Bixin Attenuates High-Fat Diet-Caused Liver Steatosis and Inflammatory Injury through Nrf2/PPARα Signals

Figure 2

Bixin upregulates Nrf2 signals by decreasing Nrf2 ubiquitination and increasing its protein stability in a Keap1 C151-dependent manner. (a) LO2 cells were treated with the indicated doses of bixin for 24 h; the mRNA expression of Nrf2 and its targets (HO-1 and NQO1) was investigated by qRT-PCR (, Ctrl vs. bixin treatments). (b) LO2 cells were treated with bixin (40 μM) for 24 h along with MG132 (10 μM) for 4 h. Anti-Nrf2 immunoprecipitates were analyzed by immunoblot analyses with anti-Ub antibody for the detection of ubiquitin-conjugated Nrf2. (c) LO2 cells were either left untreated or treated with bixin (40 μM) for 24 h. CHX (100 μM) was added for the indicated time points, and cell lysates were subjected to immunoblot analyses with anti-Nrf2 and anti-GAPDH antibodies. The intensity of Nrf2 and GAPDH bands was quantified and plotted against the time after addition of CHX, and the half-life of Nrf2 was calculated. (d) LO2 cells cotransfected with the plasmids expressing either wild-type Keap1 (Keap1 WT) or C151-mutated Keap1 (Keap1 C151S) along with mGst-ARE firefly luciferase and Renilla luciferase reporters were left untreated or treated with the indicated compounds for 16 h. Dual luciferase activities were measured, and the data are expressed as the (, Ctrl vs. compound-treated groups; ^#, Keap1 WT vs. Keap1 C151S group).

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