Effectiveness of Acanthopanax Senticosus
Harms in a Mouse Model of Nickel Allergy
Dai Tsuchida1, Tadahide Noguchi2*, Maki Tanaka3, Yusuke Takahashi3, Masaki Fujishima4, Eri Okumura4, Hideo Takekoshi4, Hidekatsu Takeda5 and Masahiro Iijima1
1Division of Orthodontics and Dentofacial Orthopedics, Department of Oral Growth and Development, School of Dentistry, Health Sciences University of Hokkaido, Japan
2Department of Dentistry, Oral and Maxillofacial Surgery, Jichi Medical University, Japan
3Department of Clinical Laboratory Science, School of Medical Technology, Health Sciences University of Hokkaido, Japan
4Sun Chlorella Corp., Production & Development Department, Japan
5Department of Physical Therapy, Sapporo Medical University of Medicine, Japan
Submission: August 08, 2022; Published: August 16, 2022
*Corresponding author: Tadahide Noguchi, Department of Dentistry, Oral and Maxillofacial Surgery, Jichi Medical University, Tochigi, Japan
How to cite this article: Dai T, Tadahide N, Maki T, Yusuke T, Masaki F, et al. Effectiveness of Acanthopanax Senticosus Harms in a Mouse Model of Nickel
Allergy. Adv Dent & Oral Health. 2022; 15(4): 555918. DOI: 10.19080/ADOH.2022.15.555918
Although metal allergies are thought to develop in a T cell-dependent manner, no appropriate animal model currently exists. Further, since the molecular mechanisms of the allergic reactions are not completely understood, a fundamental treatment method has not been established. Acanthopanax senticosus Harms (ASH) is a plant known for its anti-inflammatory and antioxidant effects. This study aimed to investigate the therapeutic effects of ASH using a mouse model of nickel allergy. Nickel was selected since it is frequently used for dental metals and metal ornaments. The mice were divided into a group to which a regular solid diet (control group) was given and a group to which ASH was added to the regular solid diets (ASH group). A mixed solution of Lipopolysaccharide (LPS) and nickel chloride (NiCl2) was intraperitoneally administered to sensitize. Seven days after sensitization, a NiCl2 solution was injected intradermally into the pinna to induce nickel allergy. For the evaluation of allergic reactions, the thickness of the pinna, the histopathological evaluation of the pinna, and the measurement of cytokines in the serum of mice were performed. In the ASH group, pinna swelling was significantly reduced at 24 and 48 h time points and lymphocyte infiltration was attenuated, compared with those in the control group. In vitro examinations showed that ASH suppressed TNF-α and interleukin-6 (IL-6) production in human peripheral blood. These results of this study suggest that ASH has a preventive effect on nickel allergy.
Keywords:Acanthopanax senticosus Harms; Metal Allergy; Nickel Allergy Mouse Model
Allergic contact hypersensitivity to metals is classified as a delayed type of hypersensitivity.
Although various metals are known to cause allergic reactions, the precise molecular mechanisms responsible for this allergy remain unclear, and no treatment for metal allergy has been established. The metal most commonly involved in such allergies is nickel, which is frequently used in dental metals and metallic ornaments, causing contact dermatitis. It occurs when metals come in direct contact with the skin and induce palmoplantar pustulosis, lichen planus, and pompholyx eczema due to the absorption of metals into the body. Oral lichen planus-like lesions may appear as lace-like keratotic lesions in the vicinity of metals in the oral cavity, which may be difficult to diagnose and treat.
Metal allergy is a disorder in which certain metallic elements act as haptens to cause localized or systemic contact dermatitis. Metal-derived contact allergic reactions can progress to allergic contact dermatitis (ACD) when exposure exceeds an individual’s threshold. Studies on the prevalence of metal allergies have been conducted in various countries, and in all of them, nickel, cobalt, and chromium have shown high favorable rates in patch tests. It is estimated that up to 17% of women and 3% of men are allergic to nickel . The prevalence of metal allergy is even higher in patients with dermatitis . Nickel exposure primarily occurs through consumer metal products. The use of accessories
such as necklaces and earrings, belt hardware, jeans buttons
and zippers, and cell phones increases the risk of sensitization
to nickel and other metals [1-4]. Metal restorations in dentistry
are also essential factors in causing metal allergies, and reports
of such allergies are rising. The removal of metal restorations
can be quite time-consuming and financially burdensome. Since
CD4 and CD8-positive T cells are involved in the development of
metal allergy, it is thought that modulating signal transduction to
immune cells, such as epithelial cells, may inhibit the progress of
allergies . Acanthopanax senticosus HARMS (ASH) has various
medicinal effects such as anti-anxiety, reduction of sexual behavior,
the proliferation of learning function, suppression of lipid
peroxidation, lowering blood pressure, induction of β-endorphin
and growth hormone, increase in natural killer (NK) cell activity,
and suppression of tumor cell proliferation [6-10]. This study
aims to investigate the preventive and therapeutic effects of ASH
by using the nickel (Ni) allergy mouse model, which is frequently
used for dental metals and metal ornaments.
4-6 weeks old female BALB/c mice were purchased from
HOKUDO (Hokkaido, Japan). Drinking water was provided ad
libitum. The mice were acclimatized to our facility for at least seven
days before the experiment. All experiments were conducted in accordance with The Animal Experiment Board and approved by
the Health Sciences University of Hokkaido animal experiments.
(Approval number, 19-022).
ASH was obtained from Sun Chlorella Corp. (Kyoto, Japan).
Nickel (II) chloride was purchased by Wako (Osaka, Japan).
Lipopolysaccharide (LPS, derived from Escherichia coli serotype
055: B5) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
All reagents used in this experiment were adjusted to the specified
concentrations using saline solution, immediately before dosing.
Solid diets (Oriental Yeast Co., Ltd.) containing 5% of ASH
extract were fed freely to animals of treatment group, while
the control group was fed regular solid diets. Body weight was
measured every week. After 28 days of treatment, nickel was
Sensitization phase. 9-11 weeks old BALB/c female mice
(after the treatment) were intraperitoneally injected with 250
uL of 5.0 μg/mL LPS and 5.0 mM nickel chloride (NiCl2) solution
(LPS-NiCl2), using a 26G needle. Elicitation phase. Seven days after
sensitization, 20 uL of 5 mM NiCl2 solution was intradermally
injected into the right pinna, under 5% isoflurane inhalation
anesthesia to induce Ni allergy, using a 33G needle.
The thickness of the pinna was measured using a peacock dial
thickness gauge. Three measurements were taken at 24, 48, and 72 hours after the challenge (Figure 1). The rate of swelling was
calculated as the increase in ear thickness after attraction, relative to the thickness immediately before attraction (mean ± standard
error, n=7). We decided to use ipsilateral pinna thickness before
attraction, as the control data because the pathogenesis involves a
systemic immune response, despite inducing a local inflammatory
response. The mice were then anesthetized with 5% isoflurane,
euthanized using CO2 gas, and blood was collected by cardiac
puncture. The blood was stored at -80°C after serum separation.
The right pinnae were excised and immersed in a fixative solution
(10% formalin) for 12 hours, embedded in paraffin blocks.
Samples for all histopathological evaluations were prepared
72 hours after elicitation using the pinna. Paraffin-embedded
blocks were sliced and stained with hematoxylin-eosin according
to the standard method. Immunostaining was performed using
the Goat anti-Rat IgG antibody Vectastain ABC-HRP kit (Vector
Laboratories, Burlingame, CA) and diaminobenzidine (DAB) as
a chromogen according to the manufacturer’s protocols. CD4
monoclonal antibody as anti CD4 antibody (4SM95, CD4:1/100,Invitrogen, Carlsbad, CA) was used. Histopathological studies
were performed by the New Drug Research Center (Hokkaido,
Blood samples from mice were centrifuged at 3000 rpm for
10 min at 4 °C, and serum was collected. Quantitative evaluation
of proinflammatory cytokines (TNF-α and IL-6) in the serum of
all mice was performed using a commercially available ELISA kit
(R&D Systems). The absorbance (primary wavelength 450 nm,
reference wavelength 620 nm) of all wells was measured using an
absorptiometer (Infinite® F200, Tecan).
Statistical analysis was performed using statistical analysis
software (SPSS Statics 25, IBM). Student’s t-test was used to test
the difference between the means of each group. P-values <0.05
were considered statistically significant.
Table 1 shows the composition of the 5 % ASH extract
blended chow. The content of each active ingredient in 100 g of
feed was 6.2 mg of isofraxidin, 18.7 mg of eleutheroside B, 41.8
mg of eleutheroside E, 6.3 mg of eleutheroside B1, and 70.7 mg of
Influence on pinna swelling: No dead mice were identified
during all experimental periods, and no significant changes in
body weight were observed (Figure 2). Significant inhibition of
swelling at 24 and 48 h time points was observed in the ASH group
(Figure 3A). Photographs of representative pinna from each group
at the 24-h time point after nickel allergy challenge are shown
in Figure 3B & 3C. The extent of redness in the ASH group was
limited and milder, compared with that in the control group.
Histopathological evaluation of the pinna: H&E staining
(×200 magnification) revealed chronic inflammatory cell
infiltration with predominance of lymphocytes and subcutaneous
edema, but both were milder in the ASH group, compared with
those in the control group (Figure 4A, B).
Immunohistochemical staining revealed lesser infiltration
of CD4 positive T cells in the blood vessels of the ASH group,
compared with that in the control group (Figure 5A & 5B).
Cytokine levels in the blood serum: The inflammatory
cytokine levels in murine blood serum are shown in Figure 6. The
mean IL-6 and TNF-α production in the control group was 82.7
pg/mL and 16.9 pg/mL, respectively, and in the ASH group it was
53.7 pg/mL and 12.6 pg/mL, respectively. A significant (p<0.05)
reduction of approximately 65.0 % for IL-6 and 15.2 % for TNF-α
was observed in the ASH group, on comparison with the control
Upon direct contact with metal accessories, the metal ions
eluted from the product by sweat, penetrate directly into the
skin. Dental metals, restored in the oral cavity, are eluted and
ionized by food, saliva and metabolites of oral bacteria and are
taken up by the oral mucosa. Ionized metals bind to self-proteins
in the body and are antigenized. These ions are then taken up
by antigen-presenting cells, transported to regional lymph
nodes, and recognized and sensitized by hapten-specific T cells.
Subsequent exposure to the same metal activates T cells and
produces inflammatory cytokines, leading to allergic symptoms.
Contact dermatitis (mucositis) is followed by systemic contact
dermatitis, in which the antigen is transported to the wholebody
via blood circulation [1,11-15]. However, no treatment has
been established yet due to difficulties in identifying the allergen
metals. The removal and re-restoration of the metal restoration
are accompanied by considerable time and economic burden, with
no guaranteed improvement in the disease state.
CD4 and CD8 positive T cells contribute to the cellular immunity
to the disease state manifestation during a metallic allergy [16-
18]. This can be suppressed by altering the signal transduction of
these immunocompetent cells. We investigated the therapeutic
effects of ASH against metal allergy using a mouse model of Ni
allergy. Takahashi et al.  reported the therapeutic effects of
ASH in a mouse model of collagen-induced arthritis (CIA); they
observed a delay in the onset of CIA and reduction in its severity.
They concluded the enhanced effects of anti-TNF-α-antibody
therapy in CIA mouse models. ASH suppressed the production of
TNF-α, IL-6, superoxide, and hydroxyl radicals. The production
of both cytokines was suppressed at the transcriptional level.
Therefore, ASH enhanced the therapeutic effect of the anti-TNFα
antibody on rheumatoid arthritis through the anti-inflammatory
and antioxidative effect .
Our study showed that ASH significantly alleviated pinna
swelling and inhibited the invasion of CD4 positive cells in a nickel
allergy mouse model. Cytokine levels were significantly reduced in
murine serum. Liriodendrin, isolated using ethyl acetate (EtOAc)
from the bark of ASH by activity-induced fractionation, inhibits
vascular permeability . This may have resulted in alleviated
edema and CD4 invasion by vascular permeability inhibition
in the ASH group. Oral mucosal diseases in which cell-mediated
immunity is thought to be involved in the pathogenesis, includes
oral lichen planus and GVHD besides metallic allergy. Lichen planus
is a recalcitrant chronic inflammatory keratosis that appears
on the skin and mucous membranes. In the oral cavity, lichen
planus occurs frequently on the buccal mucosa and gingiva and
may become cancerous by several percent. Histopathologically,
thickening with keratinization of the epithelium and zonal
infiltration of inflammatory cells (mainly T cells) just beneath
the basal cells are characteristic findings, and the interaction
between infiltrated lymphocytes and epithelial cells seems to
contribute to the pathogenesis [20-22]. Various modifiers are
complicatedly concerned in the disease state formation of oral
lichen planus besides the cell-mediated immunity mechanism,
and the scavenger such as free radical and antioxidant enzyme is
also considered to contribute to this .
ASH inhibited the generation of superoxide and hydrogen
peroxide in murine peritoneal macrophages in vitro and in vivo
, and it acts as a radical scavenger [9,25-27]. In addition,
GVHD, expressed in the oral mucosa, has clinicopathological and
histological findings like oral lichen planus, which occur when
donor-derived T cells are activated and attack the oral mucosal
epithelium of the transplant donor . Furthermore, ASH extract
is commercially available in many countries, and no obvious side
effects have been reported when administered as a single agent
. Therefore, we would like to expect ASH to be clinically applied
as a treatment for such refractory oral mucosal diseases.
In modern society, where we have the opportunity to come
into contact with many metals, many people around the world
are suffering from the symptoms of contact hypersensitivity. In
the current situation where a fundamental treatment method
has not been established for metal allergies, our research results
suggest that administration of ASH is useful for the prevention and
treatment of metal allergies.