N-methyl pyrrolidone promotes ankle fracture healing by inhibiting inflammation via suppression of the mitogen-activated protein kinase signaling pathway.

Abstract

N-methyl pyrrolidone (NMP), a small bioactive molecule, has the potential to stimulate bone formation and inhibit osteoclast differentiation. The aim of the present study was to investigate the effect of NMP on the inflammatory response and underlying molecular mechanisms in MG-63 cells. The mRNA and protein expression of cytokines from peripheral blood in children with or without ankle fracture were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA, respectively. MG-63 cells were pre-treated with/without NMP and stimulated with 1 µM bradykinin (BK). The production of cytokines from MG-63 cells was assessed by western blotting and RT-qPCR. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein were measured using western blotting and/or RT-qPCR. Western blotting was used to examine the activation level of mitogen activated protein kinase. Compared with healthy children, levels of tumor necrosis factor (TNF-α), interleukin (IL)-1β and IL-6 mRNA and protein were upregulated in children with ankle fracture. NMP treatment did not induce cytotoxicity in MG-63 cells. The BK-induced upregulation of TNF-α, IL-1β, IL-6, iNOS and COX-2 mRNA and protein was reversed in a dose-dependent manner by NMP. Furthermore, NMP downregulated the activation of c-Jun NH2-terminal kinase and p38 pathways, but not the extracellular signal-related kinase pathway. Therefore, the results of the current study demonstrate that NMP inhibits inflammation dependent on the mitogen-activated protein kinase pathway in MG-63 cells, indicating that it may be beneficial in the healing of fractures.