Acetaminophen overdose-induced acute liver injury can be alleviated by static magnetic field

Abstract

Acetaminophen (APAP), the most frequently used mild analgesic and antipyretic drug worldwide, is implicated in causing 46% of all acute liver failures in the USA and between 40% and 70% in Europe. The predominant pharmacological intervention approved for mitigating such overdose is the antioxidant N-acetylcysteine (NAC); however, its efficacy is limited in cases of advanced liver injury or when administered at a late stage. In the current study, we discovered that treatment with a moderate intensity static magnetic field (SMF) notably reduced the mortality rate in mice subjected to high-dose APAP from 40% to 0%, proving effective at both the initial liver injury stage and the subsequent recovery stage. During the early phase of liver injury, SMF markedly reduced APAP-induced oxidative stress, free radicals, and liver damage, resulting in a reduction in multiple oxidative stress markers and an increase in the antioxidant glutathione (GSH). During the later stage of liver recovery, application of vertically downward SMF increased DNA synthesis and hepatocyte proliferation. Moreover, the combination of NAC and SMF significantly mitigated liver damage induced by high-dose APAP and increased liver recovery, even 24 h post overdose, when the effectiveness of NAC alone substantially declines. Overall, this study provides a non-invasive non-pharmaceutical tool that offers dual benefits in the injury and repair stages following APAP overdose. Of note, this tool can work as an alternative to or in combination with NAC to prevent or minimize liver damage induced by APAP, and potentially other toxic overdoses.

INTRODUCTION

Acetaminophen (N-acetyl-p-aminophenol, paracetamol, APAP) is a common over-the-counter (OTC) medication for fever reduction and a first-line treatment for various pain conditions, as recommended by the World Health Organization (WHO) (Ennis et al., 2016). Concurrently, misuse or accidental overdose of APAP represents the leading cause of acute liver failure (ALF), a phenomenon prevalent in Western countries and exacerbated during the COVID-19 pandemic (Ortiz et al., 2021; Saha et al., 2022). In both Europe and North America, the incidence of acute liver injury attributed to APAP overdose exceeds all other etiologies combined.

Following APAP overdose, the body undergoes several phases, including the initial injury stage and later repair stage. During the injury stage, a fraction of APAP is metabolized into N-acetyl-p-benzoquinone imine (NAPQI), which triggers oxidative stress and subsequent hepatocyte necrosis. The formation of NAPQI and reactive oxygen species (ROS) are the main contributors to APAP-induced hepatotoxicity (Du et al., 2016). At present, N-acetylcysteine (NAC) remains the sole approved treatment for APAP overdose, functioning by replenishing glutathione (GSH) levels to detoxify NAPQI and effectively mitigate oxidative stress (Barbier-Torres et al., 2017; González-Recio et al., 2022; Jaeschke et al., 2012; Polson & Lee, 2005; Prescott et al., 1977).

During the repair stage, immune response and liver cell proliferation play important roles. The remarkable regenerative capacity of the liver not only enables the growth of new liver lobes after excision but also allows for regeneration after acute drug-induced toxicity. As hepatocytes metabolize drugs such as APAP, they may succumb to toxicity from metabolic intermediates, leading to cell death, while the residual liver tissue initiates regeneration processes, facilitating liver repair (Yanger et al., 2014). Neutrophils can promote the development of reparative macrophages through ROS-mediated mechanisms to induce liver repair (Yang et al., 2019). Inhibition of platelet C-type lectin-like receptor (CLEC-2) signaling can enhance liver recovery by increasing tumor necrosis factor-α (TNF-α) production and reparative hepatic neutrophil recruitment (Chauhan et al., 2020). While exosomes do not directly mitigate hepatocyte injury by modulating oxidative stress, they can elicit hepatoprotective effects through activation of proliferative and regenerative responses (Tan et al., 2014). Moreover, Schisandrol B can also protect against acetaminophen-induced hepatotoxicity by increasing liver regeneration (Jiang et al., 2015).

Although APAP overdose-induced acute liver failure is prevalent in many developed countries, the antioxidant NAC remains as the only therapeutic option recommended by the United States Food and Drug Administration (FDA) (Larowe et al., 2006; Rumack & Bateman, 2012; Saito et al., 2010). While NAC can effectively counteract early oxidative stress-induced liver injury, its efficacy diminishes during the later repair stage, functioning within a limited timeframe after APAP overdose and falling short in treating advanced liver injuries or when administered during later stages critical for liver repair (Licata et al., 2022; Rumack & Bateman, 2012). To date, however, only a few studies have explored novel or combinational therapies for APAP overdose, such as prostaglandin E2 (PGE2) combined with NAC, which has been shown to synergistically reduce APAP-associated liver toxicity in zebrafish (Nam et al., 2017; North et al., 2010). Thus, the development of new strategies to overcome toxicity caused by this ubiquitous pain reliever remains urgent (Lee, 2017).

Research has demonstrated the impact of static magnetic fields (SMFs) on both oxidative stress and tissue regeneration. For example, exposure to 200 μT SMF has been found to promote radical recombination, inhibiting ROS formation and affecting stem cell proliferation, differentiation, and new tissue growth in the planarian regeneration models (Van Huizen et al., 2019). Similarly, moderate, near-homogenous SMFs ranging from 0.1 T to 0.2 T have been shown to lower oxidative stress, thereby reducing cisplatin and alcohol-induced tissue toxicity and prolonging mouse survival after high doses of these substances (Song et al., 2023; Yu et al., 2023b). Furthermore, the effects of moderate and high SMFs on DNA synthesis are dependent on magnetic field direction, due to the divergent Lorentz forces acting on negatively charged DNA in motion, which differentially affects DNA supercoiling and synthesis (Song et al., 2023; Yang et al., 2020, 2021).

Here, we investigated the effects of moderate SMFs on APAP-induced acute liver injury. Results showed that SMFs reduced NAPQI and ROS levels in the liver injury stage to minimize APAP-induced toxicity. Moreover, in the later liver repair stage, vertically downward SMFs promoted liver regeneration by increasing DNA synthesis. Importantly, SMFs combined with NAC demonstrated promising therapeutic effects on high-dose APAP-induced liver toxicity, even when SMF+NAC was applied 24 h after APAP overdose.

MATERIALS AND METHODS

Magnetic field setup

The magnetic field exposure devices used for animal experiments, as shown in Supplementary Figure S1, followed our previous studies (Fan et al., 2023; Song et al., 2023; Yu et al., 2023b). The sham (placebo exposure) group was placed on an unmagnetized neodymium plate. The upward and downward quasi-uniform SMFs were provided by permanent magnet plates (length×width×height=250 mm×160 mm×45 mm) consisting of 12 neodymium magnet cubes (length×width×height=60 mm×50 mm×30 mm) purchased from Zhongxin Magnetoelectricity (China).

Field distributions were measured using a magnet analyzer (FE-2100RD, Forever Elegance, China) with a Hall sensor (HG-106A, Asahi Kasei, Japan). The data at each point represented measured magnetic field intensity along the Z direction. The magnetic flux densities on the surface of the magnet plates were approximately 0.2 T, with the distributions of the magnetic field at 1.5 cm above the magnet plate, at the approximate location of the liver, shown in Supplementary Figure S1B. At this position, the magnetic flux density was about 0.1 T (measured density 116–131 mT) and the gradient was about 3.0 T/m along the Z axis.

The magnetic field exposure devices used for cell experiments are different from animal experiments. Magnetized neodymium N38 (length×width×height: 60 mm×50 mm×35 mm) was used to generate a magnetic field of up to 0.5 T on its surface. Cell culture dishes were positioned on the magnet surface to generate downward or upward SMFs. Control and sham groups were placed at a distance from the magnet, where the magnetic field strength was significantly lower, approximately 0.001 T. The magnets were placed in a cell incubator (Thermo Fisher Scientific, USA) at 37°C and 5% CO₂.

Mouse model

Male C57BL/6J mice (4–8 weeks of age) were purchased from GemPharmatech Co., Ltd. (Nanjing, China). All mice were kept in an air-conditioned room (21–25°C) under 50%–60% humidity and a 12 h light-dark cycle. Before the experiments, the mice were subjected to an adaptive feeding period of one week. All animal experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication, 8th Edition, 2011) and approved by the Association of Laboratory Animal Sciences at Anhui Medical University (permit No. LLSC20232244).

Mice in the multiple lethal APAP dose group were intraperitoneally injected with 550 mg/kg, 550 mg/kg, 800 mg/kg, and 1 g/kg APAP (HY-66005, MedChemExpress, China) in sequence. SMF was applied immediately or 3 h after APAP injected and the survival rate of the mice was recorded. APAP was dissolved in phosphate-buffered saline (PBS) using ultrasonication and heating methods. The control group was administered the same volume of PBS.

In the acute liver injury model, 350 mg/kg APAP was administered to mice. Mice were treated with SMFs for 33 days or 2 days to assess whether the ameliorating effect of SMFs on liver injury was related to the duration of magnetic field treatment. The control group was administered the same volume of PBS. NAC-treated mice received an intraperitoneal injection of 1 200 mg/kg NAC (HY-B0215, MedChemExpress, China), with the pH adjusted to 7.4 before the injection.

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assay

Blood samples were collected in 1.5 mL centrifuge tubes and centrifuged at 4°C for 15 min at 3 500 r/min (centrifugation radius of 8 cm) to collect serum. Serum ALT and AST levels were measured using ALT (C009-2-1, Nanjing Jiancheng Bioengineering Institute, China) and AST assay kits (C010-2-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.

Hematoxylin and eosin (H&E) staining and immunohistochemistry

Liver tissues were fixed in 4% neutral-buffered formalin (Servicebio, China) and embedded in paraffin following standard procedures. Embedded liver tissues were sliced into 5-μm-thick sections, and stained with H&E or subjected to immunohistochemical staining using anti-NRF2 (GB113808-100, Servicebio, China, 1:500), anti-PCNA (13110, Cell Signaling Technology, USA, 1:500), anti-Ki67 (12202, Cell Signaling Technology, USA, 1:500), anti-TOP2A (GB111031-100, Servicebio, China, 1:500), anti-MPO (M33064, ImmunoWay, USA, 1:500), anti-F4/80 (30325, Cell Signaling Technology, USA, 1:500), and HO-1 antibodies (10701-1-AP, Proteintech, USA, 1:500) according to the manufacturers’ protocols.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)

A TUNEL detection kit (C1098, Beyotime, China) was used to detect the apoptosis in liver sections. Cells were stained with 3,3’-diaminobenzidine (DAB) reagent and incubated at room temperature for 5–30 min. Three random fields were selected for quantification from each slide.

Oil Red O staining

Lipid accumulation in liver sections was detected using Oil Red O staining solution (G1261, Solarbio, China). Frozen liver sections were first fixed in 10% formalin for 10 min, followed by immersion in 100% propylene glycol for 5 min. Then incubating slide in heated (60°C) Oil Red O solution for 6–10 min or overnight at room temperature.

The sections were then transferred to 85% propylene glycol for 2 min and rinsed in deionized water for 1 min.

Immunofluorescence staining

Frozen liver sections were treated with PBS for 10 min, followed by tissue autofluorescence quencher. Subsequently, 100 μL of staining working solution (DHE dilution, 1:1 000) was added to the sections, which were then incubated at 37°C for 60 min in the dark. Finally, the sections were stained with 4’,6-diamidino-2-phenylindole and sealed with antifade mounting medium.

Serum NAPQI detection

Serum NAPQI levels were analyzed using a mouse NAPQI enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (YJ663215, China). Firstly, 5-fold diluted mouse serum was added to micro-ELISA strips. Horseradish peroxidase (HRP)-conjugate reagent (100 µL) was then added to each well and incubated for 60 min at 37°C. After washing in wash solution, chromogen solution A (50 µL) and solution B (50 µL) were added, followed by incubation for 15 min at 37°C. Finally, stop solution (50 µL) was added and absorbance was measured at 450 nm using a microplate reader.

Measurement of GSH, malondialdehyde (MDA), and superoxide dismutase (SOD)

Liver tissue was accurately weighed, followed by the addition of saline at a ratio of 1:9. The tissue homogenate was then centrifuged at 4°C, 2 500 r/min for 10 min (centrifugation radius of 8 cm), with the resulting supernatant taken for detection of GSH, MDA, and SOD using a GSH kit (A006-2-1, Nanjing Jiancheng Bioengineering Institute, China), MDA kit (A003-1-2, Nanjing Jiancheng Bioengineering Institute, China), and SOD kit (A001-3, Nanjing Jiancheng Bioengineering Institute, China), respectively.

Western blotting

Protein was extracted from liver tissue and subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% non-fat dried milk, and incubated with NRF2 (GTX103322, GeneTex, USA), PCNA (13110, Cell Signaling Technology, USA), NQO-1 (GTX100235, GeneTex, USA), and β-actin antibodies (HC201-01, TransGen Biotech, China).

Real-time reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was purified from samples using TRIzol reagent (15596018, Thermo Fisher, USA) according to the manufacturer’s instructions. The cDNA was synthesized using a HiScript Ⅲ 1st strand cDNA synthesis kit (R312-01, Vazyme, China), with ChamQ SYBR qPCR master mix (Q311-02, Vazyme, China) used to amplify target genes under the action of specific primers. Detailed information on primers is shown in Supplementary Table S1.

Complete blood count test

The mice were sacrificed to collect blood samples, which were mixed with EDTA-2K anticoagulant. A BC-2800vet automatic hematology analyzer (Mindray China) was used for complete blood cell count analysis.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

LC-MS/MS (Triple Quad 5500+, USA) was used to detect APAP concentrations at different time points. After they were completely melted, the samples were mixed for 10–30 s and centrifuged at 4 000 r/min and 4°C for 0.5 min. Subsequently, 20 μL of plasma (blank sample and internal standard blank sample plus 20 μL of blank plasma) was added to a 96-well plate, followed by the addition of 200 μL of 50% methanol-acetonitrile solution containing internal standard (100 ng/mL). After vortexing for 5 min, the samples were centrifuged at 14 000 r/min and 4°C for 5 min, after which 100 μL of the supernatant was removed and mixed evenly with 100 μL of pure water for LC-MS/MS analysis.

Cell culture

HepG2 cells were cultivated in Dulbecco’s Modified Eagle’s Medium (Corning, USA) supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (Invitrogen, USA) at 37°C under a humidified atmosphere of 5% CO₂.

Cell counting kit‐8 assays (CCK-8)

Cells (5×10³ cells) were seeded in 96-well plates containing 100 μL of complete culture medium and different concentrations of APAP for 48 h. The Cell Counting Kit-8 assay (HY-K0301, MedChemExpress, China) was used to measure cell viability in accordance with the manufacturer’s instructions. Absorbance was then measured at 450 nm using a microplate reader.

Cell number and apoptosis analysis

Cells (8×10⁴ cells/mL) were seeded in 3.5 cm cell dishes for 12 h, after which 6 mmol/L APAP was added and the dishes were placed on the magnet. The cells were harvested after 12 h or 24 h and counted by flow cytometry (CytoFLEX, Beckman Coulter, USA).

Annexin V/propidium iodide (PI) staining (0076884, BD Biosciences, USA) was performed to assess cell apoptosis. The harvested cells were washed twice with cold PBS, resuspended in binding buffer (500 μL), and stained with annexin V-fluorescein isothiocyanate conjugate (10 μL) and PI (5 μL) at room temperature for 15 min in the dark. The proportion of apoptotic cells was determined by flow cytometry.

Measurement of cellular ROS levels

The fluorogenic probe 2’,7’-dichlorofluorescin diacetate (DCFH-DA, D6883, Sigma, USA) was used to detect cellular ROS levels. Similar to cell number and apoptosis analyses, the harvested cells were washed with PBS, then incubated with 10 μmol/L DCFH-DA at 37°C for 30 min in serum-free medium. Fluorescent intensities were tested using a flow cytometer.

Cell DNA synthesis assay

To explore the combined effects of the drug and magnetic field on DNA synthesis, a DNA synthesis assay was performed following previously described protocols (Yang et al., 2021), except 3 mmol/L APAP was added at the same time as 10 μmol/L 5-bromo-2’-deoxyuridine (BrdU) antibody (5292, Cell Signaling Technology, USA).

Statistical analysis

Significant differences between two groups were assessed using unpaired or paired two-tailed t-tests. All experimental data are expressed as mean±standard error of the mean (SEM). A P-value of less than 0.05 was regarded as statistically significant. All statistical analyses were performed using GraphPad Prism software v.8.0 (GraphPad Software, USA). To reduce potential experimenter bias and unreproducible results, all experiments were performed in a blinded manner or repeated by at least two independent researchers.

RESULTS

SMFs reduce lethality and liver damage induced by repeated high-dose APAP

We initially examined the effects of magnetic fields on mice subjected to lethal doses of APAP using neodymium magnetic plates, which generate SMFs in orientations either vertically upward (opposite to gravity) or downward (aligned with gravity), to assess their effects on APAP-induced liver injury (Figure 1A; Supplementary Figure S1). An unmagnetized neodymium plate served as the sham control. The entire cage containing mice was positioned on the magnetic plate, exposing mice to a magnetic field intensity of 0.1–0.2 T (Figure 1A; Supplementary Figure S1B). Two treatment methods were used, applying SMFs immediately after APAP administration or 3 h after each APAP administration (Figure 1B). Body weight and survival were monitored for 48 h after final drug administration, with the surviving mice ultimately sacrificed for further examinations.

Figure 1.

SMFs reduce lethality and liver damage induced by repeated high-dose APAP

A: Mouse cage was placed on magnetic plate, and mice were subjected to SMFs (0.1–0.2 T). B: Experimental design. Experiment was divided into six groups: control, sham, immediate magnetic field treatment (downward and upward), and magnetic field treatment 3 h after administration (post-downward and post-upward) groups. Injection of PBS or APAP was carried out every 72 h, and mice were dissected 48 h after final injection. n=10 mice per group. C: Body weight curves of mice. Comparisons were made with sham group. D: Survival curves. P: Sham vs. downward group. E: Survival percentage at different time points after APAP injection. F: Representative liver H&E staining. Necrotic areas are outlined using dotted lines. G, H: Quantification of necrosis area in mouse liver and red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT) in mouse plasma. F–H were performed on surviving mice at the end of the experiments (Control, n=3 mice; Sham, n=6 mice; Downward SMF, n=10 mice; Upward SMF, n=6 mice; Post-downward SMF, n=9 mice; Post-upward SMF, n=7 mice). Data represent mean±SEM. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001.

As expected, mice injected with high-dose APAP showed a reduction in body weight (Figure 1C). The vertically downward SMFs mildly alleviated this weight reduction, but not the upward SMFs. Moreover, SMFs, whether applied early or late and in either upward or downward directions, alleviated high-dose APAP-induced lethality. The vertically downward SMFs exhibited more obvious beneficial effects, especially when the SMF was applied immediately after APAP, but not 3 h later (Figure 1D, E). After the first APAP dose, the sham group had a mortality rate of 40%, while mice in the downward and post-downward SMF groups were all alive (Figure 1E). Upon completion of all four APAP doses, all mice in the downward SMF group were still alive.

As liver necrosis is a typical consequence of APAP toxicity, we examined liver tissues by H&E staining (Figure 1F). Results showed that high-dose APAP-induced liver cell necrosis was markedly reduced by multiple SMFs (Figure 1F, G), indicating reduced liver damage. The downward SMFs reduced liver necrosis by approximately 50%. Moreover, the blood analysis results (Figure 1H; Supplementary Figure S2) showed that the red blood cell abnormalities induced by high-dose APAP were significantly reduced by SMFs applied immediately after APAP injection, indicating that prolonged and/or immediate SMF application has a beneficial effect on APAP toxicity.

SMF pretreatment reduces sublethal APAP dose-induced liver injury

Given the importance of SMF application timing in its alleviation effects on APAP-induced liver damage, we subsequently examined the efficacy of SMF pretreatment by exposing mice to SMFs for one month prior to administering a single dose of 350 mg/kg APAP, with analyses conducted 2 days post-injection (Figure 2A). Results showed that SMF pretreatment did not affect food or water consumption (Supplementary Figure S3), nor did it lower the elevated APAP-induced liver index (Supplementary Figure S4A). However, significant alterations were observed in the H&E, TUNEL, and Oil Red O-stained liver sections from the SMF-treated groups (Figure 2B). Both upward and downward SMFs were effective at reducing liver necrosis and apoptosis (Figure 2B–D). Notably, the area of liver necrosis in the APAP group was 31.8%±3.9%, which decreased to 18.0%±2.1% (P<0.01) and 21.8%±2.3% (P<0.01) under downward and upward SMFs, respectively (Figure 2C). TUNEL assays revealed that the downward and upward SMFs diminished the apoptotic cell number to 67.2%±13.3% (P=0.1417) and 58.3%±8.3% (P<0.05), respectively (Figure 2D). Oil Red O staining highlighted a significant decline in lipid accumulation after downward SMF treatment, indicating improved liver cell function (Figure 2E). Blood cell count and blood biochemical analyses revealed no significant modifications following SMF treatment, except for a slight, non-significant improvement in AST, ALT, and platelet count (PLT) levels in the downward SMF group (Figure 2F–H; Supplementary Figure S4B). These findings suggest that SMF pretreatment can reduce APAP-induced acute liver tissue injury, with downward SMFs showing potentially superior efficacy.

Figure 2.

SMFs reduce single high-dose APAP-induced liver injury

A: Experimental design. B: Representative liver images, H&E, TUNEL, and Oil Red O staining. Necrotic areas are outlined using dotted lines. C–E: Quantification of necrosis area (Control, n=3 mice, 3 fields were quantified from one slice per mouse; Sham, Downward and Upward, n=6 mice per group, 3 fields were quantified from one slice per mouse), apoptosis cell number (Control, n=3 mice, 3 fields were quantified from one slice per mouse; Sham, Downward and Upward, n=6 mice per group, 3 fields were quantified from one slice per mouse), and lipid accumulation (n=3 mice per group). F, G: Relative serum levels of ALT and AST (Control, Sham and Downward, n=6 mice per group; Upward, n=5 mice). H: PLT in mouse plasma (Control, Sham and Downward, n=6 mice per group; Upward, n=5 mice). Data represent mean±SEM. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001; ^****: P<0.0001.

SMFs reduce APAP-induced liver oxidative stress in vivo and in vitro

To investigate how SMFs alleviate liver injury, we measured plasma APAP concentrations at different time points (Supplementary Figure S5A). Results showed that plasma APAP concentrations were higher in the downward and upward SMF groups compared to the sham group (Supplementary Figure S5B). The rate at which the drug was eliminated from the body per unit of time was calculated, revealing that the clearance (CL) rates in the SMF-exposed groups were lower than those in the sham group (Supplementary Figure S5C). As NAPQI is the principal toxic metabolite of APAP responsible for liver injury, NAPQI levels were next assessed. Results showed a reduction in plasma levels of NAPQI under SMF exposure, suggesting that SMFs hinder the conversion of APAP to toxic NAPQI (Supplementary Figure S5D). The mRNA levels of cytochrome P450 2E1 (CYP 2E1) and cytochrome P450 3A11 (CYP3A11) in the liver, major enzymes involved in APAP metabolism, were also assessed. Results showed that SMFs did not alter the expression of CYP2E1 and CYP3A11 in the liver (Supplementary Figure S5E, F). Thus, these findings suggest that the SMFs modulate NAPQI production, without affecting the expression of CYP450 enzymes.

Given that liver injury induced by APAP typically results from NAPQI-triggered oxidative stress, the levels of oxidative stress in mouse liver tissues were next measured, first examining nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor that plays a key role in antioxidation, and dihydroethidium (DHE), a marker for ROS levels (Figure 3A–E). Results indicated that both indicators declined markedly following SMF exposure. Quantitative analysis demonstrated that SMF treatment lowered NRF2 and especially ROS to levels comparable to those of the control group (Figure 3C). Moreover, oxidative stress marker proteins NQO-1 NAD(P)H quinone oxidoreductase 1) and MDA were both reduced following SMF treatment, indicating lower oxidative stress (Figure 3D–G). Notably, consistent with the lower NAPQI levels in plasma (Supplementary Figure S5D), levels of GSH, the principal intracellular antioxidant that reacts with toxic NAPQI, were significantly increased after SMF treatments (P<0.0001) (Figure 3H), underscoring their antioxidative effects.

Figure 3.

SMFs reduce APAP-induced liver oxidative stress both in vivo and in vitro

A–C: Liver immunohistochemical staining and quantification for NRF2 (Control, n=3 mice, 3 fields were quantified from one slice per mouse; Sham, Downward and Upward, n=6 mice per group, 3 fields were quantified from one slice per mouse) and immunofluorescent analysis of DHE-positive cells and quantification (n=3 mice per group). D–F: Representative western blot images for NRF2 and NQO-1 and their quantifications (n=3 biological replicates over three independent experiments). G, H: GSH and MDA levels in liver tissues (n=6 mice per group). I: Cellular experimental set up and magnetic field distribution at 1 mm above the magnet. HepG2 cells were treated with or without SMFs for 12 h (cell number and ROS detection), 24 h (apoptosis detection), and 48 h (cell viability detection) before analysis. J: Relative cell viability of different concentrations of APAP-treated HepG2 cells. K–M: Relative cell number, apoptosis, and ROS levels for cells treated with or without 6 mmol/L APAP and/or upward or downward SMFs. Data represent mean±SEM. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001; ^****: P<0.0001.

Next, cell culture plates were placed on the surface of the magnet, exposing cells to an approximately SMF of 0.4 T (Figure 3I). The direct effects of SMFs on oxidative stress were then examined. Results confirmed that APAP administration reduced liver cell viability in a concentration-dependent manner (Figure 3J). Moreover, consistent with the mouse liver tissue observations, SMF exposure, especially downward SMFs, mitigated APAP-induced cell number reduction, apoptosis, and ROS elevation (Figure 3K–M).

Downward SMF promotes hepatocyte proliferation

The process of liver regeneration after APAP overdose is initiated by hepatocyte death, which triggers an immune response marking the priming phase of regeneration. We quantified neutrophils by MPO and macrophages by F4/80 staining (Supplementary Figure S6A). Results showed that APAP treatment significantly increased neutrophil number, although SMF exposure did not induce marked effects (Supplementary Figure S6B). Similarly, macrophage counts were not substantially affected (Supplementary Figure S6C). Analysis of TNF-α and IL-6 mRNA levels showed no significant differences between the SMF groups compared to the sham group (Supplementary Figure S6D). These findings suggest that the beneficial impact of SMFs on the liver is not mediated through modulation of the immune response during the transitional phase of liver injury and repair.

Beyond the immune response, we also investigated the effects of SMFs on hepatocyte proliferation, a critical aspect of liver regeneration. Examination of cell proliferation markers, including PCNA, Ki67, and TOP2A, revealed significant differences among experimental groups (Figure 4A). Notably, the levels of PCNA, Ki67, and TOP2A all increased in the APAP group, indicating active liver cell proliferation after acute liver injury. Moreover, cell proliferation was promoted by downward SMFs, but not upward SMFs (Figure 4B, C). Immunohistochemical and western blot analyses also indicated that downward SMF exposure increased APAP-treated liver cell proliferation, while upward SMF exposure had an inhibitory effect (Figure 4B, D, E).

Figure 4.

Downward SMF promotes hepatocyte proliferation, while upward SMF does not

A: Respective intrahepatic staining of PCNA, Ki67, and TOP2A in mice. B, C: Quantification of relative PCNA and Ki67-positive cell number (Control, n=3 mice, 3 fields were quantified from one slice per mouse; Sham, Downward and Upward, n=6 mice per group, 3 fields were quantified from one slice per mouse). D, E: Representative western blot images for PCNA and its quantification (n=3 biological replicates over three independent experiments). F: In vitro BrdU incorporation cellular experiment showing increased DNA synthesis in downward SMF-treated cells. Data represent mean±SEM. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001; ^****: P<0.0001.

DNA synthesis, an essential event in cell proliferation, is differentially affected by vertically upward and downward SMFs due to the Lorentz force exerted on the negatively charged moving DNA (Song et al., 2023; Yang et al., 2020, 2021). In this study, in vitro cellular assays were conducted to assess the direct impact of differently oriented SMFs on DNA synthesis in cells treated with APAP. Although these assays cannot fully replicate the injury and repair phases observed in mouse models, they can offer insights into the direct influences of SMFs. The findings revealed that downward SMFs enhanced DNA synthesis in APAP-treated cells, a response not observed with upward SMFs, aligning with outcomes from the mouse studies (Figure 4F).

SMFs immediately alleviate APAP-induced acute liver injury

While downward SMF pretreatment exhibited an evident ameliorating effect on APAP-induced acute liver injury, patients require interventions post-overdose, not before APAP ingestion. Given that SMF application after lethal doses of APAP improved mouse survival, albeit less effectively than pretreatment, we examined the effects of short-term SMF application immediately after a single dose of sublethal APAP, mimicking clinical circumstances, as well as a long-term SMF pretreatment (33 days) (Figure 5A). H&E and TUNEL staining showed that SMF pretreatment and SMF treatment immediately after APAP administration both reduced acute liver injury (Figure 5B–D). Although there was no significant change in ALT levels (Figure 5E), AST levels exhibited a marked reduction, aligning with the immunohistochemical findings (Figure 5F). Furthermore, both SMF treatments exerted beneficial effects by reducing oxidative stress levels (Figure 5G–I). The observed increase in SOD after APAP treatment indicated elevated oxidative stress, as noted in previous reports (Abdulkhaleq et al., 2018; Aycan et al., 2014), and was mitigated by SMF exposure (Figure 5G). Similarly, the HO-1 increase induced by APAP was also effectively reversed by SMF, regardless of whether it was applied as a 33-day pretreatment or only for 2 days post-APAP administration (Figure 5I), demonstrated the capability of SMFs to effectively reduce APAP-induced oxidative stress within a short application period.

Figure 5.

SMF exerts an immediate alleviation effect on APAP-induced acute liver injury

A: Experimental design. B: Respective intrahepatic H&E and TUNEL staining in mice. Necrotic areas are outlined using dotted lines. C, D: Quantification of necrosis area and relative apoptosis cell number (Sham PBS, SMF 33 days PBS and Sham APAP, n=6 mice per group, 3 fields were quantified from one slice per mouse; SMF 33 days APAP and SMF 2 days APAP, n=8 mice per group, 3 fields were quantified from one slice per mouse). E, F: Relative serum levels of ALT and AST in mice. G, H: SOD and MDA levels in liver tissues. I: Respective intrahepatic staining of HO-1 and quantification of HO-1-positive cell numbers. Data represent mean±SEM (Sham PBS, SMF 33 days PBS and Sham APAP, n=6 mice per group; SMF 33 days APAP and SMF 2 days APAP, n=8 mice per group). *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001.

Downward SMFs work less efficiently than NAC during the liver injury stage, but exert significant beneficial effects at the later recovery stage

NAC is the sole drug approved by the FDA to treat APAP-induced acute liver injury; however, its effectiveness is contingent on administration within a very short time window. To explore the efficacy of SMFs, either independently or in combination with NAC, we compared NAC treatment, SMF treatment, and their combined application in single high-dose APAP-treated mice. This comparison included evaluations at two different time points: 6 h and 24 h post-APAP overdose, when the effectiveness of NAC typically diminishes in clinical settings (Figure 6A). Body weights were recorded before and after the experiments (Figure 6B; Supplementary Figure S7A). Additionally, analyses were performed on relative serum levels of ALT and AST, as well as liver tissue sections (Figure 6C–G; Supplementary Figure S7B, C).

Figure 6.

Downward SMF demonstrates reduced efficacy compared to NAC during the liver injury phase, but provides significant benefits during the recovery stage

A: Experimental design. B: Body weight of mice at the end of the experiment. C: Relative serum level of AST (n=6 mice per group). D: Representative H&E, TUNEL, and Oil Red O staining in control, sham, and SMF, NAC, and SMF+NAC 24 h after APAP treatment groups. Necrotic areas are outlined using dotted lines. E–G: Quantification of necrosis area, apoptosis cell number, and lipid accumulation (n=6 mice per group, 3 fields were quantified from one slice per mouse). Data represent mean±SEM. ^*: P<0.05; ^**: P<0.01; ^***: P<0.001; ^****: P<0.0001.

Treatment with NAC effectively alleviated APAP overdose-induced weight loss and AST elevation when administered at 6 h, but not at 24 h, consistent with clinical outcomes. In contrast, SMF and NAC in combination ameliorated APAP overdose-induced weight loss, AST increase, liver necrosis, apoptosis, and lipid accumulation, even when administered 24 h post-APAP overdose (Figure 6B–G). At this time point, NAC alone failed to reduce TUNEL-positive cells or lipid deposition in the liver, while SMF and NAC together reduced TUNEL-positive cells by 41% (P<0.05) (Figure 6F) and lipid deposition by 55% (P<0.0001) (Figure 6G). These findings demonstrate that while SMF or NAC alone can mitigate APAP-induced liver injury when applied early, their combination represents a promising treatment strategy with enhanced efficacy and a broader treatment time window (Figure 7).

Figure 7.

Schematic illustrating the impact of SMFs on injury and repair stages following APAP overdose

During the early liver injury stage, SMFs decrease free radicals and increase GSH levels to reduce oxidative stress and liver cell death. During the later liver repair stage, vertically downward SMFs (in gravity direction, g) exert Lorentz forces (F_L) on negatively charged DNA to relax the DNA supercoil, increase DNA synthesis, and promote liver cell proliferation. SMF and NAC in combination can significantly alleviate high-dose APAP-induced acute liver injury, even when used 24 h after APAP overdose.

DISCUSSION

Previous research on APAP overdose-induced acute liver injury has primarily focused on counteracting oxidative stress and managing the subsequent immune response, achieving very good clinical outcomes. However, liver regeneration, driven by cellular proliferation following injury (Michalopoulos, 2017), also plays an essential role in recovery from APAP overdose. In the present study, we demonstrated that a moderate intensity downward SMF immediately alleviated APAP overdose-induced liver toxicity, playing dual roles at both the earlier liver injury stage and later liver repair stage. Notably, it significantly reduced oxidative stress and increased liver cell proliferation, thereby contributing to liver recovery. When administered alongside NAC, this strategy significantly reduced liver damage 6 h after APAP overdose. Additionally, the advantageous effects of the combined treatment persisted even 24 h post-overdose, a period during which the efficacy of NAC alone typically diminishes.

DNA synthesis is a crucial phase of cell proliferation and tissue regeneration. Our earlier studies have suggested that negatively charged DNA within cells is influenced by Lorentz forces in the presence of moderate to strong SMFs, with the direction of the magnetic field playing a pivotal role (Song et al., 2023; Yang et al., 2020, 2021). A vertically upward SMF, which acts antiparallel to gravitational force, applies Lorentz forces inward to the double helix of the DNA, tightening its supercoiled structure and consequently inhibiting DNA synthesis when the magnetic field is sufficiently strong. Conversely, a vertically downward SMF, aligned with gravitational force, tends to relax the DNA structure, facilitating DNA synthesis. In this orientation, the Lorentz force opposes the centripetal force acting on the DNA, leading to a relaxation of the supercoiled structure, which is conducive to DNA replication and subsequent cell proliferation.

In our previous study, an upward SMF exaggerated the lethal effects of high-dose alcohol (Song et al., 2023), while in the current study, it demonstrated some alleviating effects. This discrepancy may be partially attributed to the distinct roles of ROS in APAP versus alcohol-induced liver toxicity. In APAP overdose, excessive oxidative stress is the primary reason for liver failure, underscoring the reason why NAC remains the only approved medicine in clinical settings. In alcohol overdose, oxidative stress plays a very important role, but it is not as critical as in APAP overdose. Consequently, for APAP-exposed mice, ROS reduction outweighs the negative impact on DNA synthesis, unlike in mice exposed to high levels of alcohol. In alcohol-treated mice, DNA synthesis and liver regeneration, marked by proliferation marker Ki67, are substantially greater than in APAP-treated mice and are notably reduced by upward SMF treatment (Song et al., 2023). The timing of treatment is also crucial, with alcohol-exposed mice undergoing SMF treatment for approximately three weeks, significantly longer than the brief, acute intervention required for overdose with highly toxic APAP. Notably, in the initial phase post-APAP overdose, mice treated with an upward SMF exhibited favorable survival rates, aligning with the anticipated reduction in oxidative stress. However, after 200 h, survival rates began to resemble those of the sham control (Figure 1D), consistent with its beneficial effects on reducing oxidative stress, but harmful effects on DNA synthesis.

Multiple studies have demonstrated the importance of magnetic fields on ROS (Wang & Zhang, 2017; Yu et al., 2023a; Zhang et al., 2017). As previously discussed, exposure to a 200 μT SMF can inhibit ROS formation, affecting new tissue growth (Van Huizen et al., 2019). The same SMF exposure used in our study has been shown to decrease oxidative stress, thereby reducing cisplatin and alcohol-induced tissue toxicity (Song et al., 2023; Yu et al., 2023b). Conversely, negating the geomagnetic field can significantly impair adult hippocampal neurogenesis and hippocampus-dependent learning, processes strongly correlated with ROS fluctuations (Zhang et al., 2021). While it is suggested that SMFs may modulate radical recombination processes through singlet and triplet transition, thereby potentially regulating ROS formation (Van Huizen et al., 2019), the precise mechanism by which specific magnetic field parameters influence ROS generation and elimination in living organisms, which involves multiple steps, remains unclear.

Due to instrument constraints, we did not investigate the effects of horizontal SMFs in the present study. While we anticipate that the direction of SMFs may not significantly impact oxidative stress, the influence on DNA synthesis by horizontal SMFs remains speculative, given uncertainties regarding DNA orientation and the possibility of intermediate effects between upward and downward SMFs. As such, efforts are currently underway to develop horizontal SMFs for further study. Additionally, in our experiments, the magnetic field strength at the liver location was approximately 0.1 T. Considering the greater distance between the abdominal skin and the liver in humans compared to mice, small magnets may not be strong enough to achieve sufficient penetration to elicit the observed effects. Nonetheless, we believe that a scaled-up magnet plate design could replicate the magnetic field conditions effective in the mouse model. Both scaled-up magnet plate design and horizontal SMFs, commonly utilized in MRIs, should be explored in future research.

Nevertheless, given their affordability, non-invasiveness, high penetration ability, and ease of application and removal, moderate SMFs possess considerable advantages and promising application potential. Our research demonstrated that SMFs are effective during both the injury and repair stages of APAP toxification. Utilizing downward SMFs alone or in combination with NAC showed substantial benefits, with combination treatment showing efficacy up to 24 h post-APAP overdose, broadening the therapeutic window of NAC and providing a promising treatment strategy for APAP overdose-induced acute liver injury in clinical settings.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

Funding Statement

This study was supported by the National Key R&D Program of China (2023YFB3507004), National Natural Science Foundation of China (U21A20148), International Partnership Program of Chinese Academy of Sciences (116134KYSB20210052), Anhui Provincial Natural Science Foundation (2308085QE183, 2308085QE181), CASHIPS Director’s Fund (YZJJ2024QN44, YZJJ2023QN43), Heye Health Technology Chong Ming Project (HYCMP2021010), China Post-doctoral Science Foundation (2023M743536), and Science Research Fund for Post-doctoral in Anhui Province (2023B669)