However, NOX1/4 inhibition prevented macrophage infiltration and activation to levels similar to those observed in mice untreated with CCl4 (Fig. 4A-C). Hepatic mRNA expression of tumor necrosis factor alpha (TNF-α) was also increased in SOD1mu mice after CCl4 treatment, but this increase was suppressed by GKT137831 (Fig. 4D). Serum ALT levels were increased in CCl4-treated SOD1mu mice, compared

to CCl4-treated WT mice, which was also reduced by NOX1/4 inhibition by check details GKT137831 (Fig. 4E). These results indicate that increased liver inflammation and injury in CCl4-treated SOD1mu and WT mice was inhibited to similar lower levels by GKT137831 treatment. To investigate ROS mediated LPO, we measured the LPO products, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), as indicators of oxidative stress in the liver. Hepatic 4-HNE levels were increased in SOD1mu mice, compared to WT mice, after CCl4 treatment, but this increase was suppressed by inhibition of NOX1/4 (Fig. 5A). Measurement of MDA using TBARS showed that increased

hepatic MDA levels in SOD1mu mice were suppressed by GKT137831 treatment after Selleck FDA approved Drug Library CCl4 injections (Fig. 5B). In agreement with liver fibrosis and inflammation results, the levels of hepatic LPO in CCl4-treated SOD1mu mice were decreased to the same low level as WT mice by Nox 1/4 inhibition. We used activation of primary cultures of HSCs as a model of activated myofibroblasts.26, 27 Primary Anacetrapib HSCs were isolated from both WT and SOD1mu mice. In quiescent HSCs, mRNA expression of collagen α1(I) and Acta2 are at the same low level. mRNA levels in activated SOD1mu HSCs were significantly greater than in activated WT HSCs (Fig. 5C). Incubating with GKT137831 reduced expression levels of collagen α1(I) and Acta2 mRNA to the same low levels in both activated SOD1mu and WT HSCs (Fig. 5C). Interestingly, mRNA expression of NOX4 and, to a lesser extent, NOX1 was increased in activated SOD1mu HSCs, compared

to WT HSCs, and this increase was also suppressed by GKT137831 (Fig. 5D). Next, we assessed HSC activation by measuring GFP fluorescence in quiescent HSCs purified from the ColI-GFP reporter mouse, in which the collagen α1(I) promoter/enhancer drives GFP. Ang II induced higher GFP fluorescence in SOD1mu HSCs than in WT HSCs, but this GFP enhancement was suppressed by GKT137831 (Fig. 6A,B). To assess the effect of SOD1mu on ROS generation, we measured the quantity of ROS in DCFDA-loaded HSCs after Ang II treatment. Ang II induced excessive ROS production in SOD1mu HSCs, compared to WT HSCs. GKT137831 suppressed ROS generation in SOD1mu HSCs to the same levels as WT HSCs treated with the NOX1/4 inhibitor (Fig. 6C).