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Rho iso-alpha acids from hops inhibit the GSK-3/NF-kappaB pathway and reduce inflammatory markers associated with bone and cartilage degradation.

Konda VR, Desai A, Darland G, Bland JS, Tripp ML - J Inflamm (Lond) (2009)

Bottom Line: To understand the mechanisms, we evaluated the effect of RIAA in cell signaling pathways and inflammatory markers using various in vitro models.The inhibitory effect of RIAA on inflammatory markers was assessed by measuring nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-alpha/IL-1beta-mediated MMP-13 expression in SW1353 cells.RIAA selectively inhibited the NF-kappaB pathway while having no effect on ERK1/2, p38 and JNK phosphorylation in LPS-stimulated RAW 264.7 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: MetaProteomics Nutrigenomics Research Center (a subsidiary of Metagenics, Inc), 9770 44th Avenue N,W,, Gig Harbor, WA, 98332, USA. vrkonda@metagenics.com.

ABSTRACT

Background: Rho iso-alpha acids (RIAA) from hops have been shown to have anti-inflammatory properties. To understand the mechanisms, we evaluated the effect of RIAA in cell signaling pathways and inflammatory markers using various in vitro models. We also investigated their therapeutic effect in mice with collagen-induced arthritis.

Methods: The LPS-stimulated RAW 264.7 macrophages were used to evaluate the effect of RIAA on the NF-kappaB and MAPK signaling pathways; phosphorylation of ERK1/2, p38 and JNK was assessed by western blotting and NF-kappaB binding by electrophoretic mobility shift assays. Effect on the NF-kappaB activity was evaluated by the luciferase reporter assays in LPS-stimulated RAW 264.7 cells. GSK-3alpha/beta kinase activity was measured in cell-free assays. The inhibitory effect of RIAA on inflammatory markers was assessed by measuring nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-alpha/IL-1beta-mediated MMP-13 expression in SW1353 cells. Mice with collagen-induced arthritis were fed with RIAA for 2 weeks. Symptoms of joint swelling, arthritic index and joint damage were assessed.

Results: RIAA selectively inhibited the NF-kappaB pathway while having no effect on ERK1/2, p38 and JNK phosphorylation in LPS-stimulated RAW 264.7 cells. RIAA also inhibited GSK-3alpha/beta kinase activity and GSK-3beta dependent phosphorylation of beta-catenin in RAW 264.7 cells. In addition, RIAA inhibited NF-kappaB-mediated inflammatory markers in various cell models, including nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-alpha/IL-1beta-mediated MMP-13 expression in SW1353 human chondrosarcoma cells. Finally, in a mouse model of collagen-induced arthritis, RIAA ameliorated joint damage as evidenced by significant reduction of the arthritis index and histology score; at 250 mg/kg-body weight, RIAA had efficacy similar to that of 20 mg/kg-body weight of celecoxib.

Conclusion: RIAA may have potential as an anti-inflammatory therapeutic.

No MeSH data available.


Related in: MedlinePlus

Effect of RIAA on LPS-induced inflammatory signaling pathways in RAW 264.7 cells. (A) Cells were incubated for 1 h with RIAA (0, 10 and 20 μg/ml) followed by LPS stimulation (1 μg/ml) for 2 h. Nuclear extract were analyzed for NF-κB binding by EMSA. (B) Cells were pre-incubated with RIAA (5 and 20 μg/ml) for 1 h and stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were analyzed for phosphorylation of ERK1/2, p38 and JNK using western blot. (C) Cells transiently transfected with NF-κB firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) or NF-κB inhibitor parthenolide (10 μM) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and normalized with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times. (D) Cells transiently transfected with CRE firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and corrected with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times.
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Figure 1: Effect of RIAA on LPS-induced inflammatory signaling pathways in RAW 264.7 cells. (A) Cells were incubated for 1 h with RIAA (0, 10 and 20 μg/ml) followed by LPS stimulation (1 μg/ml) for 2 h. Nuclear extract were analyzed for NF-κB binding by EMSA. (B) Cells were pre-incubated with RIAA (5 and 20 μg/ml) for 1 h and stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were analyzed for phosphorylation of ERK1/2, p38 and JNK using western blot. (C) Cells transiently transfected with NF-κB firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) or NF-κB inhibitor parthenolide (10 μM) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and normalized with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times. (D) Cells transiently transfected with CRE firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and corrected with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times.

Mentions: First, the effect of RIAA on NF-κB in vitro was investigated. RAW 264.7 cells were treated with LPS alone (1 μg/ml) or RIAA (10 and 20 μg/ml) followed by LPS incubation. Nuclear extracts were then analyzed by EMSA. RIAA at 20 μg/ml inhibited NF-κB to its consensus sequence as evidenced by reduced band intensity (Fig. 1A, lane 4). Next, the effect of RIAA on MAPK was investigated. Western blot analysis of total extract showed that, in the presence or absence of LPS stimulation, RIAA (5 and 20 μg/ml) had no effects on phosphorylation of ERK1/2, p38 and JNK in RAW 264.7 cells (Fig. 1B). Next, RAW cells were transfected with either NF-κB or CRE firefly luciferase construct, treated without or with RIAA (1, 5 and 10 μg/ml), and followed by LPS (1 μg/ml) stimulation. The luciferase reporter assays revealed that RIAA inhibited transactivation of NF-κB (Fig. 1C), but had no effects on that of CRE (Fig. 1D).


Rho iso-alpha acids from hops inhibit the GSK-3/NF-kappaB pathway and reduce inflammatory markers associated with bone and cartilage degradation.

Konda VR, Desai A, Darland G, Bland JS, Tripp ML - J Inflamm (Lond) (2009)

Effect of RIAA on LPS-induced inflammatory signaling pathways in RAW 264.7 cells. (A) Cells were incubated for 1 h with RIAA (0, 10 and 20 μg/ml) followed by LPS stimulation (1 μg/ml) for 2 h. Nuclear extract were analyzed for NF-κB binding by EMSA. (B) Cells were pre-incubated with RIAA (5 and 20 μg/ml) for 1 h and stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were analyzed for phosphorylation of ERK1/2, p38 and JNK using western blot. (C) Cells transiently transfected with NF-κB firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) or NF-κB inhibitor parthenolide (10 μM) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and normalized with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times. (D) Cells transiently transfected with CRE firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and corrected with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times.
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Figure 1: Effect of RIAA on LPS-induced inflammatory signaling pathways in RAW 264.7 cells. (A) Cells were incubated for 1 h with RIAA (0, 10 and 20 μg/ml) followed by LPS stimulation (1 μg/ml) for 2 h. Nuclear extract were analyzed for NF-κB binding by EMSA. (B) Cells were pre-incubated with RIAA (5 and 20 μg/ml) for 1 h and stimulated with LPS (1 μg/ml) for 1 h. Cell lysates were analyzed for phosphorylation of ERK1/2, p38 and JNK using western blot. (C) Cells transiently transfected with NF-κB firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) or NF-κB inhibitor parthenolide (10 μM) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and normalized with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times. (D) Cells transiently transfected with CRE firefly luciferase construct were incubated with RIAA (1, 5 and 10 μg/ml) for 1 h followed by LPS (1 μg/ml) stimulation for 8 h. The luciferase activities were determined and corrected with Renilla expression, and expressed as fold change compared to vehicle control. Data shown is representative of the experiment repeated several times.
Mentions: First, the effect of RIAA on NF-κB in vitro was investigated. RAW 264.7 cells were treated with LPS alone (1 μg/ml) or RIAA (10 and 20 μg/ml) followed by LPS incubation. Nuclear extracts were then analyzed by EMSA. RIAA at 20 μg/ml inhibited NF-κB to its consensus sequence as evidenced by reduced band intensity (Fig. 1A, lane 4). Next, the effect of RIAA on MAPK was investigated. Western blot analysis of total extract showed that, in the presence or absence of LPS stimulation, RIAA (5 and 20 μg/ml) had no effects on phosphorylation of ERK1/2, p38 and JNK in RAW 264.7 cells (Fig. 1B). Next, RAW cells were transfected with either NF-κB or CRE firefly luciferase construct, treated without or with RIAA (1, 5 and 10 μg/ml), and followed by LPS (1 μg/ml) stimulation. The luciferase reporter assays revealed that RIAA inhibited transactivation of NF-κB (Fig. 1C), but had no effects on that of CRE (Fig. 1D).

Bottom Line: To understand the mechanisms, we evaluated the effect of RIAA in cell signaling pathways and inflammatory markers using various in vitro models.The inhibitory effect of RIAA on inflammatory markers was assessed by measuring nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-alpha/IL-1beta-mediated MMP-13 expression in SW1353 cells.RIAA selectively inhibited the NF-kappaB pathway while having no effect on ERK1/2, p38 and JNK phosphorylation in LPS-stimulated RAW 264.7 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: MetaProteomics Nutrigenomics Research Center (a subsidiary of Metagenics, Inc), 9770 44th Avenue N,W,, Gig Harbor, WA, 98332, USA. vrkonda@metagenics.com.

ABSTRACT

Background: Rho iso-alpha acids (RIAA) from hops have been shown to have anti-inflammatory properties. To understand the mechanisms, we evaluated the effect of RIAA in cell signaling pathways and inflammatory markers using various in vitro models. We also investigated their therapeutic effect in mice with collagen-induced arthritis.

Methods: The LPS-stimulated RAW 264.7 macrophages were used to evaluate the effect of RIAA on the NF-kappaB and MAPK signaling pathways; phosphorylation of ERK1/2, p38 and JNK was assessed by western blotting and NF-kappaB binding by electrophoretic mobility shift assays. Effect on the NF-kappaB activity was evaluated by the luciferase reporter assays in LPS-stimulated RAW 264.7 cells. GSK-3alpha/beta kinase activity was measured in cell-free assays. The inhibitory effect of RIAA on inflammatory markers was assessed by measuring nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-alpha/IL-1beta-mediated MMP-13 expression in SW1353 cells. Mice with collagen-induced arthritis were fed with RIAA for 2 weeks. Symptoms of joint swelling, arthritic index and joint damage were assessed.

Results: RIAA selectively inhibited the NF-kappaB pathway while having no effect on ERK1/2, p38 and JNK phosphorylation in LPS-stimulated RAW 264.7 cells. RIAA also inhibited GSK-3alpha/beta kinase activity and GSK-3beta dependent phosphorylation of beta-catenin in RAW 264.7 cells. In addition, RIAA inhibited NF-kappaB-mediated inflammatory markers in various cell models, including nitric oxide in LPS-stimulated RAW 264.7 cells, RANKL-mediated TRAP activity in transformed osteoclasts, and TNF-alpha/IL-1beta-mediated MMP-13 expression in SW1353 human chondrosarcoma cells. Finally, in a mouse model of collagen-induced arthritis, RIAA ameliorated joint damage as evidenced by significant reduction of the arthritis index and histology score; at 250 mg/kg-body weight, RIAA had efficacy similar to that of 20 mg/kg-body weight of celecoxib.

Conclusion: RIAA may have potential as an anti-inflammatory therapeutic.

No MeSH data available.


Related in: MedlinePlus