Fig. 4

Fucoidan activates the PI3K/AKT/Nrf2 signaling pathway. (A-B) Fucoidan promotes the phosphorylation of PI3K and AKT. HepG2 cells were stimulated with FFAs (0.5 mM) for 24 h, further treatment with DMSO (control) or fucoidan (100 µg/ml) for additional 24 h. The phosphorylation levels of PI3K and AKT were analyzed by western blotting. (C) Fucoidan increases the expression and phosphorylation of Nrf2. HepG2 cells were treated as described in (A-B), and the expression and phosphorylation of Nrf2 were analyzed by western blotting. (D-E) Fucoidan promotes the nuclear translocation of Nrf2. HepG2 cells were treated as described in (A-B), and the protein levels of Nrf2 in nuclear and cytoplasm were measured by western blotting. (F-G) Nrf2 knock-down in HepG2 cells and spheroids. HepG2 cells were transfected with Adenovirus contains antisense of Nrf2 shRNA for 5 h, then supplemented with fresh medium, and continuously cultured for an additional 48 h. Spheroids were constructed with Adenovirus transfected HepG2 cells. The expression of Nrf2 in HepG2 cells and spheroids were analyzed by qRT-PCR and western blotting. (H) Nrf2 shRNA abolished the overexpression of Nrf2 induced by Fucoidan in spheroids. Spheroids were aggregated with Adenovirus transfected HepG2 cells for 48 h, then stimulated with FFAs (0.5 mM) for 24 h, further treatment with DMSO (control) or fucoidan (100 µg/ml) for additional 24 h. The expression of Nrf2 in spheroids was analyzed by western blotting. Data were analyzed by student’s t test and expressed as the mean ± SD (n = 3). * represents the differences between the two groups. *** P < 0.001