Fig 6.

The extract of Rhizoma Acori tatarinowii (AT) and asarones activate ERK cascade but not Akt cascade to promote neural progenitor cell (NPC) proliferation. (A) Embryonic neural precursors were cultured in reduced growth factor medium (1 ng mL⁻¹ EGF and 1 ng mL⁻¹ bFGF) for 24 h and were treated with 1 mg mL⁻¹ AT, 1 μm α-asarone, or 1 μm β-asarone for the indicated time. Protein samples were analyzed on Western blots using antibodies against the phosphorylated ERK1/2 and Akt. Blots were probed for total ERK and Akt as loading controls. Four independent experiments showed similar results. (B) MEK inhibitor U0126 (U; 0.1 μm), ERK inhibitor FR180204 (F; 10 μm), PI3K inhibitor LY294002 (L; 10 μm), or Akt inhibitor MK-2206 (M; 10 μm) was added to the embryonic neural precursor culture. After 30 min, cells were treated with AT or asarones for 5 min. Blots of the phosphorylated ERK1/2, total ERK, phosphorylated Akt, and Akt were shown. Four independent experiments showed similar results. (C–E) Embryonic neural precursors cultured in reduced growth factor medium were pretreated with MEK inhibitor U0126 (U; 0.1 μm), ERK inhibitor FR180204 (F; 10 μm), PI3K inhibitor LY294002 (L; 10 μm), or Akt inhibitor MK-2206 (M; 10 μm) for 30 min. (C) AT, (D) α-asarone or (E) β-asarone was then added, and the cells were incubated for additional 24 h. EdU incorporation on the last 2 h was visualized with staining and quantified. All the groups in (C–E) were compared with the cell culture treated with (C) AT, (D) α-asarone, or (E) β-asarone alone, respectively. N = 5 independent experiments. (F–G) Mice were treated with vehicle (Ctrl), AT, or asarones for 5 days. DG sections were immunostained for phosphorylated ERK1/2 (F, red) and Sox2 (F, green), and the p-ERK⁺Sox2⁺ cells were quantified as in (G). N = 4–6 per group. Scale bars, 20 μm. (H) Schematic diagram shows that asarones promote NPC proliferation through the activation of ERK cascades. Quantifications are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001; analyzed by one-way ANOVA followed by Fisher’s protected least significant difference test.