Chronic-plus-binge alcohol intake induces production of proinflammatory mtDNA-enriched extracellular vesicles and steatohepatitis via ASK1/p38MAPKα-dependent mechanisms
Jing Ma, … , Jian Sun, Bin Gao
Jing Ma, … , Jian Sun, Bin Gao
Published June 16, 2020
Citation Information: JCI Insight. 2020;5(14):e136496. https://doi.org/10.1172/jci.insight.136496.
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Research Article Hepatology

Chronic-plus-binge alcohol intake induces production of proinflammatory mtDNA-enriched extracellular vesicles and steatohepatitis via ASK1/p38MAPKα-dependent mechanisms

Abstract

Alcohol-associated liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting release by hepatocytes of proinflammatory mitochondrial DNA–enriched (mtDNA-enriched) extracellular vesicles (EVs). The aim of the present study was to investigate the role of the stress kinase apoptosis signal–regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol–induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the greatest hepatic upregulation of metallothionein 1 and 2 (Mt1/2), which encode 2 of the most potent antioxidant proteins. Genetic deletion of the Mt1 and Mt2 genes aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress, and ASK1/p38 activation in the liver. Inhibition or genetic deletion of Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, ROS levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.

Authors

Jing Ma, Haixia Cao, Robim M. Rodrigues, Mingjiang Xu, Tianyi Ren, Yong He, Seonghwan Hwang, Dechun Feng, Ruixue Ren, Peixin Yang, Suthat Liangpunsakul, Jian Sun, Bin Gao

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Figure 1

Mt1/2–/– mice are more susceptible to chronic-plus-binge ethanol–induced liver injury.

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Mt1/2–/– mice are more susceptible to chronic-plus-binge ethanol–induce...
(A) C57BL/6N mice were fed an ethanol diet for 8 weeks (E8w) or 8 weeks plus 1 binge (E8w+1B). Liver tissues were collected 9 hours after binge in the E8w+1B group and subjected to microarray analysis. A heat map of oxidative stress–related genes is shown. (B) C57BL/6N mice were pair-fed with control diet (pair-fed, n = 4) or fed an ethanol diet for 8 weeks (E8w), or E8w+1B, and were euthanized 9 hours after binge. C57BL/6N mice were fed a control diet (pair-fed) or ethanol diet for 10 days plus 1 binge (E10d+1B) and were euthanized 3, 6, or 9 hours later. Hepatic mRNA levels of Mt1 and Mt2 were examined by RT-qPCR. (C–F) WT and Mt1/2–/– mice were pair-fed or fed an ethanol diet for 10 days, followed by gavage of a single dose of maltose or ethanol, respectively and were euthanized 9 hours later. Serum ALT levels and hepatic TG levels were measured (C). Hepatic neutrophils were examined by IHC staining with an anti-MPO antibody or RT-qPCR analysis of Ly6g mRNA (D). Hepatic macrophages were examined by immunostaining with an anti-F4/80 antibody or RT-qPCR analysis of F4/80 mRNA (E). Liver tissues were also subjected to TUNEL staining, and the TUNEL+ hepatocytes were counted (F). Values represent mean ± SEM (each dot represents 1 mouse sample). Statistical evaluation was performed by Student’s t test or 1-way ANOVA with Tukey’s post hoc test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.