Analysis of the mechanisms of liver inflammation and liver fibrosis, and of the effects of NF-kB activity inhibitor on iNOS knockout/HFD mice livers at 48 weeks. (A) Liver samples were stained with α-SMA antibodies. Pericellular staining was visible only in iNOS−/−/HFD mice at 48 weeks. Scale bar, 50 μm. (B) Liver IL-1, TGF-β, PDGF, TIMP-1, TIMP-2 and collagen I mRNA expression levels were significantly higher in iNOS−/−/HFD mice than in iNOS+/+/HFD mice at 48 weeks. (C) Analysis of liver NF-kB activity by EMSA showed that the NF-kB band of iNOS−/−/HFD mice at 48 weeks was enhanced compared with iNOS+/+/HFD mice. P.C.: positive control using HeLa cells. N.C.: negative control. (D) NF-kB protein was expressed in the nucleus of hepatic nonparenchymal cells and injured hepatocytes in iNOS−/−/HFD mice at 48 weeks. (E) Confocal microscopic images showed that NF-kB-activated cells (red) were almost completely merged with F4/80-positive cells (green) in iNOS−/−/HFD mice. The nuclei were stained with 4′-diamidine-2′-phenylindole hydrocholoride (DAPI: blue). (Data are expressed as the mean ± SEM. * p < 0.05, represents significant difference between iNOS+/+/HFD mice and iNOS−/−/HFD mice).