Nutr Res Pract.  2014 Oct;8(5):501-508. 10.4162/nrp.2014.8.5.501.

Anti-inflammatory effects of Rubus coreanus Miquel through inhibition of NF-kappaB and MAP Kinase

• Lee JE
• Cho SM
• Park E
• Lee SM
• Kim Y
• Auh JH
• Choi HK
• Lim S
• Lee SC
• Kim JH

• KMID: 2313770
• DOI: http://doi.org/10.4162/nrp.2014.8.5.501

Abstract

BACKGROUND/OBJECTIVES
Rubus Coreanus Miquel (RCM), used as a traditional Korean medicine, reduces chronic inflammatory diseases such as cancer and rheumatoid arthritis. However, its mechanism has not been elucidated. In this study, we examine the anti-inflammatory effects of RCM and their possible mechanisms using RAW 264.7 cells.
MATERIALS/METHODS
Unripe RCM ethanol extract (UE), unripe RCM water extract (UH), ripe RCM ethanol extract (RE), and ripe RCM water extract (RH) were prepared. Inflammatory response was induced with LPS treatment, and expression of pro-inflammatory mediators (iNOS, COX-2, TNF-alpha, IL-1beta, and IL-6) and NO and PGE2 productions were assessed. To determine the anti-inflammatory mechanism of RCM, we measured NF-kappaB and MAPK activities.
RESULTS
UE and UH treatment significantly reduced NF-kappaB activation and JNK and p38 phosphorylation and reduced transcriptional activities decreased iNOS, COX-2, and pro-inflammatory cytokines expressions, and NO and PGE2 productions. RE and RH treatments reduced IL-1beta and IL-6 expressions through suppressions of JNK and p38 phosphorylation.
CONCLUSIONS
In this study, we showed that RCM had anti-inflammatory effects by suppression of pro-inflammatory mediator expressions. Especially, unripe RCM showed strong anti-inflammatory effects through suppression of NF-kappaB and MAPK activation. These findings suggest that unripe RCM might be used as a potential functional material to reduce chronic inflammatory responses.

Keyword

Cytokines; inflammation; MAPK; NF-kappaB; Rubus coreanus Miquel

MeSH Terms

Arthritis, Rheumatoid
Cytokines
Dinoprostone
Ethanol
Inflammation
Interleukin-6
NF-kappa B*
Phosphorylation
Phosphotransferases*
Tumor Necrosis Factor-alpha
Water
Cytokines
Dinoprostone
Ethanol
Interleukin-6
NF-kappa B
Phosphotransferases
Tumor Necrosis Factor-alpha
Water

Figure

• 

  Fig. 1 Effects of Rubus coreanus Miquel (RCM) extracts on cell viability in RAW 264.7. Cells were treated with UE, UH, RE and RH (0, 12.5, 25, 50, 100, 200 and 400 µg/ml) for 24 h then percentage of cell viability was measured by MTT assay. Values not sharing the same letter were significantly different (P < 0.05). (UE: unripe Rubus coreanus Miquel ethanol extract, UH: unripe Rubus coreanus Miquel water extract, RE: ripe Rubus coreanus Miquel ethanol extract, and RH: ripe Rubus coreanus Miquel water extract)

• 

  Fig. 2 Effects of RCM extracts on NO production and iNOS expression in LPS induced RAW 264.7. RAW 264.7 cells were treated with UE, UH, RE and RH (400 µg/ml) for 24 h then stimulated with LPS (1 µg/ml) for 4 h (B) or 18 h (A, C). Relative NO production, iNOS mRNA expression and iNOS protein expression were measured by nitrite/nitrate kit (A), PCR (B), and western blot (C). Values not sharing the same letter were significantly different (P < 0.05). (UE: unripe Rubus coreanus Miquel ethanol extract, UH: unripe Rubus coreanus Miquel water extract, RE: ripe Rubus coreanus Miquel ethanol extract, and RH: ripe Rubus coreanus Miquel water extract)

• 

  Fig. 3 Effects of RCM extracts on PGE2 production and COX-2 expression in LPS induced RAW 264.7. RAW 264.7 cells were treated with UE, UH, RE and RH (400 µg/ml) for 24 h then stimulated with LPS (1 µg/ml) for 4 h (B) or 18 h (A, C). Relative PGE2 production, COX-2 mRNA expression and COX-2 protein expression were measured by PGE2 EIA kit (A), PCR (B), and western blot (C). Values not sharing the same letter were significantly different (P < 0.05). (UE: unripe Rubus coreanus Miquel ethanol extract, UH: unripe Rubus coreanus Miquel water extract, RE: ripe Rubus coreanus Miquel ethanol extract, and RH: ripe Rubus coreanus Miquel water extract)

• 

  Fig. 4 Effect of RCM on TNF-α (A and B), IL-1β (C and D), and IL-6 (E and F) mRNA and protein expression. Cells were pretreated with 400 µg/ml concentration of RCM for 24 h then stimulated with LPS (1 µg/ml) for 4 or 16 h. Total RNA and protein lysates were subjected to semi-quantitative PCR or Western blot analysis. The values are expressed as the mean ± S.D. of three individual experiments. Values not sharing the same letter were significantly different (P < 0.05). (UE: unripe Rubus coreanus Miquel ethanol extract, UH: unripe Rubus coreanus Miquel water extract, RE: ripe Rubus coreanus Miquel ethanol extract, and RH: ripe Rubus coreanus Miquel water extract)

• 

  Fig. 5 Effects of RCM extracts on NF-κB activity (A), IκB phosphorylation (B) and MAP kinase activity (C) in LPS induced RAW 264.7. Cells were treated with UE, UH, RE and RH (400 µg/ml) for 24 h then stimulated with LPS (1 µg/ml) for 20 min (A, C) or 15 min (B). NF-κB activity and relative IκB phospholylation were measured by NF-κB (p65) transcription factor assay kit (A) and western blot (B) and MAP kinase activity was measured by western blot (C). Values not sharing the same letter were significantly different (P < 0.05). (UE: unripe Rubus coreanus Miquel ethanol extract, UH: unripe Rubus coreanus Miquel water extract, RE: ripe Rubus coreanus Miquel ethanol extract, and RH: ripe Rubus coreanus Miquel water extract)

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