Andrographolide interferes with binding of nuclear factor-kappaB to DNA in HL-60-derived neutrophilic cells

Abstract

1. Andrographolide, the major active component from Andrographis paniculata, has shown to possess anti-inflammatory activity. Andrographolide inhibits the expression of several proinflammatory proteins that exhibit a nuclear factor kappa B (NF-kappaB) binding site in their gene. 2. In the present study, we analyzed the effect of andrographolide on the activation of NF-kappaB induced by platelet-activating factor (PAF) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) in HL-60 cells differentiated to neutrophils. 3. PAF (100 nM) and fMLP (100 nM) induced activation of NF-kappaB as determined by degradation of inhibitory factor B alpha (IkappaB alpha) using Western blotting in cytosolic extracts and by binding to DNA using electrophoretic mobility shift assay (EMSA) in nuclear extracts. 4. Andrographolide (5 and 50 microM) inhibited the NF-kappaB-luciferase activity induced by PAF. However, andrographolide did not reduce phosphorylation of p38 MAPK or ERK1/2 and did not change IkappaB alpha degradation induced by PAF and fMLP. 5. Andrographolide reduced the DNA binding of NF-kappaB in whole cells and in nuclear extracts induced by PAF and fMLP. 6. Andrographolide reduced cyclooxygenase-2 (COX-2) expression induced by PAF and fMLP in HL-60/neutrophils. 7. It is concluded that andrographolide exerts its anti-inflammatory effects by inhibiting NF-kappaB binding to DNA, and thus reducing the expression of proinflammatory proteins, such as COX-2.

Figure 1

Inhibition of NF-κB-luciferase activity by andrographolide. (a) Schematic representation of NF-κB-luc construct. (b) HL-60/neutrophils were transiently transfected with the NF-κB-luc construct (Firefly luciferase) and a control gene (Renilla luciferase). The cells were preincubated with andrographolide (5 and 50 μM) for 30 min and stimulated with 100 nM PAF for 60 min. Luciferase activity was measured in a luminometer, and expressed as fold from control group; each bar represents mean±s.e.m., n=3. ^*P<0.05 vs PAF group.

Figure 2

Andrographolide does not affect MAPK phosphorylation and IkBα degradation. (a) HL-60/neutrophils were pretreated with andrographolide (10 or 50 μM) or vehicle for 15 min, and then stimulated with PAF or fMLP (100 nM) for 2 min. Western blot analysis was made with p-ERK1/2, ERK1/2, p-p38 and p38 antibodies. (b) HL-60/neutrophils were pretreated with andrographolide (50 μM) or vehicle for 15 min, and then stimulated with PAF or fMLP (100 nM) for 30 min. IkBα was analyzed in the cytosolic extract by Western blot with anti-IkBα antibody. The amount of proteins was normalized with anti-β-actin antibody.

Figure 3

Andrographolide reduces DNA NF-κB binding in vivo. (a) HL-60/neutrophils stimulated with 100 nM PAF increased the DNA NF-κB binding and 100 μM andrographolide was able to inhibit this DNA binding. (b) fMLP (100 nM) increased the DNA binding of NF-κB and 50 and 100 μM andrographolide inhibited this effect. The arrow indicates specific binding of NF-κB. (c) EMSA assay. Lane 1, binding in the presence of 100-fold excess specific probe; lane 2, binding in the absence of nuclear extract; lane 3, binding in the presence of 500-fold excess mutated probe. For all experiments, 2 μg of nuclear proteins was assayed for the binding of DNA, resolved in 6% PAGE and analyzed (Methods). Each bar represents the mean±s.e.m., n=at least 3. ^*P<0.05 vs PAF or fMLP group.

Figure 4

Andrographolide inhibition of DNA NF-κB binding in vitro. To analyze the effect of andrographolide in vitro, HL-60/neutrophils were stimulated with 100 nM PAF (a) or fMLP (b), and nuclear extracts were obtained as described in Methods. The nuclear extracts were incubated with andrographolide (10, 50 or 100 μM) for 15 min, and then the DNA binding reaction was carried out and analyzed as in Figure 3. The arrow indicates specific binding of NF-κB. Each bar represents the intensity fold of control, mean±s.e.m., n=at least 3. ^*P<0.05 vs PAF or fMLP group.

Figure 5

Andrographolide reduces COX-2 expression in HL-60/neutrophils. Cells were preincubated with 50 μM andrographolide for 15 min, and then stimulated with PAF or fMLP for 2 h. Total protein extracts were obtained and 80 μg of protein was used to analyze COX-2 expression and β-actin by Western blot with specific antibodies.