Flufenamic Acid Inhibits Adipogenic Differentiation of Mesenchymal Stem Cells by Antagonizing the PI3K/AKT Signaling Pathway

Figure 5

FFA inhibits adipogenic differentiation of hASCs by inhibiting AKT signaling pathway. (a–c) FFA at 25 μM downregulated the expression of FOXO1, FOXO3, and CEBPα of hASCs grown in both PM and AM conditions, as determined by qRT-PCR. Human ASCs were treated with PM, PM+FFA at 25 μM, AM, or AM+FFA at 25 μM for 7 days. (d) Western blot of protein expression of p-PI3K, PI3K, p-AKT, AKT, PPARγ, and the internal control, GAPDH. Human ASCs were treated with PM, PM+FFA at 25 μM, AM, or AM+FFA at 25 μM for 7 days. (e, f) Phosphorylated PI3K/PI3K (e) and p-AKT/AKT (f) of protein bands showed in (d). (g, h) FFA at 25 μM inhibited lipid droplet formation in hASCs, but the inhibitory effect was attenuated in the presence of IGF-1(100 ng/mL), an activator of AKT signaling. Human ASCs were treated with PM, AM, AM+25 μM FFA, or AM+25 μM FFA, and IGF-1 for 21 days for Oil red O staining and quantification. . (i) Relative mRNA expression of CEBPα. Human ASCs were cultured in PM, AM, AM+25 μM FFA, or AM+FFA at 25 μM and IGF-1 for 7 days. (j) Western blot of protein expression of p-PI3K, PI3K, p-AKT, AKT, PPARγ, and the internal control, GAPDH. Human ASCs were cultured in PM, AM, AM+25 μM FFA, or AM+FFA at 25 μM and IGF-1 for 7 days. (k, l) Phosphorylated PI3K/PI3K (k) and p-AKT/AKT (l) of proteins bands in (j). All data are presented as the , . and compared with CTRL (a–c, e, f). compared with AM+FFA (h, i, k, l). FFA: flufenamic acid; hASC: human adipose derived stem cell; PM: proliferation media; AM: adipogenic media; FOXO1: forkhead box protein 1; FOXO3: forkhead box protein 3; PI3K: phosphatidylinositol-3-kinase; CEBPA: CCAAT enhancer binding protein alpha; qRT-PCR: quantitative real-time reverse transcription PCR; IGF-1,: insulin-like growth factor-1.

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