Journal of Pharmacological Sciences 147 (2021) 294e304

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Journal of Pharmacological Sciences

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Guanabenz inhibits alveolar bone resorption in a rat model of

periodontitis
Ryujiro Muramatsu a, Takuma Sato a, Kazunori Hamamura b, Ken Miyazawa a, *,
Atsushi Takeguchi a, Masako Tabuchi a, Akifumi Togari b, Shigemi Goto a
a
Department of Orthodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Japan
b
Department of Pharmacology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Increase of sympathetic activity has been known to exacerbate osteoporosis through promotion of bone
Received 14 June 2021 resorption. However, it is largely unknown about involvement of sympathetic activity in exacerbation of
Received in revised form periodontitis. In this study, we investigated whether a2-adrenergic receptor (a2-AR) agonist guanabenz
3 August 2021
which decreases sympathetic activity, attenuates alveolar bone resorption in rats having high sympa-
Accepted 10 August 2021
thetic activity with periodontitis. Volumes of residual alveolar bone and attachment levels in perio-
Available online 14 August 2021
dontium were examined using micro-computed tomography and hematoxylin-eosin staining,
respectively. Furthermore, osteoclast numbers per bone surface and osteoclast surface per bone surface
Keywords:
Periodontitis
were measured using tartrate-resistant acid phosphatase staining. To examine the suppressive effects of
Sympathetic activity guanabenz on pro-inflammatory cytokines, expression levels of tyrosine hydroxylase (TH), TNF-a, IL1-b,
Guanabenz and IL-6 in periodontium were measured using immunohistostaining. Administration of guanabenz
Alveolar bone resorption attenuated loss of alveolar bone and attachment levels in rats having high sympathetic activity.
Pro-inflammatory cytokine Furthermore, its administration suppressed osteoclast numbers in rats having high sympathetic activity.
TH, TNF-a, IL-1b, and IL-6 positive cells in periodontium in rats treated with guanabenz for 12 weeks,
were lower than those in control rats having high sympathetic activity. This study demonstrated
administration of a2-AR agonist guanabenz attenuates alveolar bone resorption through decrease of
sympathetic activity in rats.
© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological
Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/
4.0/).

1. Introduction plasma, dopamine b-hydroxylase, and adrenal tyrosine hydroxylase

It has been known that increase of sympathetic activity activates
osteoclastogenesis resulting in the promotion of bone resorption.
For instance, inactivation of the sympathetic system by treated rats
with guanethidine, impairs bone resorption through suppression of
osteoclast formation.1 However, it is largely unknown whether
sympathetic activity exacerbates periodontitis via increase of
alveolar bone resorption.
Spontaneously hypertensive rats (SHRs) used in this study, is
known to have high sympathetic activity.2e4 Therefore, SHRs shows
increase of heart rate, blood pressure, levels of catecholamine in
* Corresponding author. Department of Orthodontics, School of Dentistry, Aichi
Gakuin University, 2-11 Suemori-Dori, Chikusa-ku, Nagoya 464-8651, Japan. Fax:
þ81 52 751 8900.
E-mail address: miyaken@dpc.agu.ac.jp (K. Miyazawa).
Peer review under responsibility of Japanese Pharmacological Society.
(TH) activity.2e4 TH is the rate-limiting enzyme in the synthesis of
catecholamine neurotransmitters such as dopamine, adrenaline,
and noradrenaline, and it is a representative marker of sympathetic
activity.5e7 Since SHRs also show increase of bone turnover and
decrease of cortical and cancellous bone mass,8,9 it has been used as
an animal model of osteoporosis with high sympathetic activity.10
Furthermore, it was also reported about the experimental model
of periodontitis caused by the ligature wire placed around the
contact point between the maxillary first molar (M1) and second
molar (M2) in SHRs.11 These shows that SHR is useful as an animal
model of periodontitis with high sympathetic activity.
Here, we address a question using SHRs with periodontitis: does
selective a2-adrenergic receptor (a2-AR) agonist guanabenz which
decreases sympathetic activity, attenuate periodontitis in rats
having high sympathetic activity? To examine the inhibitory effect
of guanabenz on periodontitis, we evaluated loss of alveolar bone
and attachment levels in periodontium. Furthermore, we measured
osteoclast numbers per bone surface (Oc.N/BS) and osteoclast

https://doi.org/10.1016/j.jphs.2021.08.003
1347-8613/© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
R. Muramatsu, T. Sato, K. Hamamura et al. Journal of Pharmacological Sciences 147 (2021) 294e304

surface per bone surface (Oc.S/BS) using tartrate-resistant acid
phosphatase (TRAP) staining for bone resorption. To assess the
suppressive effects of guanabenz on pro-inflammatory cytokines,
we measured positive cells and scores of TNF-a, IL1-b, and IL-6 in
periodontium using immunohistostaining.
(approval number: AGUD 417), and animal care and experimental
procedures were performed in accordance with the animal exper-
imentation guidelines of the School of Dentistry, Aichi Gakuin
University.

2.2. Animals and reagents

2. Materials and methods
Seven-week-old male SHR/Izm and WKY/Izm rats were pur-
2.1. Ethical approval chased from Japan SLC Inc. (Shizuoka, Japan). Rats were housed in
cages (3 rats per cage) under an automatically controlled constant
All experiments were approved by the Animal Care and Use environment (23 ± 1  C, 50 ± 10% humidity) and a 12-hr light/dark
Committee at the School of Dentistry of Aichi Gakuin University cycle, with ad libitum access to tap water and standard laboratory

Fig. 1. Schema of Experimental Design. (A) Position of ligature wire placement, buccal view (/: Ligature wire, M1: Maxillary first molar, M2: Maxillary second molar, M3: Maxillary
third molar). (B) Position of ligature wire placement, occlusal view (/: Ligature wire). (C) Schematic diagram of the ration of residual alveolar bone (a: residual amount of alveolar
bone, b: total volume). (D) Formula for calculating of the ratio of residual alveolar bone. (E) Schematic diagram of the ratio of attachment level in a rat. (a: area of attachment level, b:
length of cementoenamel junction [CEJ] to root apex). (F) Formula for calculating of the ratio of attachment levels.

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Fig. 2. Suppression of sympathetic activity in periodontium by 2.0 mg/kg guanabenz(GUA). (A) TH-stained histological sections of periodontium at 8 weeks after ligature placement
(Scale bar ¼ 200 mm). (B) TH positive cells in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (C) TH
staining scores in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (D) TH-stained histological sections
of periodontium at 12 weeks after ligature placement (Scale bar ¼ 200 mm). (E) TH positive cells in periodontium at 12 weeks after ligature placement (WKY control; n ¼ 4, WKY
GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (F) TH staining scores in periodontium at 12 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control;
n ¼ 4, SHR GUA; n ¼ 4).

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chow. All rats were acclimatized for 1 week to the housing con-
ditions. Guanabenz was purchased from Sigma-Aldrich (St. Louis,
MO, USA). Animals in SHR guanabentz and WKY guanabentz
groups were administered guanabentz at 2.0 mg/kg by s.c. once
daily for 8 and 12 weeks for structural analysis of the alveolar
bone (8 weeks; n ¼ 6, 12 weeks; n ¼ 6), Hematoxylin-eosin (HE)
staining (8 weeks; n ¼ 6, 12 weeks; n ¼ 6), TRAP staining (8
weeks; n ¼ 4, 12 weeks; n ¼ 4), and immunostaining of TNF-a, IL-
1b, and IL-6 (8 weeks; n ¼ 4, 12 weeks; n ¼ 4). SHR control and
WKY control groups were administered the same volume of saline
as in the treatment groups.
2.3. Development of a rat model of experimental periodontitis
Rats were anesthetized by intraperitoneal administration of a
mixture of 3 anesthetic agents, namely, medetomidine hydro-
chloride (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), midazolam
(Astellas Pharma Inc., Tokyo, Japan), and butorphanol tartrate (Meiji
Seika Pharma Co., Ltd.). A 0.2-mm-diameter stainless steel ligature
wire (Tomy International Inc., Tokyo, Japan) was placed around the
contact point between the left maxillary first molar (M1) and sec-
ond molar (M2), following the method by Takeguchi et al.11 (Fig. 1A
and B).
2.4. Structural analysis of the alveolar bone
Maxillae were harvested at the end of the 8 and 12-week period
after ligature placement, and subjected to three-dimensional
structural analysis using a micro-computed tomography (mCT)
(Rigaku, Tokyo, Japan) with a tube voltage of 90 kV, tube current of
150 mA, scanning time of 2 min, and pixel size of 20  20  20 mm.
Residual alveolar bone mass was measured as described by Take-
guchi et al.,10 defined as the residual alveolar bone mass in the area
between the cementeenamel junction (CEJ) between the left
maxillary first and second molars and the root apex relative to the
volume of the entire area (Fig. 1C and D).

Fig. 3. Effect of 2.0 mg/kg guanabenz (GUA) on ratio of remaining alveolar bone. (A) Ratio of residual alveolar bone between the first and second molar at 8 weeks after ligature wire
placement (WKY control; n ¼ 6, WKY GUA; n ¼ 6, SHR control; n ¼ 6, SHR GUA; n ¼ 6). (B) mCT images between the first and second molars at 8 weeks after ligature wire placement.
(C) Ratio of residual alveolar bone between the first and second molar at 12 weeks after ligature wire placement (N ¼ 6) (WKY control; n ¼ 6, WKY GUA; n ¼ 6, SHR control; n ¼ 6,
SHR GUA; n ¼ 6). (D) mCT images between the first and second molars at 12 weeks after ligature wire placement.

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2.5. Histological analysis
Alveolar bone tissue specimens were fixed using 10% neutral
buffered formalin and decalcified in 10% ethylenediaminetetra-
acetic acid (pH 7.2) for 4 weeks. The specimens were embedded in
paraffin, sectioned transversely at a 5-mm thickness. Tissue obser-
vation sites were selected at points where molar roots could be
observed. HE staining was performed. The ratio of the distance
from the CEJ on the distal aspect of the first molar to the bottom of
the gingival sulcus relative to the distance from the CEJ to the root
apex was calculated as the ratio of the attachment levels (Fig. 1E
and F). TRAP staining with an acid phosphatase leukocyte TRAP kit
(FUJIFILM Wako Pure Chemical Corporation, St. Osaka, Japan) for
measurement of osteoclast numbers per bone surface (Oc.N/BS)
and osteoclast surface per bone surface (Oc.S/BS) was performed.
The TRAP-stained slides were analyzed within a selected area
(1.0 mm  0.8 mm) 200 mm below the alveolar crest, between the
first and second molars of the left maxilla. Next, immunostaining of
TH, TNF-a, IL-1b, and IL-6 were performed. Immunostaining was
conducted using the histofine simple stain rat MAX-PO and histo-
fine simple stain DAB substrate kits (Nichirei Bioscience Inc., Tokyo,
Japan) with anti-Tyrosine hydroxylase antibody (ab75875, TH:1/
200, Abcam Inc., Waltham, MA, USA), anti-TNF alpha antibody
(GTX110520, TNF-a:1/100, GeneTex., LosAngeles, USA), anti-IL-1b
antibody (ab205924, IL-1b:1/500, Abcam Inc.), and anti-IL6 anti-
body (GTX110527, TNF-a:1/100, GeneTex., Los Angeles, USA).
Following Rogers et al.,12 the sections were scored as 1, 2, 3, or 4,
indicating 0e20%, 21e40%, 41e60%, and >60% positive staining,
respectively. Positive cells of TNF-a, IL-1b, and IL-6 in periodontium
were counted.

Fig. 4. Effect of 2.0 mg/kg guanabenz (GUA) on ratio of attachment levels. (A) Ratio of the attachment levels at 8 weeks after ligature wire placement (WKY control; n ¼ 6, WKY
GUA; n ¼ 6, SHR control; n ¼ 6, SHR GUA; n ¼ 6). (B) HE-stained histological sections of periodontium at 8 weeks after ligature placement (Scale bar ¼ 1000 mm). (C) Ratio of the
attachment levels at 12 weeks after ligature wire placement (WKY control; n ¼ 6, WKY GUA; n ¼ 6, SHR control; n ¼ 6, SHR GUA; n ¼ 6). (D) HE-stained histological sections of
periodontal tissue at 12 weeks after ligature placement (Scale bar ¼ 1000 mm).

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2.6. Statistical analysis 3. Results

Experimental data are expressed as the mean ± standard devi-
ation (S.D.), and statistically significant differences (p < 0.05) were
analyzed using Tukey's multiple comparison test. All statistical
analyses were conducted using GraphPad Prism v.6 (GraphPad
Software Inc.).
3.1. Suppression of sympathetic activity by guanabenz
Positive cells and scores of TH in periodontium were measured
using their immuno-stained histological sections (Fig. 2A and D).
The numbers of TH positive cells and its score decreased in SHRs

Fig. 5. Effect of 2.0 mg/kg guanabenz (GUA) on osteoclast numbers and surface. (A) TRAP-stained histological sections of periodontium at 8 weeks after ligature placement (Scale
bar ¼ 200 mm). (B) Osteoclast numbers per bone surface at 8 weeks after ligature placement (Oc.N/BS) (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4).
(C) Osteoclast surface per bone surface at 8 weeks after ligature placement (Oc.S/BS) (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (D) TRAP-stained
histological sections of periodontium at 12 weeks after ligature placement (Scale bar ¼ 200 mm). (E) Osteoclast numbers per bone surface at 12 weeks after ligature placement
(Oc.N/BS) (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (F) Osteoclast surface per bone surface at 12 weeks after ligature placement (Oc.S/BS) (WKY
control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4).

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Fig. 6. Effect of 2.0 mg/kg guanabenz (GUA) on expression of TNF-a in periodontium. (A) TNF-a-stained histological sections of periodontium at 8 weeks after ligature placement
(Scale bar ¼ 200 mm). (B) TNF-a positive cells in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (C)
TNF-a staining scores in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (D) TNF-a-stained his-
tological sections of periodontium at 12 weeks after ligature placement (Scale bar ¼ 200 mm). (E) TNF-a positive cells in periodontium at 12 weeks after ligature placement (WKY
control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (F) TNF-a staining scores in periodontium at 12 weeks after ligature placement (WKY control; n ¼ 4, WKY
GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4).

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Fig. 7. Effect of 2.0 mg/kg guanabenz (GUA) on expression of IL-1b in periodontium. (A) IL-1b-stained histological sections of periodontium at 8 weeks after ligature placement
(Scale bar ¼ 200 mm). (B) IL-1b positive cells in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (C) IL-
1b staining scores in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (D) IL-1b-stained histological
sections of periodontium at 12 weeks after ligature placement (Scale bar ¼ 200 mm). (E) IL-1b positive cells in periodontium at 12 weeks after ligature placement (WKY control;
n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (F) IL-1b staining scores in periodontium at 12 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4,
SHR control; n ¼ 4, SHR GUA; n ¼ 4).

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Fig. 8. Effect of 2.0 mg/kg guanabenz (GUA) on expression of IL-6 in periodontium. (A) IL-6-stained histological sections of periodontium at 8 weeks after ligature placement (Scale
bar ¼ 200 mm). (B) IL-6 positive cells in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (C) IL-6
staining scores in periodontium at 8 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (D) IL-6-stained histological
sections of periodontium at 12 weeks after ligature placement (Scale bar ¼ 200 mm). (E) IL-6 positive cells in periodontium at 12 weeks after ligature placement (WKY control; n ¼ 4,
WKY GUA; n ¼ 4, SHR control; n ¼ 4, SHR GUA; n ¼ 4). (F) IL-6 staining scores in periodontium at 12 weeks after ligature placement (WKY control; n ¼ 4, WKY GUA; n ¼ 4, SHR
control; n ¼ 4, SHR GUA; n ¼ 4).

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treated with guanabenz for 8 weeks compared with SHRs (Fig. 2B
and C). Administration of guanabenz for 12 weeks decreased the
numbers of TH positive cells and score of TH in SHR rats and score
of TH in WKY rats (Fig. 2E and F).
3.2. Suppression of periodontitis-decreased alveolar bone by
guanabenz
The ratio of the remaining alveolar bone in rats was measured
by mCT (Fig. 3B and D). The remaining alveolar bone in SHRs were
significantly lower than that in WKY rats at 8 and 12 weeks after
induction of periodontitis, and the decrease in SHRs was sup-
pressed by treated with 2.0 mg/kg guanabenz (Fig. 3A and C).
Administration of 2.0 mg/kg guanabenz for 12 weeks into WKY rats
increased the remaining alveolar bone (Fig. 3C). Of note, effect of
guanabenz on suppression of alveolar bone loss was dose-
dependent manner (Supplemental Fig. 1).
3.3. Suppression of periodontitis-lost attachment levels by
guanabenz
The attachment levels in periodontium were measured using HE
stained histological sections (Fig. 4B and D). Administration of
guanabenz for 8 and 12 weeks into SHRs and for 12 weeks into
WKY rats attenuated loss of attachment levels induced by the
placing of the wire (Fig. 4A and C).
3.4. Suppression of periodontitis-increased osteoclast numbers by
guanabenz
Osteoclast numbers were measured using TRAP-stained histo-
logical sections (Fig. 5A and D). Oc.N/BS and Oc.S/BS in SHRs were
significantly higher than those in WKY rats at 8 and 12 weeks after
induction of periodontitis, and the increase in SHRs was suppressed
by treated with guanabenz (Fig. 5B, C, E, and F). Administration of
guanabenz for 12 weeks into WKY rats also decreased Oc.S/BS
(Fig. 5F).
3.5. Suppression of periodontitis-induced pro-inflammatory
cytokines by guanabenz
Positive cells and scores of TNF-a, IL1-b, and IL-6 in perio-
dontium were measured using their immuno-stained histological
sections (Figs. 6e8). Although there was no difference of positive
cells and scores of TNF-a in the presence or absence of guanabenz
for 8 weeks (Fig. 6A, B, and C), they decreased in SHRs treated with
guanabenz for 12 weeks compared with SHRs (Fig. 6D, E, and F). The
score of IL-1b decreased in SHRs treated with guanabenz for 8
weeks compared with SHRs (Fig. 7A and C). Administration of
guanabenz for 12 weeks decreased the numbers of IL-1b positive
cells in both WKY rats and SHRs (Fig. 7D and E). The numbers of IL-6
positive cells and its score decreased in SHRs treated with guana-
benz for 8 weeks compared with SHRs (Fig. 8A and B). Guanabenz
decreased the numbers of IL-6 positive cells and score of IL-6 in
both WKY rats and SHRs (Fig. 8D, E and F).
4. Discussion
This study demonstrated that administration of a2-AR agonist
guanabenz reduces sympathetic activity and attenuates periodon-
titis in rats. Residual alveolar bone in SHRs decreased compared to
WKY rats. Administration of guanabenz suppressed loss of alveolar
bone and attachment levels in SHRs. Its administration also
reduced osteoclast numbers in SHRs. Furthermore, pro-
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Journal of Pharmacological Sciences 147 (2021) 294e304
inflammatory cytokines in periodontium were decreased in SHRs
treated with guanabenz.
It was reported that administration of nonselective b-adrenergic
receptor (b-AR) antagonist propranolol and b2-AR antagonist
butoxamine suppressed alveolar bone resorption in a rat of peri-
odontal disease.11,13,14 This improvement may be caused by the
suppression of bone resorption based on acting of propranolol and
butoxamine on b2-AR expressed in alveolar bone for the following
reasons. First, b2-AR is expressed in osteoclasts and pre-osteo-
clasts,15,16 and b2-AR agonist promotes the differentiation of human
pre-osteoclasts.15 Second, b-AR agonist enhances bone resorbing
activity in mature osteoclasts.17 Third, b2-AR expressed in osteo-
blasts promotes osteoclast formation through increase of produc-
tion of RANKL.18 In the present study, a2-AR agonist guanabenz was
used for treatment of periodontitis in rats having high sympathetic
activity. Guanabenz binds to a2-ARs on presynaptic membranes,
leading to the suppression of noradrenaline secretion from sym-
pathetic nerves.19 Therefore, guanabenz attenuates sympathetic
activity, resulting in the suppression of osteoclast formation.
It has been known that periodontitis is exacerbated by endo-
plasmic reticulum (ER) stress. ER stress causes alveolar bone
resorption and is involved in immune response.20,21 Guanabenz is
known to attenuate endoplasmic reticulum stress through inhibi-
tion of de-phosphorylation of eukaryotic translational initiation
factor 2a (eIF2a).22 eIF2a-mediated signaling has been known to be
involved in osteoclastogenesis, and guanabenz decreases osteoclast
formation via suppression of cytoplasmic 1 (NFATc1).23,24 Further-
more, guanabenz was reported to downregulate inflammatory re-
sponses.25 For instance, guanabenz suppresses pro-inflammatory
cytokines induced by activation of macrophages and T-cells. a2-
ARs are expressed in pre-osteoclasts and osteoclasts.16,26 Guana-
benz which is selective a2-AR agonist, was reported to suppress
osteoclastogenesis markers nuclear factor of activated T-cells,
NFATc1, TRAP, and cathepsin K, leading to attenuation of osteoclast
formation.26 Our study is consistent with these previous studies
that guanabenz decreased osteoclast numbers and pro-
inflammatory cytokines.
In summary, this study showed the administration of a2-AR
agonist guanabenz attenuates periodontitis through suppression of
alveolar bone resorption, loss of the attachment levels, pro-
inflammatory cytokines. Suppression of sympathetic activity by
guanabenz may contribute to deterioration of periodontitis. Gua-
nabenz also may suppress alveolar bone resorption through
attenuation of ER stress and action of a2-AR expressed in pre-
osteoclasts.
Author contributions
R.M.: Writing the original draft, Data curation, Formal analysis,
Methodology, Visualization, K.M.: Writing the original draft,
Conceptualization, Methodology, Project administration, K.H.:
Writing the original draft, Conceptualization, Project administra-
tion, T.S.: Writing the original draft, Conceptualization, Data cura-
tion, Formal analysis, Methodology, Project administration, A.T.
(Atsushi Takeguchi): Data curation, Methodology, Revising the draft
critically, M.T.: Methodology, Revising the draft critically, A.T.
(Akifumi Togari): Conceptualization, Revising the draft critically,
S.G.: Conceptualization, Revising the draft critically, Funding
acquisition, Supervision. All authors read and approved the final
manuscript.
Declaration of competing interest
The authors declare no conflict of interest.
R. Muramatsu, T. Sato, K. Hamamura et al. Journal of Pharmacological Sciences 147 (2021) 294e304

Acknowledgment 11. Takeguchi A, Miyazawa K, Sato T, et al. Effects of a b2-adrenergic receptor

blocker on experimental periodontitis in spontaneously hypertensive rats. Life
Sci. 2021;277:119593. https://doi.org/10.1016/j.lfs.2021.119593.
This work was supported by JSPS KAKENHI Grant Number 12. Rogers JE, Li F, Coatney DD, et al. Actinobacillus actinomycetemcomitans
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13. Okada Y, Hamada N, Kim Y, et al. Blockade of sympathetic b-receptors inhibits
Appendix A. Supplementary data Porphyromonas gingivalis-induced alveolar bone loss in an experimental rat
periodontitis model. Arch Oral Biol. 2010;55(7):502e508. https://doi.org/
Supplementary data to this article can be found online at 10.1016/j.archoralbio.2010.04.002.
14. Rodrigues WF, Madeira MFM, da Silva TA, et al. Low dose of propranolol down-
https://doi.org/10.1016/j.jphs.2021.08.003. modulates bone resorption by inhibiting inflammation and osteoclast differ-
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j.1476-5381.2011.01686.x.
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