Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/p38MAPK pathways in RAW264.7 macrophages

Abstract

Aim: The aim of this study was to investigate the effect of the squamosamide derivative FLZ (N-2-(4-hydroxy-phenyl)-ethyl-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) on lipopolysaccharide (LPS)-induced inflammatory mediator production and the underlying mechanism in RAW264.7 macrophages.

Methods: RAW264.7 cells were preincubated with non-toxic concentrations of compound FLZ (1, 5, and 10 micromol/L) for 30 min and then stimulated with 10 microg/L LPS. The production of nitric oxide (NO), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and the activation of nuclear factor kappa-B (NF-kappaB) and mitogen-activated protein kinase (MAPK) pathways were examined.

Results: FLZ significantly inhibited the LPS-induced production of NO, as well as the expression of iNOS and COX-2 at both the RNA and the protein levels in RAW264.7 cells. The LPS-induced increase in the DNA binding activity of NF-kappaB and activator protein 1 (AP-1), the nuclear translocation of NF-kappaB p65, the degradation of the inhibitory kappaBalpha protein (IkappaBalpha) and the phosphorylation of IkappaBalpha, IkappaB kinase (IKK) alpha/beta, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPKs were all suppressed by FLZ. However, the phosphorylation of extracellular signal-regulated kinase (ERK) was not affected. Further study revealed that FLZ inhibited the phosphorylation of transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1), which is an upstream signaling molecule required for IKKalpha/beta, JNK and p38 activation.

Conclusion: FLZ inhibited the LPS-induced production of inflammatory mediators at least partly through the downregulation of the TAK-IKK and TAK-JNK/p38MAPK pathways.

Figure 1

The chemical structure of FLZ.

Figure 2

Effect of FLZ on NO production. (A) RAW264.7 cells were cultured with 10 μg/L LPS in the presence or absence of FLZ for 36 h. The nitrite in supernatants was determined. (n=4). ^cP<0.01 compared with the normal group; ^fP<0.01 compared with the normal LPS-treated group. (B) FLZ (10 μmol/L) and sodium nitroprusside (10 mmol/L in PBS) were incubated at 25 °C for 180 min. At 30 min intervals, samples of the reaction were removed and the nitrite was detected. n=4. Data are presented as the mean±SD.

Figure 3

Effect of FLZ on LPS-induced iNOS protein and mRNA expression in RAW264.7 cells. (A) iNOS protein was detected by Western blot. RAW264.7 cells were pretreated with various concentrations (1, 5, and 10 μmol/L) of FLZ for 30 min; then LPS (10 μg/L) was added and the cells were incubated for 24 h. Total cellular proteins (40 μg) were subjected to Western blot analysis using an antibody against iNOS. (B) RT-PCR detection of iNOS mRNA. RAW264.7 cells were pretreated with different concentrations (1, 5, and 10 μmol/L) of FLZ for 30 min; then LPS (10 μg/L) was added and the cells were incubated for 8 h. iNOS mRNA was detected with RT-PCR and agarose gel electrophoresis as described under Methods. n=3. Values are presented as the mean±SD. ^cP<0.01 compared with the normal group; ^fP<0.01 compared with the LPS-treated group.

Figure 4

Effect of FLZ on LPS-induced COX-2 protein and mRNA expression in RAW264.7 cells. (A) COX-2 protein expression was detected by Western blot. The cells were treated with various concentrations (1, 5, and 10 μmol/L) of FLZ for 30 min; then LPS (10 μg/L) was added and the cells were incubated for 24 h. Cellular proteins (40 μg) were subjected to Western blot analysis using an antibody against COX-2. Experiments were repeated three times. (B) COX-2 mRNA detection. RAW264.7 cells were pretreated with various concentrations (1, 5, and 10 μmol/L) of FLZ for 30 min; then LPS (10 μg/L) was added and the cells were incubated for 8 h. COX-2 mRNA was detected by RT-PCR and agarose gel electrophoresis as described under Methods. n=3. Values are presented as the mean±SD. ^cP<0.01 compared with the normal group; ^eP<0.05; ^fP<0.01 compared with the LPS-treated group.

Figure 5

Effects of FLZ on the LPS-induced DNA binding activity of NF-κB and AP-1, nuclear translocation of NF-κB p65 and degradation of IκBα in RAW264.7 cells. (A) Cells were treated with the indicated concentrations of FLZ for 30 min before stimulation with LPS for 1 h. The nuclear proteins were extracted and tested for DNA binding to NF-κB or AP-1 consensus binding sequences by EMSA. (B) NF-κB p65 assay. The cells were treated with 1, 5, or 10 μmol/L of FLZ for 30 min and then with LPS (10 μg/L) for 1 h. Nuclear proteins (40 μg) were analyzed by Western blot. (C) IκB assay. RAW264.7 cells were treated with 1, 5, or 10 μmol/L of FLZ for 30 min and then with LPS 10 μg/L for 30 min. Total cellular proteins (40 μg) were analyzed by Western blot using an antibody against IκBα. n=3. Values are presented as the mean±SD. ^cP<0.01 compared with the normal group; ^fP<0.01 compared with the LPS-treated group.

Figure 6

Effect of FLZ on the LPS-induced phosphorylation of IκBα and IKKα/β in RAW264.7 cells. RAW264.7 cells were treated with FLZ for 30 min and then with LPS (10 μg/L) for 5 min. Total cellular proteins (40 μg) were subjected to Western blot analysis using an antibody against p-IκBα (A) and p-IKKα/β (B). n=3. Values are presented as the mean±SD. ^bP<0.05, ^cP<0.01 compared with the normal group; ^eP<0.05, ^fP<0.01 compared with the LPS treated group.

Figure 7

Effect of FLZ on the LPS-induced phosphorylation of ERK, JNK, and p38 MAPK in RAW264.7 cells. RAW264.7 cells were treated with FLZ for 30 min and then with LPS (10 ng/mL) for 20 min. Total cellular proteins (40 μg) were subjected to Western blot analysis using the indicated antibody.

Figure 8

Effect of FLZ on the LPS-induced phosphorylation of TAK1 in RAW264.7 cells. RAW264.7 cells were treated with FLZ for 30 min and then with LPS (10 μg/L) for 5 min. Total cellular proteins (40 μg) were subjected to Western blot analysis using an antibody against p-TAK1. n=3. Values are presented as the mean±SD. ^bP<0.05 compared with the normal group; ^eP<0.05 compared with the LPS treated group.