Abstract: Objective The aim of this experiment was to explore the changes of NADPH oxidase 4 (NOX4) gene and its protein in liver tissues of mice with carbon tetrachloride (CCl₄)-induced liver fibrosis and hepatic stellate HSC-T6 cells.Methods The liver fibrosis model was established by intraperitoneal injection of CCl₄ in ten mice, and the NOX4 mRNA and its protein in liver tissues were detected by qRT-PCR and Western bloting. The HSC-T6 cells were normally cultured and divided into blank, nonsense and NOX4-siRNA-intervened groups, which were transfected by liposome 2000-coated meaningless sequence or NOX4-siRNA in the two latter groups. The expression ofNOX4, α-smooth muscle actin (α-SMA), type I collagen (Col1a I), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-2 (MMP-2), transforming growth factor-β1 (TGF-β1), Smad2 and Smad3 in HSC-T6 cells was detected by qRT-PCR and Western bloting. The intracellular reactive oxygen species (ROS) content was detected by DCFH-DA fluorescent probe, the cell proliferation was detected by MTT assay, and the cell cycles and apoptosis were detected by flow cytometry.Results There was a significant pathological damage, with a large amount of collagen fiber deposition in liver tissues of mice in model; the NOX4 mRNA level in liver tissues of mice in model was significantly higher than that in control group (P<0.05); the NOX4 mRNA and its protein, ROS, proliferation activity, percentage of cells in S phase, the α-SMA, Col1a I, TIMP-1, MMP-2, TGF-β1, p-Smad2/Smad2 and p-Smad3/Smad3 expression were significantly decreased, while the percentage of cells in G0/G1 phase, and apoptosis rate were significantly increased (P<0.05) in NOX4-siRNA-intervened group. Conclusion The NOX4 is highly expressed in liver fibrotic tissues, and the down-regulation of NOX4 could inhibit proliferation and activation of HSCs, and promote their apoptosis, which mmight be related to the inhibition of TGF-β/Smad signaling pathway.

Key words: Liver fibrosis, HSC-T6 cells, NADPH oxidase 4, TGF-β/Smad signaling pathway, Mice