Sericin promotes chondrogenic proliferation and differentiation via glycolysis and Smad2/3 TGF-β signaling inductions and alleviates inflammation in three-dimensional models

Abstract

Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-β signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1β, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-β signaling pathways, and exhibiting anti-inflammatory properties.

Keywords: Differentiation; Glycolysis; Proliferation; Sericin; TGF-β signaling.

Figure 1

The ATDC5 cell viability and GAGs production (H-score) expression after incubation with sericin in several concentrations. (A) Assessment of ATDC5 cell viability treated with various concentrations of sericin for 48 h. The GAGs expression of the negative control, Se1, Se25, Se50, positive control, CM1, CM25, and CM50 (B) on day 7, (C) day 14, (D) day 21, and (E) day 28. (F) The line graph of GAGs expression in all periods. (G) The macroscopic image of control groups on days 7, 14, 21, and 28. (H) The alcian blue staining microscopic images of negative control, Se50, positive control, and CM50 groups on day 7, 14, 21, and 28. The abbreviations used in this study were as follows: Se1, Se25, and Se50, which referred to culture medium with 1, 25, and 50 μg/ml of sericin solution, respectively. The CM1, CM25, and CM50 referred to chondrogenic differentiation medium containing 1, 25, and 50 μg/ml of sericin solution, respectively.

Figure 2

The immunolocalized microscopic images and H-score expression graphs of cartilage-specific markers (COL2A1, ALP, COL X, and aggrecan). The immunolocalized images of the ATDC5 pellet in negative control, Se50, positive control, and CM50 (A) on day 7, (B) day 14, (C) day 21, and (D) day 28. (E) Bar graphs showing the H-score expression of four specific cartilage markers (COL2A1, ALP, COL X, and aggrecan) on day 7, 14, 21, and 28. (F) The line graph of the COL2A1, ALP, COL X, and aggrecan markers from day 7 to 28 across all pellet groups.

Figure 3

The immunogold labeling of ultrastructural cytoskeleton proteins (F-actin and β-tubulin) and COL2A1 in ATDC5 pellets. The transmission electron microscopic images of pellets from the negative control, Se50, positive control, and CM50 on day 28 were stained with (A–D) F-actin, (F–I) β-tubulin, and (K–N) COL2A1. The bar graphs illustrated the expression of each marker as a labeling/field: (E) F-actin, (J) β-tubulin, and (O) COL2A1.

Figure 4

The cartilage specific-gene expression (SOX-9, COL2A1, ALP, aggrecan, and PCNA) and a proteomic analysis in pellets on day 28. (A) The relative gene expression of SOX-9, COL2A1, ALP, aggrecan, and PCNA in pellets from the negative control, Se50, positive control, and CM50 on day 28. (B–C) The proteomic results were illustrated as follows: (B) A pie chart classifying 106 upregulated proteins into five groups associated with chondrogenesis: 1. proliferation of chondrocytes (19%), 2. differentiation of chondrocytes (15%), 3. extracellular matrix proteins (13%), 4. cytoskeletal proteins (8%), and 5. others (45%) and (C) A diagram of protein–protein interactions within the 4 categorized groups. The interactions of these proteins are associated with various biological processes: collagen biosynthesis and modifying enzymes (purple; MMU-1650814), glycolysis (red; MMU-70171), ECM proteoglycans (green; MMU-3000178), and extracellular matrix organization (yellow; MMU-1474244).

Figure 5

The schematic of glycolysis, exploration of chondrogenic differentiation via gene expression of TGF-β and BMP signaling, and illustration of TGF-β and BMP signaling pathways. (A) The presence of glycolytic enzymes (PGAM1 and TPI) in the glycolysis pathway, slightly adapted from the Reactome database (MMU-70171). (B) Relative gene expression of Smad2 and Smad3 (TGF-β signaling), and RUNX2, Smad1, BMP2 and BMP4 (BMP signaling). (C) Illustrations of Smad2 and Smad3 (green boxes) in TGF-β signaling pathway (MMU-2173789.1) and the Smad1, BMP2 and BMP4 (yellow and orange boxes) in BMP signaling pathway (MMU-201451.1), adapted from the Reactome database.

Figure 6

The scanning electron microscopic images of the gelatin scaffold structure and culture. (A and B) The structure of porous gelatin scaffolds without cells, (C and D) The scaffold culture with ATDC5 chondrogenic cells on day 7, and (E and F) day 21.

Figure 7

Immunolocalized microscopic images with H-score expression graphs and gene expression analysis of early inflammation markers in scaffold cultures under normal condition, including positive control (inflammation without treatment), Dex treatment, GS treatment, and Se1, Se25, and Se50 treatments. (A) The immunolocalized microscopic images of different treatments with early inflammation-related markers (IL1-β, TNF-α, MMP-1, and COL2A1). (B) Bar graphs of the H-score expression of IL1-β, TNF-α, MMP-1, and COL2A1 for each treatment group. (C) Relative gene expression of IL-1β, TNF-α, MMP-13, and COL2A1 in each treatment group. The abbreviations used in this study were as follows: Pos control was the positive control, Dex treatment referred to dexamethasone treatment, GS treatment indicated glucosamine sulfate treatment, and Se1, Se25, and Se50 referred to sericin treatment at 1, 25, and 50 μg/ml concentrations, respectively.