Asiaticoside inhibits TGF-β1-induced mesothelial-mesenchymal transition and oxidative stress via the Nrf2/HO-1 signaling pathway in the human peritoneal mesothelial cell line HMrSV5

Figures

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  Fig. 1. Chemical structure of asiaticoside. (Abbreviated AS in the figures)

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  Fig. 2. Effect of asiaticoside on HPMCs viability. Notes: Cell viability was evaluated using CCK-8 assay. Data were expressed as the percentages of living cells versus the control. (A) HPMCs were treated with various dose of asiaticoside (0, 50, 100, 150 and 200 μM) for 24 h or (B) with 150 μM of asiaticoside for various times (0, 12, 24, 36 and 48 h). Abbreviations: HPMCs, human peritoneal mesothelial cells; CCK-8, cell counting kit-8

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  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…

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  Fig. 4. Effect of asiaticoside on TGF-β1-induced migration ability of HPMCs. Notes: HPMCs were exposed to asiaticoside (150 μM) with or without TGF-β1 (10 ng/cm3) and subjected to microscope to visualize the lateral (A) and longitudinal (B) migration of cells. Quantitative measurements of the fluorescence intensities were conducted using ImageJ software. Five random fields for each insert were counted, and three independent experiments were performed in each group. n = 3. Abbreviations: AS, asiaticoside; TGF-β1, transforming growth factor-β1

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  Fig. 5. Effect of asiaticoside on reactive oxygen species (ROS) production in TGF-β1-treated HPMCs. Notes: 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), an NAC group (treated with 5 mM NAC), an NAC + TGF-β1 group (pretreated with NAC at 5 mM 2 h prior to 10 ng/cm3 TGF-β1), a LY2109761 group (treated with 4 μM LY2109761), 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, 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. Abbreviations: AS, asiaticoside; TGF-β1, transforming growth factor-β1; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; NAC, n-acetylcysteine; LY2109761, TGF-β receptor kinase inhibitor

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  Fig. 6. Effect of asiaticoside on Nrf2 activation in HPMCs. Notes: (A) After asiaticoside (150 μM) treatment for various times (0, 1, 3, 6, 12, 24 h), the expression of Nrf2 in the nucleus and HO-1 expression were detected by immunoblotting. Histone H3 and β-actin were used as the loading controls for the nucleus and cytosol, respectively. (B) After treatment with various doses of asiaticoside (0, 50, 100, 150 μM) for 6 h, Nrf2 and HO-1 expression were detected by immunoblotting. (C) After treated with asiaticoside at various concentrations (0, 50, 100, 150 μM) and TGF-β1 (10 ng/cm3) treatment for 6 h, Nrf2 and HO-1 expression were detected by immunoblotting. β-actin was used as a loading control. Densitometric analysis of the expression of Nrf2 and HO-1 are shown as unique figures. Data are expressed 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. (D) Immunofluorescence assays were performed to observe the subcellular translocation of Nrf2. Nrf2 expression in the membrane, cytoplasm and nucleus were visualized based on the green fluorescent signal obtained by fluorescence microscopy (scale bar = 50 μm).…

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  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.…

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  Fig. 8. Schematic diagram illustrating the mechanism by which asiaticoside inhibited MMT and ROS generation