Anti-inflammatory effect of glycyrrhizin with Equisetum arvense extract

概要

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 are
produced as a host defense response, play an important role in the onset and
progression of the disease. Therefore, appropriate control of inflammatory cytokines is
necessary to prevent the onset of periodontitis and to inhibit the progression of the
disease.
Glycyrrhizin (GL) has been widely used as an ingredient with anti-inflammatory
properties and inhibits TNF-α production by lipopolysaccharide (LPS) derived from
periodontal pathogenic bacteria, but there is a maximum dose for the use of GL. In the
present study, I first explored plant extracts that enhance the anti-inflammatory effect
of GL and elucidated the mechanism of enhancement. Next, I investigated the
inhibitory 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 and
elucidation of the mechanism of enhancement
The effects of extracts from six different plants (Crataegus oxyacantha, Salvia

officinalis, Equisetum arvense, Hamamelis virginiana, Paeonia lactiflora and Betula
alba) on GL-suppressed TNF-α expression levels in THP-1 macrophages stimulated
with LPS were examined. Next, the inhibitory effect of GL with EA supplementation on
TNF-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 from
mouse 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 and
elucidation of the mechanism of enhancement
Screening experiments demonstrated that EA had the strongest additive effect on the
suppression 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 by
suppressing 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 and
GL 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-induced
periodontitis model rat was significantly reduced by EA administration to the same
level as that in the Control group. Immunohistochemistry of osteoclastogenesis related
factors showed that RANKL expression in the periodontal ligament was increased and
OPG 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, and
rather 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 in
decreasing osteoclastogenesis. This study suggests the possibility of developing a new
periodontitis prevention/treatment product by combined GL and EA.

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