Figure 4. Butyrate and propionate might inhibit Sa.LPP-induced NO production by inhibiting HDAC. (A) RAW 264.7 cells (3×105 cells/ml) were pre-treated with HDAC inhibitors (0.5 μM SAHA or 0.05 μM TSA) or SCFAs (3 mM of acetate, propionate, or butyrate) for 1 h and subsequently stimulated with 5 μg/ml of Sa.LPP for additional 23 h. Nitrite in the culture supernatant was measured to determine NO concentration using the Griess reagent. (B) RAW 264.7 cells (3×105 cells/ml) were pre-treated with 0.01 or 0.05 μM of TSA for 1 h and subsequently stimulated with 5 μg/ml of Sa.LPP for additional 3 h. Cell lysates were analyzed to measure phosphorylated STAT1, nonphosphorylated STAT1, phosphorylated NF-κB p65 or β-actin by Western blotting. (C) RAW 264.7 cells (5×105 cells/ml) were pre-treated with 0.01 or 0.05 μM of TSA for 1 h and subsequently stimulated with 5 μg/ml of Sa.LPP for an additional 6 h. Total RNA was isolated, and the mRNA expression level of IFN-β was examined by RT-PCR. (D) RAW 264.7 cells (3×105 cells/ml) were treated with 5 μg/ml of Sa.LPP in the presence or absence of acetate, propionate, or butyrate for 4 h. Cell lysates were obtained and analyzed by Western blotting to measure histone acetylation at lysine residues. (E, F) RAW 264.7 cells (3×105 cells/ml) were pre-treated with (E) 0.01 μM of MPN, or (F) 0.01 μg/ml of PTX for 1 h and subsequently stimulated with 5 μg/ml of Sa.LPP for an additional 23 h. Nitrite in the culture supernatant was measured to determine NO concentration using the Griess reagent. Data are mean values±standard deviation of 3 independent experiments.
*Asterisks indicate significant differences (p<0.05) compared with the appropriate controls.