Invest Clin 66(1): 4 - 15, 2025 https://doi.org/10.54817/IC.v66n1a01
Corresponding author: Waiping Zhou. Department of Pain, Wuhan Fourth Hospital, No. 473 Hanzheng Street, Wu-
han, 430030, China. Tel.: 86-027-83353510. Email: zh_wp3442@126.com
Phillygenin reduced neuropathic pain by
inhibiting the rats’ TLR4/MyD88/NF-κB
pathway.
Lei Hua, Waiping Zhou, Mengjie Li and Rongchun Li
Department of Pain, Wuhan Fourth Hospital, Wuhan, China.
Keywords: neuropathic pain; phillygenin; proinflammatory cytokines; TLR4, MyD88.
Abstract. To elucidate the effects of phillygenin (PHI) and the potential
mechanism on TLR4 and MyD88/NF-κB signalling in neuropathic pain in an-
imal studies, chronic constriction injury (CCI) models were constructed for
neuropathic pain induction using male Sprague-Dawley rats. PHI (20 mg/kg)
was delivered through intragastric administration. Von Frey and Hargreaves
tests were implemented to determine the 50% paw-withdrawal threshold (PWT)
and paw-withdrawal latency (PWL). A nitric oxide (NO) assay was used for NO
level detection, and an ELISA assay was employed to measure the expression of
proinflammatory cytokines. Western blotting and RT-qPCR were conducted for
protein and mRNA level detection. Treatment with PHI significantly enhanced
50% of PWT and PWL. PHI significantly decreased the levels of NO and reduced
the levels of TNF-α, IL-1β, and IL-6. PHI also downregulated TLR4 and MyD88
expressions and inhibited the phosphorylation of NF-κB. PHI ameliorated in-
flammatory status and alleviated neuropathic pain in CCI rats, targeting TLR4
and suppressing MyD88/NF-κB signalling.
Phillygenin reduced neuropathic pain by inhibiting the rats’ TLR4/MyD88/NF-κB pathway 5
Vol. 66(1): 4 - 15, 2025
Filigenina reduce el dolor neuropático en ratas inhibiendo
la vía del TLR4/MyD88/NF-κB.
Invest Clin 2025; 66 (1): 4 – 15
Palabras clave: dolor neuropático, filigenina, citocinas proinflamatorias, TLR4, MyD88.
Resumen. Para dilucidar los efectos de la filigenina (PHI) y el mecanismo
potencial en la señalización de TLR4 y MyD88/NF-κB en el dolor neuropático
en estudios con animales, se construyeron modelos de lesión por constricción
crónica (CCI) para la inducción del dolor neuropático utilizando ratas Sprague-
Dawley macho. La PHI (20 mg/kg) se administró por vía intragástrica. Se im-
plementaron las pruebas de von Frey y Hargreaves para determinar el 50% del
umbral de retracción de la pata (PWT) y la latencia de retracción de la pata
(PWL). Se utilizó un ensayo de óxido nítrico (NO) para la detección del nivel de
NO, y se empleó un ensayo ELISA para medir la expresión de citocinas proinfla-
matorias. Se realizaron transferencia Western y RT-qPCR para la detección del
nivel de proteína y ARNm. El tratamiento con PHI mejoró significativamente el
50% del PWT y la PWL. La PHI disminuyó significativamente los niveles de NO
y redujo los niveles de TNF-α, IL-1β e IL-6. PHI también disminuyó la expresión
de TLR4 y MyD88 e inhibió la fosforilación de NF-κB. PHI mejoró el estado in-
flamatorio y alivió el dolor neuropático en ratas con CCI, actuando sobre TLR4
y suprimiendo la señalización MyD88/NF-κB.
Received: 27-09-2024 Accepted: 18-01-2025
INTRODUCTION
Neuropathic pain usually has a chronic
progression and affects a significant number
of individuals globally, leading to physical,
emotional, and economic burdens 1. It arises
due to a dysfunction in the nervous system,
causing abnormal signalling and processing
of pain signals 2. Patients with neuropathic
pain experience various symptoms such as
shooting or burning pain, numbness, tin-
gling, and hypersensitivity to stimuli 3. Vari-
ous factors can cause neuropathic pain,
including injury, infection, diabetes, or che-
motherapy. This condition causes a substan-
tial burden to the patients, and the current
treatments for neuropathic pain are often
limited in efficacy and associated with ad-
verse effects 4. Hence, the development of
alternative treatment agents for the clini-
cal therapy of neuropathic pain is urgently
needed.
Toll-like receptor 4 (TLR4) belongs to
the TLR family that initiates the innate im-
mune response 5. TLR4 has been found in
various cells, including immune cells and
neurons. It was reported that TLR4 could
be activated by lipopolysaccharides (LPS)
and damage-associated molecular patterns
(DAMPs) 6. Activation of TLR4 leads to the
upregulation of myeloid differentiation fac-
tor 88 (MyD88). Moreover, the activated
TLR4 could promote the nuclear factor-κB
(NF-κB) pathway, resulting in the upregu-
lation of various inflammatory factors and
chemokine production 7. TLR4 participates
in the occurrence and progression of neu-
ropathic pain. Publications have shown that
6 Hua et al.
Investigación Clínica 66(1): 2025
TLR4 increases in the spinal cord in animal
models of neuropathic pain 8, and TLR4-
deficient mice exhibit reduced pain behav-
ior in various neuropathic pain models 9.
Therefore, implementing TLR4 antagonists
or inhibitors might have analgesic effects in
neuropathic pain treatment.
Phillygenin (PHI) is a bioactive com-
pound found in Forsythia suspensa, tradi-
tionally used for treating inflammatory and
infectious diseases 10. PHI exhibits a wide
range of bioactive capabilities, including im-
proving the inflammatory status, preventing
cancer progression, and protecting against
neuron injury 11. The modulation of differ-
ent cellular processes, such as NF-κB-related
inflammation and PI3K/Akt-mediated tu-
mor cell proliferation, accounts for the di-
verse properties of the substance 12. Recent
research indicates that PHI could suppress
TLR4 activation, further inhibiting the NF-
κB signalling pathway downstream, suggest-
ing its potential as a treatment target for the
therapy of inflammatory and neuropathic
conditions 13. However, the mechanism of
action underlying the potential use of PHI in
treating neuropathic pain remains unclear.
This study explored the potential effects
of PHI in neuropathic pain progression and
its underlying mechanism. We implemented
rat chronic constriction injury (CCI) models
for in vivo studies on neuropathic pain. We
hypothesized that treatment with PHI could
significantly inhibit TLR4 expression and
suppress MyD88/NF-κB signalling, reducing
the neuropathic behavior in CCI rats. Our
study aimed to provide new insights into the
potential use of PHI for the clinical therapy
of neuropathic pain.
METHODS
Animal models
Male Sprague-Dawley rats (210-250 g)
were purchased from the Hunan Sileck Jing-
da Experimental Animal Co., Ltd. All rats
were kept in individual cages with a 12 h
light/dark cycle, constant temperature (22
± 2°C) and humidity (50 ± 10%), and food
and water were freely available. Both male
and female rats were included in the sample.
The final allocation of 40 rats comprised an
equal representation of both sexes to ensure
a comprehensive assessment of the experi-
mental outcomes. The acclimatization of
animals (at least seven days) was performed
before they underwent various treatments
and experiments. The Animal Care and Use
Committee of the Wuhan Fourth Hospital
reviewed and authorized all animal experi-
ments.
The CCI model was implemented as per
a previous study 14. During surgery, the rats
received continuous anesthesia with 2% iso-
flurane in oxygen, and the exposition of the
left sciatic nerve was made at the mid-thigh
level. Subsequently, 4-0 chromic gut was used
as loose ligatures for making ties around the
nerve with a 1-mm interval. The tightened
ligatures were further made to produce a
mild nerve constriction, which induced the
CCI condition. Layers of 4-0 silk sutures were
used to close the wound, and all rats were
then kept in a warm environment for recov-
ery. Rats that exhibited motor dysfunction
or showed signs of infection were excluded
from the study. A total of 40 rats were then
randomly allocated into four groups (10 rats
in each, according to previous studies)15, 16:
(1) Sham group: rats underwent the same
surgical process as the CCI group, but with-
out performing the nerve ligation; (2) CCI
group, where CCI surgery was implemented;
(3) CCI+PHI group, where CCI surgery was
implemented and rats received daily intra-
gastric administration of PHI (20 mg/kg, us-
ing DMSO as vehicle, accordingly to previous
studies ) 11, 17 for 14 consecutive days. PHI was
obtained from a commercial supplier, spe-
cifically the Shanghai Yuanye Biotechnology
Co., Ltd., which provided a high-purity grade
of PHI suitable for our experimental needs;
(4) CCI+NC group, where CCI surgery was
implemented and rats were treated with the
same volume of normal saline. Once the CCI
model was established and the rats had re-
Phillygenin reduced neuropathic pain by inhibiting the rats’ TLR4/MyD88/NF-κB pathway 7
Vol. 66(1): 4 - 15, 2025
covered from the anesthesia, they received
daily intragastric administration of PHI (20
mg/kg) or the same volume of normal saline
in the CCI+NC group for 14 consecutive
days. Researchers performing data analysis
and histological assessments were blinded to
the treatment groups. After model establish-
ment, the rats were euthanized, and their
spinal cords were collected for subsequent
experiments. Researchers performing data
analysis and histological assessments were
blinded to the treatment groups.
Von Frey Test
Von Frey assay (Ugo Basile, Italy) was
used to assess mechanical allodynia before
surgery and on postoperative days 0, 2, 4, 6,
8 10, 12 and 14. All rats were maintained
in boxes that contained a wire mesh bottom
and acclimatized for 30 minutes. After ac-
climation, the von Frey test was conducted
on the mid-plantar surface of the ipsilateral
and contralateral hind paw. The cutoff was
set at 26 g, and the data were collected au-
tomatically. Pain-like responses, such as an
abrupt withdrawal of the paw, licking, or vig-
orously shaking, were noted. The 50% paw
withdrawal threshold (PWT) was determined
through the up-down approach as described
in a previous study 18.
Hargreaves Test
The Hargreaves test was performed on
rats before surgery and on postoperative
days 0, 2, 4, 6, 8, 10, 12, and 14 to assess
thermal hyperalgesia. The Plantar Test Ap-
paratus (Ugo Basile, Italy) was obtained for
this purpose. A total of 30 minutes of accli-
matization were first made. A radiant heat
source treated the mid-plantar surface of the
right hind paw, and the paw withdrawal la-
tency (PWL) was recorded. A 20-second cut-
off threshold was set for the heat stimulation
test to prevent injury. A decrease in PWL
was considered a sign of heat hyperalgesia.
The behavioral tests were accomplished by
experimenters blinded to the group assign-
ments 18.
Nitric oxide (NO) assay
The expression of NO was detected us-
ing a 2,3-diaminonaphthalene (DAN) assay
kit. First, the tissue samples were first isolat-
ed and homogenized in PBS at 4°C and then
centrifuged at 4°C for 15 minutes (12,000
rpm). Next, the supernatant was obtained,
and 50 μL of the sample and 50 μL of DAN
solution (5 mM in 0.62 M HCl) were added
and maintained for 15 minutes at room tem-
perature. Afterward, NaOH (100 μL, 2 M)
was used to terminate the reaction. The in-
tensity was measured using a fluorescence
spectrophotometer (excitation wavelength:
365 nm, emission wavelength: 450 nm).
ELISA assay
Following the manufacturer’s pro-
tocol, TNF-α, IL-1β, and IL-6 expressions
were measured using a commercial ELISA
kit (R&D Systems, USA). Briefly, the spinal
cord tissue samples were obtained as de-
scribed above. Then, 200 μL of detection
reagent was added and maintained for 2
h, after which 200 μL of substrate solution
was added and maintained for 1 h in the
dark. A 50 μL stop solution was added to
terminate the reaction, and the absorbance
was monitored through a microplate reader
(450 nm).
RT-qPCR
TRIzol reagent (Invitrogen, USA) was
obtained to isolate total RNA from the spinal
cord tissues, following the manufacturer´s
protocol. Then, total RNA was reverse tran-
scribed into cDNA using a PrimeScript RT
Reagent Kit (Takara Bio, Japan). The ex-
pression levels of mRNAs were quantified
using an SYBR Premix kit (Takara Bio, Ja-
pan) and a StepOnePlus Real-Time PCR sys-
tem (Applied Biosystems, USA). The mRNA
levels were normalized to that of GAPDH,
and the calculation was performed as per
the 2-ΔΔCt method. The primers used are list-
ed as follows:
8 Hua et al.
Investigación Clínica 66(1): 2025
TLR4-F: 5’- GAATGCTAAGGTTGGCAC
TCTC -3’
TLR4-R: 5’- CTCAGGCAGGAAAGGAA-
CAATG -3’
MyD88-F: 5’- GCTGAGAGGAAGAGTTC-
TAC -3’
MyD88-R: 5’- CAGTGATAACCCTGGAC-
TAC -3’
NF-κB: 5’- AGACCTGGAGCAAGCCATT
AG -3’
NF-κB: 5’- CGGACCGCATTCAAGTCAT
AG -3’
GAPDH: 5’- TTCAACGGCACAGTCAAG
G -3’
GAPDH: 5’- GTCTTCTGAGTGGCAGT-
GATG -3’
Western blotting
For Western blot analysis, spinal cord
tissue samples underwent homogenization
in RIPA buffer (Roche, Switzerland), which
was added to the protease inhibitor cocktail.
Then, quantification was performed via BCA
Protein Assay kit (Thermo Scientific, USA).
After separation on 10% SDS-PAGE, pro-
teins were transferred to PVDF membranes
(Millipore, USA). Then, 5% non-fat milk was
obtained for blocking. Subsequently, the
membranes were incubated with primary
antibodies against TLR4 (ab22048, Ab-
cam, 1:1000), MyD88 (ab219413, Abcam,
1:1000), p-NF-κB (#3039, CST, 1:1000),
NF-κB (#6956, CST, 1:1000), and β-actin
(ab8226, Abcam, 1:1000) overnight at 4°C.
The membranes were further incubated with
secondary antibodies (1:1000) for 2 hours
at room temperature. The protein levels of
target genes were quantified by visualizing
protein bands using enhanced chemilumi-
nescence reagents (Millipore, USA) and ana-
lyzed through ImageJ (National Institutes of
Health, USA). Normalization to β-actin ex-
pression levels was performed.
Statistical analysis
All data were presented as means ±
standard deviation (SD) and analyzed us-
ing GraphPad Prism 8. One-way analysis of
variance (ANOVA) followed by Dunnett’s
post-hoc test was used to determine the sta-
tistical significance of differences among
multiple groups. P<0.05 was considered sta-
tistically significant.
RESULTS
The data illustrated in the behavioral
tests revealed that rats subjected to the CCI
model had significantly lower 50% PWT and
PWL (Figs 1A and B) compared to the Sham
group. However, when compared with CCI rats,
the additional administration of PHI dramati-
cally enhanced 50% of PWT and PWL. More-
over, the implementation of NO and ELISA as-
says revealed, respectively, that the levels of NO
and the inflammatory cytokines TNF-α, IL-1β,
and IL-6 significantly increased after establish-
ing the CCI model (Fig 2). In contrast, admin-
istration of PHI significantly reduced these ex-
pression levels. Western blotting and RT-qPCR
(Figs. 3A and 3B) results showed that the levels
of TLR4, MyD88, and p-NF-κB were significant-
ly increased by CCI induction, while treatment
with PHI significantly reversed the change in
these expression levels. And the results will be
presented as “relative quantification (RQ) us-
ing the 2^(-ΔΔCt) method,” indicating the
fold change in expression levels compared to
the control group.
DISCUSSION
In our study, treatment with Phillygenin
(PHI) significantly enhanced the 50% paw with-
drawal threshold (PWT) and paw withdrawal
latency (PWL), indicating an effective analge-
sic effect. Notably, PHI treatment resulted in
a marked decrease in nitric oxide (NO) levels,
alongside a reduction in proinflammatory cy-
tokines, including TNF-α, IL-1β, and IL-6. Fur-
thermore, our findings demonstrated that PHI
downregulated the expression of TLR4 and
MyD88, key components in the inflammatory
pathway, and inhibited the phosphorylation of
NF-κB, a critical factor in inflammation and
pain signalling.
Phillygenin reduced neuropathic pain by inhibiting the rats’ TLR4/MyD88/NF-κB pathway 9
Vol. 66(1): 4 - 15, 2025
These results suggest that PHI may al-
leviate pain by reducing inflammation and
modulating key signalling pathways, high-
lighting its potential as a therapeutic agent
in pain management.
In our study, we observed that treatment
with Phillygenin (PHI) significantly enhanced
the 50% paw withdrawal threshold (PWT) and
paw withdrawal latency (PWL) in CCI rats, in-
dicating its potential efficacy in alleviating neu-
Fig. 1. PHI treatment significantly attenuated mechanical allodynia and thermal hyperalgesia. (A) The time
course effect of PHI treatment on mechanical allodynia was assessed using the 50% paw withdrawal
threshold (PWT). (B) The time course effect of PHI treatment on thermal hyperalgesia was deter-
mined by measuring paw withdrawal latency (PWL). Data are presented as means ± SD. Statistical
analyses were performed using a One-way variance analysis (ANOVA). Significant differences are indi-
cated as **P<0.01 vs Sham group and ##P<0.01 vs CCI group (n=10). Abbreviations: CCI, chronic
constriction injury; PWT, paw withdrawal threshold; PWL, paw withdrawal latency.
Fig 2. PHI treatment significantly reduced the inflammatory status in CCI rats, as indicated by the levels of
nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6
(IL-6) measured after treatment. Data are presented as means ± SD. Statistical analyses were conduc-
ted using a One-way analysis of variance (ANOVA). Significant differences are indicated as **P<0.01
vs Sham group and ##P<0.01 vs CCI group (n=10). Abbreviations: CCI, chronic constriction injury;
NO, nitric oxide; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6.
The Y-axis of the graphs denotes the “Levels” of these inflammatory markers.
A
B
10 Hua et al.
Investigación Clínica 66(1): 2025
ropathic pain. This finding aligns with recent
literature suggesting that neuropathic pain is
closely associated with elevated inflammatory
factors and increased TLR4 expression 18. Spe-
cifically, TLR4 signalling has been implicated
in mediating neuropathic pain and exacerbat-
ing inflammatory responses, as demonstrated
in studies indicating that partial sciatic nerve
ligation leads to heightened TLR4 levels and
subsequent pain sensations 19. Another study
suggested that the increased TLR4 level was
linked to partial sciatic nerve ligation-mediat-
ed neuropathic pain 20. Moreover, our findings
support Zhang et al.’s observations that opioid
receptor agonists can non-selectively activate
TLR4 and increase proinflammatory cytokines
like TNF-α, IL-1β, and IL-6, contributing to
neuroinflammation.
Fig. 3. PHI treatment inhibited CCI rats’ TLR4/MyD88/NF-κB signalling pathway. (A) Protein expression le-
vels of TLR4, MyD88, and phosphorylated NF-κB (p-NF-κB) relative to total NF-κB were determined by
western blotting. (B) mRNA expression levels of TLR4 and MyD88 were assessed using RT-qPCR. Data
are presented as means ± SD. And the results will be presented as “relative quantification (RQ) using
the 2^(-ΔΔCt) method,” indicating the fold change in expression levels compared to the control
group. Statistical analyses were performed using a One-way analysis of variance (ANOVA). Significant
differences are indicated as **P<0.01 vs Sham group and ##P<0.01 vs CCI group (n=10). Abbre-
viations: CCI, chronic constriction injury; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation
primary response 88; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; p-NF-κB,
phosphorylated NF-κB.
A
B
Phillygenin reduced neuropathic pain by inhibiting the rats’ TLR4/MyD88/NF-κB pathway 11
Vol. 66(1): 4 - 15, 2025
In contrast, our results demonstrate
that PHI significantly decreased the levels
of these inflammatory cytokines, suggest-
ing a potential mechanism for its analgesic
effects 21. Li et al. reported enhanced TLR4
and MyD88 levels in the dorsal root ganglion
following paclitaxel treatment, which corre-
lated with diminished PWT and PWL 18. Our
study extends these findings by showing that
PHI not only modulates TLR4 expression but
also reduces inflammation, thereby improv-
ing pain thresholds in CCI rats. Overall, our
results suggest that targeting TLR4 could be
a viable strategy for alleviating neuropathic
pain. The integration of our findings with ex-
isting literature underscores the importance
of inflammatory pathways in neuropathic
pain and positions PHI as a promising can-
didate for further investigation in pain man-
agement strategies.
PHI has been found to participate in var-
ious aspects of inflammatory status relief and
in reducing TLR4 expression. For instance,
in our study, we noted that administration
of PHI inhibited LPS-induced inflammatory
responses and apoptosis in BEAS-2B cells.
This inhibition was linked to the subsequent
activation of PPAR-γ signalling due to the
downregulation of MMP-8. By reducing MMP-
8 levels, PHI effectively alleviated acute lung
injury 22. Additionally, a previous study found
that dietary PHI supplementation reduced
malondialdehyde and inflammatory mediator
production, increased antioxidant enzyme
contents and Bcl-2 levels, and ameliorated
aflatoxin B1-induced liver damage 23. Xue et
al. found that PHI reversed the expression of
SOD and MDA and downregulated the levels
of TNF-α, IL-1β, IL-6, and IL-10 in a model
of colitis in mice, and reduced the propor-
tion of tyrosine kinase Src activated by TLR4,
suggesting that PHI might be a potential
drug candidate that could effectively safe-
guard against colitis 13. Our study found that
PHI administration might ameliorate neuro-
pathic pain in CCI rats, potentially through
mechanisms such as the downregulation of
TLR4, inhibition of MyD88/NF-κB signalling,
and reduction of proinflammatory cytokines.
Similarly, our findings demonstrated that PHI
played a beneficial role in reducing inflam-
mation in CCI rats, suggesting it may serve
as an alternative therapeutic strategy for
neuropathic pain. In our study, we observed
that treatment with Phillygenin (PHI) sig-
nificantly enhanced the 50% paw withdrawal
threshold (PWT) and paw withdrawal latency
(PWL) in CCI rats, indicating its potential as
an analgesic agent.
Our results suggest that PHI may al-
leviate neuropathic pain by targeting the
TLR4 and MyD88/NF-κB signalling path-
ways. While these findings are promising,
a more detailed mechanistic explanation is
warranted to elucidate how PHI exerts its ef-
fects. One potential mechanism involves the
downregulation of TLR4 expression by PHI,
which could reduce the activation of down-
stream inflammatory pathways. By inhibiting
TLR4, PHI may prevent the subsequent ac-
tivation of MyD88 and the phosphorylation
of NF-κB, leading to a decrease in proinflam-
matory cytokines such as TNF-α, IL-1β, and
IL-6. This reduction in inflammatory me-
diators may alleviate pain and contribute to
restoring normal neuronal function in the
context of neuropathic pain. Additionally,
PHI may modulate oxidative stress pathways.
By decreasing nitric oxide (NO) levels, PHI
could help mitigate oxidative damage that
often accompanies neuropathic conditions,
further supporting neuronal health and func-
tion. This dual action-reducing inflammation
while protecting neurons–could provide a
comprehensive approach to pain manage-
ment. While our findings align with existing
literature, further investigation is needed
to explore these proposed mechanisms. Fu-
ture studies should aim to clarify the specific
interactions of PHI with TLR4 and MyD88/
NF-κB, potentially utilizing molecular and
cellular assays to provide insight into the
therapeutic implications of targeting these
pathways in neuropathic pain. This enhanced
mechanistic understanding would strength-
en the rationale for PHI as a viable therapeu-
12 Hua et al.
Investigación Clínica 66(1): 2025
tic strategy in clinical settings. We utilized a
nitric oxide (NO) assay to assess NO levels,
a key signalling molecule involved in various
physiological and pathological processes, in-
cluding inflammation and pain. Our results
indicated that Phillygenin (PHI) treatment
significantly reduced NO levels in CCI rats.
This reduction is particularly noteworthy, as
elevated NO levels are often associated with
increased pain sensitivity and inflammatory
responses in neuropathic pain models. The
decrease in NO levels suggests that PHI may
exert an analgesic effect by modulating the
nitric oxide signalling pathway. Elevated NO
contributes to pain by enhancing nociceptive
signalling and promoting neuroinflamma-
tion. By lowering NO levels, PHI may help re-
store the balance of neuroinflammatory me-
diators, thereby alleviating pain sensations.
Furthermore, the reduction of NO could also
imply potential protective effects on neuro-
nal health, as excessive NO can lead to oxida-
tive stress and neuronal damage. This aspect
is crucial, as it highlights PHI’s dual role in
alleviating pain through anti-inflammatory
effects and promoting neuronal survival in a
neuropathic context.
Previous publications have investigated
the role of the TLR4 and its downstream
MyD88/NF-κB signalling in inflammation
and neuropathic pain. Wang et al. indicat-
ed that the upregulation of the TLR4 and
MyD88/NF-κB signalling participated in the
development of inflammation and contrib-
uted to vascular dementia 24. MicroRNA-27a
modulated the TLR4 and MyD88/NF-κB sig-
nalling, further decreasing proinflammatory
cytokine levels and ameliorating acute lung
injury 25. We referenced the findings of Liu et
al., who suggested that activating the GABA
receptor, specifically the GABAA receptor,
might inhibit TLR4 and MyD88/NF-κB sig-
nalling pathways, thereby ameliorating the
progression of diabetic neuropathic pain 26.
Our results indicate that PHI influences the
TLR4 expression and inhibits MyD88/NF-κB
signalling, highlighting its crucial role in
neuropathic pain.
In our study, we conducted behavioral
tests to assess mechanical allodynia and
thermal hyperalgesia in CCI rats following
PHI treatment. A one-way ANOVA was initial-
ly applied to establish differences between
the groups, and this analysis is represented
in the bar graphs of Fig. 1. These graphs il-
lustrate each group’s total magnitude of
mechanical allodynia and thermal hyperal-
gesia. Additionally, the line graphs in the
same figure display intriguing patterns of
changes in nociceptive variables over time.
Given the temporal nature of these data, we
agree that a time-oriented analysis would
enhance the depth of our results. Thus, we
will perform a two-way ANOVA to analyze
the time-dependent effects of PHI on these
behavioral measures in our future studies.
This approach will allow us to examine both
the treatment and time factors, providing a
more comprehensive understanding of the
dynamics of pain response. The initial analy-
sis using one-way ANOVA provided valuable
insights into the overall effects of PHI on
mechanical allodynia and thermal hyperal-
gesia. However, the rich temporal data dis-
played in the line graphs suggested that a
more nuanced approach could be beneficial.
A two-way ANOVA is more appropriate for
analyzing changes across time points and
treatment conditions.
By incorporating this analysis, we can
better interpret the interaction effects of
treatment and time on pain responses, en-
hancing our understanding of the pharma-
codynamics of PHI. In acknowledging the
potential limitations of our study, several
factors warrant consideration. Although we
used 40 rats divided into four groups, a larg-
er sample size may have provided more ro-
bust data and enhanced the statistical pow-
er of our findings. Smaller sample sizes can
lead to variability and may not fully capture
the effects observed. Other factors, such as
environmental stressors, the time of day,
and individual animal differences, could in-
fluence pain responses. While we attempted
Phillygenin reduced neuropathic pain by inhibiting the rats’ TLR4/MyD88/NF-κB pathway 13
Vol. 66(1): 4 - 15, 2025
to control for these variables, they may still
introduce bias or variability in our results.
Our study demonstrated that PHI treat-
ment could reduce TLR4 levels and attenu-
ate the inflammatory status in CCI rats,
indicating that targeting the TLR4 and its
downstream MyD88/NF-κB signalling might
be a viable therapeutic strategy for neuro-
pathic pain. However, our study needed fur-
ther investigation on other inflammatory
factors and molecular pathways related to
PHI and neuropathic pain to explore its un-
derlying mechanism further.
Conflicts of interest
All authors declare no conflict of interest.
Ethics Approval
The ethics approval was obtained from
the Ethics Committee of Wuhan Fourth Hos-
pital.
Funding
None.
ORCID number authors
• Lei Hua (LH):
0000-0002-6129-0174
• Waiping Zhou (WPZ):
0009-0000-6866-2607
• Mengjie Li (MJL):
0009-0001-5248-0988
• Rongchun Li (RCL):
0009-0007-1040-4892
Author´s contributions
LH conceived and designed experi-
ments; performed experiments and data
analysis; provided technical support, data
collection and analysis; and wrote the
manuscript. WPZ conceived and designed
experiments; provided technical support,
data collection and analysis; and wrote the
manuscript. MJL conceived and designed
experiments; provided technical support,
data collection and analysis; and wrote the
manuscript. RCL conceived and designed
experiments; provided technical support,
data collection and analysis; and wrote the
manuscript.
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