Phytomedicine : international journal of phytotherapy and phytopharmacology · 2026
Roburic acid inhibits glucocorticoid-induced osteonecrosis of the femoral head associated osteoclast activity via targeting PTP1B.
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Wang Gang, Mo Liang, Wang Chao, Zhang Yuting, Li Yaning, Yuan Jinbo, Jin Haiming, Chen Peng, Chen Di, Zhou Jianpeng, He Wei, Xu Jiake
Аннотация
BACKGROUND: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) has unclear pathogenesis and lacks effective therapies. Protein tyrosine phosphatase 1B (PTP1B) regulates osteoclastogenesis, but its role and translational value in GIONFH remain unknown. Roburic acid (RA), a triterpenoid from Gentiana macrophylla Pall., exhibits anti-inflammatory activity, yet whether it protects against GIONFH by targeting PTP1B to inhibit osteoclast hyperactivity is elusive.
METHODS: For clinical experiments, femoral heads from GIONFH patients were subjected to H&E staining, CTSK immunohistochemistry (IHC), and CTSK/PTP1B co-immunofluorescence in order to analyze PTP1B protein level and its colocalization with osteoclasts. For animal experiments, mouse bone marrow-derived macrophages (BMMs) were induced with dexamethasone (DEX) and RANKL to mimic GIONFH-related osteoclast hyperactivity, and RA was then administered. Target validation was performed via molecular docking, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA). For animal experiments, a methylprednisolone (MPS)-induced GIONFH rat model was used to investigate RA's effect on PTP1B protein expression and osteoclast activity.
RESULTS: In femoral head samples from GIONFH patients, IHC analysis revealed marked upregulation of PTP1B in necrotic regions, while co-immunofluorescence staining confirmed colocalization of PTP1B with the osteoclast-specific marker CTSK, with synchronized elevation of both proteins. In vitro, RA dose-dependently inhibited osteoclast formation induced by co-stimulation with DEX and RANKL. Target prediction and multidimensional validation (molecular docking, SPR, CETSA) identified PTP1B as a direct RA binding target, with high binding affinity verified by SPR. RA suppressed PTP1B protein level in both DEX-treated osteoclast precursors and mature osteoclasts exposed to DEX. In the rat GIONFH model, Micro-CT assay revealed that RA dose-dependently alleviated GIONFH-related bone erosion, increased BV/TV and Tb.N, and decreased Tb.Sp. H&E staining indicated that RA undermined necrotic lacunae formation and mitigated bone loss, while TRAP staining and CTSK IHC staining illustrated that RA decreased osteoclast activity in GIONFH. Western blotting revealed reduced expression of PTP1B, RANK, NFATc1, MMP9, and CTSK after RA intervention. IHC analysis of RhoA, NF-κB, and NFATc1 in GIONFH demonstrated that RA inhibited expression of these key proteins downstream of PTP1B.
CONCLUSION: RA protects against GIONFH by targeting PTP1B to inhibit osteoclast overactivation, with a correlative suppression of the RhoA/NF-κB/NFATc1 pathway. RA represents a potential preclinical candidate for further investigation in GIONFH.
METHODS: For clinical experiments, femoral heads from GIONFH patients were subjected to H&E staining, CTSK immunohistochemistry (IHC), and CTSK/PTP1B co-immunofluorescence in order to analyze PTP1B protein level and its colocalization with osteoclasts. For animal experiments, mouse bone marrow-derived macrophages (BMMs) were induced with dexamethasone (DEX) and RANKL to mimic GIONFH-related osteoclast hyperactivity, and RA was then administered. Target validation was performed via molecular docking, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA). For animal experiments, a methylprednisolone (MPS)-induced GIONFH rat model was used to investigate RA's effect on PTP1B protein expression and osteoclast activity.
RESULTS: In femoral head samples from GIONFH patients, IHC analysis revealed marked upregulation of PTP1B in necrotic regions, while co-immunofluorescence staining confirmed colocalization of PTP1B with the osteoclast-specific marker CTSK, with synchronized elevation of both proteins. In vitro, RA dose-dependently inhibited osteoclast formation induced by co-stimulation with DEX and RANKL. Target prediction and multidimensional validation (molecular docking, SPR, CETSA) identified PTP1B as a direct RA binding target, with high binding affinity verified by SPR. RA suppressed PTP1B protein level in both DEX-treated osteoclast precursors and mature osteoclasts exposed to DEX. In the rat GIONFH model, Micro-CT assay revealed that RA dose-dependently alleviated GIONFH-related bone erosion, increased BV/TV and Tb.N, and decreased Tb.Sp. H&E staining indicated that RA undermined necrotic lacunae formation and mitigated bone loss, while TRAP staining and CTSK IHC staining illustrated that RA decreased osteoclast activity in GIONFH. Western blotting revealed reduced expression of PTP1B, RANK, NFATc1, MMP9, and CTSK after RA intervention. IHC analysis of RhoA, NF-κB, and NFATc1 in GIONFH demonstrated that RA inhibited expression of these key proteins downstream of PTP1B.
CONCLUSION: RA protects against GIONFH by targeting PTP1B to inhibit osteoclast overactivation, with a correlative suppression of the RhoA/NF-κB/NFATc1 pathway. RA represents a potential preclinical candidate for further investigation in GIONFH.