Depletion of PTGS1 Inhibits IL-1β-Induced Inflammatory Responses and Osteoblast Dysfunction in MC3T3-E1 Cells via the Wnt/β-Catenin Pathway
TL;DR
In vitro findings suggest that PTGS1 may modulate osteoblastic responses under inflammatory conditions; however, further validation is required before any conclusion regarding TMJ-OA can be drawn.
Abstract
: Objectives: Temporomandibular joint osteoarthritis (TMJ-OA) is a chronic disorder of the mandibular joint characterized by severe symptoms and extensive damage. This study aims to investigate the role of Prostaglandin-Endoperoxide Synthase 1 (PTGS1) in a mouse calvaria-derived pre-osteoblast cell line (MC3T3-E1 cells) under Interleukin-1 beta (IL-1 β )-induced inflammatory conditions, using TMJ-OA as the clinical context for this in vitro mechanistic investigation. Methods: The association between the IL-1 β -mediated inflammatory environment and PTGS1 expression was assessed in vitro . PTGS1 expression in IL-1 β -stimulated MC3T3-E1 cells was determined by Western blot and quantitative real-time polymerase chain reaction (qPCR). Interleukin-6 (IL-6) levels in cell supernatants were quantified by Enzyme-linked immunosorbent assay (ELISA), and cell behaviors were analyzed by ELISA and flow cytometry. The regulatory role of PTGS1 in the IL-1 β pathway was further examined through Western blot. Results: Both PTGS1 mRNA and protein levels were significantly upregulated in IL-1 β -treated MC3T3-E1 cells compared to the control group ( p < 0.01). Knockdown of PTGS1 markedly reduced IL-1 β -induced cell apoptosis, decreased the secretion of IL-6 and tumor necrosis factor-alpha (TNF-α ), and inhibited cell migration ( p < 0.01). Moreover, PTGS1 depletion reversed IL-1 β -mediated changes in bone metabolism-related molecules: it restored the IL-1 β -induced downregulation of osteoprotegerin (OPG) and suppressed the IL-1 β -induced upregulation of receptor activator of nuclear factor kappa-B ligand (RANKL) ( p < 0.01). Mechanistically, PTGS1 silencing inhibited the activation of the wingless-related integration site/catenin beta 1 (Wnt/ β -catenin) pathway, as indicated by reduced expression of β -catenin, c-Myc, and Cyclin D1 ( p < 0.01). Treatment with SKL2001 counteracted the effects of PTGS1 depletion, restoring IL-1 β -induced apoptosis, inflammation, migration, and the abnormal expression of OPG/RANKL and Wnt/ β -catenin pathway components ( p < 0.05). Conclusion: PTGS1 knockdown attenuated IL-1 β -induced apoptosis, inflammation, and migration in MC3T3-E1 cells, effects that were associated with suppression of the Wnt/ β -catenin pathway. These in vitro findings suggest that PTGS1 may modulate osteoblastic responses under inflammatory conditions; however, further validation is required before any conclusion regarding TMJ-OA can be drawn