Figure 5.

Madecassic acid regulated ACC1-mediated shift of T helper type 17 (Th17) toward regulatory T cells via the activation of AMPK. Naive T cells were cultured with or without compound C and AMPK siRNA under Th17-inducing conditions in the presence of madecassic acid for 4 days. (a) Naive T cells were treated with madecassic acid (1, 3, 10 μM) under Th17-inducing conditions for 4 days. The protein expression of p-AMPK was analyzed by western blot. (b) Naive T cells were cultured with madecassic acid (3 μM), oleic acid (50 μM) and ACC1 siRNA under Th17-polarizing conditions for 4 days. The protein expression of p-AMPK was measured by western blot. (c and d) Naive T cells were cultured with madecassic acid (3 μM), compound C (1 μM) and AMPK siRNA under Th17-polarizing conditions for 4 days. The proportions of CD4⁺IL-17⁺ T cells and CD4⁺Foxp3⁺ T cells were evaluated by flow cytometry. (e) Naive T cells were cultured with madecassic acid (3 μM) and compound C (1 μM) under Th17-polarizing conditions for 4 days. The relative expression of p-AMPK, AMPK, ACC1 and Foxp3 was analyzed by western blot. (f) Naive T cells were cultured with madecassic acid (3 μM) and AMPK siRNA under Th17-polarizing conditions for 4 days. The relative expression of ACC1 and Foxp3 was analyzed by western blot. (g) Naive T cells were cultured with madecassic acid (3 μM) and compound C (1 μM) under Th17-polarizing conditions for 4 days. The relative expression of ACC1 and Foxp3 was analyzed by immunofluorescence. (h) Naive T cells were cultured with madecassic acid (3 μM) and AMPK siRNA under Th17-polarizing conditions for 4 days. The relative expression of ACC1 and Foxp3 was analyzed by immunofluorescence. GAPDH was used as a cytoplasm marker. The data were expressed as the means±S.E.M., n=3. ^#P<0.05, ^##P<0.01 versus Th0 group; *P<0.05, **P<0.01 versus Th17 group; ^$P<0.05, ^$$P<0.01 versus madecassic acid group