Dual protective role of velutin against articular cartilage degeneration and subchondral bone loss via the p38 signaling pathway in murine osteoarthritis

Abstract

Osteoarthritis (OA) is a common degenerative joint condition associated with inflammation and characterized by progressive degradation of the articular cartilage and subchondral bone loss in the early stages. Inflammation is closely associated with these two major pathophysiological changes in OA. Velutin, a flavonoid family member, reportedly exerts anti-inflammatory effects. However, the therapeutic effects of velutin in OA have not yet been characterized. In this study, we explore the effects of velutin in an OA mouse model. Histological staining and micro-CT revealed that velutin had a protective effect against cartilage degradation and subchondral bone loss in an OA mouse model generated by surgical destabilization of the medial meniscus (DMM). Additionally, velutin rescued IL-1β-induced inflammation in chondrocytes and inhibited RANKL-induced osteoclast formation and bone resorption in vitro. Mechanistically, the p38 signaling pathway was found to be implicated in the inhibitory effects of velutin. Our study reveals the dual protective effects of velutin against cartilage degradation and subchondral bone loss by inhibiting the p38 signaling pathway, thereby highlighting velutin as an alternative treatment for OA.

Keywords: chondrocyte; osteoarthritis; p38 pathway; subchondral bone; velutin.

Figure 1

Velutin reduces osteoarthritis (OA) progression in a DMM-induced OA mouse model in vivo. (A) Hematoxylin and eosin staining in different experimental groups (control, DMM, and DMM+velutin groups). The blue rectangle represents the local magnification of the articular cartilage and the red rectangle represents the local magnification of the subchondral bone. (B–F) Statistics of each parameter of articular cartilage and subchondral bone. (G–J) Immunohistochemical (IHC) staining of aggrecan (G) and type II Collagen (H) in different experimental groups (control, DMM, and DMM+velutin groups). Scale bar: 100 μm; n = 6 per group. Data are presented as mean ± SD; ^# P < 0.01 vs. control group; *P < 0.05, **P < 0.01.

Figure 2

Velutin treatment reduces articular cartilage degeneration and subchondral bone deterioration in mice with DMM-induced OA. (A) Representative Safranin O staining of articular cartilage in distinct experimental groups; scale bar: 100 μm. (B) Osteoarthritis Research Society International (OARSI) scores of the cartilage. (C, D) Representative photos of fast green-stained subchondral bones and TRAP-stained osteoclasts in subchondral bone of the knee joint; the yellow triangle represents osteoclasts; scale bar: 100 μm. (E-I) Three-dimensional micro-CT images of the medial compartment of the tibial subchondral bone in sagittal views (control, DMM, and DMM+velutin groups); scale bar: 100 μm. Three-dimensional structural characteristics of the tibial subchondral bone histogram: (BV/TV) trabecular bone volume/tissue volume, (Tb.N) trabecular number, (Tb. Th) trabecular thickness, (Tb.Sp) trabecular separation, and trabecular bone volume/tissue volume, (Tb. Th) trabecular thickness. Data are presented as mean ± SD; ^# P < 0.01, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Velutin protects against IL-1β–induced chondrocyte inflammation and extracellular matrix degradation in vitro. (A, B) CCK-8 experiments revealing the cytotoxic effect of velutin on chondrocytes during 24 and 48 h at various doses (n = 5). (C) Safranin O staining in high-cell density culture of primary mouse chondrocytes. (D) Safranin O staining in human cartilage tissues. (E, F) qPCR of the mRNA expression levels of TNF-α and IL-6. (G–I) Velutin inhibited IL-1β-induced protein and mRNA expression of COX-2 in chondrocytes in a dose-dependent manner, according to Western blot and qPCR analysis. (J–O) Western blot and qPCR analyses showing that IL-1β treatment induced collagen II degradation and increased the expression of collagen X, MMP3, and ADAMTS5, whereas velutin treatment rescued these effects. Expression of target genes was normalized to β-actin and expressed as fold change in comparison to the controls group (n = 3). Data are presented as mean ± SD; ^# P < 0.01 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.01 vs. IL-1β alone (n = 3).

Figure 4

Velutin inhibits nuclear factor-κB receptor activator ligand (RANKL)-induced osteoclast formation and bone resorption in vitro. (A) Bone marrow-derived macrophages (BMMs) were grown for 6 days with M-CSF and RANKL, as well as varied velutin concentrations, before being stained for TRAP detection. Osteoclasts were defined as cells with three nuclei or less. (n = 3); scale bar: 200 μm. (B) CCK-8 assays showing the cytotoxicity of velutin in BMMs (n = 5). (C-D) Analysis of number and size (area) of TRAP‐positive multinucleated (nuclei > 3) cells (n = 3). (E) Schematic diagram of the timeline of BMM cells treated with velutin. (F) BMMs were cultivated in complete medium, then treated with velutin (4 μM) and stained for TRAP on the days indicated; scale bar: 200 μm. (G-H) Analysis of number and size (area) of TRAP‐positive multinucleated (nuclei > 3) cells (n = 3). (I-J) Bone tissue samples were used to cultivate M-CSF-dependent BMMs, then stimulated with RANKL and the specified velutin doses. SEM examination observed bone resorption lacunae after 15 days, which were calculated as percentages using Image J program (n = 3). (K–P) qPCR result of RANKL-induced osteoclast-related genes’ relative expression levels. Expression of target genes was adjusted to β-actin and expressed as fold change compared to the controls group (n = 3). (Q–S) M-CSF-dependent BMMs were serum-starved and pretreated for 2 h with velutin (4 μM) or vehicle control, before being stimulated with RANKL for the durations indicated times (0, 5, 15, 30, and 60 min). Western blot analysis was used to extract total cellular protein for protein expression levels (n = 3). Data are presented as mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001, in comparison to cells treated only with RANKL.

Figure 5

Velutin mediates its inhibitory effects through the p38 signaling pathway blockage. (A, B) Immunofluorescence DAPI nuclear staining showing that IL-1β promoted the expression of p-p38, velutin treatment reversed this effect. (C, D) Immunohistochemical staining of p-p38 MAPK in cartilage and percentages of p-p38 MAPK positive cells; the yellow triangle indicates positive cells. (E, F) Chondrocytes were pretreated with velutin (4 μM) for 2 h, then stimulated with or without IL-1β for 15 min. IL-1β promoted p38 phosphorylation, while velutin reduced it in a dose-dependent manner. The expression of p-p38 MAPK relative to total p38 MAPK was determined by ImageJ software. (G, H) M-CSF-dependent bone marrow-derived macrophages (BMMs) were serum-starved and given velutin (4 M) or a vehicle control for 2 h before being activated with nuclear factor-κB receptor activator ligand (RANKL) for the indicated times (0, 5, 15, 30, and 60 min). RANKL promoted p38 phosphorylation, whereas velutin reduced in a time-dependent way. ImageJ software was used to calculate the expression of p-p38 in comparison to total p38. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001, n = 3.