Journal: Stem Cell Research & Therapy
Article Title: Low concentration flufenamic acid enhances osteogenic differentiation of mesenchymal stem cells and suppresses bone loss by inhibition of the NF-κB signaling pathway
doi: 10.1186/s13287-019-1321-y
Figure Lengend Snippet: FFA in low concentrations promoted the osteogenic differentiation of hMSCs by inhibiting the NF-κB pathway. a FFA at 50 μM downregulated the expression of TNF , IL6 , IL8 , and ICAM1 both in PM and OM in hBMMSCs, as determined by qRT-PCR. b FFA at 50 μM could not increase ALP activity in hBMMSCs in the presence of TNF-α or LPS. Yet FFA at 50 μM increased ALP activity more in hBMMSCs in the presence of Bay117082. Human BMMSCs were treated with PM, OM, OM with FFA at 50 μM, or OM with FFA at 50 μM and TNF-α or LPS or Bay117082 for 7 days before ALP staining. c FFA at 50 μM no longer accelerated mineralization in hBMMSCs in the presence of TNF-α or LPS. Yet FFA at 50 μM accelerated mineralization more in hBMMSCs in the presence of Bay117082. Cells were treated with PM, OM, OM with FFA at 50 μM, or OM with FFA at 50 μM and TNF-α or LPS or Bay117082 for 14 days, and calcium deposition was then tested using ARS staining. d The result of quantification of ALP activity was consistent with the result of ALP staining. e The result of quantification of ARS was consistent with the result of ARS staining. f TNF-α and LPS downregulated the expression of RUNX2 and BGLAP in hBMMSCs, which had been increased by 50 μM FFA, as determined by qRT-PCR. Bay117082 further upregulated the expression of RUNX2 and BGLAP in hBMMSCs, which had been increased by 50 μM FFA, as determined by qRT-PCR. g Western blot of protein expression of p-IKK, p-IκBα, IκBα, p-P65, P65, and the internal control GAPDH. Human BMMSCs were cultured in PM or PM with FFA for 7 days before treated with TNF-α or LPS for 30 min. h Western blot of protein expression of p-IKK, p-IκBα, IκBα, p-P65, P65, RUNX2, and the internal control GAPDH. Human BMMSCs were cultured in OM or OM with FFA for 7 days before treated with TNF-α or LPS for 30 min. All data are shown as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01, and *** p < 0.001. FFA, flufenamic acid; hBMMSC, human bone marrow-derived mesenchymal stem cell; PM, proliferation media; OM, osteogenic media; ALP, alkaline phosphatase; ARS, alizarin red S; RUNX2, runt-related transcription factor 2; BGLAP, bone gamma-carboxyglutamate protein; qRT-PCR, quantitative real-time reverse transcription PCR; hMSC, human mesenchymal stem cell; NF-κB, nuclear factor kappa B; TNF-α, tumor necrosis factor alpha
Article Snippet: Primary human BMMSCs and human adipose-derived mesenchymal stem cells (hASCs) were purchased from ScienCell Company (San Diego, CA, USA).
Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Staining, Western Blot, Control, Cell Culture, Derivative Assay, Reverse Transcription
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