Role of nuclear factor-kappaB and heme oxygenase-1 in the mechanism of action of an anti-inflammatory chalcone derivative in RAW 264.7 cells

Abstract

The synthetic chalcone 3',4',5',3,4,5-hexamethoxy-chalcone (CH) is an anti-inflammatory compound able to reduce nitric oxide (NO) production by inhibition of inducible NO synthase protein synthesis. In this work, we have studied the mechanisms of action of this compound. CH (10-30 microm) prevents the overproduction of NO in RAW 264.7 macrophages stimulated with lipopolysaccharide (1 microg ml(-1)) due to the inhibition of nuclear factor kappaB (NF-kappaB) activation. We have shown that treatment of cells with CH results in diminished degradation of the NF-kappaB-IkappaB complex leading to inhibition of NF-kappaB translocation into the nucleus, DNA binding and transcriptional activity. We also demonstrate the ability of this compound to activate NfE2-related factor (Nrf2) and induce heme oxygenase-1 (HO-1). Our results indicate that CH determines a rapid but nontoxic increase of intracellular oxidative species, which could be responsible for Nrf2 activation and HO-1 induction by this chalcone derivative. This novel anti-inflammatory agent simultaneously induces a cytoprotective response (HO-1) and downregulates an inflammatory pathway (NF-kappaB) with a mechanism of action different from antioxidant chalcones.

Figure 1

(a) Effect of CH on iNOS and HO-1 mRNA expression in LPS-stimulated cells. Cells were treated with CH for 30 min and stimulated with LPS (1 μg ml⁻¹) for 4 h. Northern blot was performed as indicated in Methods. Results are representative of three separate experiments. (b) Inhibition of NF-κB reporter activity. RAW 264.7 cells grown in six-well plates were transfected with the NF-κB reported plasmid and assayed for luciferase activity. Data are expressed as mean±s.e.m. (n=3). ^**P<0.01 compared with LPS-treated cells.

Figure 2

Effect of CH on NF-κB–DNA binding activity (a) and p65 translocation (b). Cells were preincubated with different concentrations of CH or MG-132 (10 μM) for 30 min and then stimulated with LPS (1 μg ml⁻¹) for 1 h. Nuclear fractions were then obtained for the analyses. Results are representative of three separate experiments.

Figure 3

Effect of CH on IκB-α expression and phosphorylation. (a) IκB-α and IκB-α-P protein expression. Cells were preincubated with CH (30 μM) and/or MG-132 (10 μM) for 30 min and then stimulated with LPS (1 μg ml⁻¹) for 30 min. (b, c) IKK activity. Cells were stimulated as above and IKK protein was immunoprecipitated as indicated in Methods. The formation of GST-IκB-α-P was assessed by Western blot and that of GST-IκB-α-³²P by autoradiography. Results are representative of four separate experiments.

Figure 4

In vitro interaction with DNA and inhibition of NF-κB release. (a) In vitro interaction of CH with DNA binding. Cells were stimulated with LPS (1 μg ml⁻¹) for 1 h and then nuclear fractions were obtained. CH was incubated with nuclear fractions for 30 min and EMSA was carried out. In some experiments, 2-mercaptoethanol (mET, 140 mM) was added to the reaction tube and incubated for 30 min: a (mET was added after the 30 min incubation of nuclear fractions with CH) or b (mET was added before the 30 min incubation of nuclear fractions with CH). Results are representative of three separate experiments. (b) Release of NF-κB from the IκB complex. Unstimulated cells were treated with CH and then cytoplasmic extracts were obtained. Aliquots of these extracts were treated with detergents as indicated in Methods to release NF-κB, and then EMSA was performed. In some experiments, antibody against either p65 or p50 was incubated with the released NF-κB before performing EMSA.

Figure 5

Concentration-dependent induction of HO-1 by CH. (a) Protein expression. (b) Bilirubin accumulation in the culture medium as an index of enzyme activity. RAW 264.7 macrophages were incubated with different concentrations of CH for 8 h. After centrifugation, cell pellets were used for Western blot analysis and bilirubin was measured in the supernatants as indicated in Methods. (a) Results are representative of three separate experiments. (b) Data are the mean±s.e.m. of six determinations. ^**P<0.01.

Figure 6

Time course of HO-1 induction by CH. (a) mRNA expression. (b) Protein expression. (c) Bilirubin accumulation in the culture medium. RAW 264.7 macrophages were incubated with 30 μM CH for different times. After centrifugation, cell pellets were used for Western blot analysis and bilirubin was measured in the supernatants as indicated in Methods. In another series of experiments, mRNA was extracted from cell pellets as indicated in Methods. (a, b) Results are representative of three separate experiments. (c) Data are the mean±s.e.m. of six determinations. ^**P<0.01.

Figure 7

(a, b) Effect of agents modulating oxidative stress and GSH on HO-1 mRNA expression. Cells were pretreated with Tiron (500 μM), NAC (10 mM) or GSH (2 or 10 mM) for 30 min and then incubated with 30 μM CH for 2 h. In the case of BSO (200 μM), cells were pretreated for 6 h to deplete GSH and then incubated with CH as above. Northern blot analysis was performed as indicated in Methods. Results are representative of three separate experiments.

Figure 8

LSC analysis of intracellular generation of ROS and cytotoxicity. (a) Histogram of rhodamine (Rho) fluorescence in the presence or absence of CH (30 μM). RAW 264.7 cells were incubated with DHR (5 μM) for 10 min at 37°C. After washing, cells were incubated with or without CH (30 μM) for 20 min. (b) Determination of apoptotic (A)/necrotic(N) and viable (V) cells after incubation with CH (30 μM) for 20 min. Fluorescein isothiocyanate–annexin V (horizontal axis), propidium iodide (vertical axis). (c) Concentration-dependent increase of Rho fluorescence after CH treatment. Cells were incubated with 10, 20 or 30 μM CH for 20 min under the same conditions as in (a). Results are representative of four similar experiments. ^*P<0.05, ^**P<0.01.

Figure 9

(a) Time course of CH modification of GSH levels. CH was incubated with cells at 20 and 30 μM for different times. Intracellular GSH levels were determined as indicated in Methods. ^*P<0.05, ^**P<0.01 with respect to basal. (b, c) Nrf2 activation. Cells were incubated with CH (30 μM) for different times and then nuclear and cytoplasmic fractions were obtained. (b) Nrf2–DNA binding in nuclear fractions. EMSA was performed as indicated in Methods. (c) Nrf2 translocation to the nucleus. Western blot analysis was performed as indicated in Methods. Results are representative of three separate experiments.