Arvelexin from Brassica rapa suppresses NF-κB-regulated pro-inflammatory gene expression by inhibiting activation of IκB kinase

Abstract

Background and purpose: Brassica rapa species constitute one of the major sources of food. In the present study, we investigated the anti-inflammatory effects and the underlying molecular mechanism of arvelexin, isolated from B. rapa, on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and on a model of septic shock induced by LPS.

Experimental approach: The expression of Inducible nitric oxide synthase (iNOS) and COX-2, TNF-α, IL-6 and IL-1β were determined by Western blot and/or RT-PCR respectively. To elucidate the underlying mechanism(s), activation of NF-κB activation and its pathways were investigated by electrophoretic mobility shift assay, reporter gene and Western blot assays. In addition, the in vivo anti-inflammatory effects of arvelexin were evaluated in endotoxaemia induced with LPS.

Key results: Promoter assays for iNOS and COX-2 revealed that arvelexin inhibited LPS-induced NO and prostaglandin E(2) production through the suppression of iNOS and COX-2 at the level of gene transcription. In addition, arvelexin inhibited NF-κB-dependent inflammatory responses by modulating a series of intracellular events of IκB kinase (IKK)-inhibitor κBα (IκBα)-NF-κB signalling. Moreover, arvelexin inhibited IKKβ-elicited NF-κB activation as well as iNOS and COX-2 expression. Serum levels of NO and inflammatory cytokines and mortality in mice challenged injected with LPS were significantly reduced by arvelexin.

Conclusion and implications: Arvelexin down-regulated inflammatory iNOS, COX-2, TNF-α, IL-6 and IL-1β gene expression in macrophages interfering with the activation of IKKβ and p38 mitogen-activated protein kinase, and thus, preventing NF-κB activation.

Figure 1

Chemical structures of indole compounds isolated from B. rapa and effects of arvelexin on the lipopolysaccharide (LPS)-induced NO production, iNOS expression and iNOS promoter activity in RAW 264.7 macrophages. (A) The chemical structures of arvelexin, caulilexin and indole-3-acetonitrile from the root skin of B. rapa. (B) Following pretreatment with arvelexin (25, 50, 100 µM) for 1 h, cells were treated with LPS (1 µg·mL⁻¹) for 24 h. Controls were not treated with LPS and arvelexin. L-NIL (10 µM) was used as a positive control. (C) Lysates were prepared from control, 24 h LPS (1 µg·mL⁻¹) alone or LPS plus with arvelexin (25, 50 or 100 µM). Total cellular proteins (30 µg) were resolved by SDS-PAGE, transferred to PVDF membranes and detected with specific antibodies. Total RNA was prepared for the RT-PCR analysis of iNOS from RAW 264.7 macrophages stimulated with LPS (1 µg·mL⁻¹) with/without arvelexin (25, 50 or 100 µM) for 4 h. The experiments were repeated three times and similar results were obtained. (D) Cells were transfected with a pGL3-iNOS promoter (−1592/+185) vector and the phRL-TK vector as an internal control. The level of luciferase activities was determined. Data are presented as the means ± SD of three independent experiments. #P < 0.05 versus the control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated group.

Figure 2

Effects of arvelexin on the LPS-induced PGE₂ production, COX-2 expression and COX-2 promoter activity in RAW 264.7 macrophages. (A) Following pretreatment with arvelexin (25, 50, 100 µM) for 1 h, cells were treated with LPS (1 µg·mL⁻¹) for 24 h. Controls were not treated with LPS and arvelexin. NS-398 (5 µM) was used as a positive control. (B) Lysates were prepared from control, 24 h LPS (1 µg·mL⁻¹) alone or LPS plus with arvelexin (25, 50 or 100 µM). Total cellular proteins (30 µg) were resolved by SDS-PAGE, transferred to PVDF membranes and detected with specific antibodies. Total RNA was prepared for the RT-PCR analysis of COX-2 from RAW 264.7 macrophages stimulated with LPS (1 µg·mL⁻¹) with/without arvelexin (25, 50 or 100 µM) for 4 h. The experiments were repeated three times and similar results were obtained. (C) Cells were transfected with a pGL3-COX-2 promoter (−965/+39) vector and the phRL-TK vector as an internal control. The level of luciferase activities was determined as described in Methods. Data are presented as the means ± SD of three independent experiments. #P < 0.05 versus the control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated group.

Figure 3

Effects of arvelexin on LPS-induced NF-κB activation. (A) Cells were transiently transfected with pNF-κB-luc reporter construct with the phRL-TK vector as an internal control. The level of luciferase activities was determined. Nuclear extracts from cells were prepared and used for analysis of NF-κB-DNA binding by electrophoretic mobility shift assay. The arrow indicates the position of the NF-κB band. Specificity of binding was examined by competition with 80-fold excess of unlabeled NF-κB oligonucleotide (cp). (B) Nuclear extracts were prepared for Western blot of p65 and p50 of NF-κB using specific anti-p65 and anti-p50 monoclonal antibodies. (C) Following pretreatment with arvelexin (25, 50 or 100 µM) for 1 h, cells were treated with LPS for 10 min. Total proteins were prepared and Western blot was performed using specific IκBα and pIκBα antibodies. (D) Following pretreatment with arvelexin (25, 50 or 100 µM) for 1 h, cells were treated with LPS (1 µg·mL⁻¹) for 5 min. Total cellular proteins (80 µg) were resolved by SDS-PAGE, transferred to PVDF membranes and detected with specific pIKKα/β, IKKα, IKKβ antibodies. (E) Cells were transiently transfected with pNF-κB-luc reporter construct and the phRL-TK vector as an internal control in combination with expression vector encoding IKKβ. The level of luciferase activities was determined. The data shown are representative of three independent experiments. Data are presented as the mean ± SD of three independent experiments. #P < 0.05 versus the control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated or IKK-overexpressed group.

Figure 4

Effects of arvelexin on IKKβ-elicited iNOS and COX-2 expression in RAW 264.7 macrophages. (A,B) Cells were transiently transfected with pGL3-iNOS promoter vector or pGL3-COX-2 promoter vector and the phRL-TK vector as an internal control in combination with expression vector encoding IKKβ. The level of luciferase activities were determined as described in Methods section. (C) Cells were transfected with expression vector encoding IKKβ. After 24 h of transfection, cells were treated with arvelexin for 24 h and then collected for Western Blot analysis. The data shown are representative of three independent experiments. Data are presented as the mean ± SD of three independent experiments. #P < 0.05 versus. the control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus the IKK overexpressed group.

Figure 5

Involvement of MAPK with anti-inflammatory effect of arvelexin in RAW 264.7 macrophages. (A) Following pretreatment with arvelexin (25, 50 or 100 µM) for 1 h, cells were treated with LPS (1 µg·mL⁻¹) for 10 min. Whole cell lysates were analysed by Western blot using antibodies against activated MAPKs. (B) Cells were transiently transfected with pGL3-iNOS promoter vector or pGL3-COX-2 promoter vector and the phRL-TK vector as an internal control. After 4 h of transfection, cells were pretreated with SB203580 or SP600125 and then stimulated with LPS (1 µg·mL⁻¹) for 18 h. The level of luciferase activities was determined as described in Methods. (C) Cells were transiently transfected with pNF-κB-luc reporter construct and the phRL-TK vector as an internal control. After 4 h of transfection, cells were pretreated with SB203580, SP600125 or PD98059 and then stimulated with LPS (1 µg·mL⁻¹) for 18 h. The level of luciferase activities was determined. Data are presented as the mean ± SD of three independent experiments. #P < 0.05 versus the control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus the LPS-stimulated group.

Figure 6

Effects of arvelexin on LPS-induced production and the mRNA expression of TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages. (A) Following pretreatment with arvelexin (25, 50 or 100 µM) for 1 h, the cells were treated with LPS (1 µg·mL⁻¹) for 24 h. Control values were obtained in the absence of LPS and arvelexin. (B) Total RNA was prepared for the RT-PCR analysis of TNF-α, IL-6 and IL-1β gene expression from RAW 264.7 macrophage cells pretreated with different concentrations (25, 50 or 100 µM) of arvelexin for 1 h followed by LPS (1 µg·mL⁻¹) for 4 h. TNF-α-specific sequences (351 bp), IL-6-specific sequences (142 bp) and IL-1β-specific sequences (387 bp) were detected by agarose gel electrophoresis. PCR of β-actin was performed to verify that the initial cDNA contents of samples were similar. Data are presented as the means ± SD of three independent experiments. #P < 0.05 versus the control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus the LPS-stimulated group.

Figure 7

Effect of arvelexin on in vivo production of inflammatory mediators and lethality inLPS-induced septic shock model. (A) Arvelexin (50, 75 or 100 mg·kg⁻¹, p.o.) given to mice 1 h before LPS (25 mg·kg⁻¹, i.p.) injection. Serum was collected 12 h after LPS, the levels of NO and cytokines were determined by Griess reaction assay and Bio-Plex assays (n = 5–6). (B) Six mice per group were treated with vehicle only or arvelexin (50, 75 or 100 mg·kg⁻¹, p.o.) and after 1 h, LPS injected (25 mg·kg⁻¹, i.p.). Survival rates of these mice were observed over the next 84 h. *P < 0.05, **P < 0.01, ***P < 0.001 versus the LPS-injected group.