Ursolic Acid: A Novel PTP-oc Inhibitor that Suppresses Osteoclastogenesis and Root Resorption

Abstract

Background: Root resorption, an unwanted side effect often seen in orthodontics, depends largely on osteoclasts. Osteoclastic protein tyrosine phosphatase (PTP-oc) plays a major role in controlling osteoclast activity. This study aimed to investigate the effects of Ursolic acid (UA), a novel PTP-oc inhibitor, on osteoclastogenesis and root resorption. Methods: UA was pinpointed using a PTP inhibition assay. Its inhibitory characteristics, inhibitor constant (Ki) against ∆PTP-oc, and selectivity were through an Inhibition Kinetics assay. The effects of UA on osteoclastogenesis were examined by treating human U937 histiocytic lymphoma cells with or without UA and then assessing osteoclastogenesis mediated by phorbol 12-myristate 13-acetate /1α, 25 di-hydroxy vitamin D₃ (1,25(OH)₂D₃). The cell counting kit-8 (CCK-8) evaluated how a UA affects U937 cell proliferation in vitro. Osteoclast-like cell development was examined using tartrate-resistant acid phosphatase (TRAP) Staining, while Real-time-polymerase chain reaction (qPCR) was used to assess the expression of tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor-κB (RANK), matrix metalloproteinase-9 (MMP-9), cathepsin K (CK), and calcitonin receptor (CTR). Tyrosine kinase c-Src (c-Src) protein was measured using Western Blot. A rat model was used to study the levels of root resorption and tyrosine phosphorylation of c-Src (PY-527) by histology and immunohistochemistry. Results: UA displayed a strong and reversible inhibitory effect on PTP-oc in a competitive manner, showing selective in vitro (p < 0.001). The compound did not impact the viability of U937 cells at concentrations between 1–5 μM UA. UAs inhibition of osteoclastogenesis depends on its concentration, and the compound effectively suppresses the expression of osteoclast marker genes, TRAP, RANK, MMP-9, CK, and CTR. Additionally, the area of TRAP-positive cells was reduced. This effect is facilitated by inhibiting enzymatically active PTP-oc, which elevates the levels of c-Src tyrosine phosphorylation, thereby decreasing the activity of c-Src Protein Tyrosine Kinases (PTK). In animal studies, as UA concentration increased, there was a noticeable delay and weakening in root resorption. Finally, IHC staining results revealed that compared to the 0 μM UA (control) group, c-Src PY-527 in the model groups increased alongside the rise in UA concentration (p < 0.05). Conclusions: This study demonstrates that UA application effectively suppresses osteoclastogenesis and root resorption by inhibiting the enzymatic activity of PTP-oc. This effect is dose-dependent and may be mediated by the regulation of c-Src signaling pathway.