Diacerein counteracts acetaminophen-induced hepatotoxicity in mice via targeting NLRP3/caspase-1/IL-1β and IL-4/MCP-1 signaling pathways

Abstract

The current study aims at repurposing the anti-arthritic drug diacerein (DCN) for the treatment of acetaminophen hepatotoxicity and investigating the potential underlying mechanisms. Mice were randomly divided into six groups receiving either no treatment (control group), 20 mg/kg DCN i.p, 400 mg/kg acetaminophen i.p, DCN 4 h before acetaminophen, DCN 2 h after acetaminophen, or 400 mg/kg N-acetylcysteine (NAC) i.p, 2 h after acetaminophen. Biomarkers of liver dysfunction, oxidative stress, and apoptosis were assessed. Hepatic necroinflammatory changes were evaluated along with hepatic expression of NF-κB and caspase-1. The levels of NLRP3, IL-1β, IL-4, MCP-1, and TNF-α in the liver, as well as CYP2E1 mRNA expression, were measured. Diacerein significantly reduced biomarkers of liver dysfunction, oxidative stress, hepatocyte necrosis, and infiltration of neutrophils and macrophages whether administered 4 h before or 2 h after acetaminophen. Further, the effects were comparable to those of NAC. Diacerein also counteracted acetaminophen-induced hepatocellular apoptosis by increasing Bcl-2 and decreasing Bax and caspase-3 expression levels. Moreover, DCN normalized hepatic TNF-α and significantly decreased NF-κB p65 expression. Accordingly, DCN can prevent or reverse acetaminophen hepatotoxicity in mice, suggesting potential utility as a repurposed drug for clinical treatment.

Keywords: Acetaminophen; Diacerein; IL-1β; IL-4; MCP-1; NLRP3.

Fig. 1

Effect of diacerein (DCN) and N-acetyl cysteine (NAC) on A alanine aminotransferase (ALT), B aspartate aminotransferase (AST), and C lactate dehydrogenase (LDH) serum levels in acetaminophen-injected mice. Data are expressed as mean ± SEM (n = 8). #p < 0.05, ##p < 0.01 compared to control group, *p < 0.05, **p < 0.01 compared to acetaminophen-injected group, ɸp < 0.05, ɸɸp < 0.01 compared to DCN 4 h prior to acetaminophen group, •p < 0.05, ••p < 0.01 compared to DCN 2 h post-acetaminophen group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test

Fig. 2

A Representative images of hematoxylin–eosin-stained liver sections showing the effect of diacerein (DCN) and N-acetyl cysteine (NAC) on acetaminophen-induced hepatic histopathological changes in mice (figures a, c, e, g, i and k: X 100, bar = 100 µm, figures b, d, f, h, j and l: X 400, bar = 50 µm). (a, b) Liver of control group showing normal arrangement of hepatic cords around central vein (CV) with normal sinusoids; (c, d) Liver of DCN group showing normal arrangement of hepatic cords around CV with normal sinusoids; (e, f) Liver of acetaminophen group showing confluent areas of necrosis (black arrows) with neutrophils infiltration (white arrows) around congested blood vessels (red arrows) and in area of hepatocytes necrosis; (g, h) Liver of DCN 4 h prior to acetaminophen group showing few cell deaths (black arrows) with few neutrophils infiltration (white arrows); (i, j) Liver of DCN 2 h post-acetaminophen group showed few neutrophil infiltration (white arrows) around CV; (k, l) Liver of NAC 2 h post acetaminophen-group showing smaller areas of necrosis with neutrophil infiltration (white arrows) around congested blood vessels (red arrows) and area of hepatocytes necrosis. B Scatter dot plots of the histopathological assessment of hepatic necrosis scores. C Scatter dot plots of the histopathological assessment of hepatic neutrophil infiltration scores. $p < 0.05, $$p < 0.01 compared to control group, +p < 0.05, ++p < 0.01 compared to acetaminophen group using Kruskal–Wallis followed by Dunn's Multiple comparison test

Fig. 3

Effect of diacerein (DCN) on hepatic levels of monocyte chemoattractant protein-1 (MCP-1) in acetaminophen-injected mice. Data are expressed as mean ± SEM (n = 8). #p < 0.05, ##p < 0.01 compared to control group, *p < 0.05, **p < 0.01 compared to acetaminophen-injected group, ɸp < 0.05, ɸɸp < 0.01 compared to DCN 4 h prior to acetaminophen group, •p < 0.05, ••p < 0.01 compared to DCN 2 h post-acetaminophen group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test

Fig. 4

A Effect of diacerein (DCN) on hepatic F4/80 assessed by immunohistochemistry (figures a, c, e, g, i and k: X 100, bar = 100 µm, figures b, d, f, h, j and l: X 400, bar = 50 µm). (a, b) hepatic sections of control group showed negative staining; (c, d) hepatic sections of DCN group showed negative staining; (e, f) hepatic sections of acetaminophen group showed marked positive brown expression appears in hepatocytes (black arrows); (g, h) hepatic sections of DCN 4 h prior to acetaminophen group showed a significant reduction of F4/80 expression (black arrows); (i, j) hepatic sections of DCN 2 h post-acetaminophen group showed a significant reduction of F4/80 expression (black arrows); (k, l) hepatic sections of NAC 2 h post-acetaminophen-group showed a significant reduction of F4/80 expression (black arrows). B scatter dot plots of the immunohistopathological assessment of hepatic F4/80 scores. $$p < 0.01 compared to control group, +p < 0.05 compared to acetaminophen group using Kruskal–Wallis followed by Dunn's Multiple comparison test

Fig. 5

Effect of diacerein (DCN) on hepatic levels of A B-cell leukemia/lymphoma 2 (Bcl-2), B Bcl2 associated X (Bax), and C caspase-3 in acetaminophen-injected mice. Data are expressed as mean ± SEM (n = 8). #p < 0.05, ##p < 0.01 compared to control group, *p < 0.05, **p < 0.01 compared to acetaminophen group, ɸp < 0.05, ɸɸp < 0.01 compared to DCN 4 h prior to acetaminophen group, •p < 0.05, ••p < 0.01 compared to DCN 2 h post-acetaminophen group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test

Fig. 6

Effect of diacerein (DCN) and N-acetyl cysteine (NAC) on A reduced glutathione (GSH), B glutathione reductase (GR), C glutathione peroxidase (GPx), D glutathione-S-transferase (GST), E superoxide dismutase (SOD), and F malondialdehyde (MDA) in hepatic tissue of acetaminophen-injected mice. Data are expressed as mean ± SEM (n = 8). #p < 0.05, ##p < 0.01 compared to control group, *p < 0.05, **p < 0.01 compared to acetaminophen group, ɸp < 0.05, ɸɸp < 0.01 compared to DCN 4 h prior to acetaminophen group, •p < 0.05, ••p < 0.01 compared to DCN 2 h post-acetaminophen group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test

Fig. 7

Effect of DCN on mRNA expression of CYP2E1 in hepatic tissue of acetaminophen- injected mice. Data are expressed as mean ± SEM (n = 4). CYP2E1: cytochrome P450 2E1, GAPDH: glyceraldehyde-3-phosphate dehydrogenase, #p < 0.05, ##p < 0.01 compared to control group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test

Fig. 8

A Effect of diacerein (DCN) on hepatic tumor necrosis factor alpha (TNF-α) level. Data are expressed as mean ± SEM (n = 8). #p < 0.05, ##p < 0.01 compared to control group, *p < 0.05, **p < 0.01 compared to acetaminophen group, ɸp < 0.05, ɸɸp < 0.01 compared to DCN 4 h prior to acetaminophen group, •p < 0.05, ••p < 0.01 compared to DCN 2 h post-acetaminophen group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. B Effect of diacerein (DCN) on hepatic nuclear factor kappa-B p65 subunit (NF-κB p65) expression assessed by immunohistochemistry (X 400, bar = 50 µm); (a) hepatic sections of control group showing negative staining; (b) hepatic sections of DCN group showing negative staining; (c) hepatic sections of acetaminophen group showing marked positive brown nuclear and cytoplasmic expressions in hepatocytes (black arrows) associated with area of lesions around central veins; (d) hepatic sections of DCN 4 h prior to acetaminophen group showing a significant reduction of NF-κB p65 nuclear and cytoplasmic expressions (black arrows); (e) hepatic sections of DCN 2 h post-acetaminophen group showing a significant reduction of NF-κB p65 nuclear and cytoplasmic expressions (black arrows), and (f) scatter dot plots of the immunohistopathological assessment of hepatic NF-κB p65 score. $p < 0.05, $$p < 0.01 compared to control group, +p < 0.05, ++p < 0.01 compared to acetaminophen group using Kruskal–Wallis followed by Dunn's Multiple comparison test

Fig. 9

Effect of diacerein (DCN) on A NLR family pyrin domain containing 3 (NLRP3) and B Interlukin-1 beta (IL-1β) levels in hepatic tissue of acetaminophen-injected mice. Data are expressed as mean ± SEM (n = 8). #p < 0.05, ##p < 0.01 compared to control group, *p < 0.05, **p < 0.01 compared to acetaminophen-injected group, ɸp < 0.05, ɸɸp < 0.01 compared to DCN 4 h prior to acetaminophen group, •p < 0.05, ••p < 0.01 compared to DCN 2 h post-acetaminophen group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. C Effect of diacerein (DCN) on hepatic caspase-1 assessed by immunohistochemistry (X 400, bar = 50 µm); (a) hepatic sections of control group showing negative staining; (b) hepatic sections of DCN group showing negative staining; (c) hepatic sections of acetaminophen group showing marked positive brown cytoplasmic expression in hepatocytes (black arrows) associated with area of lesions around central veins; (d) hepatic sections of DCN 4 h prior to acetaminophen group showing a significant reduction of caspase-1 cytoplasmic expression (black arrows); (e) hepatic sections of DCN 2 h post-acetaminophen group showing a significant reduction of caspase-1 cytoplasmic expression (black arrows), and (f) scatter dot plots of the immunohistopathological assessment of hepatic caspase-1 scores. $p < 0.05, $$p < 0.01 compared to control group, +p < 0.05, ++p < 0.01 compared to compared to acetaminophen group using Kruskal–Wallis followed by Dunn's Multiple comparison test

Fig. 10

Effect of diacerein (DCN) on hepatic levels of A interleukin (IL)-4, and B IL-10 in acetaminophen-injected mice. Data are expressed as mean ± SEM (n = 8). #p < 0.05, ##p < 0.01 compared to control group, *p < 0.05, **p < 0.01 compared to acetaminophen group, ɸp < 0.05, ɸɸp < 0.01 compared to DCN 4 h prior to acetaminophen group, •p < 0.05, ••p < 0.01 compared to DCN 2 h post-acetaminophen group using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test

Fig. 11

Schematic representation of the proposed underlying mechanisms of DCN against acetaminophen-induced hepatotoxicity in mice