Fig. 7
The role of Nrf2 activation in the inhibitory effect of asiaticoside on TGF-β1-induced MMT and ROS production in HPMCs. Notes: (A) After HPMCs treatment with two concentration of ML385 (5, 10 μM) and subsequently incubation for 48 h and 72 h, Nrf2 was analyzed by immunoblotting. Data are shown as the mean ± SEM, *p < 0.05 vs. control, **p < 0.01 vs. Control. (B) HPMCs were divided into a vehicle group, a TGF-β1 group (treated with 10 ng/ml TGF-β1), a TGF-β1 + asiaticoside group (treated with 150 μM asiaticoside + 10 ng/cm3 TGF-β1), a ML385 group (treated with 10 μM ML385 for 72 h), a ML385 + TGF-β1 group (pretreated with ML385 at 10 μM 72 h prior to 10 ng/cm3 TGF-β1), and a ML385 + TGF-β1 + asiaticoside group (pretreated with ML385 at 10 μM 72 h prior to 150 μM asiaticoside + 10 ng/cm3 TGF-β1). After incubation for 24 h, DCFH-DA fluorescence in cultured cells was analyzed by fluorescence microscopy (scale bar = 50 μm). Quantitative measurements of the fluorescence intensities were conducted using ImageJ software. (C) Immunoblot was performed to detect MMT markers, Smad-related proteins and Nrf2 expression. Data are shown as the mean ± SEM, *p < 0.05 vs. control, **p < 0.01 vs. control; #p < 0.05 vs. TGF-β1 treatment; ##p < 0.01 vs. TGF-β1 treatment. Abbreviations: AS, asiaticoside; TGF-β1, transforming growth factor-β1; Nrf2, nuclear factor erythroid-2-related factor 2; p-Smad2/3, phosphorylated Smad2/3; ML385, Nrf2 inhibitor; ROS, reactive oxygen species