Pharmacological inhibition of NOX4 ameliorates alcohol-induced liver injury in mice through improving oxidative stress and mitochondrial function

• Published in: Biochimica et biophysica acta Vol. 1861; no. 1; pp. 2912 - 2921
• Main Authors: Sun, Qian, Zhang, Wenliang, Zhong, Wei, Sun, Xinguo, Zhou, Zhanxiang
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017
• Subjects: adenosine triphosphate; Adenosine Triphosphate - metabolism; Alcoholic liver disease; Animals; apoptosis; Apoptosis - drug effects; beta oxidation; Biomarkers - blood; blood serum; caspase-9; diet; DNA, Mitochondrial - metabolism; Electron Transport - drug effects; electron transport chain; Enzyme Inhibitors - pharmacology; ethanol; Ethanol - toxicity; Gene Knockdown Techniques; GKT137831; Hepatocyte Nuclear Factor 4 - metabolism; hydrogen peroxide; In Situ Nick-End Labeling; Lipid Metabolism - drug effects; lipids; liver; Liver - drug effects; Liver - injuries; Liver - pathology; liver diseases; Male; membrane potential; Membrane Potential, Mitochondrial - drug effects; mice; Mice, Inbred C57BL; Mitochondria; Mitochondria - drug effects; Mitochondria - metabolism; mitochondrial DNA; mitochondrial membrane; NAD - metabolism; NAD(P)H oxidase (H2O2-forming); NADP (coenzyme); NADPH oxidase; NADPH Oxidase 4; NADPH Oxidases - antagonists & inhibitors; NADPH Oxidases - metabolism; NOX4; Oxidation-Reduction; oxidative stress; Oxidative Stress - drug effects; Protective Agents - pharmacology; Pyrazoles - pharmacology; Pyridines - pharmacology; Rats, Wistar; Reactive Oxygen Species - metabolism; ROS; Signal Transduction - drug effects; Subcellular Fractions - drug effects; Subcellular Fractions - enzymology; NOX4; Mitochondria; GKT137831; NADPH oxidase; ROS; Alcoholic liver disease
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/j.bbagen.2016.09.009

Background: Oxidative stress plays a crucial role in the development of alcoholic liver disease (ALD), however effective pharmacological treatment for oxidative injury is still lacking. The objective of this study was to determine whether inhibition of NADPH oxidase activity could reverse alcohol-induced liver injury via protecting mitochondrial functions. Methods: C57BL/6J mice were pair-fed with Lieber-DeCarli control or ethanol diet for four week with or without administration with 30mg/kg/d GKT137831, a NOX4 inhibitor for the last two weeks. H4IIEC3 cells were transfected with scrambled or NOX4 shRNA. Cells were then treated with 200mM ethanol for 48h. Results: Alcohol exposure induced NOX4 expression in the liver and mitochondrial fraction. GKT137831 partially reversed alcohol-induced liver injury and elevation of serum H2O2. The levels of mitochondrial ROS, mitochondrial DNA, respiratory chain complex IV, and hepatic ATP were partially reversed by GKT137831 after alcohol exposure. Furthermore GKT137831 ameliorated alcohol-induced lipid accumulation and increased HNF-4α and β-oxidation enzymes. GKT137831 also decreased alcohol-induced apoptosis coupled with decreased insertion of Bax into mitochondria and decreased activation of cleaved caspase-9 and cleaved PARP. Mechanistic study shows that ethanol induced expression of NOX4 in H4IIEC3 cells. Knockdown of NOX4 caused an increased mitochondrial membrane potential, decreased mitochondrial superoxide levels, reduced number of apoptotic cells, decreased lipid accumulation, and improved ATP levels and NAD+/NADH ratio after ethanol treatment. Conclusion: Pharmacological inhibition of NOX4 activity protects against alcohol-induced fat accumulation and activation of intrinsic apoptosis via improving mitochondrial function. General significance: Pharmacological inhibition of NOX4 could be a promising treatment for ALD. [Display omitted] •NOX4 predominantly localizes in the mitochondria in the liver of mice, and chronic alcohol exposure increases hepatic NOX4.•Inhibition of NOX4 activity decreases alcohol-elevated serum hydrogen peroxide levels.•Inhibition of NOX4 activity decreases alcohol-induced fat accumulation but increases HNF-4α and CPT-1 in the liver.•Inhibition of NOX4 activity decreases alcohol-induced mitochondrial apoptotic cell death pathway.•Knockdown of NOX4 protects against ethanol-induced loss of mitochondrial membrane potential and generation of ROS.