J Periodontal Implant Sci.  2018 Apr;48(2):70-83. 10.5051/jpis.2018.48.2.70.

Magnoliae Cortex and maize modulate Porphyromonas gingivalis-induced inflammatory reactions

• Kim JY
• Kim KH
• Kwag EH
• Seol YJ
• Lee YM
• Ku Y
• Rhyu IC

• KMID: 2410686
• DOI: http://doi.org/10.5051/jpis.2018.48.2.70

Abstract

PURPOSE
The aim of this study was to evaluate the capacity of single and combined applications of the bark of the stems and roots of Magnolia officinalis Rehd. et Wils. (Magnoliae Cortex) and Zea mays L. (maize) to modulate inflammation in RAW 264.7 cells stimulated with Porphyromonas gingivalis.
METHODS
RAW 264.7 cells were stimulated with P. gingivalis, and Magnoliae Cortex and/or maize was added. Cytotoxicity and the capacity to modulate inflammation were determined with a methylthiazol tetrazolium (MTT) assay, nitrite production, enzyme-linked immunosorbent assay (ELISA), and western blotting.
RESULTS
Treatment with Magnoliae Cortex and/or maize inhibited nuclear transcription factor κB (NF-κB) pathway activation and nuclear p44/42 mitogen-activated protein kinase (MAPK) and inducible nitric oxide synthase (iNOS) protein expression in P. gingivalis-stimulated RAW 264.7 cells. Moreover, the treatments suppressed cytokines (prostaglandin E2 [PGE2], interleukin [IL]-1β, and IL-6) and nitrite production.
CONCLUSIONS
Both Magnoliae Cortex and maize exerted an anti-inflammatory effect on P. gingivalis-stimulated RAW 264.7 cells, and this effect was more pronounced when the extracts were combined. These findings show that these extracts may be beneficial for slowing the progression of periodontal disease.

Keyword

Cytokines; Magnolia; Mitogen-activated protein kinase 3; Periodontal diseases; Porphyromonas gingivalis; Zea mays

MeSH Terms

Blotting, Western
Cytokines
Enzyme-Linked Immunosorbent Assay
Inflammation
Interleukins
Magnolia*
Mitogen-Activated Protein Kinase 3
Nitric Oxide Synthase Type II
Periodontal Diseases
Porphyromonas gingivalis
Porphyromonas*
Protein Kinases
RAW 264.7 Cells
Transcription Factors
Zea mays*
Cytokines
Interleukins
Mitogen-Activated Protein Kinase 3
Nitric Oxide Synthase Type II
Protein Kinases
Transcription Factors

Figure

• 

  Figure 1 Effects of M and Z on Pam3CSK4-induced NO production in RAW 264.7 cells. Cells (5×104/cm2) were treated with M, Z, MZ, or IBU and 10 μg/mL Pam3CSK4 for 24 hours at 37°C. In experiments without Pam3CSK4, the medium alone was used as a negative control, and IBU was used as a positive control. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, NO: nitric oxide, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide.a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison with the control; c)P<0.05 in comparison with Z.

• 

  Figure 2 Cytotoxicity of M and Z treatments in RAW 264.7 cells. Cells (3.3×104/cm2) were treated with M, Z, or IBU for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide, MZ: the combination treatment of M and Z.a)P<0.05 in comparison with the control.

• 

  Figure 3 Effects of M and Z on Pam3CSK4-induced PGE2 formation in RAW 264.7 cells. Cells (5×104/cm2) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, PGE2: prostaglandin E2, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide.a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison with the control.

• 

  Figure 4 Effects of M and Z on Pam3CSK4-induced IL-1β formation in RAW 264.7 cells. Cells (5×104/cm2) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, IL-1β: interleukin-1β, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide.a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison with the control; c)P<0.05 in comparison with both Z and IBU.

• 

  Figure 5 Effects of M and Z on Pam3CSK4-induced IL-6 formation in RAW 264.7 cells. Cells (5×104/cm2) were treated with M, Z, MZ, or IBU for 30 minutes before treatment with 10 μg/mL Pam3CSK4 for 24 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, IL-6: interleukin-6, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide.a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison with the control; c)P<0.05 in comparison with M.

• 

  Figure 6 Effects of M and Z on nuclear p44/42 MAPK expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of p44/42 MAPK and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×104/cm2) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect p44/42 MAPK expression, Pam3CSK4 was added for 30 minutes at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of p44/42 MAPK, P-p44/42 MAPK and β-actin expression; (B) ratio of P-p44/42 MAPK to p44/42 MAPK expressed as a percentage.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), MAPK: mitogen-activated protein kinase, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide.a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison with the control.

• 

  Figure 7 Effects of M and Z on nuclear iNOS and COX-2 expression levels in Pam3CSK4-induced RAW 264.7 cells. The levels of iNOS, COX-2, and β-actin expression were detected by western blotting using specific antibodies. Cells (1.9×104/cm2) were treated with M, Z, MZ, or IBU for 30 minutes before being treated with 10 μg/mL Pam3CSK4. To detect iNOS and COX-2 expression, Pam3CSK4 was added for 24 hours at 37°C. The blots shown here are representative of 3 independent experiments. Each sample contained 10 μg of total protein. (A) The levels of iNOS, COX-2, and β-actin expression; (B) ratio of iNOS to β-actin expressed as a percentage.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide.a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison with the control.

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  Figure 8 Effects of M and Z on Pam3CSK4-induced NF-κB transactivation in RAW 264.7 cells. Cells (5×104/cm2) were treated with M, Z, MZ, or IBU for 30 minutes prior to treatment with 10 μg/mL Pam3CSK4 for 2 hours at 37°C. The data are expressed as mean±standard deviation, as obtained from 6 separate experiments.M: soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, NF-κB: nuclear transcription factor κB, MZ: the combination treatment of M and Z, IBU: ibuprofen (10 mM in 1% DMSO), DMSO: dimethyl sulfoxide, p65: transcription factor p65.a)P<0.05 in comparison with Pam3CSK4; b)P<0.05 in comparison with the control; c)P<0.05 in comparison with M and Z.

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  Figure 9 Schematic model for the anti-inflammatory mechanism of M and Z in Pam3CSK4-induced RAW 264.7 cells.ML soft 75% ethanol Magnoliae Cortex extract (60 μg/mL in 1% DMSO), Z: titrated unsaponifiable maize extract fraction (300 μg/mL in 1% DMSO), Pam3CSK4: synthetic Toll-like receptors of Porphyromonas gingivalis, MZ: the combination treatment of M and Z, JNK 1/2: c-Jun N-terminal kinase 1/2, p38: p38 mitogen-activated protein kinase, ERK 1/2: extracellular signal-regulated kinase 1/2, NF-κB/IκBα: nuclear transcription factor κB pathway, JAK/STAT3: Janus kinase/signal transduction and activator of transcription 3 signaling pathway, iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, IL: interleukin; PGE2: prostaglandin E2, NO: nitric oxide, DMSO: dimethyl sulfoxide.

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