NADPH oxidase 4 deficiency promotes hepatocellular carcinoma arising from hepatic fibrosis by inducing M2-macrophages in the tumor microenvironment

Hepatocellular carcinoma (HCC) often arises in the cirrhotic livers, highlighting the intricate link between hepatic fibrosis and carcinogenesis. Reactive oxygen species produced by NADPH oxidase 4 (NOX4) contribute to liver injury leading to hepatic fibrosis. Paradoxically, NOX4 is known to inhibit HCC progression. This study aims to elucidate the role of NOX4 in hepatocarcinogenesis in the background of hepatic fibrosis. We established the mouse model of HCC arising from the fibrotic liver by administering diethylnitrosamine and carbon tetrachloride to wild-type (WT) or NOX4^-/- mice. Hepatic fibrogenesis, tumorigenesis, and macrophage polarization were assessed by immunohistochemistry, PCR, and flow cytometry using in vivo and in vitro models. In NOX4^-/- mice, hepatic fibrosis was attenuated, while the number of tumors and the proliferation of HCC cells were increased compared to WT mice. Notably, a significant increase in M2-polarized macrophages was observed in NOX4^-/- mice through immunohistochemistry and PCR analysis. Subsequent experiments demonstrated that NOX4-silenced HCC cells promote macrophage polarization toward M2. In addition to attenuating hepatic fibrogenesis, NOX4 deficiency triggers macrophage polarization towards the M2 phenotype in the fibrotic liver, thereby promoting hepatocellular carcinogenesis. These findings provide novel insights into the mechanism of NOX4-mediated tumor suppression in HCC arising from fibrotic livers.