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. 2025 Jul 21;15:26395. doi: 10.1038/s41598-025-10764-x

IL-37 ameliorates acetaminophen-induced acute liver injury by limiting MAPK/NFκB signaling-mediated liver inflammation

Juan Fu 1,2,#, Qiuhong Huang 1,#, Changfeng Sun 2, Shuyi Li 1, Qingsong Wang 1, Yunjian Sheng 2,✉, Binfeng He 1,3,✉, Zaichun You 1,3,✉
PMCID: PMC12280191  PMID: 40691258

Abstract

Acetaminophen (APAP) overdose is a common cause of drug-induced liver injury (DILI), which can lead to sterile inflammation and progress to acute liver failure and even death. However, there are currently limited therapeutic options available. Iinterleukin-37 (IL-37) is considered as an anti-inflammatory cytokine. The role and novel mechanism of IL-37 on DILI are still unknown. Male C57BL/6 mice were pretreated with IL-37 for 2 h prior to intraperitoneal injection of acetaminophen (APAP). Hepatic function was assessed by measuring serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). Liver tissue damage was evaluated via hematoxylin and eosin (H&E) staining. The inflammatory response was characterized by immunohistochemical (IHC) analysis of myeloperoxidase (MPO) and lymphocyte antigen 6 complex locus G (Ly6G), and the levels of interleukin-6 (IL-6), IL-10, transforming growth factor-beta 1 (TGF-β1), and tumor necrosis factor-alpha (TNF-α) in liver tissue. Oxidative stress status was determined by measuring superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) levels. CYP2E1 mRNA expression was analyzed using qPCR. Protein expression of phosphorylated p38 (pP38), phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), and phosphorylated p65 (pP65) was evaluated by Western blotting. Compared to the model group without recombinant human IL-37 treatment, the model + IL-37 group exhibited significantly attenuated liver injury, characterized by reduced neutrophil infiltration, decreased levels of pro-inflammatory mediators IL-6 and TNF-α, and elevated levels of the anti-inflammatory cytokines IL-10 and TGF-β1. The levels of pp38, pERK1/2, and pp65 in liver tissue were significantly suppressed in the Model + IL-37 group compared to the Model group at 24 h. Furthermore, MDA levels were significantly lower in the IL-37-treated group relative to the model group, while SOD activity showed no significant difference. Our results also indicate that neither CYP2E1 mRNA relative expression nor GSH levels differed significantly between the model group and the IL-37-treated group at either 4 h or 24 h after APAP exposure. IL-37 has a significant protective effect against acetaminophen-induced liver injury (AILI) by suppressing the inflammatory response involved in the MAPK-NF-κB/p65 signalling pathway. Our study suggests IL-37 as a potential therapeutic strategy for DILI.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-10764-x.

Keywords: IL-37, APAP, Liver injury, Sterile inflammation, Oxidative stress

Subject terms: Hepatology, Hepatotoxicity

Introduction

Drug-induced liver injury (DILI) is the second most common type of non-infectious liver disease in China1. In European and American countries, it is the primary cause of acute liver failure, with a high incidence (about 60% or more)2–4. APAP overdose constitutes a predominant etiological factor in DILI, with clinical progression to severe hepatotoxicity representing a frequent therapeutic challenge in clinical hepatology.

APAP overdose induces severe hepatotoxicity through a pathophysiological cascade initiated by glutathione (GSH) depletion, followed by exacerbated oxidative stress and consequent mitochondrial dysfunction, ultimately culminating in acute liver failure5. Excessive APAP is metabolized by cytochrome P450 2E1 (CYP2E1), resulting in overproduction of N-acetyl-p-benzoquinone imine (NAPQI), which depletes hepatic glutathione (GSH) reserves6,7. Excessive NAPQI covalently binds to mitochondrial proteins, forming hepatotoxic protein adducts that trigger oxidative stress and profoundly disrupt the mitochondrial electron transport chain (ETC). These sequential events culminate in severe mitochondrial dysfunction, widespread necrotic cell death, and ultimately mediate fulminant hepatotoxicity, progressing to life-threatening hepatic injury or acute liver failure8–10.

Currently, N-acetylcysteine (NAC) remains the only FDA-designated specific antidote for acetaminophen (APAP)-induced hepatotoxicity, functioning through glutathione (GSH) replenishment in hepatocytes11. Clinical evidence demonstrates that optimal therapeutic outcomes are achieved when NAC administration is initiated within 8 h post-overdose12,13. However, delayed administration significantly compromises its therapeutic efficacy, as most hospitalized patients with APAP-induced acute liver injury typically present with advanced-stage hepatic damage or even liver failure at initial clinical evaluation14. In addition, poor bioavailability of NAC means that DILI patients need to receive relatively high doses and longer treatment times, as well as being labor-intensive and costly15. These clinical realities underscore the urgent need to develop novel therapeutic strategies that can extend the critical treatment window and improve clinical outcomes in delayed presentation cases of drug-induced liver injury.

IL-37, a unique anti-inflammatory cytokine within the IL-1 family, exists as five splice variants (IL-37a-e) with distinct functional characteristics. This cytokine exerts potent anti-inflammatory effects through dual mechanisms: (1) direct suppression of signaling kinase activation (e.g., MAPK and NF-κB pathways), and (2) downregulation of pro-inflammatory mediators including TNF-α, IL-6, and IL-1β16,17. Experimental evidence from concanavalin A-induced acute hepatitis models reveals that endogenous IL-37 modulates inflammatory responses by balancing Th1/Th17 cytokine production while enhancing IL-10 expression, thereby attenuating both hepatic and systemic inflammation18. In a mouse model of hepatic ischemia/reperfusion injury, it has been demonstrated that the activation of liver parenchymal cells and Kupffer cells is inhibited by IL-3719. Meanwhile, IL-37 can reduce the release of pro-inflammatory cytokines and chemokines20, and also weaken the infiltration of neutrophils, monocytes, and macrophages into the liver21,22, alleviating local and systemic inflammation, and thus relieving the degree of liver injury.

This study investigated the protective effect of IL-37 against APAP-induced liver injury by assessing hepatic function and histopathological changes. Furthermore, further explore potential mechanisms underlying IL-37’s protection against DILI, we further evaluated the levels of inflammatory mediators, oxidative stress status, CYP2E1 mRNA expression, and MAPK-NF-κB/p65 signaling pathway.

Materials and methods

Material and reagents

The materials and reagents used in the study are as follows: (a) Acetaminophen (APAP): Purchased from TOCRIS bio-techno (America) and dissolved in a warmed sterile PBS solution. (b) Recombinant human IL-37b: Purchased from R&D SYSTEMS (America). (c) TGF-β1 ELISA kit, , IL-6 ELISA kit, TNF-α ELISA kit, and IL-10 ELISA kit: Purchased from Invitrogen (America). (d) Rabbit anti-mouse Ly-6G and MPO antibodies: Purchased from serotct. € Alanine transaminase (ALT) detection kit, aspartate aminotransferase (AST) detection kit, lactate dehydrogenase (LDH) detection kit, Malondialdehyde (MDA) test kit, superoxide dismutase (SOD) test kit, and glutathione (GSH) assay kit: Purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). (f) Hieff UNICON® Power qPCR SYBR Green Master Mix (11197ES03) and RNA reverse transcription kit: Purchased from Yeasen (Shanghai, China). These materials and reagents were used for various experimental procedures, including APAP administration, IL-37 treatment, ELISA assays for cytokine detection, immunohistochemistry with lymphocyte antigen 6 complex locus G6D (LY-6G) and myeloperoxidase (MPO) antibodies, and biochemical assays for liver function markers (ALT, AST, LDH), oxidative stress markers (MDA, SOD, GSH), and gene expression analysis (qPCR). All experiments were conducted in accordance with the relevant guidelines.

Acute liver injure model

In this study, male C57BL/6 mice aged 5–8 weeks with a body weight of 18–20 g were employed as experimental subjects. The mice were maintained under specific-pathogen-free (SPF) conditions in a controlled environment featuring 60% relative humidity, a consistent 12-h light/dark cycle, and room temperature. Throughout the experimental period, animals had unrestricted and libitum access to standard rodent chow and fresh drinking water.

Male mice were randomly divided into four experimental groups with the following treatment protocols: (1) Sham group: Received an intravenous injection of 0.2 mL PBS, followed 2 h later by an intraperitoneal injection of 0.2 mL PBS; (2) IL-37/Sham group: Received 2 μg IL-37β dissolved in 0.2 mL sterile PBS by intravenous injection, followed 2 h later by an intraperitoneal injection of 0.2 mL PBS; (3) Model group: Received an intravenous injection of 0.2 mL PBS, followed 2 h later by an intraperitoneal injection of 250 mg/kg APAP solution (dose determined by preliminary experiments); (4) IL-37/model group: Treated with 2 μg IL-37β dissolved in 0.2 mL sterile PBS by intravenous injection, followed 2 h later by an intraperitoneal injection of 250 mg/kg APAP solution23,24. The mice were euthanized using sodium pentobarbital (150 mg/kg) via intraperitoneal injection. Evidence of liver injury was assessed, and hepatic physiological and biochemical parameters were evaluated to assess liver function. All animal protocols were performed in strict accordance with the regulations approved by the Experimental Animal Welfare Ethics Committee of Southwest Medical University, and reported following the ARRIVE guidelines (Supplementary file, No. 2020419).

Serum ALT, AST and LDH analysis

The levels of serum ALT, AST and LDH each sample was determined using an automated biochemical analyzer according to manufacturer instructions.

Enzyme-linked immunosorbent assay and colorimetric assay

Liver tissue samples (100 mg) were processed with a homogenizer in a lysis buffer comprising 30 mM Tris, 7 M urea, 2 M thiourea, and 4% (w/v) CHAPS at pH 8.5. The homogenized samples were kept on ice for a 2-h incubation. Following this, the supernatant was harvested after centrifugation at 12,000 rpm for 15 min at 4 °C and stored at − 20 °C until analysis. All procedures were conducted on ice to maintain sample integrity. The concentrations of  , IL-6, IL-10, TNF-α, and TGF-β1 in the supernatant were determined using enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, malondialdehyde (MDA) and reduced glutathione (GSH) levels, as well as superoxide dismutase (SOD) activity, were assessed using commercially sourced MDA, GSH, and SOD assay kits, respectively23.

RT-qPCR

Total RNA of liver tissues was isolated by Trizol reagent according to the standard protocol. Reverse transcription of 2 μg total RNA to cDNA using the Servicebio® 1st Strand cDNA Synthesis Kit. The quantitative real-time PCR (qPCR) was performed on a Step-OnePlus system using Servicebio® 2 × Fast SYBR Green qPCR Master Mix (High ROX) on the Step-One Plus system. GAPDH serves as an internal reference gene. The relative expression of the target gene was calculated by the 2−ΔΔ Ct method. The primer used in this study was listed in Supplementary Table S1.

Hematoxylin and eosin (H&E) staining and IHC25

The left outer lobe of the mouse liver was fixed in 4% formaldehyde solution and then embedded in paraffin. Prepare sections with a thickness of 4 μm and fix them on glass slides. Samples were stained with hematoxylin and eosin (H&E) and analyzed by microscope26.

The levels of LY-6G and myeloperoxidase (MPO) in liver tissues were assessed using immunohistochemistry (IHC) according to the manufacturer’s instructions25. The positive staining area of LY-6G and MPO was quantified using Image Processing and Analysis in Java (ImageJ 1.53a; Java 1.8.0_112[64-bit], https://imagej.net/software/fiji/) software for quantitative analysis.

Western blotting

Total proteins were extracted from liver tissues using RIPA lysis buffer, separated by SDS-PAGE, and subsequently transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature (RT), then incubated overnight at 4 °C with primary antibodies (dilution 1:5000 in blocking buffer), followed by 1-h incubation with HRP-conjugated secondary antibodies at RT. Protein bands were visualized using ECL reagents.

Statistical analysis

Continuous variables are presented as mean ± standard error of the mean (SEM). Statistical analyses were conducted using SPSS 22.0 (IBM Corporation, www.ibm.com) with graphical representations generated through GraphPad Prism 6.0 (GraphPad Software, www.graphpad.com). Comparative analyses between experimental groups employed parametric unpaired Student’s t-tests. Multigroup comparisons utilized one-way analysis of variance (ANOVA) with post hoc Bonferroni correction. Statistical significance was defined as p < 0.05.

Results

Determination of the therapeutic dose of IL-37 and its ability to reduce transaminase levels in an APAP-induced mouse model of acute liver injury

To evaluate the hepatoprotective effects of IL-37 against APAP-induced acute liver injury, we established a DILI murine model (Fig. 1A). To investigate the retention effect of exogenous IL-37 (rhIL-37) in the liver and determine the appropriate therapeutic dose, we injected mice with 0.5, 1.0, and 2.0 μg of IL-37 via caudal vein injection, and detected IL-37 levels in liver tissues at 4 h and 24 h after injection. The results showed that at 24 h post-injection, the concentration of IL-37 in the liver of the 2.0 μg dose group was significantly higher than that in other dose groups (Fig. 1B) (p < 0.01). Furthermore, we found that dosages of 0.5–2 μg of IL-37 improved serum ALT and AST levels caused by APAP in a dose-dependent manner (Supplementary Fig. 1). Based on these findings, we further compared the IL-37 levels in liver tissues at 0, 4, 12, 24, and 48 h after injection between the Sham + IL-37 group and the Model + IL-37 group. Compared with the sham operation group, the results showed that the levels of IL-37 in the liver of the model group were significantly higher at 4 and 12 h after injection (p < 0.01) (Fig. 1C). Serum levels of ALT, AST, and LDH, well-characterized biomarkers of hepatocellular injury, were quantified in Fig. 1D–F. These data showed that APAP challenge alone induced significant elevations in all three markers at 24 h post-administration (p < 0.05). In contrast, the IL-37-treated group demonstrated a substantial reduction in the levels of these markers, particularly evident at the 24-h time point (p < 0.01). Results indicate that IL-37 administration effectively mitigates APAP-induced liver injury in mice.

Fig. 1.

Fig. 1

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IL-37 has the potential to improve acute liver injury induced by APAP. C57BL/6J mice (n = 5 per group) were administered APAP (250 mg/kg, i.p.) and IL-37 (2 μg, i.v.) for specified durations (4 h and 24 h). The experimental setup is depicted in Figure (A). (B) The levels of IL-37 in the liver at 4 h and 24 h after tail vein injection of 0.5, 1, 2 μg recombinant human IL-37 in mice. (C) The supernatant of freshly isolated mouse liver homogenate was collected at 0, 4, 12, 24 and 48 h after tail vein injection of 2 μg recombinant human IL-37, and the level of IL-37 was detected. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and Lactate dehydrogenase (LDH) were measured as indicators of liver injury (D–F), respectively. Statistical significance was denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. p > 0.05.

IL-37 demonstrates the ability to alleviate hepatocyte necrosis in acute liver injury induced by APAP

To determine the effects of IL-37 pretreatment on APAP-induced hepatic damage, we evaluated gross and histological changes in murine livers. The results suggested that at 4 h post-APAP challenge, livers in the model group had little discoloration and congestion grossly, with histology showing patchy hepatocyte necrosis and focal inflammatory cell infiltration; lobular architecture remained mostly intact. Conversely, model + IL-37 group livers appeared rosy grossly without petechiae/ecchymosis, and histology showed minimal inflammatory infiltration with absence of necrosis; lobular structure was fully preserved. At 24 h, model group livers demonstrated marked pallor, increased volume with significant congestion grossly, and histology revealed extensive confluent necrosis with dense inflammatory infiltration; hepatic lobules were extensively disrupted. In contrast, model + IL-37 group livers maintained rosy coloration grossly with minor congestion, while histology indicated focal necrosis and localized inflammatory infiltration; normal lobular architecture was predominantly retained (Fig. 2A,B). In addition, the liver-to-body weight ratio, a validated hepatotoxicity index, was higher in the model group than in the IL-37 + model group (p < 0.05) (Fig. 2C).

Fig. 2.

Fig. 2

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IL-37 demonstrates the ability to alleviate hepatocyte necrosis in acute liver injury induced by APAP. The impact of IL-37 on hepatocyte necrosis was assessed using the following approaches. (A) The appearance of fresh liver tissues of experimental mice was observed at 4 and 24 h after APAP administration. (B) Pathological changes in liver tissues were examined through HE staining at 40× magnification. (C) The liver index of mice in each group was calculated by dividing the liver weight by body weight. Statistical significance was denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. p > 0.05.

IL-37 mitigates the inflammatory response in the liver induced by APAP

We investigated the anti-inflammatory effect of IL-37 in liver tissue from DILI model mice. Our data showed that the levels of IL-6 and TNF-α mRNA were significantly decreased, while the levels of the anti-inflammatory cytokines IL-10 and TGF-β1 mRNA were upregulated in the IL-37/model group compared to the model group at 24 h time-point (all p < 0.05) (Fig. 3A). Consistent with these results, ELISA analysis showed a significant reduction in the levels of IL-6 and TNF-α in liver tissue homogenates 24 h after IL-37 intervention. In addition, there was a significant increase in the levels of IL-10 and TGF-β1 compared to the model group (all p < 0.05) (Fig. 3B).

Fig. 3.

Fig. 3

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IL-37 suppresses the inflammatory response in APAP-induced acute liver injury. (A) The hepatic mRNA levels of IL-6, IL-10, TNF-α, and TGF-β1 genes were measured in the indicated groups. (B) The serum levels of inflammatory cytokines IL-6, IL-10, TNF-α, and TGF-β1 were quantified using ELISA. The data are presented as mean ± SEM (n = 5 per group). Statistical significance was indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. p > 0.05.

To investigate the potential of IL-37 to inhibit inflammatory cell recruitment in APAP-induced acute liver injury, the expression of myeloperoxidase (MPO) and lymphocyte antigen 6 complex locus G6D (LY-6G) in liver tissue was assessed by immunohistochemistry (IHC). LY-6G is a marker for monocytes, granulocytes and neutrophils, while MPO is a marker for neutrophil tissue infiltration. The data showed no significant difference in the expression of MPO and LY-6G between the treatment group and the model group after 4 h of APAP treatment (p > 0.05). After 24 h of APAP treatment, a significant reduction in the levels of MPO and LY-6G was observed in the therapy group compared to the model group with statistical significance (p < 0.01). Notably, at both 4 and 24 h after APAP treatment, no significant difference in the expression of MPO and LY-6G was observed between the sham group and the sham + IL-37 group (p > 0.05) (Fig. 4A–C).

Fig. 4.

Fig. 4

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IL-37 suppresses the recruitment of neutrophils in APAP-induced acute liver injury. (A) Immunohistochemical staining was performed to visualize Ly-6G and MPO in liver tissue. Arrows indicate MPO+ and Ly-6G+ neutrophils. (B) The quantification of Ly-6G-positive cells (n = 5) is presented. (C) The quantification of MPO-positive cells (n = 5) is presented. Statistical significance was indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. p > 0.05.

The hepatoprotective effect of IL-37 is independent of APAP metabolism and antioxidant stress

APAP is metabolised by cytochrome P450 2E1 (CYP2E1) into toxic compounds that promote oxidative stress through glutathione (GSH) depletion in hepatocytes, thereby inducing liver tissue damage27. Herein, we investigate whether IL-37 exerts a hepatoprotective effect by suppressing CYP2E1 expression and improving oxidative stress status. The data showed that IL-37 did not suppress CYP2E1 expression or improve GSH levels in this context (Fig. 5A,B, and Supplement Fig. 2) (p > 0.05).

Fig. 5.

Fig. 5

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The hepatoprotective effect of IL-37 is independent of APAP metabolism and antioxidant stress. (A) The content of malondialdehyde (MDA) in liver tissue was measured at 4 and 24 h after exposure to APAP. (B) Superoxide dismutase (SOD) activity in liver tissue was assessed after 4 and 24 h of exposure to APAP. (C) The relative expression levels of CYP2E1 mRNA in liver tissue were determined at 4 and 24 h after exposure to APAP. (D) The level of total glutathione (T-GSH) in liver tissue homogenate suspension was analyzed at 4 and 24 h after exposure to APAP. Statistical significance was indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. p > 0.05.

As shown in Fig. 5C and D, the levels of MDA and SOD were significantly elevated in the liver tissues of DILI model compared to the sham group (p < 0.01). Notably, IL-37 significantly suppressed APAP-induced MDA production. However, there was no significant difference in SOD levels between the model group and the IL-37-treated group (p > 0.05). These data suggested that the hepatoprotective effect of IL-37 is independent of APAP metabolism and storage levels of GSH.

IL-37 suppressed the activation of the MAPK-NF-κB/p65 signalling pathway induced by DILI in liver tissue.

Our results revealed that protein expression levels of pP38, pERK1/2, and pP65 in liver tissue were significantly down regulated in the Model + IL-37 group compared to the Model group at 24 h (Fig. 6) (p < 0.01). Notably, at 48 h post-treatment, protein expression levels of ERK and p65 in the Model + IL-37 group exhibited a rebound compared to the Model group (Fig. 6) (p < 0.001). These data suggested that IL-37 confers significant anti-inflammatory protection through inhibiting the activity of the MAPK-NF-κB/p65 signaling pathway. This effectively attenuates hepatic inflammation and reduces hepatocyte necrosis.

Fig. 6.

Fig. 6

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IL-37 inhibits the MAPK-NF-κB/P65 signalling pathway in APAP-induced acute liver injury in mice. (A) Western blot analysis of the expression of pERK, pP38 and pP65 proteins in the livers of mice in the Model and Model + IL-37 groups at 0, 4, 24 and 48 h. (B) Semi-quantification of the above bands in (A). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. p > 0.05.

Discussion

Liver inflammation is recognized as a critical event in acetaminophen (APAP)-induced acute liver injury28,29. IL-37, as an endogenous immunoregulatory cytokines, exhibits potent immunosuppression and anti-inflammatory properties. Growing evidence indicates that IL-37 participates in the regulation of inflammatory responses in various diseases30,31. Our findings demonstrate that IL-37 exerts significant protective effects against APAP-induced acute liver injury in mice by regulating the MAPK signaling pathway, thereby inhibiting the activation of the NF-κB/p65, resulting in suppressing the inflammatory response and alleviating hepatocyte necrosis.

It was found that IL-37 exerted anti-inflammatory effects by down-regulating the levels of pro-inflammatory mediators IL-6 and TNF-α, enhancing the levels of anti-inflammatory mediators IL-10 and TGF-β1, and inhibiting the recruitment of neutrophils in the liver. These findings align with prior investigations highlighting IL-37’s immunomodulatory properties across diverse inflammatory disorders20,32–34. IL-37 exerts anti-inflammatory properties mainly by inhibiting the activation of signaling kinase35. Our further results showed that IL-37 exerts anti-inflammatory and protective effects against APAP-induced acute liver injury by modulating the p38/MAPK/ERK signaling axis and suppressing the activation of the NF-κB/p65 signaling pathway, thereby attenuating hepatic inflammation and reducing hepatocellular necrosis. This finding is consistent with the conclusion of temporomandibular arthritis, psoriasis and liver fibrosis disease models36–38. Accumulating evidence indicates that IL-37 exerts its anti-inflammatory effects by specifically interacting with the Smad3 protein and inflammasomes39,40. In addition, IL-37 elicits an unexpected pseudo-starvation effect on the mTOR signaling pathway, inhibits TAK1 and Fyn kinase activity, and activates downstream signaling molecules such as STAT3, and PTEN and up-regulates the expression of p62 protein, ultimately blocking the progress of inflammatory response41. These potential links or molecular targets are expected to become an important entry point for further exploration of its mechanism of action.

Our results demonstrate that IL-37 does not alter the expression of the hepatic metabolic enzyme CYP2E1 or affect the hepatic reserves of the antioxidant glutathione (GSH). This study demonstrates that IL-37 confers protection against liver injury by directly suppressing the inflammatory response, without interfering with the generation or metabolic processing of APAP’s toxic metabolites. In contrast, previous studies have shown that the hepatoprotective effect of N-acetylcysteine (NAC) against APAP-induced liver injury primarily relies on enhancing glutathione (GSH) biosynthesis and accelerating the efficient detoxification of toxic metabolites42,43. The distinct mechanisms underlying the hepatoprotective effects of IL-37 and NAC highlight their divergent therapeutic pathways. This discovery may pave the way for the development of more targeted therapeutic strategies for drug-induced liver injury. Further analysis showed that the level of malondialdehyde (MDA) decreased after IL-37 pretreatment, while the level of superoxide dismutase (SOD) did not change significantly. It shows that IL-37 only shows a limited regulatory role in the oxidative stress pathway. Oxidative stress is involved in the process of APAP liver injury44,45, and IL-37 has been confirmed to be involved in the regulation of oxidative stress in other disease models46. Therefore, it is necessary to further explore its specific regulatory mechanism in APAP liver injury.

NAC remains the only drug approved by the FDA for treating acute liver injury caused by APAP overdose. However, its clinical efficacy is limited to the first 8 h after overdose.

Our study confirmed that IL-37 has a significant protective effect on APAP-induced acute liver injury through strong anti-inflammatory activity and independent the biological process of APAP metabolites. Several studies21,33,47 have demonstrated that low doses of exogenous recombinant IL-37 can rapidly exert anti-inflammatory effects within 2 h of administration48–50. While IL-37 has not yet been clinically applied for inflammatory diseases, our findings suggest its potential for synergistic use with NAC in AILI management. Specifically, IL-37 provides rapid early hepatoprotection, minimizing initial hepatocyte injury before NAC exerts its therapeutic effects. This creates a critical time window for NAC therapy. Furthermore, IL-37’s anti-inflammatory action complements NAC’s primary mechanism of improving oxidant stress by replenishing GSH stores.

This study has the following limitations. First, the lack of cytological verification reduces the robustness of the research results. Secondly, together research mechanism is needed to elucidate the new way of IL-37 promoting the treatment of drug-induced liver injury. Thirdly, there is a lack of relevant research on the clinical application of IL-37, and its clinical transformation potential needs further experimental verification.

In summary, our results suggest that IL-37 plays an anti-inflammatory protective role in APAP-induced acute liver injury mouse model, while significantly retaining the metabolic detoxification process associated with APAP hepatotoxicity. It is confirmed that IL-37 can be used as a new molecular target for the treatment of drug-induced liver injury, which provides a theoretical basis and intervention direction for optimizing the clinical management strategy of drug-induced liver injury.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1MB, docx)
Supplementary Material 2 (61.1KB, pdf)
Supplementary Material 3 (10.4KB, docx)
Supplementary Material 4 (10.6KB, docx)
Supplementary Material 5 (10.4KB, docx)

Author contributions

All authors participated in the conception and design of the study and contributed to data extraction, analysis, and interpretation. S.-Y. J. and Y.-Z. C. were responsible for validating the manuscript. H.-B. F. oversaw the revision and final review of the manuscript. F.-J and H.-Q.H. drafted the manuscript, while H.-Q.H., L.-S. Y. and S.-C. F. undertook the experimental design and performed data proofreading. All authors provided final approval of the manuscript. Full access to all study data, including statistical reports and tables, was available to all authors, who took responsibility for data integrity and the accuracy of data analysis.

Funding

This study was funded by the Foundation of The Affiliated Hospital of Southwest Medical University under Grant Number 15050.

Data availability

All data relevant to the study are included in the article. Primary data are available upon request from Binfeng He (ldhbf@tmmu.edu.cn).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Juan Fu and Qiuhong Huang contributed equally to this work.

Contributor Information

Yunjian Sheng, Email: sheng200410@163.com.

Binfeng He, Email: ldhbf@tmmu.edu.cn.

Zaichun You, Email: youzaichun619@163.com.

References

  • 1.Zhongping, D., PengFei, Y., Qiao, W. & Yu, C. Research advances in the mechanism of drug-induced liver injury due to paracetamol. J. Clin. Hepatobiliary Dis.35(9), 2108–2112 (2019). [Google Scholar]
  • 2.Larson, A. et al. Acetaminophen-induced acute liver failure: Results of a United States multicenter, prospective study. Hepatology (Baltimore, MD)42(6), 1364–1372. 10.1002/hep.20948 (2005). [DOI] [PubMed] [Google Scholar]
  • 3.Lee, W. Acetaminophen-related acute liver failure in the United States. Hepatol. Res. Off. J. Jpn. Soc. Hepatol.38, S3-8. 10.1111/j.1872-034X.2008.00419.x (2008). [DOI] [PubMed] [Google Scholar]
  • 4.Bhushan, B. & Apte, U. Liver regeneration after acetaminophen hepatotoxicity: Mechanisms and therapeutic opportunities. Am. J. Pathol.189(4), 719–729. 10.1016/j.ajpath.2018.12.006 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kon, K. et al. Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes. Hepatology40(5), 1170–1179. 10.1002/hep.20437 (2004). [DOI] [PubMed] [Google Scholar]
  • 6.Hartmut, J., Tamara, R. K. & Mary Lynn, B. The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol. Lett.144(3), 279–288. 10.1016/s0378-4274(03)00239-x (2003). [DOI] [PubMed] [Google Scholar]
  • 7.Laura, P. J., Philip, R. M. & Jack, A. H. Acetaminophen-induced hepatotoxicity. Drug Metab. Dispos.31(12), 1499–1506. 10.1124/dmd.31.12.1499 (2003). [DOI] [PubMed] [Google Scholar]
  • 8.Donnelly, P. J., Walker, R. M. & Racz, W. J. Inhibition of mitochondrial respiration in vivo is an early event in acetaminophen-induced hepatotoxicity. Arch. Toxicol.68(2), 110–118. 10.1007/s002040050043 (1994). [DOI] [PubMed] [Google Scholar]
  • 9.Burcham, P. C. & Harman, A. W. Mitochondrial dysfunction in paracetamol hepatotoxicity: In vitro studies in isolated mouse hepatocytes. Toxicol. Lett.50(1), 37–48. 10.1016/0378-4274(90)90250-p (1990). [DOI] [PubMed] [Google Scholar]
  • 10.Jaeschke, H. A. Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo: The protective effect of allopurinol. J. Pharmacol. Exp. Ther.255(3), 935–941 (1990). [PubMed] [Google Scholar]
  • 11.Manakkat Vijay, G. K. et al. Neutrophil toll-like receptor 9 expression and the systemic inflammatory response in acetaminophen-induced acute liver failure. Crit. Care Med.44(1), 43–53. 10.1097/CCM.0000000000001309 (2016). [DOI] [PubMed] [Google Scholar]
  • 12.European Association for the Study of the Liver. EASL clinical practice guidelines: Drug-induced liver injury. J. Hepatol.70(6), 1222–1261. 10.1016/j.jhep.2019.02.014 (2019). [DOI] [PubMed] [Google Scholar]
  • 13.Smilkstein, M. J. et al. Efficacy of oral N-acetylcysteine in the treatment of acetaminophen overdose. Analysis of the national multicenter study (1976 to 1985). N. Engl. J. Med.319(24), 1557–1562. 10.1056/nejm198812153192401 (1988). [DOI] [PubMed] [Google Scholar]
  • 14.Anna, L. et al. N-Acetylcysteine for preventing acetaminophen-induced liver injury: A comprehensive review. Front. Pharmacol.13, 828565. 10.3389/fphar.2022.828565 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Shakila, T. et al. Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells. Exp. Biol. Med. (Maywood)240(2), 261–272. 10.1177/1535370214549520 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Schofield, Z. V. et al. Neutrophils—A key component of ischemia-reperfusion injury. Shock40(6), 463–470. 10.1097/shk.0000000000000044 (2013). [DOI] [PubMed] [Google Scholar]
  • 17.Teng, X. et al. IL-37 ameliorates the inflammatory process in psoriasis by suppressing proinflammatory cytokine production. J. Immunol.192(4), 1815–1823. 10.4049/jimmunol.1300047 (2014). [DOI] [PubMed] [Google Scholar]
  • 18.Bulau, A. M. et al. In vivo expression of interleukin-37 reduces local and systemic inflammation in concanavalin A-induced hepatitis. Sci. World J.11, 2480–2490. 10.1100/2011/968479 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wu, B. et al. Interleukin-37 ameliorates myocardial ischaemia/reperfusion injury in mice. Clin. Exp. Immunol.176(3), 438–451. 10.1111/cei.12284 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Mingsheng, Z. et al. IL-37 isoform D downregulates pro-inflammatory cytokines expression in a Smad3-dependent manner. Cell Death Dis.9(6), 582. 10.1038/s41419-018-0664-0 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Nold, M. F. et al. IL-37 is a fundamental inhibitor of innate immunity. Nat. Immunol.11(11), 1014–1022. 10.1038/ni.1944 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Suzhao, L. et al. Role for nuclear interleukin-37 in the suppression of innate immunity. Proc. Natl. Acad. Sci. U.S.A.116(10), 4456–4461. 10.1073/pnas.1821111116 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Naden, T. K. et al. Distinct subclades of Aux/IAA genes are direct targets of ARF5/MP transcriptional regulation. New Phytol.204(3), 474–483. 10.1111/nph.12994 (2014). [DOI] [PubMed] [Google Scholar]
  • 24.Emma, B., Eugenia, H. T. & Ian, M. F. In vitro and clinical studies examining the expression of osteopontin in cigarette smoke-exposed endothelial cells and cigarette smokers. BMC Cardiovasc. Disord.12, 1–10. 10.1186/1471-2261-12-75 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Agata, B. Heterogeneity of hepatitis C virus particles and their evolution during infection. Cell. Mol. Gastroenterol. Hepatol.4(3), 443–444. 10.1016/j.jcmgh.2017.09.001 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Mitchell, R. M. et al. The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation. J. Clin. Investig.122(4), 1574–1583. 10.1172/jci59755 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Hongli, G. et al. Shikonin attenuates acetaminophen-induced acute liver injury via inhibition of oxidative stress and inflammation. Biomed. Pharmacother.112, 108704. 10.1016/j.biopha.2019.108704 (2019). [DOI] [PubMed] [Google Scholar]
  • 28.Dan, X., Rong, M. & Xiaofan, W. The roles of IL-1 family cytokines in the pathogenesis of systemic sclerosis. Front. Immunol.10, 225. 10.3389/fimmu.2019.02025 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Charalambos Gustav, A. et al. Secretory leukocyte protease inhibitor: A pivotal mediator of anti-inflammatory responses in acetaminophen-induced acute liver failure. Hepatology59(4), 1564–1576. 10.1002/hep.26933 (2013). [DOI] [PubMed] [Google Scholar]
  • 30.Lawson, J. A. et al. The hepatic inflammatory response after acetaminophen overdose: Role of neutrophils. Toxicol. Sci.54(2), 509–516. 10.1093/toxsci/54.2.509 (2000). [DOI] [PubMed] [Google Scholar]
  • 31.Hartmut, J., Mitchell, R. M. & Anup, R. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: Lessons learned from acetaminophen hepatotoxicity. Drug Metab. Rev.44(1), 88–106. 10.3109/03602532.2011.602688 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Nozomu, S. et al. Interleukin-37 reduces liver inflammatory injury via effects on hepatocytes and non-parenchymal cells. J Gastroenterol. Hepatol.27(10), 1609–1616. 10.1111/j.1440-1746.2012.07187.x (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yan, L. et al. Interleukin-37 attenuates bleomycin-induced pulmonary inflammation and fibrosis in mice. Inflammation41(5), 1772–1779. 10.1007/s10753-018-0820-9 (2018). [DOI] [PubMed] [Google Scholar]
  • 34.Qingwei, J. et al. Exogenous interleukin 37 ameliorates atherosclerosis via inducing the Treg response in ApoE-deficient mice. Sci. Rep.7(1), 3310. 10.1038/s41598-017-02987-4 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Giulio, C. & Charles, A. D. Suppression of inflammation and acquired immunity by IL-37. Immunol. Rev.281(1), 179–190. 10.1111/imr.12605 (2017). [DOI] [PubMed] [Google Scholar]
  • 36.Luo, P. et al. IL-37b alleviates inflammation in the temporomandibular joint cartilage via IL-1R8 pathway. Cell Prolif.52(6), e12692. 10.1111/cpr.12692 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Xiu, T. et al. IL-37 ameliorates the inflammatory process in psoriasis by suppressing proinflammatory cytokine production. J. Immunol.192(4), 1815–1823. 10.4049/jimmunol.1300047 (2014). [DOI] [PubMed] [Google Scholar]
  • 38.Feng, X. X. et al. IL-37 suppresses the sustained hepatic IFN-γ/TNF-α production and T cell-dependent liver injury. Int. Immunopharmacol.69, 184–193. 10.1016/j.intimp.2019.01.037 (2019). [DOI] [PubMed] [Google Scholar]
  • 39.Marcel, F. N. et al. IL-37 is a fundamental inhibitor of innate immunity. Nat. Immunol.11(11), 1014–1022. 10.1038/ni.1944 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Amy, H. C. & Kate, S. Inflammasome signaling and regulation of interleukin-1 family cytokines. J. Exp. Med.217(1), e20190314. 10.1084/jem.20190314 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Claudia, A.N.-P. et al. IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction. Nat. Immunol.16(4), 354–365. 10.1038/ni.3103 (2015). [DOI] [PubMed] [Google Scholar]
  • 42.Mitchell, J. R. et al. Acetaminophen-induced hepatic injury: Protective role of glutathione in man and rationale for therapy. Clin. Pharmacol. Ther.16(4), 676–684. 10.1002/cpt1974164676 (1974). [DOI] [PubMed] [Google Scholar]
  • 43.Lauterburg, B. H., Corcoran, G. B. & Mitchell, J. R. Mechanism of action of N-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo. J. Clin. Investig.71(4), 980–991. 10.1172/jci110853 (1983). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Yi, D. et al. Attenuating oxidative stress by paeonol protected against acetaminophen-induced hepatotoxicity in mice. PLoS ONE11(5), e0154375. 10.1371/journal.pone.0154375 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.McMillin, M. et al. The TGFbeta1 receptor antagonist GW788388 reduces JNK activation and protects against acetaminophen hepatotoxicity in mice. Toxicol. Sci.170(2), 549–561. 10.1093/toxsci/kfz122 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Ruggeri, R. M. et al. Increased serum interleukin-37 (IL-37) levels correlate with oxidative stress parameters in Hashimoto’s thyroiditis. J. Endocrinol. Investig.42(2), 199–205. 10.1007/s40618-018-0903-3 (2018). [DOI] [PubMed] [Google Scholar]
  • 47.Giulio, C. et al. Interleukin 37 reverses the metabolic cost of inflammation, increases oxidative respiration, and improves exercise tolerance. Proc. Natl. Acad. Sci. U.S.A.114(9), 2313–2318. 10.1073/pnas.1619011114 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Sakai, N. et al. Interleukin-37 reduces liver inflammatory injury via effects on hepatocytes and non-parenchymal cells. J. Gastroenterol. Hepatol.27(10), 1609–1616. 10.1111/j.1440-1746.2012.07187.x (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.McCann, R. L. & Wolfe, W. G. Splenorenal anastomosis during resection of juxtarenal aortic aneurysm. Surg. Gynecol. Obstet.168(3), 271–272 (1989). [PubMed] [Google Scholar]
  • 50.Zhiwei, X. et al. Protective effects of interleukin-37 expression against acetaminophen-induced hepatotoxicity in mice. Evid. Based Complement. Alternat. Med.10.1155/2022/6468299 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (1MB, docx)
Supplementary Material 2 (61.1KB, pdf)
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Supplementary Material 4 (10.6KB, docx)
Supplementary Material 5 (10.4KB, docx)

Data Availability Statement

All data relevant to the study are included in the article. Primary data are available upon request from Binfeng He (ldhbf@tmmu.edu.cn).

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