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

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, pERK_1/2, 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.

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

Fig. 1

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.

Fig. 2

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.

Fig. 3

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.

Fig. 4

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.

Fig. 5

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.

Fig. 6

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.