1E). TNF-�� has selleck Brefeldin A been suggested as a central proinflammatory cytokine that is produced by activated inflammatory cells and mediates insulin resistance and hepatocyte apoptosis in liver disease (7, 38). Consistent with activation of the inflammatory cascade, serum TNF-�� level was increased in MCD diet-fed control genotype mice compared with the MCS diet-fed controls (Fig. 1F). In contrast, MCD-induced TNF-�� was significantly lower in MD-2- or TLR4-deficient MCD diet-fed mice (Fig. 1F). These data suggested that TLR4-MD-2 complex deficiency is partially protective against MCD-induced liver inflammation and damage. MD-2 and TLR4 deficiency attenuates oxidative stress. Increased lipid peroxidation and oxidative stress are key in development of steatosis in NAFLD (20).

We identified significantly higher levels of liver TBARS, indicative of lipid peroxidation, in MCD diet- compared with the MCS diet-fed genotype control mice (Fig. 2A). Consistent with our hypothesis that MD-2-TLR4 complex plays a role in NASH, we found significantly reduced induction of TBARS in the livers of MCD diet-fed MD-2- and TLR4-deficient mice (Fig. 2A). NADPH oxidases play an important role in the generation of reactive oxygen radicals (25, 30). The classic NADPH complex is composed of at least six components, which include two trans-membrane flavocytochrome b components (gp91phox and p22phox) and four cytosolic components (p47phox, p67phox, p40phox, and Rac-1 protein) (30). TLR4-mediated signals are strong inducers of NADPH transcription and functional activity (25).

Investigation of NADPH oxidase expression revealed a significant upregulation of the cytoplasmic components of the NADPH oxidase, including p47phox (Fig. 2B) and p67phox (Fig. 2C), in MCD diet-fed animals of control genotypes. The membrane-associated components of the NADPH complex, gp91phox (Fig. 2D) and p22phox (Fig. 2E), were also upregulated at the mRNA level in the livers of MCD diet- compared with the MCS diet-fed mice of control genotypes. Deficiency in MD-2 or TLR4 abrogated the MCD-induced upregulation of all of the NADPH oxidase subunits (Fig. 2, B-E), suggesting that NADPH-mediated oxidative stress is dependent on MD-2 and TLR4 expression in this model. To test for the biological significance of the mRNA increase in the NADPH subunits, we evaluated the NADPH oxidase activity.

Consistent with the increased mRNA levels of NAPDH oxidase complex components, NADPH oxidase activity was elevated, as suggested by the increased NADP+-to-NADPH ratio in livers of MCD-fed compared with MCS-fed mice of control genotypes (Fig. 2F). More importantly, we identified that both TLR4 KO and MD-2 KO mice were protected from the MCD diet-induced activation of NADPH oxidase (Fig. 2F). Collectively, these results indicated that MD-2-TLR4 complex-induced signals Cilengitide contribute to liver pathology via NADPH-dependent lipid peroxidation and oxidative stress in the MCD diet-induced NASH model.