Biochemical and Biophysical Research Communications 645 (2023) 55e60

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Anti-inflammatory and analgesic effect of Forsythiaside B on complete

Freund's adjuvant-induced inflammatory pain in mice
Yu-ting Wang, Kai Lu, Dan-dan Yao, Shu-xia Zhang, Gang Chen*
Department of Anesthesiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China

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

Article history: Chronic pain is frequently reported in clinical practice. Therefore, it is important to identify effective
Received 31 December 2022 therapy to relieve pain. In this work, we selected Forsythoside B (FB), a phenylethanoid glycoside isolated
Accepted 13 January 2023 from Forsythia suspensa (Thunb.) Vahl, to evaluate its effect in modulating inflammatory pain induced by
Available online 13 January 2023
complete Freund's adjuvant (CFA) and the involved mechanisms. We discovered that FB could attenuate
inflammatory pain triggered by CFA injection and exert anti-anxiety effects. In detail, proinflammatory
Keywords:
cytokines, consisting of IL-6 and TNF-a, were decreased after FB administration in the CFA-injected mice.
Forsythiaside B
Furthermore, the FB application ameliorated the activation of ionized calcium-binding adaptor molecule
Complete Freund's adjuvant
Inflammation pain
1 (Iba-1) and glial fibrillary acidic protein (GFAP), the microglia and astrocytes markers respectively.
Proinflammatory cytokines Therefore, our findings indicate that FB could be a promising treatment for chronic inflammatory pain.
© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction and/or indirectly responsible for pain [6].

Pain is an unpleasant sensory as well as an emotional experience
that serves an important physiological function in the human body
by alerting the body to the possibility of tissue damage. At least
one-third of the global population suffers from pain and costs bil-
lions of dollars annually [1]. Chronic pain is often part of the post-
intensive care syndrome in patients infected with COVID-19 [2].
The incidence of chronic pain after the ICU is expected to range
between 14% and 77%, depending on a variety of conditions [3]. The
pain appears to be a major consideration in returning to work and
maintaining the quality of life at least 5 years after discharge [4].
Furthermore, people who have did survive the critical illness of
COVID-19 are more likely to be vulnerable to chronic pain [2]. Thus,
there is an urgent need for clinical drug therapy to relieve chronic
pain. Nonetheless, the necessity for the effective treatment of
chronic pain remains unmet. Studies suggest that glial cells of the
spinal cord (such as microglia and astrocytes) are activated and
exacerbated pain under chronic inflammatory pain. Moreover, the
activated glial cells promote the secretion of proinflammatory cy-
tokines [5]. The research found that the immune system appears to
promote the establishment of pain following actual or potential
tissue damage. Immune cells release cytokines that are directly
* Corresponding author.
E-mail address: chengang120@zju.edu.cn (G. Chen).
Pain is usually treated with anti-inflammatories and opioids,
however, these medications may result in toxicities and seriously
side effects, thereby influencing both long-term use and dose level
of possible analgesia [7]. Since ancient times, natural products,
specifically those extracted from herbs promote the development
of modern medical therapy. Natural products as medication target
specific inflammation and pain mediators with limited or no
adverse side effects. In recent years, the search for novel natural
products that relieve pain has gained a research premium. Here, we
have selected Forsythoside B (FB), a phenylethanoid glycoside iso-
lated from Forsythia suspensa (Thunb.) Vahl, to estimate its effect
on inflammatory pain treatment. Research indicated that FB has a
strong neuroprotective property with a beneficial therapeutic time
window, reducing cerebral ischemia and reperfusion injury degree,
attenuating blood-brain barrier breakdown by suppressing in-
flammatory response [8]. Moreover, FB could attenuate neuro-
inflammation and memory impairment by suppressing the NF-kB
signaling pathway in Alzheimer's disease [9]. All these results
indicated that FB plays an important role in the nervous system.
Nevertheless, whether FB promotes chronic pain treatment re-
mains unclear.
As such, we analyzed the roles of FB in the treatment of chronic
inflammatory pain and the related mechanisms. FB attenuated
chronic pain and had anxiolytic-like effects on CFA-injected mice
models. After FB administration, we noted a drop in proinflammatory
cytokines, consisting of IL-6 and TNF-a. Furthermore, the FB

https://doi.org/10.1016/j.bbrc.2023.01.036
0006-291X/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Y.-t. Wang, K. Lu, D.-d. Yao et al.
application ameliorated the activation of Iba-1 and GFAP, the micro-
glia and astrocytes markers respectively. Therefore, these findings
suggest that FB could be a candidate therapy for chronic inflammatory
pain.
2. Materials and methods
2.1. Animals
All animal experiments were performed in accordance with the
guidelines approved by the Animal Care and Use Committee of the
Zhejiang University and the National Institutes of Health Guide for
the Care and Use of Laboratory Animals. Healthy male C57BL/6 mice
(8e10 weeks, 23e25 g) were used for the experiments. Mice were
housed in temperature-controlled (25 ± 1
C), humidity-controlled
(60 ± 5%), and a 12-h light/dark schedule (from 07:00 to 19:00)
rooms with enough food and freshwater available ad libitum. The
animals should adapt to the test room for about a week before the
behavioral tests and acclimate to the testing environment for at
least 30 min before each test. The lowest number of mice was used
and all experiments were conducted humanely.
2.2. Experimental designs and FB treatment
To generate an inflammatory pain model, we subcutaneously
inoculated 10 mL of 50% complete Freund's adjuvant (CFA; Sigma)
into the left hind paw. Consistently, the control animals were given
the same volume of saline in the hind paw. FB (HY-N0029; MCE)
was dissolved in saline and intraperitoneally injected into the
experimental groups. Afterward, animals received either FB (10 mg/
kg) or a similar volume of saline (Vehicle) 3 days after CFA insult for
analgesic analysis and anxiety-like behavior examinations. All
behavioral tests were performed at a fixed time on the test days and
mice were acclimatized to the testing room for 30 min before each
behavioral test. After behavioral tests, animals were sacrificed after
Fig. 1. The involvement of FB in CFA-triggered thermal hyperalgesia and mechanical allodynia. (A) FB chemical structure; (B) CFA-injected lead to left hind paw swelling, compared
to saline injection; (C) Mechanical allodynia and (D) Thermal hyperalgesia of CFA-injected mice treated with FB (10 mg/kg i.p). Data are expressed as the mean ± SEM. n ¼ 10 per
group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, CFA þ Veh vs CFA þ FB.
PWT: Paw withdrawal threshold; PWL: paw withdrawal latency; BF: Before the test.
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Biochemical and Biophysical Research Communications 645 (2023) 55e60
3 consecutive days of FB (10 mg/kg) application to obtain spinal
cord L5 samples to examine molecular events.
2.3. Mechanical allodynia
The paw withdrawal threshold (PWT) was recorded using the
von Frey filament to measure mechanical allodynia as per the up-
down algorithm documented by Chaplan [10]. Before the experi-
ment, the mice were individually placed in an opaque cage with a
raised mesh grid for half an hour. Von Frey filament was vertically
exerted to the plantar surface of the left hind paw until it bent. The
filament of the subsequent force was greater in the absence of a
withdrawal response, while a smaller force was required for a
positive response. The mechanical stimulus producing a 50%
withdrawal response was calculated and considered the PWT.
2.4. Thermal hyperalgesia (Hargreaves test)
The paw withdrawal latency (PWL) was done as documented in
the previous report [11]. Thermal stimulation intensity was modified
to obtain approximately 8e12 s average thermal withdrawal
threshold in non-inflamed animals. The planter analgesia instrument
(Ugo Basile, Italy) was utilized to determine thermal nociceptive
responses. Before each testing, mice were allowed to adapt to the
testing cage for at least 30 min. Thereafter, we used the radiant heat
source to stimulate the middle of the plantar surface of each hind
paw. The time between the heat source onset and foot withdrawal
was set as the PWL. Every trial was repeated three times at 5-min
intervals. Notably, a 20 s cutoff was set to avoid damage to the paw.
2.5. Open field test
The open-field test (OFT) was done as documented previously
[12]. The open field was a 45  45  45 cm3 white plastic box with a
camera fixed above. The mice were placed separately in the middle
Y.-t. Wang, K. Lu, D.-d. Yao et al. Biochemical and Biophysical Research Communications 645 (2023) 55e60

of the box and had a free movement for 20 min. Of note, the
movement and exploratory behaviors were analyzed with the ANY-
maze software (Stoelting, Wood Dale, USA).
2.6. Elevated plus maze test (EPM)
The elevated plus maze (EPM) test was carried out as detailed
previously [12]. The EPM was 60 cm above the ground comprising
two enclosed arms with 15 cm high walls (30  5 cm) and two open
arms without walls. Each mouse was allowed to explore the maze
for 5 min. Before each test, the maze was thoroughly cleaned with
75% ethanol. The outcomes were recorded using a camera, before
analysis using the ANY-maze software (Stoelting, Wood Dale, USA).
2.7. Western blot analysis
Mice were anesthetized with 2% sevoflurane to collect the spinal
cord L5 segments, and then homogenized in RIPA lysis buffer (HY-
K1001, MCE), containing 1% PMSF (HY-B0496, MCE). BCA assays
were conducted to quantify the extracted protein concentration.
For each sample, 30 mg of protein were fractionated on an SDS-PAGE
gel, then blotted onto PVDF membranes at 4
C. Blots were blocked
in 5% non-fat milk in TBST for 1 h at room temperature and then
inoculated overnight with the following primary antibody: TNF-a
(1:1000; A11534, ABclonal), IL-6 (1:1000; ab2594341, Abcam), IL-
1b (1:1000; A16288, ABclonal) and b-actin (1:3000; AC026,
ABclonal) at 4
C, respectively. And then the blots were inoculated
with HRP-linked secondary antibody (1:5000; HA1001, HuaBio) for
2 h. The molecular weight of the protein was labeled with a color

Fig. 2. The application of FB altered the anxiety-like behaviors in the elevated plus-maze (AeD) and open field test (EeG). (A) Representative tracks of control and CFA-injected mice
treated with FB in EPM; (B) The number of open arms entries in the EPM; (C) CFA þ veh group spent less time in open arms unlike the other three groups in the EPM; (D) There were
no remarkable differences in the total distance in the EPM; (E) Representative traces of control and CFA-injected mice treated with FB in OFT. (F) FB administration increased the
distance moved in the central squares compared to CFA-injected mice. (G) There were no remarkable differences in the total distance in the OFT. Data are given as the mean ± SEM.
n ¼ 8 per group, *P < 0.05, CFA þ Veh vs CFA þ FB.

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Y.-t. Wang, K. Lu, D.-d. Yao et al.
mixed protein marker (RM19001, ABclonal). Signals were detected
using the ECL luminescence Reagent (Absin). All the findings were
analyzed and quantified using ImageJ (NIH, Bethesda, MD).
2.8. Immunofluorescence
All mice were anesthetized by 1% sodium pentobarbital, then
intracardially perfused with PBS, then by 4% PFA. The spinal cord L5
was harvested and fixed overnight in PFA at 4
C. Tissues were
sequentially transferred to 10%, 15%, and 30% sucrose until satu-
rated. Subsequently, the spinal cord was sectioned into a coronal
section (30-mm) at 20
C in a cryostat (NX50, Thermo). All slices
were blocked for 1 h with 5% normal donkey serum in 0.3% Triton
X-100 (Sigma) in PBS buffer, and then incubated overnight with
primary antibody (Iba-1, 1:50; 17198, CST; GFAP, 1:200; 3670, CST)
at 4
C. After rewarming up, slices were incubated at room tem-
perature for 2 h with the suitable secondary antibody (donkey anti-
rabbit, ab150064 and donkey anti-mouse, ab150105, Abcam).
Nuclei were stained with DAPI (ab104139, Abcam) and the images
were obtained under a fluorescence microscope (VS120, Olympus).
2.9. Data analyses
All data were given as mean ± SEM and analyzed in Graph-pad
Prism 9. Comparison of values between different experimental
groups was performed using one-way or two-way repeat measure
ANOVA; Sidak's test or Tukey's test was adopted for post hoc
comparisons. P < 0.05 signified statistical significance.
3. Results
3.1. Analgesic effects of FB on thermal hypersensitivity and
mechanical allodynia on CFA-induced inflammatory pain mice
model
Inflammatory pain is an extreme pain-sensitive reaction to tis-
sue damage and inflammation resulting from the release of sensi-
tizing inflammatory mediators, which reduce the threshold of
nociceptors dominating the inflamed tissue. FB (Fig. 1A), a phe-
nylethanoid glycoside isolated from Forsythia suspensa (Thunb.)
Fig. 3. The treatment of FB reduced the up-regulation of TNF-a (A) and IL-6 (B) but not IL-1b (C) in the spinal cord after CFA injection. Data are provided as the mean ± SEM. n ¼ 3
per group, *P < 0.05, CFA þ Veh vs CFA þ FB.
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Biochemical and Biophysical Research Communications 645 (2023) 55e60
Vahl, is widely used in nerve-related diseases [8,9]. To investigate
the effect of FB on pain, we established a model of mice on in-
flammatory pain-triggered by CFA. As we expected, CFA injection
led to apparent swelling in the left hind paw, compared to saline
injection (Fig. 1B). Meanwhile, CFA treatment caused mechanical
allodynia and thermal hypersensitivity in the left hind paw. The
PWT and PWL of CFA-injected mice were reduced in contrast with
the saline-treated group (Fig. 1C and D). Administration of FB
(10 mg/kg) could significantly attenuate mechanical allodynia
(Fig. 1C) and thermal hypersensitivity (Fig. 1D) in mice injected
with CFA. Our findings revealed that FB relieved inflammatory pain
in CFA-injected mice.
3.2. Anxiolytic effects of FB on mice with inflammatory pain-
triggered by CFA
Chronic pain and psychiatric disorders including anxiety
frequently co-occur in clinical settings. Subsequently, we assessed
whether CFA treatment results in anxiety-like behaviors and
defined the effects of FB application. Here, we employed the open
field test (OFT) and elevated plus maze (EPM) to analysis anxiety-
like behaviors following FB treatment. The results found that CFA
elicited anxiety-like behaviors in the EPM, as evidenced by a
decrease in the number of open arms entries (Fig. 2A and B), and a
shorter duration spent in open arms relative to the control group
(Fig. 2C). In the OFT, CFA administration decreased the distance
stayed in the central area in contrast with the control ones (Fig. 2E
and F). However, the total distance of the CFA injection group had
no significant difference from others (Fig. 2G). The use of FB pro-
longed the period spent and frequency of open arms entries in the
EPM (Fig. 2B and C) and distance moved within central areas of the
OFT (Fig. 2F). No significant differences were observed in the total
distance of each group in the EPM and OFT (Fig. 2D and G). Our data
revealed that FB therapy exerted effective roles in anti-anxiety on
CFA-injected mice but with no effect on locomotion activity.
3.3. FB decreases CFA-induced proinflammatory cytokines levels in
the spinal cord
To determine whether CFA injection activates the inflammatory
Y.-t. Wang, K. Lu, D.-d. Yao et al. Biochemical and Biophysical Research Communications 645 (2023) 55e60

Fig. 4. The application of FB decreased the activation of microglia, as well as the astrocytes in the spinal cord after CFA injection.
Iba-1 (A) and GFAP (B) immunofluorescence, as the marker of microglia and astrocytes respectively, was increased in the CFA þ Veh group, while FB reduced the immunofluo-
rescence intensity of Iba-1 and GFAP. Immunofluorescence intensity of Iba-1 (C) and GFAP (D). Data are given as the mean ± SEM. n ¼ 3 per group, *P < 0.05, CFA þ Veh vs CFA þ FB.

signaling pathway, we evaluated levels of proinflammatory cyto-
kines. Using spinal cord L5 specimen, in Western blotting demon-
strated that CFA injection upregulated levels of IL-6 (Fig. 3A), TNF-a
(Fig. 3B), and IL-1b (Fig. 3C) proteins. FB treatment (10 mg/kg)
suppressed the protein contents of IL-6 (Fig. 3A) and TNF-a (Fig. 3B)
in contrast with the saline-treated group. Nevertheless, IL-1b
expression was not altered (Fig. 3C) following FB administration. All
results demonstrated that FB treatment exerts anti-inflammation
effects and alleviates inflammatory pain induced by CFA.
3.4. FB attenuates CFA-triggered microglia and astrocyte activation
Microglia are the resident macrophage-like cells of the central
nervous system. Astrocytes are abundant in the central nervous
system, accounting for 40e50% of all glial cells [13]. Under normal
circumstances, astrocytes and microglia are relatively static. How-
ever, they are activated and promote the pathogenesis of neuro-
logical disorders when faced with injury or under disease condition
[14]. Previous reports revealed that microglia and astrocytes
contribute to the development of chronic pain [15]. Sections of the
spinal cord L5 were harvested and subjected to immunostaining
against Iba-1 and GFAP to assess the activation of microglia and
astrocytes, respectively. As shown in (Fig. 4A) and (Fig. 4B), CFA-
injected mice improved the fluorescence intensity of Iba-1 and
GFAP, but not in the control groups. This indicates that CFA injection
promotes the activation of microglia and astrocytes. Moreover,
intraperitoneally administration of FB (10 mg/kg) significantly
reduced Iba-1 (Fig. 4C) and GFAP expression (Fig. 4D). This implies
that FB decreased the activation of immune cell in the spinal cord
after CFA injection to exert analgesic effects.

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Y.-t. Wang, K. Lu, D.-d. Yao et al.
4. Discussion
Herein, we explored the analgesic role of FB on inflammatory
pain-induced by CFA treatment, we discovered that FB attenuates
the mechanical allodynia and thermal hypersensitivity. Meanwhile,
FB (10 mg/kg) administration prolonged the duration spent and the
entries for open arms in EPM, as well as the distance moved in
central areas in the OFT. This illustrates that FB exerts analgesic and
anti-anxiety effects.
Accumulating evidence suggests that TNF-a and IL-6 are asso-
ciated with both neuropathic [16,17] and inflammatory [18] pain
conditions. Similarly, hindpaw CFA injection significantly increased
IL-1b, TNF-a, and IL-6 concentration in spinal cord tissues [19].
Recently, it has been discovered that the effects of spinal IL-6 and
TNF-a on neuronal excitability are interdependent, as the sensiti-
zation of spinal TNF-a could be blocked by disrupting the spinal
transduction of IL-6 [20,21]. In this work, our findings revealed that
inflammatory pain caused an increase in TNF-a, IL-1b, and IL-6
levels following CFA injection. Nonetheless, FB (10 mg/kg) poten-
tially inhibits the generation of IL-6 and TNF-a in the spinal cord.
Altogether, our results revealed that FB (10 mg/kg) inhibits
inflammation by downregulating the level of TNF-a and IL-6 in the
spinal cord and alleviates CFA-induced inflammatory pain.
Inflammatory pain is related to the activation of glial cells and
central sensitization. Activated spinal glial cells has been linked to
the development and maintenance of inflammatory pain [22]. The
spinal glial cells are actively involved in pain signaling transmission
pathways, particularly at the spinal cord dorsal horn levels [23,24].
Furthermore, astrocyte and microglia activation in the spinal cord
dorsal horn was significant in the CFA-injected mice. In the region
of the spinal dorsal horn, microglia increased and appeared hy-
pertrophied soma and thick retracted processes, whereas the
activated astrocytes exhibited hypertrophy [25,26]. Consistently,
our findings revealed that CFA treatment upregulated the expres-
sion of Iba-1 and GFAP in activated astrocytes and microglia,
respectively, and FB decreased the fluorescence intensity of Iba-1
and GFAP in the spinal cord. These findings show that FB sup-
presses the activation of microglia and astrocytes, at the same time
relieving inflammatory pain-triggered by CFA.
Data availability
The data used to support the findings of this study are included
in the article.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgment
This research was funded by the National Natural Science
Foundation of China (No.82171176 and No.82001424), and the Key
Program of the Natural Science Foundation of Zhejiang, China (No.
LZ19H090003).
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