学位論文要約Anti-inflammatory effect of glycyrrhizin with

Equisetum arvense extract（グリチルリチン酸とスギナ抽出物による抗炎症効果）

指導教員：宮内睦美

教授（医系科学研究科 口腔顎顔面病理病態学）

芝典江

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Objectives
Periodontitis is an infectious/inflammatory disease with progressive bone destruction,and inflammatory cytokines such as tumor necrosis factor α (TNF-α), which areproduced as a host defense response, play an important role in the onset andprogression of the disease. Therefore, appropriate control of inflammatory cytokines isnecessary to prevent the onset of periodontitis and to inhibit the progression of thedisease.
Glycyrrhizin (GL) has been widely used as an ingredient with anti-inflammatory
properties and inhibits TNF-α production by lipopolysaccharide (LPS) derived fromperiodontal pathogenic bacteria, but there is a maximum dose for the use of GL. In thepresent study, I first explored plant extracts that enhance the anti-inflammatory effectof GL and elucidated the mechanism of enhancement. Next, I investigated theinhibitory effect of Equisetum arvense extract (EA), which enhanced anti-inflammatory
effect of GL in the exploratory study, on osteoclastic bone resorption.

Methods
Exp. 1: Screening of plant extracts that enhance the anti-inflammatory effect of GL andelucidation of the mechanism of enhancement
The effects of extracts from six different plants (Crataegus oxyacantha, Salvia

officinalis, Equisetum arvense, Hamamelis virginiana, Paeonia lactiflora and Betulaalba) on GL-suppressed TNF-α expression levels in THP-1 macrophages stimulatedwith LPS were examined. Next, the inhibitory effect of GL with EA supplementation onTNF-α expression in junctional epithelium (JE) of LPS-induced periodontitis rat model
was examined. The effects of GL and/or EA on LPS induced signal pathways were also
analyzed by Western blotting.
Exp. 2: Investigation of the effect of EA on alveolar bone destruction
LPS-induced periodontitis model rats were used to examine the inhibitory effect of EA
on the formation of osteoclasts along the alveolar bone margin. Moreover, effects of EA
on the expression of osteoclastogenesis-related factors in cloned stromal-cell line frommouse bone marrow (ST2 cells) stimulated by LPS was analyzed by Real time PCR.

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Results
Exp. 1: Screening of plant extracts that enhance the anti-inflammatory effect of GL andelucidation of the mechanism of enhancement
Screening experiments demonstrated that EA had the strongest additive effect on thesuppression of TNF-α expression by GL at both mRNA and protein levels. In addition,
LPS-induced periodontitis rat model showed that GL with EA supplementation
significantly downregulated immunoexpression of TNF-α in JE, the front-line
epithelium directly exposed to plaque-derived irritants such as LPS. Furthermore,signal pathway analysis showed GL downregulated the production of TNF-α bysuppressing nuclear factor-kappa B (NF-κB) p65 phosphorylation, but not c-Jun
N-terminal kinase (JNK) or p38 phosphorylation. In contrast, EA decreased JNK
phosphorylation but not NF-κB p65 or p38 phosphorylation. The combination of EA andGL effectively attenuated LPS induced phosphorylation of NF-κB p65 and JNK.
Exp. 2: Investigation of the effect of EA on alveolar bone destruction
The number of osteoclasts formed along the alveolar bone margin in the LPS-inducedperiodontitis model rat was significantly reduced by EA administration to the samelevel as that in the Control group. Immunohistochemistry of osteoclastogenesis related
factors showed that RANKL expression in the periodontal ligament was increased andOPG expression was decreased in the LPS group. In the LPS/EA group, the increased
expression of RANKL was suppressed, while the expression of OPG was recovered, andrather enhanced than that in the Control group.

Conclusions
These results indicate that GL and EA additively suppressed TNF-α expression via
suppression of LPS-induced phosphorylation of NF-κB p65 and JNK, respectively. In
addition, it is indicated that EA has a novel function in regulating alveolar bone
destruction by suppressing the LPS-stimulated increase in RANKL and inflammatory
cytokine expression, restoring the suppression of OPG expression, resulting indecreasing osteoclastogenesis. This study suggests the possibility of developing a new
periodontitis prevention/treatment product by combined GL and EA.

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