J Periodontal Implant Sci.  2013 Aug;43(4):191-197. 10.5051/jpis.2013.43.4.191.

Effect of quercetin on the production of nitric oxide in murine macrophages stimulated with lipopolysaccharide from Prevotella intermedia

• Cho YJ
• Kim SJ

• KMID: 2027815
• DOI: http://doi.org/10.5051/jpis.2013.43.4.191

Abstract

PURPOSE
Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action.
METHODS
LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory kappaB (IkappaB)-alpha degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting.
RESULTS
Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of IkappaB-alpha induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling.
CONCLUSIONS
Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-kappaB and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease.

Keyword

Lipopolysaccharides; Nitric oxide; Periodontal diseases; Prevotella intermedia; Quercetin

MeSH Terms

Cell Culture Techniques
Heme Oxygenase-1
I-kappa B Proteins
JNK Mitogen-Activated Protein Kinases
Lipopolysaccharides
Macrophages
Metalloporphyrins
Nitric Oxide
Nitric Oxide Synthase
Periodontal Diseases
Phosphorylation
Prevotella
Prevotella intermedia
Protoporphyrins
Quercetin
STAT1 Transcription Factor
Tin
Heme Oxygenase-1
I-kappa B Proteins
JNK Mitogen-Activated Protein Kinases
Lipopolysaccharides
Metalloporphyrins
Nitric Oxide
Nitric Oxide Synthase
Protoporphyrins
Quercetin
STAT1 Transcription Factor
Tin

Figure

• 

  Figure 1 Effects of quercetin on Prevotella intermedia lipopolysaccharide (LPS)-induced nitric oxide (NO) production (A) and inducible NO synthase (iNOS) protein expression (B) in RAW264.7 cells. Cells were treated with different doses of quercetin (0, 0.1, 1, 10, 20, and 30 µM) in the absence or presence of P. intermedia LPS (10 µg/mL) for 24 hours. (A) NO production was assayed by measuring the accumulation of nitrite in culture supernatants. The results are mean±standard deviation of three independent experiments. *P<0.05 vs. P. intermedia LPS alone. **P<0.01 vs. P. intermedia LPS alone. (B) iNOS protein synthesis was measured by immunoblot analysis of cell lysates using an iNOS-specific antibody. A representative immunoblot from two separate experiments with similar results is shown.

• 

  Figure 2 Involvement of heme oxygenase-1 (HO-1) in the inhibitory effects of quercetin on Prevotella intermedia lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. (A) Cells were treated with different doses of quercetin (0, 0.1, 1, 10, 20, and 30 µM) in the absence or presence of P. intermedia LPS (10 µg/mL) for 6 hours. HO-1 protein synthesis was measured by immunoblot analysis of cell lysates using HO-1-specific antibody. A representative immunoblot from two separate experiments with similar results is shown. (B) Cells were treated with quercetin (30 µM) and P. intermedia LPS (10 µg/mL) for 24 hours in the presence of different doses of tin protoporphyrin IX (SnPP) (0, 10, 25, 50, and 100 µM). Supernatants were removed after 24 hours and assayed for NO. The results are mean±standard deviation of three independent experiments. **P<0.01 vs. P. intermedia LPS alone. †P<0.01 vs. P. intermedia LPS plus quercetin.

• 

  Figure 3 Effects of quercetin on Prevotella intermedia lipopolysaccharide (LPS)-induced phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in RAW264.7 cells. Cells were incubated with different doses of quercetin (0, 0.1, 1, 10, 20, and 30 µM) in the absence or presence of P. intermedia LPS (10 µg/mL) for 30 minutes (for JNK) or 15 minutes (for p38). The cell lysates were subjected to immunoblot analysis using specific antibodies. A representative immunoblot from two separate experiments with similar results is shown.

• 

  Figure 4 Effects of quercetin on Prevotella intermedia lipopolysaccharide (LPS)-induced inhibitory κB (IκB)-α degradation in RAW264.7 cells. Cells were incubated with different doses of quercetin (0, 0.1, 1, 10, 20, and 30 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 an antibody against IκB-α. A representative immunoblot from two separate experiments with similar results is shown.

• 

  Figure 5 Effects of quercetin on Prevotella intermedia lipopolysaccharide (LPS)-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) in RAW264.7 cells. Cells were incubated with different doses of quercetin (0, 0.1, 1, 10, 20, and 30 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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