Hepatocyte DDX3X protects against drug-induced acute liver injury via controlling stress granule formation and oxidative stress

Abstract

Drug-induced liver injury (DILI) is the leading cause of acute liver failure (ALF). Continuous and prolonged hepatic cellular oxidative stress and liver inflammatory stimuli are key signatures of DILI. DEAD-box helicase 3, X-linked (DDX3X) is a central regulator in pro-survival stress granule (SG) assembly in response to stress signals. However, the role of DDX3X in DILI remains unknown. Herein, we characterized the hepatocyte-specific role of DDX3X in DILI. Human liver tissues of DILI patients and control subjects were used to evaluate DDX3X expression. APAP, CCl4 and TAA models of DILI were established and compared between hepatocyte-specific DDX3X knockout (DDX3X^Δhep) and wild-type control (DDX3X^fl/fl) mice. Hepatic expression of DDX3X was significantly decreased in the pathogenesis of DILI compared with controls in human and mice. Compared to DDX3X^fl/fl mice, DDX3X^Δhep mice developed significant liver injury in multiple DILI models. DDX3X deficiency aggravates APAP induced oxidative stress and hepatocyte death by affecting the pro-survival stress granule (SG) assembly. Moreover, DDX3X deficiency induces inflammatory responses and causes pronounced macrophage infiltration. The use of targeted DDX3X drug maybe promising for the treatment of DILI in human.

Fig. 1. DDX3X expression is decreased in the livers of humans and mice with DILI.

A Serum levels of ALT of control subjects and patients with DILI. Data are expressed as the Median (25th, 75th percentiles), ***p < 0.001. B Representative H&E and IHC sections of human liver tissues. Scale bars, 10 µm; C–E mRNA and protein expression of DDX3X in multiple DILI mouse models treated with APAP, CCL4, or TAA. Data are expressed as mean ± SD, n = 3-6/group. *p < 0.05, **p < 0.01.

Fig. 2. Deficiency of hepatocyte DDX3X exacerbate APAP induced DILI in mice.

A Schematic diagram of APAP induced DILI model; B, C mRNA and protein expression of DDX3X in mice treated with PBS or APAP for 12 h. n = 3/group; D Histological evaluation of liver sections from DDX3X^fl/fl and DDX3X^Δhep mice treated with APAP or PBS for 12 h, respectively. Scale bars, 100 µm (top), 20 µm (bottom). n = 6/group; E Serum levels of ALT and AST of DDX3X^fl/fl and DDX3X^Δhep mice with APAP induction. n = 6/group; F Survival assay of DDX3X^fl/fl and DDX3X^Δhep mice treated with APAP or PBS for 72 h, respectively. n = 10/group. Data are expressed as mean ± SD. **p < 0.01.

Fig. 3. Deficiency of hepatocyte DDX3X exacerbate CCl4 and TAA induced DILI in mice.

A Schematic diagram of CCl4 induced DILI model; B histological evaluation of liver sections from DDX3X^fl/fl and DDX3X^Δhep mice treated with CCl4 for 48 h, respectively. Scale bars, 100 µm (left), 20 µm (right); C serum levels of ALT and AST of DDX3X^fl/fl and DDX3X^Δhep mice with CCl4 induction; D schematic diagram of TAA induced DILI model; E Histological evaluation of liver sections from DDX3X^fl/fl and DDX3X^Δhep mice treated with TAA for 24 h, respectively. Scale bars, 100 µm (left), 20 µm (right); F serum levels of ALT and AST of DDX3X^fl/fl and DDX3X^Δhep mice with TAA induction. Data are expressed as mean ± SD, n = 6/group. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. Hepatocytes DDX3X is required for stress granules formation and cell survival in APAP-induced liver injury.

A Negative control of primary hepatocytes were treated with PBS; and positive control of SG in primary hepatocytes were first primed with LPS for 4 h, then induced by 50 μM sodium (meta) arsenite for 30 min; B representative IF stained sections of G3BP1 (a marker of SGs) in primary hepatocytes isolated from DDX3X^fl/fl or DDX3X^Δhep mice with in vitro stimulation of APAP for 12 h. Scale bars, 10 µm; C representative IF stained sections of SYTOX green in primary hepatocytes isolated from DDX3X^fl/fl or DDX3X^Δhep mice with in vitro stimulation of APAP or PBS for 12 h, respectively. Scale bars, 20 µm; D Quantitative data of SYTOX green⁺ cells of primary hepatocytes; Data are expressed as mean ± SD, n = 3/group. ***p < 0.001.

Fig. 5. Hepatocyte DDX3X ablation induces oxidative stress in DILI.

A Representative DHE stained liver sections of DDX3X^fl/fl and DDX3X^Δhep mice treated with APAP or PBS. Scale bars, 25 µm; B calculation of DHE⁺ cells of liver sections; C, D protein expression of CYP2E1 in primary hepatocytes treated with APAP for 12 h. Data are expressed as mean ± SD, n = 3/group. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. Hepatic DDX3X deletion exacerbates macrophage infiltration and inflammation.

A mRNA expression levels of IL-1β, HMGB1 and TNF-α in liver tissues and primary hepatocytes of DDX3X^fl/fl and DDX3X^Δhep mice; B Representative IHC stained sections of F4/80 in liver tissues of APAP induced DILI. Scale bars, 100 µm (left), 25 µm (right); (C) Schematic diagram of DDX3X^fl/fl macrophage co-cultured with DDX3X^fl/fl or DDX3X^Δhep hepatocytes in APAP medium; D, E Numbers of infiltrated macrophages were evaluated by co-culture system. Scale bars, 100 µm; F Schematic diagram of DDX3X in DILI. DDX3X knockout in the hepatocytes of mice significantly exacerbated histological severity in multiple DILI murine models. This effect is through regulation of SG formation to protect hepatocytes from oxidative stress and macrophage inflammation. Data are expressed as mean ± SD, n = 3-6/group. *p < 0.05, **p < 0.01, ***p < 0.001.