Hepatocyte Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice

Abstract

Background & Aims Reactive oxidative species (ROS) are believed to be involved in the progression of non-alcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of NADPH oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. Methods Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4hepKO) and NOX4floxp+/+ C57BL/6 mice (controls) were given fast food diets (supplemented with high-fructose corn syrup) or choline-deficient L-amino acid-defined to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase (PKR), and phospho-eIF-2alpha kinase (PERK)-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. Results Levels of NOX4 were increased in patients with NASH, compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of PKR and PERK-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831had increased insulin sensitivity. Conclusion NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

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

“…In addition, it will be of interest to feed alcohol to mice with hepatocyte-specific deletion of NOX4. Previous study showed that hepatocyte-specific deletion of NOX4 attenuated oxidative stress, lipid peroxidation, and liver fibrosis in mice with dietary induced steatohepatitis 36 . Thus, mice with hepatocyte-specific deletion of NOX4 may protect from alcohol-induced liver injury via decrease of oxidative stress and apoptosis.…”

Global Deletion of Glutathione S-Transferase A4 Exacerbates Developmental Nonalcoholic Steatohepatitis

“…23 Moreover, the presence of p47phox in liver parenchymal cells has also been shown to be a key mediator of liver pathology in alcoholic liver disease. 24 In addition, recent studies from Bettaieb et al 25 suggest that hepatocyte Nox4 may also play an important role in development of oxidative stress and progression of NASH pathology to inflammation and fibrosis. In the current study, knockdown of Ppara also resulted in increased inflammation, necrosis, matrix remodeling, and evidence of stellate cell activation and fibrosis after feeding 70% HF diets relative to Wt mice.…”

Pathophysiology of gadolinium-associated systemic fibrosis

“…Interestingly, a wide array of evidence shows that Nox4-derived reactive oxygen species play a major role in the pathogenesis of renal, cardiac, lung, and liver fibrosis (9,36,37,40,45,49,63,65). Importantly, Nox4 has also been reported to be a primary target of TGF-␤ and a critical mediator of its profibrotic actions in various cell types (7,36,40,45).…”

“…Importantly, Nox4 has also been reported to be a primary target of TGF-␤ and a critical mediator of its profibrotic actions in various cell types (7,36,40,45). It should be pointed out that pharmacological inhibitors of Nox4 are available and have undergone preclinical studies in animal models of fibrotic diseases, where they successfully attenuated the pathological changes observed in renal complication of diabetes, atherosclerosis, liver fibrosis and idiopathic pulmonary fibrosis (5,9,36,37,40,49,50,63). Therefore, Nox4 may represent an attractive therapeutic target for prophylaxis or the treatment of gadolinium-associated systemic fibrosis.…”