J Periodontal Implant Sci.  2011 Jun;41(3):157-163. 10.5051/jpis.2011.41.3.157.

Curcumin suppresses the production of interleukin-6 in Prevotella intermedia lipopolysaccharide-activated RAW 264.7 cells

• Kim SJ

• KMID: 1783607
• DOI: http://doi.org/10.5051/jpis.2011.41.3.157

Abstract

PURPOSE
Curcumin is known to exert numerous biological effects including anti-inflammatory activity. In this study, we investigated the effects of curcumin on the production of interleukin-6 (IL-6) by murine macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory periodontal disease, and sought to determine the underlying mechanisms of action.
METHODS
LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time polymerase chain reaction to detect IL-6 mRNA expression. IkappaB-alpha degradation, nuclear translocation of NF-kappaB subunits, and STAT1 phosphorylation were characterized via immunoblotting. DNA-binding of NF-kappaB was also analyzed.
RESULTS
Curcumin strongly suppressed the production of IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW 264.7 cells. Curcumin did not inhibit the degradation of IkappaB-alpha induced by P. intermedia LPS. Curcumin blocked NF-kappaB signaling through the inhibition of nuclear translocation of NF-kappaB p50 subunit. Curcumin also attenuated DNA binding activity of p50 and p65 subunits and suppressed STAT1 phosphorylation.
CONCLUSIONS
Although further study is required to explore the detailed mechanism of action, curcumin may contribute to blockade of the host-destructive processes mediated by IL-6 and appears to have potential therapeutic values in the treatment of inflammatory periodontal disease.

Keyword

Curcumin; Periodontal disease; Prevotella intermedia; Lipopolysaccharides; Interleukin-6

MeSH Terms

Curcumin
DNA
I-kappa B Proteins
Immunoblotting
Interleukin-6
Lipopolysaccharides
NF-kappa B
Periodontal Diseases
Phosphorylation
Prevotella
Prevotella intermedia
Real-Time Polymerase Chain Reaction
RNA, Messenger
Curcumin
DNA
I-kappa B Proteins
Interleukin-6
Lipopolysaccharides
NF-kappa B
RNA, Messenger

Figure

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  Figure 1 Effects of curcumin on Prevotella intermedia lipopolysaccharide (LPS)-induced production of interleukin (IL)-6 in RAW 264.7 cells. Cells were incubated with different doses of curcumin (0, 1, 5, 10, and 20 µM) in the absence or presence of P. intermedia LPS (10 µg/mL). Supernatants were removed after 24 hours and assayed for IL-6. The results are means±standard deviation of three independent experiments. a)P<0.01 versus P. intermedia LPS alone.

• 

  Figure 2 Effects of curcumin on Prevotella intermedia lipopolysaccharide (LPS)-induced interleukin (IL)-6 mRNA expression in RAW 264.7 cells. Cells were incubated with different doses of curcumin (0, 1, 5, 10, and 20 µM) in the absence or presence of P. intermedia LPS (10 µg/mL) for 24 hours. Real-time polymerase chain reaction was performed with EvaGreen Supermix, β-actin being used as an endogenous control. Data are presented as percentage of P. intermedia LPS alone. The results are means±standard deviation of three independent experiments. a)P<0.05. b)P<0.01 vs. P. intermedia LPS alone.

• 

  Figure 3 Effects of curcumin on Prevotella intermedia lipopolysaccharide (LPS)-induced IκB-α degradation in RAW 264.7 cells. Cells were incubated with different doses of curcumin (0, 1, 5, 10, and 20 µM) in the absence or presence of P. intermedia LPS (10 µg/mL). After 30 minutes of incubation, IκB-α degradation was determined by immunoblot analysis of cell lysates using antibody against IκB-α. A representative immunoblot from two separate experiments with similar results is shown.

• 

  Figure 4 Effects of curcumin on nuclear translocation (A) and DNA binding activity (B) of NF-κB subunits induced with Prevotella intermedia lipopolysaccharide (LPS) in RAW 264.7 cells. (A) Cells were incubated with different doses of curcumin (0, 1, 5, 10, and 20 µM) in the absence or presence of P. intermedia LPS (10 µg/mL). After 30 minutes (for NF-κB p65) or 8 hours (for NF-κB p50) of incubation, the nuclear fraction was isolated from cells. Nuclear translocation of NF-κB subunits was assessed by immunoblot analysis using antibodies against NF-κB p65 and p50. A representative immunoblot from two separate experiments with similar results is shown. (B) Cells were incubated with different doses of curcumin (0 and 20 µM) in the absence or presence of P. intermedia LPS (10 µg/mL). After 30 minutes (for NF-κB p65) or 8 hours (for NF-κB p50) of incubation, DNA-binding activity of NF-κB in nuclear extracts was assessed by using the enzyme-linked immunosorbent assay-based NF-κB p65/NF-κB p50 transcription factor assay kits. The results are means±standard deviation of two independent experiments. a)P<0.05 vs. P. intermedia LPS alone. OD: optical density.

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  Figure 5 Effects of curcumin on Prevotella intermedia lipopolysaccharide (LPS)-induced phosphorylation of STAT1 in RAW 264.7 cells. Cells were incubated with different doses of curcumin (0, 1, 5, 10, and 20 µM) in the absence or presence of P. intermedia LPS (10 µg/mL) for 4 hours. Expression of phospho-STAT1 was measured by immunoblot analysis of cell lysates. Total-STAT1 was used as an internal control. A representative immunoblot from two separate experiments with similar results is shown.

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