Mangiferin reduces the inhibition of chondrogenic differentiation by IL-1β in mesenchymal stem cells from subchondral bone and targets multiple aspects of the Smad and SOX9 pathways

Abstract

Mangiferin is a natural immunomodulator found in plants including mango trees. The effects of mangiferin on chondrogenesis and cartilage repair have not yet been reported. This study was designed to determine the effect of mangiferin on chondrogenic differentiation in IL-1β-stimulated mesenchymal stem cells (MSCs) from subchondral bone and to explore the mechanisms underlying these effects. MSCs were isolated from the subchondral bone of rabbit and treated with mangiferin alone and/or interleukin-1β (IL-1β). Mangiferin induced chondrogenic differentiation in MSCs by upregulating transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, and BMP-4 and several key markers of chondrogenesis, including sex-determining region Y-box (SRY-box) containing gene 9 (SOX9), type 2α1 collagen (Col2α1), cartilage link protein, and aggrecan. In IL-1β-stimulated MSCs, mangiferin significantly reversed the production of TGF-β, BMP-2, BMP-4, SOX9, Col2α1, cartilage link protein, and aggrecan, as well as matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5). Mangiferin upregulated the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and SOX9 in IL-1β-stimulated MSCs. In the presence of mangiferin, SOX9 siRNA suppressed the activation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1 expression. In conclusion, mangiferin exhibits both chondrogenic and chondroprotective effects on damaged MSCs and mediates these effects by targeting multiple aspects of the Smad and SOX9 signaling pathways.

Figure 1

Histological analysis of mesenchymal stem cells from rabbit subchondral bone undergoing chondrogenic, adipogenic, and osteogenic differentiation. (A) control; (B) chondrogenic differentiation visualized by staining with alcian blue; (C) adipogenic differentiation visualized by staining with Oil Red O; and (D) osteogenic differentiation visualized by staining with Alizarin Red.

Figure 2

Effects of mangiferin on chondrogenic differentiation in mesenchymal stem cells (MSCs). (A) Histological analysis by alcian blue staining during chondrogenesis of MSCs pellet culture. Chondrogenic differentiation of MSCs was induced by mangiferin at 10 µM. Cells were then stained with alcian blue. Cartilage nodules were observed following alcian blue staining at 3, 7, and 14 days after culturing under chondrogenic conditions (upper panel) or in the presence of 10 µM mangiferin (lower panel); (B) Line graph showing the staining intensity of alcian blue; (C) The mRNA expression of chondrogenic markers in MSCs. The level of sex-determining region Y–box (SRY-box) containing gene 9 (SOX9), type 2α1 collagen (Col2α1), cartilage link protein, and aggrecan were measured by real time RT-PCR at days 3, 7, and 14, and normalized relative to β-actin; and (D) The level of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, and BMP-4 measured using enzyme-linked immunosorbent assay (ELISA) during chondrogenesis of MSCs. Results are from at least three separate experiments, and each bar represents the mean ± standard error of mean (SEM). ^## p < 0.01 and ^### p < 0.001 compared with 0 day. *** p < 0.001 compared with control.

Figure 3

Effect of mangiferin on the chondrogenic differentiation of IL-1β-stimulated mesenchymal stem cells (MSCs). (A) Histological analysis of mangiferin by alcian blue staining shows recovery of chondrogenic differentiation in IL-1β-stmulated MSCs treated with mangiferin. The bar graph shows the intensity of alcian blue staining; (B,C) Inhibitory effect of mangiferin on sulfated glycosaminoglycan (sGAG) and type II collagen degradation in IL-1β-stimulated MSCs. The release of sGAG (B) and type II collagen (C) were shown as the cumulative release into the culture medium, collected at day 7 and measured by colorimetric analysis. Results were from at least three separate experiments, and each bar represents the mean ± SEM. ^### p < 0.001 compared with control. * p < 0.05 and *** p < 0.001 compared with IL-1β.

Figure 4

Effects of mangiferin on the expression of anabolic and catabolic genes in IL-1β-stimulated mesenchymal stem cells (MSCs). (A) mRNA expression levels of the chondrogenic markers SOX9, Col2α1, cartilage link protein, and aggrecan. MSCs pellet culture was treated with mangiferin (1, 10, and 20 µM) for 7 days. mRNA expression was measured by real time RT-PCR and normalized relative to β-actin; (B) mRNA expression levels of TGF-β, BMP-2, and BMP-4; and (C) mRNA expression levels of MMP-1, MMP-13, and ADAMS5. Data were obtained from three independent experiments and are represented as mean ± SEM. ^### p < 0.001 compared with control. ** p < 0.01, and *** p < 0.001 compared with IL-1β.

Figure 5

Effects of mangiferin on the phosphorylation of the Smad and SOX9 signaling pathways in IL-1β-stimulated mesenchymal stem cells (MSCs). (A) Mangiferin upregulates the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and the SOX9 signaling pathways. MSCs were prestimulated with IL-1β (5 ng/mL) for 1 h and then treated with the indicated dose of mangiferin for 72 h. Whole cell lysates were subjected to Western blotting with the indicated antibodies; (B,C) SOX9 deficiency inhibits the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1. β-actin served as an internal control. MSCs were transfected with control siRNA or SOX9 siRNA and then treated with or without mangiferin for 72 h. Whole cell lysates were subjected to Western blotting with the indicated antibodies. siRNA targeting SOX9 was transfected into two different experiments.

Figure 6

Schematic model of the pathways by which mangiferin stimulates SOX-9, a key transcription factor of TGF-β/BMPs-induced chondrogenesis, and activates the phosphorylation of Smad 1/5/8 and Smad 2/3 signaling pathways. → activation; ┤ inhibition; ↑ increase; ↓ decrease.