J Periodontal Implant Sci.  2015 Aug;45(4):145-151. 10.5051/jpis.2015.45.4.145.

Topical application of herbal formula for the treatment of ligature-induced periodontitis

• Kim MH
• Choi YY
• Lee HJ
• Lee H
• Park JC
• Yang WM

• KMID: 2164343
• DOI: http://doi.org/10.5051/jpis.2015.45.4.145

Abstract

PURPOSE
The aim of this study was to investigate the therapeutic effects of a herbal formula, PerioH-035, containing Angelica sinensis, steamed Rehmannia glutinosa, Angelica dahurica, Cimicifuga heracleifolia, and Zanthoxylum piperitum on the periodontal breakdown in a well-established ligature-induced periodontitis model in rats.
METHODS
Sprague-Dawley rats were randomly assigned to 1 of 4 groups: NL (non-ligatured), L (ligatured), P1 (ligatured and treated with 1 mg/mL PerioH-035), P100 (ligatured and treated with 100 mg/mL PerioH-035). Periodontitis was induced by placing a ligature around the mandibular first molars. PerioH-035 was topically applied to both sides of the first molar for 2 weeks. The right side of the mandibles was retrieved for micro-computed tomography (CT) and methylene blue staining to analyze alveolar bone loss. The left side of the mandibles was histologically analyzed by TRAP and H&E staining. The MMP-9 mRNA level in gingival tissue was investigated by RT-PCR.
RESULTS
Alveolar bone resorption was significantly reduced in the PerioH-035-treated groups. The number of dense multi-nucleated cells found to be TRAP-positive by staining in the ligatured rats was markedly decreased by PerioH-035 application. In addition, periodontal tissue destruction, especially cementum demineralization, was ameliorated in the P1 and P100 groups. Moreover, gingival tissue from the PerioH-035-treated group showed a decrease in the MMP-9 mRNA level, resulting in recovery of collagen degradation.
CONCLUSIONS
These results suggest that PerioH-035 has therapeutic effects on periodontitis, and thus, PerioH-035 shows promise as a treatment for periodontitis. GRAPHICAL ABSTRACT:

Keyword

Matrix metalloproteinase 9; Medicinal herbs; Osteoclasts; Periodontitis

MeSH Terms

Alveolar Bone Loss
Angelica
Angelica sinensis
Animals
Bone Resorption
Cimicifuga
Collagen
Dental Cementum
Ligation
Mandible
Matrix Metalloproteinase 9
Methylene Blue
Molar
Osteoclasts
Periodontitis*
Plants, Medicinal
Rats
Rats, Sprague-Dawley
Rehmannia
RNA, Messenger
Steam
Zanthoxylum
Collagen
Matrix Metalloproteinase 9
Methylene Blue
RNA, Messenger
Steam

Figure

• 

  Figure 1 The effect of PerioH-035 on alveolar bone loss indicated by three-dimensional reconstruction from micro-CT images. PerioH-35 is an herbal formula, containing Angelica sinensis, steamed Rehmannia glutinosa, Angelica dahurica, Cimicifuga heracleifolia, and Zanthoxylum piperitum. Representative micro-computed tomography images are shown (A) and the measurement of the alveolar bone resorption is illustrated (B). The scale bar is 1 mm. Data shown are mean±S.E.M (NL = 785.67±208.5 µm; L = 1,197.07±234.22 µm; P1 = 1,140.03±261.30 µm; P100 = 1,014.04±221.81 µm). Statistically significant differences between groups (a) P<0.001). NL, non-ligatured; L, ligatured; P1, ligatured and treated with 1 mg/mL PerioH-035; P100, ligatured and treated with 100 mg/mL PerioH-035.

• 

  Figure 2 The effect of PerioH-035 on alveolar bone loss indicated by methylene blue staining. PerioH-35 is an herbal formula, containing Angelica sinensis, steamed Rehmannia glutinosa, Angelica dahurica, Cimicifuga heracleifolia, and Zanthoxylum piperitum. (A) Representative images demonstrate the alveolar bone resorption. The scale bar is 1 mm. (B) The measurement of vertical alveolar bone resorption is illustrated, and the application of P100 has significantly inhibited the alveolar bone resorption. The red lines represent the distance from the cementoenamel junction to the alveolar bone crest. Data shown are mean ±S.E.M (NL = 2,720±130 µm; L = 3,610±280 µm; P1 = 3,003±140 µm; P100 = 2,920±310 µm). Mean values were significantly different among groups. (a)P<0.05, b)P<0.01). NL, non-ligatured; L, ligatured; P1, ligatured and treated with 1 mg/mL PerioH-035; P100, ligatured and treated with 100 mg/mL PerioH-035.

• 

  Figure 3 The effect of PerioH-035 on osteoclastic activity indicated by tartrate-resistant acid phosphatase (TRAP) staining. PerioH-35 is a herbal formula, containing Angelica sinensis, steamed Rehmannia glutinosa, Angelica dahurica, Cimicifuga heracleifolia, and Zanthoxylum piperitum. The arrows indicate TRAP-positive cells. Scale bar = 100 µm. NL, non-ligatured; L, ligatured; P1, ligatured and treated with 1 mg/mL PerioH-035; P100, ligatured and treated with 100 mg/mL PerioH-035.

• 

  Figure 4 The effect of PerioH-035 on histological changes of the periodontium. PerioH-35 is an herbal formula, containing Angelica sinensis, steamed Rehmannia glutinosa, Angelica dahurica, Cimicifuga heracleifolia, and Zanthoxylum piperitum. The yellow arrows indicate root cementum (A). The red arrows indicate gingival tissue (B). The thickness and the structure appears intact in the P100 group. Significant destruction of gingival tissue is also visible in the experimental periodontitis groups; however, the P100 group shows the pattern with the least destruction. C, cementum; D, dentin; PL, periodontal ligament; G, gingiva; NL, non-ligatured; L, ligatured; P1, ligatured and treated with 1 mg/mL PerioH-035; P100, ligatured and treated with 100 mg/mL PerioH-035. Scale bar = 100 µm (A) and 400 µm (B). H&E staining.

• 

  Figure 5 The effect of PerioH-035 on the expression of MMP-9 mRNA. PerioH-35 is an herbal formula, containing Angelica sinensis, steamed Rehmannia glutinosa, Angelica dahurica, Cimicifuga heracleifolia, and Zanthoxylum piperitum. Polymerase chain reaction results are shown (A), and a dose-dependent decreasing pattern of inverse relationship is observed. The ratio of the expression of MMP-9 in comparison to GAPDH is illustrated (B). The data shown are mean±S.E.M (NL = 1±0.08; L = 14.1±0.11; P1 = 8.7±0.29; P100 = 7.3±0.34). Application of P1 and P100 has significantly reduced the expression of MMP-9 (a)P<0.05). MMP-9, matrix metalloproteinase-9; NL, non-ligatured; L, ligatured; P1, ligatured and treated with 1 mg/mL PerioH-035; P100, ligatured and treated with 100 mg/mL PerioH-035.

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