Anti-Inflammatory Effect of 1,3,5,7-Tetrahydroxy-8-isoprenylxanthone Isolated from Twigs of Garcinia esculenta on Stimulated Macrophage

Figure 5

Effect of TIE on NF-κB, MAPK activation, and miR155 expression in LPS/IFNγ-stimulated RAW264.7 cells. (a) RAW264.7 cells were transiently cotransfected with a pNF-κB reporter vector. Cells were incubated with TIE (3.125, 6.25, 12.5, and 25 μM) and L-NIL (50 μM) for 1 h before stimulation with LPS and IFNγ for 18 h. Luciferase activities were measured by Dual Luciferase Reporter reagents following manufacturer’s instruction. Luciferase activity was normalized to transfection efficiency as monitored by Renilla luciferase expression. (b, c) DNA-binding activity of p65 and p50 proteins in nuclear extracts was assessed using NF-κBp50/p65 EZ-TFA transcription factor assay. Absorbance was measured at 450 nm in a microplate spectrophotometer. Results were normalized to absorbance/mg protein. (d) RAW264.7 cells (1 × 10⁶ cells/dish) were treated with varying doses of TIE with IFNγ (10 U/mL) plus LPS (100 ng/mL) for 4 h. Total RNA was isolated and subjected to qRT-PCR to determine the level of TBK1 mRNA. (e) RAW264.7 cells were plated at a density of 1 × 10⁶ cells/well in 30 mm dish overnight. TIE was added to cells followed by 30 min stimulation of IFNγ (10 U/mL) plus LPS (100 ng/mL). Whole cell lysates were prepared and subjected to Western blotting. The ratios of immunointensity of p-ERK1/2, p-JNK, and p-p38 were calculated, respectively. Total ERK1/2, JNK, and p38 (T-ERK1/2, T-JNK, and T-p38) were used as a control of the protein amount in the same samples. Data shown are the representative of three independent experiments. (f) The cells were stimulated by IFNγ (10 U/mL) plus LPS (100 ng/mL) with or without 12.5 and 25 μM concentrations of TIE for 24 h. Total RNA was isolated and the expression of miR155 was determined by qRT-PCR. RNU6B was used here as an endogenous control. The data represent the mean ± SD of triplicate experiments. , versus control group; , versus model group.

(a)

(b)

(c)

(d)

(e)

(f)