Investigation of the molecular mechanism of quercetin in inhibiting ankylosing spondylitis ossification via the bone morphogenetic protein/smad signaling pathway.

Abstract

Ankylosing spondylitis (AS) is a chronic inflammatory disease involving the spine and bone joints, which is characterized by hyperosteogeny, ossification of ligaments, and ankylosis. Quercetin is a natural polyphenolic compound with various biological activities such as antioxidant, anti-inflammatory, and anti-tumor. It was to explore the effect of quercetin on AS ossification and its molecular mechanism. In vitro culture of AS mesenchymal stem cells was conducted. Cells were treated with 0, 10, 30, 60, and 80 μM quercetin, divided into control, 10 μM, 30 μM, 60 μM, and 80 μM groups. Alkaline phosphatase (ALP) staining, Alizarin Red staining, real-time quantitative polymerase chain reaction (qRT-PCR), and Western blot (WB) were employed to investigate the effect of quercetin on the expression of osteogenic-related genes and proteins. Additionally, bone morphogenetic protein (BMP) and Smad genes were knocked out to explore quercetin's regulation of BMP/Smad. In vivo experiments were conducted using 50 mice, including 10 in the normal group. An AS model was established in 36 mice, divided into negative control (n = 18, 0.9% saline) and quercetin groups (n = 18, quercetin). Safranin O-fast green (HE) staining and MicroCT scanning were performed before and 4 weeks after injection. In the 60 μM and 80 μM quercetin groups, ALP activity, Ca²⁺ deposition area, and relative protein/mRNA levels of BMP-1, BMP-2, Smad1, Smad4, and Smad5 in AS mesenchymal stem cells were significantly lower compared to the control, 10 μM, and 30 μM groups (P < 0.05). The 80 μM group exhibited lower levels than the 60 μM group (P < 0.05). In the siRNA + 80 μM group, the reduction in mRNA expression of BMP1, BMP2, Smad1, Smad4, and Smad5 was significantly greater compared to the siRNA group and the 80 μM group (P < 0.05). At 4 weeks post-injection, mice in the quercetin group showed significantly reduced severity of articular cartilage lesions, lymphocyte infiltration, and tissue edema, with no significant increase in sacroiliac joint fusion. Quercetin downregulates the expression of BMP and Smad-related proteins, inhibiting osteogenic differentiation of AS mesenchymal stem cells and effectively reducing ALP activity and Ca²⁺ deposition levels. These findings suggest that quercetin holds potential application value in the control and treatment of AS disease.