Hcy accumulation resulted in oxidative stress and HO-1 downregulation. (A) Hcy concentrations of plasma were determined by an ELISA assay and indicated for each group of C57BL/6 J mice (n = 7 for each group). The livers were homogenized and the total protein concentrations were assessed to determine the enzyme activity units of SOD by WST-1 method (B) and the MDA concentrations using an ELISA assay (C). (D) The total RNA of livers were extracted and subjected to qRT-PCR for the assessment of HO-1 mRNA level. The bar graph shows mRNA levels of HO-1 after normalization to GAPDH. Protein levels of HO-1, Bach1 and Nrf2 were analyzed using Western blotting (E and F). β-actin was used as an internal control to ensure equal loading in all lanes of the gel. Representative results of 3 independent experiments are shown. Data are presented as means ± SEM from 3 three independent experiments. *P < 0.05 vs. mice fed with chow diet.