Since the inflammatory marker OPN, the pro-inflammatory cytokine TNFα, and the marker Kupffer cells F4/80, play important roles in NASH [11,21–23], the levels of these proteins were evaluated in mice fed HFD. As shown in Fig. 3A, immunoblot analysis revealed that the cleaved (active form) OPN levels were only increased in the WT-HFD. Furthermore, quantitative immunohistochemistry analysis revealed that F4/80 positive cells were markedly increased in the WT-HFD compared with the other three groups (Fig. 3B–F). However, intrahepatic levels of TNFα and MCP-1 were lower in the WT-HFD than their corresponding WT-LFD groups, while intrahepatic MCP-1 increased in HFD-null group compared to their corresponding control (Fig. 3G and H). We did not detect significant changes in apoptosis among the groups evaluated by TUNEL assay, and the levels of pro-apoptotic proteins such as Bax, Bcl2, and cleaved caspase 3 (data not shown).
