N-methyl pyrrolidone (NMP) inhibits lipopolysaccharide-induced inflammation by suppressing NF-κB signaling

Abstract

OBJECTIVE: N-methyl pyrrolidone (NMP), a small bioactive molecule, stimulates bone formation and inhibits osteoclast differentiation and bone resorption. The present study was aimed to evaluate the anti-inflammatory potentials of NMP on the inflammatory process and the underlying molecular mechanisms in RAW264.7 macrophages. MATERIALS AND METHODS: RAW264.7 macrophages and mouse primary bone marrow macrophages (mBMMs) were used as an in vitro model to investigate inflammatory processes. Cells were pre-treated with or without NMP and then stimulated with lipopolysaccharides (LPS). The productions of cytokines and NO were determined by proteome profiler method and nitrite analysis, respectively. The expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were measured by Western blotting and/or qPCR. Western blot, ELISA-base reporter assay, and immunofluorescence were used to evaluate the activation of MAP kinases and NF-B. RESULTS: LPS-induced mRNA expressions of TNF-, IL-1, IL-6, iNOS, and COX-2 were inhibited by NMP in a dose-dependent manner. NMP also suppressed the LPS-increased productions of iNOS and NO. The proteome profiler array showed that several cytokines and chemokines involved in inflammation and up-regulated by LPS stimulation were significantly down-regulated by NMP. Additionally, this study shows that the effect of NMP is mediated through down-regulation of NFB pathway. CONCLUSIONS: Our results show that NMP inhibits the inflammatory mediators in macrophages by an NFB-dependent mechanism, based on the epigenetical activity of NMP as bromodomain inhibitor. In the light of its action on osteoblast and osteoclast differentiation process and its anti-inflammatory potential, NMP might be used in inflammation-related bone loss. CONCLUSIONS:Our results show that NMP inhibits the inflammatory mediators in macrophages by an NFκB-dependent mechanism, based on the epigenetical activity of NMP as bromodomain inhibitor. In the light of its action on osteoblast and osteoclast differentiation process and its anti-inflammatory potential, NMP might be used in inflammation-related bone loss.

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

“…C0036; Beyotime Institute of Biotechnology, Haimen, China), a colorimetric assay for nonradioactive quantification of cellular proliferation, viability and cytotoxicity, according to the manufacturer's protocol. Briefly, MG-63 cells were cultured in 96-well plates for 24 h and then incubated with different concentrations of NMP (0, 5 or 10 mM) (17) for 48 h at 37˚C. Following stimulation, WST-1 (1/10 of total volume) was added to each well and incubated for a further 2 h at 37˚C in the dark.…”

“…It has been demonstrated that NMP inhibits inflammation by repressing the NF-kB pathway (17). This indicates that NMP may be used as an adjuvant therapy alongside established methods of bone fracture treatment.…”

N-methyl pyrrolidone (NMP) ameliorates the hypoxia-reduced osteoblast differentiation via inhibiting the NF-κB signaling

“…Our study confirmed the upregulated NF-κB level and the increased transcriptional activity of NF-κB, and confirmed the downregulated IκB expression in the hypoxia-treated hFOB 1.19 cells. The downregulation by NMP on the NF-κB pathway has been rec- ognized in macrophages (Ghayor et al, 2015) and in osteoclasts . And the NF-κB pathway is indicated to regulate the expression of multiple genes involved in inflammatory and immune response (McKay and Cidlowski, 1999;Caamaño and Hunter, 2002).…”

“…Therefore, NMP might be useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption. In addition, it exerts an anti-inflammatory potential on the lipopolysaccharide (LPS)-induced inflammatory process (Ghayor et al, 2015). The LPS-promoted levels of TNF-α, IL-1β, IL-6, iNOS and COX-2 were inhibited by NMP in a dosedependent manner.…”

“…The LPS-promoted levels of TNF-α, IL-1β, IL-6, iNOS and COX-2 were inhibited by NMP in a dosedependent manner. And the effect of NMP is mediated through the downregulation of nuclear factor κB (NF-κB) pathway (Ghayor et al, 2015).…”