Sequential Changes in NOX4 Expression, Oxidative Stress Indices, PIIINP, and Liver Histopathology During Hepatocellular Carcinogenesis Induced in Mice

Abstract

Background and aim: Hepatocellular carcinoma (HCC) is a chronic disease caused by complex histological and biochemical changes related to oxidative stress leading to fibrosis, cirrhosis, and malignancy. Knowing the sequential changes in different stages of HCC development is essential for understanding the mechanisms of HCC pathogenesis.

Methods: This study was designed to evaluate alterations in NADPH oxidase 4 (NOX4) expression and oxidative stress during HCC progression in mice, induced with administration of diethylnitrosamine (DEN, 50 mg/kg) and phenobarbitone (PB, 500 mg/L via drinking water). The correlation of N-terminal propeptide type III collagen (PIIINP) as a serum indicator of fibrosis with HCC progression was also assessed. Newborn C57/bl6 mice were divided into four groups (n = 12/group): control, PB, DEN, and HCC. Then they were euthanized at different time schedules 2, 4, and 7 months (n = 4/subgroup). Blood and liver tissues were collected for estimation of serum PIIINP and total antioxidant capacity (TAC) liver NOX4 mRNA and protein expression, total oxidative stress, and glutathione (GSH).

Results: The results showed that NOX4 protein expression increased in the first months of HCC induction. Accordingly, liver NOX4-specific mRNA was substantially elevated (2.4 fold). Circulating fibrosis marker, the PIIINP levels together with total oxidative stress increased during HCC induction. TAC and GSH were increased over time during HCC induction.

Conclusions: Based on the sequential changes observed following HCC induction by DEN, we conclude that increased expression of NOX4 in the liver precedes other changes such as other oxidative stress factors and fibrosis markers during HCC progression.

Keywords: HCC; NOX4; PIIINP; diethylnitrosamine; glutathione; liver fibrosis; oxidative stress.