Fig. 3
Effect of asiaticoside on TGF-β1-induced activation of Smad2/3 and MMT in HPMCs. Notes: (A) After treatment with TGF-β1 (10 ng/cm3) or/and asiaticoside (150 μM), the morphologic alterations of HPMCs were observed using a microscope (scale bar = 50 μm). (B) HPMCs were treated with TGF-β1 (10 ng/cm3) for various times (0, 6, 12, 24 h) and subjected to immunoblot for Smad-related proteins. (C) HPMCs were treated with asiaticoside at various concentrations (0, 50, 100, 150 μM) with or without TGF-β1 (10 ng/cm3) treatment for 24 h. The expression of Smad2/3 and p-Smad2/3 was detected by immunoblotting. (D) Immunoblot showed relative levels of Smad2/3 phosphorylation at 1 h and 24 h post-treatment with LY2109761. (E) HPMCs were divided into a vehicle group, an asiaticoside group (treated with 150 μM asiaticoside), a TGF-β1 group (treated with 10 ng/ cm3 TGF-β1), a TGF-β1 + asiaticoside group (treated with 150 μM asiaticoside + 10 ng/cm3 TGF-β1) and a LY2109761 + TGF-β1 group (pretreated with LY2109761 at 4 μM 1 h prior to 10 ng/cm3 TGF-β1). After incubation for 24 h, immunoblot was performed to detect relative proteins. β-actin was used as a loading control. The densitometric analysis of the expression of MMT markers, Smad2/3 and p-Smad2/3 are shown as unique figures. Data are expressed as 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; LY, TGF-β receptor kinase inhibitor (LY2109761); p-Smad2/3, phosphorylated Smad2/3