered at a dose of 100 mg/kg body weight/day in drinking water as the vehicle. LPS was administered at a dose of 5 mg/kg body weight/day intraperitoneal injection. At the end of the study, 24 h after VRC administration, the mice were euthanized using an overdose of CO₂. Liver samples were collected and subjected to immunofluorescence staining. All animal procedures and post-treatment were approved by the Animal Ethics Committee of Nanfang Hospital (Approval No. NFYY-2020–73).

Blood samples were collected (into heparinized tubes) 4 h after a single 20 mg/kg dose of VRC. Plasma was separated by centrifugation at 13,000 rpm for 10 min, and the supernatant was collected. Additional plasma samples were centrifuged at 3,500 rpm and 4°C for 15 min. The supernatant was stored at –80°C until analysis. VRC metabolic analysis followed the methodology described in a previous study (20). The concentrations of VRC and its N-oxide derivative were measured using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The transparent supernatant was evaporated under nitrogen gas, redissolved in 100 µL acetonitrile with 0.1% formic acid (40/60, v/v), and analyzed through HPLC using standard solutions and quality control samples. Data were analyzed using PK Solver software. The VRC metabolic ratio was calculated using the following formula:

Livers of mice were processed through a 70 µm cell strainer (BD Biosciences, San Jose, CA, USA). Mononuclear cells were washed with phosphate-buffered saline (PBS), minced into small pieces, and resuspended in 40% Percoll (Sigma-Aldrich). The cell suspension was carefully layered onto 70% Percoll and centrifuged at 400 × g for 20 min. For hepatocyte or KC isolation, livers of mice were perfused with collagenase as previously described.

For enzyme-linked immunosorbent assay (ELISA) testing, reagents were prepared and samples were diluted at a 1:2 ratio. IL levels were measured using a standard curve. Liver levels of cytokines were quantified using ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Serum and supernatant concentrations of Arg-1, IL-6, IL-10, TNF-α, IL-17A, and IL-1β were measured. Cytokine levels are expressed as mass per milligram of tissue.

Liver microsomes were prepared as described in a previous study (23). Freshly collected Livers of mice were homogenized in ice-cold normal saline at a 1:3 (m/V) ratio to create a liver homogenate. The tissue was homogenized in a buffer containing sucrose and a protease inhibitor cocktail (1 mL per 400 mg tissue). The homogenate was centrifuged twice at 10,800 × g for 15 min to remove cell debris. Then the supernatant was centrifuged at 800 × g for 20 min to isolate pink translucent microsomal pellets. All procedures were performed at 4°C. The microsomal pellet was resuspended in a storage buffer and stored at –80°C until use.

The liver microsome incubation system consisted of 5 mL microsomes (20 mg/mL), 2 mL VRC (1 mM), 20 mL NADPH (1.3 mM), and 0.05 mM Tris/HCl buffer (pH 7.4), up to a total volume of 200 mL. The negative control group, excluding liver microsomes, included 2 mL 1 mM VRC, 20 mL 1.3 mM NADPH, and 0.05 mM Tris/HCl buffer at pH 7.4, also totaling 200 mL.

Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO₂ atmosphere, with treatments involving small interfering RNA (siRNA) oligonucleotides or control siRNA. The medium was changed every 3 days until the cells reached confluence. Hepatocytes were incubated for 30 min with or without LPS or IL-10. For mechanism studies, cells were harvested at different time points for polymerase chain reaction or immunoblot analysis. Each assay was repeated at least three times.

Cell viability was assessed using a CCK8 assay (Boster, Wuhan, China). Cells were rinsed with PBS. A specified quantity of cells (200 μL or 1 × 10⁴) was centrifuged, resuspended in fresh medium, and 10 µL CCK8 reagent was added to each well of a 96-well plate. The plates were incubated at 37°C for 90 min. The control well contained no cells but included the medium and CCK8 reagent. Absorbance at 490 nm was measured after 48 h using a Biotek ELISA plate reader (Winooski, VT, USA). Cell viability was calculated using a formula from a previous study (20):

qRT-PCR was used to analyze the expressions of Cyp2c38, Cyp2c29, Cyp3a11, and genes encoding specific inflammatory cytokines and transcription factors (20). Samples were homogenized for mRNA extraction using TRIzol reagent (Invitrogen). mRNA concentration was measured with an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Highly homologous and comparable product profiles between human Cyp2c19, Cyp2c9, and Cyp3a4, and mouse Cyp2c isoforms Cyp2c38, Cyp2c29, Cyp3a11, respectively, have been established (24, 25). Supplementary Table S1 provides the primer sequences for the target genes and the internal control gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The 2^–ΔΔCt method was used to quantify fold changes in relative gene expression, normalized to GAPDH, based on three replicates.

IL-10 selective siRNA oligonucleotides (20 nM) were obtained from Santa Cruz Biotechnology (catalog number: sc-39634). Cells were transfected with IL-10 siRNA using Lipofectamine™ 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Then they were cultured to 85% confluence. Control siRNA sequences (catalog number: sc-37007; Santa Cruz) were transfected into cells using the same protocol. The cells were serum-starved for 12 h prior to transfection with 20 nM siRNAs. Transfected cells underwent either protein extraction or immunofluorescence staining.

KCs were efficiently isolated from liver samples using the three-step method (26). The cells were stained with surface antibodies according to the manufacturer’s guidelines. Each group of KCs was fixed in 4% buffered formaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 30 min, and blocked with 1% bovine serum albumin (BSA) for 30 min, all at room temperature. Then the KCs were rinsed with PBS at room temperature for 5 min. They were incubated overnight at 4°C, shielded from light, with primary antibodies targeting F4/80, CD206, and iNOS, each at a dilution of 1:200. The fluorescent antibodies are listed in Supplementary Table S2 . Next, the KCs were incubated with a secondary antibody (1:1,000) at room temperature for 1 h, shielded from light. Finally, they were incubated with DAPI at 37°C for 30 min, protected from light, and kept at room temperature for 10 min. After sealing with a fluorescent quenching agent, KC staining was examined via fluorescence microscopy and analyzed using the FlowJo software (27). Cells exhibiting an M1 phenotype were characterized by F4/80⁺ iNOS⁺ CD206^– markers, while the M2 phenotype was indicated by F4/80⁺ iNOS^– CD206⁺ markers.

ChIP assays were performed using a SimpleChIP Plus Enzymatic ChIP kit as previously described (20, 28). Livers of mice were preserved using formaldehyde. Following sample lysis, chromatin was sheared and subjected to immunoprecipitation with either anti-p65 or normal IgG. DNA fragments were purified and quantified using qPCR with specific primers ( Supplementary Table S1 ).

Each group of KCs and hepatocytes was fixed in 4% buffered formaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 30 min, and blocked with 1% BSA, all at room temperature. KCs or hepatocytes were washed with PBS at room temperature for 5 min. The KCs were incubated overnight at 4°C, shielded from light, with primary antibodies targeting iNOS and IL-10, both at a dilution of 1:200. The hepatocytes were incubated with primary antibodies against p65 (1:200). Both sample types were washed and kept in the dark at room temperature for 5 min. They were incubated with a secondary antibody (1:1,000) at room temperature for 1 h, protected from light and subsequently washed and shielded from light at room temperature for 5 min. Finally, they were incubated with DAPI at 37°C for 30 min, protected from light. They were stained and observed using fluorescence microscopy after sealing with a fluorescent quenching agent.

Experimental data are reported as means and standard deviations (SDs). GraphPad Prism 8.0 was used for data analysis. The Student’s t-test was used to assess differences between two groups, while the Kruskal-Wallis test followed by the Dunn’s post hoc test were used for comparisons among multiple groups. Linear regression analysis was performed using the loss minimization function in R 3.5.3 (R Core Team, Vienna, Austria). Other statistical methods are reported accordingly. Results with P < 0.05 were considered statistically significant.

The systemic inflammatory response model induced by LPS suppresses the expression of hepatic drug-metabolizing enzymes. In this study, we investigated the impact of 1-MNA on the inflammatory response in the liver and its regulation of drug-metabolizing enzymes, particularly in relation to VRC metabolism. Liver microsomes, hepatocytes, and KCs were isolated from liver tissues following intragastric administration of 1-MNA (Figure 1A). Given that VRC metabolism is influenced by three key drug-metabolizing enzymes, CYP2C38, CYP2C29, and CYP3A11, we sought to elucidate the effects of 1-MNA on these enzymes and the potential mechanisms involved (Figure 1B).

To assess the inflammatory response, we quantitatively analyzed the expression of four pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-17A) and one anti-inflammatory cytokine (IL-10) in the liver tissues across experimental groups. The LPS-induced model group showed a marked increase in pro-inflammatory cytokine expression ( Figures 1C, D, G, H ) and a significant reduction in anti-inflammatory cytokines (IL-10) and the M2 macrophage marker Arg-1 ( Figures 1E, F ). Following 1-MNA administration, there was a significant reduction in pro-inflammatory cytokines and an increase in anti-inflammatory markers in the liver tissues of both the LPS model and normal rats ( Figures 1C–H ).

Furthermore, we analyzed the relationship between inflammatory cytokine levels and the metabolic rate of VRC. An increase in IL-10 levels was positively correlated with an enhanced metabolic rate of VRC in the liver ( Figures 1I, J ). These findings indicate that IL-10 may facilitate improved VRC metabolism in an inflammatory hepatic environment, although the precise mechanism underlying this effect remains unclear.

Gene expression analysis of the drug-metabolizing enzymes revealed that 1-MNA significantly upregulated the expression of Cyp2c38 in an LPS-induced inflammatory environment, while having no observable effect on Cyp2c29, Cyp3a11, or P-glycoprotein gene expression. In addition, we assessed the expression of nuclear transcription factors involved in the regulation of drug-metabolizing enzymes. The expression of the pregnane X receptor (PXR) was significantly increased following 1-MNA administration, suggesting that 1-MNA may enhance Cyp2c38 gene expression by upregulating PXR expression under inflammatory conditions.

To investigate the anti-inflammatory effects of 1-MNA and its role in upregulating CYP2C38 expression, KCs were isolated from liver tissue, cultured, and analyzed in vitro. Immunofluorescence analysis revealed that 1-MNA significantly increased the expression of the M1 macrophage marker protein iNOS ( Figure 2A ) and the M2 macrophage marker protein IL-10 ( Figure 2B ).

Flow cytometric analysis of liver tissue showed that, compared to a water-treated group, the number of F4/80⁺iNOS⁺CD206^– cells (M1 macrophages) induced by LPS was significantly higher. However, administration of 1-MNA resulted in a substantial reduction in these pro-inflammatory cells compared to the LPS group ( Figure 2C ). Conversely, the number of F4/80⁺iNOS^-CD206⁺ cells (M2 macrophages) was significantly lower in the LPS-induced group than in controls but increased markedly following 1-MNA administration ( Figure 2D ).

Comparative analysis of KC polarization types revealed that the M1/M2 ratio in LPS-induced inflammatory rats was significantly reduced following 1-MNA administration. This was accompanied by a significant increase in IL-10 levels in liver tissue ( Figure 2E ). Correlation analysis further demonstrated a significant negative association between the M1/M2 ratio and IL-10 levels, with R² = 0.61 ( Figure 2F ). These findings suggest that 1-MNA mitigates the pro-inflammatory response by promoting KC polarization toward the anti-inflammatory M2 phenotype.

Mechanistic experiments were conducted on isolated primary cells to elucidate how 1-MNA and IL-10 enhance hepatic enzyme activity, correlated with in vivo outcomes. The CCK8 assay was used to determine the optimal concentrations of 1-MNA, IL-10, and LPS for direct administration to liver cells. As shown in Figure 3 , 1-MNA concentrations exceeding 10 μmol/L significantly reduced hepatocyte viability. IL-10 did not significantly affect liver cell viability within the range of 1–50 ng/mL. The effects of combining LPS with either 1-MNA or IL-10 on cell viability were also assessed. In combination with LPS (1 μg/mL), 1-MNA (0.1 μg/mL) did not significantly affect cell viability (P > 0.05), IL-10 (10 μg/mL) significantly reduced cell viability (P < 0.05), and IL-10 (5 μg/mL) had no effect on hepatocyte viability.

To create an efficient inflammatory cell model without compromising cell viability, the in vitro LPS concentration was established at 1 μg/mL. These findings guided further exploration of the effects of LPS combined with 1-MNA or IL-10 on CYP450 activity and expression. The optimal concentrations for the in vitro model were determined as 1 μg/mL LPS (inflammatory stimulus), 0.1 μg/mL 1-MNA, and 5 ng/mL IL-10 ( Figure 3D ).

Following the combined administration of 1-MNA and LPS to hepatocytes, the expression of CYP450 enzymes (CYP2C38, CYP3A11, and CYP2C29) did not significantly differ from hepatocytes treated with LPS alone ( Figure 3E ), indicating that 1-MNA does not directly upregulate CYP450 enzyme expression. Conversely, the combined administration of IL-10 with LPS resulted in a significant increase in the expressions of CYP2C19 and CYP3A4 ( Figure 3F ) and upregulation of PXR transcriptional expression under LPS-induced pro-inflammatory conditions ( Figure 3G ).

NF-κB-p65 in hepatocyte nuclei significantly inhibits the transcription of CYP2C19 (20). To further explore the effect of IL-10 on p65 expression in hepatocytes, immunofluorescence experiments were conducted on primary hepatocytes. IL-10 administration significantly reduced p65 expression in hepatocytes, an effect that was abolished when IL-10 expression was silenced ( Figure 4A ). These findings suggest that IL-10 inhibits p65 expression in LPS-induced inflammatory hepatocytes.

Furthermore, IL-10 administration upregulated CYP2C38 expression in inflammatory hepatocytes ( Figure 4B ), indicating a direct regulatory role of IL-10 in enhancing CYP2C38 expression under inflammatory conditions.

To elucidate the underlying mechanism, Western blotting analysis was performed to assess p65 expression in the cytoplasm and nucleus. IL-10 administration inhibited nuclear p65 expression without affecting cytoplasmic levels ( Figure 4C ). In addition, cytoplasmic PXR expression was significantly increased ( Figures 4C, D ). These findings demonstrate that IL-10 specifically suppresses nuclear p65 activity in inflammatory hepatocytes.

ChIP analysis was used to further investigate the regulatory role of NF-κB on PXR transcription in vivo ( Figure 4E ). The results demonstrated that activated NF-κB-p65 was recruited to PXR-binding sites in hepatocytes, suppressing its expression. However, IL-10 administration significantly reduced p65 recruitment at the PXR-binding site, while no significant differences were observed in p65 accumulation at distal non-binding regions (NRs) ( Figure 4E ).

These results suggest that activated NF-κB specifically inhibits PXR transcription in hepatocytes and that IL-10 administration reverses this effect, highlighting the critical regulatory role of IL-10 in modulating the NF-κB/PXR pathway in inflammatory hepatocytes.

In vivo validation of the findings in animal models is crucial for understanding potential drug interactions. To confirm the results, IL-10 gene knockdown (IL-10^–/–) mice were used for further investigation. The experimental design included four groups, with Group 3 involving IL-10^–/– mice and a 1-week combined treatment of LPS and 1-MNA. On the first day following the week-long treatment, liver microsomes were isolated for pharmacokinetic testing to assess the effects of different treatments on VRC metabolism ( Figure 5A ).

As anticipated from the in vitro data ( Figures 5B ), IL-10 knockdown in vivo led to a reduction in PXR expression in the cytoplasm, indicating that IL-10 upregulates PXR transcriptional expression. The mechanism likely involves IL-10 inhibiting the nuclear transcription of p65.

Regarding VRC metabolism, we measured VRC nitrogen oxide levels 4 h after administering the drug to isolated liver microsomes ( Figure 5C ). In IL-10 knockdown mice, the concentration of VRC nitrogen oxides was significantly lower than in the other three groups. By contrast, LPS-treated hepatitis mice administered 1-MNA exhibited significantly higher nitrogen oxide levels compared to both the LPS and IL-10 knockdown groups.

Based on these findings, a mechanistic model was proposed: IL-10 upregulates the expression of CYP2C38, which metabolizes VRC, by reducing the inhibitory effects of nuclear p65 on PXR transcription. This ultimately leads to reduced accumulation of the parent compound, VRC, in the liver ( Figure 5D ).