Triptolide inhibits the function of TNF-α in osteoblast differentiation by inhibiting the NF-κB signaling pathway

Abstract

Chronic inflammation often delays fracture healing or leads to bone nonunion. Effectively suppressing pathological inflammation is crucial for fracture healing or bone remodeling. Triptolide, which is a diterpenoid epoxide, is the major active component of the Thunder God Vine, Tripterygium wilfordii. The aim of the present study was to investigate the role of triptolide in osteoblast differentiation and explore the molecular mechanisms of triptolide in fracture healing. Alkaline phosphatase (ALP) activity was used to evaluate osteoblast differentiation. ALP activity was measured via histochemical staining and western blotting was used to determine the expression of factors associated with inflammation. C2C12 cells were initially treated with 200 ng/ml bone morphogenetic protein (BMP)-2 alone for 3 days, which caused a significant increase in ALP activity (P<0.01). However, treatment with tumor necrosis factor (TNF)-α significantly decreased the ALP activity (P<0.05). Notably, treatment with the chronic inflammatory cytokine TNF-α significantly decreased the effect of BMP-2 in C2C12 cells compared with BMP-2 treatment alone (P<0.01). C2C12 cells were treated with increasing concentrations of BMP-2 or TNF-α for 3 days. The results demonstrated that TNF-α treatment significantly inhibited BMP-2-induced osteoblast differentiation in a dose-dependent manner (P<0.01). The role of triptolide in BMP-2-induced osteoblast differentiation was also examined. Cells were treated with BMP-2, BMP-2 + TNF-α alone, or BMP2 + TNF-α with increasing concentrations of triptolide (4, 8 or 16 ng/ml). After 3 days, the results of ALP activity revealed that triptolide significantly reversed the TNF-α-associated inhibition of osteoblast differentiation (P<0.01). Western blotting analysis demonstrated that triptolide markedly inhibited the phosphorylation of nuclear factor-κB, therefore suppressing the effects of TNF-α. In summary, triptolide is able to reverse the TNF-α-associated suppression of osteoblast differentiation, suggesting that triptolide treatment may have a positive effect on bone remodeling and fracture repairing.