Abstract
Objective
The purpose of this study was to analyze the impact of Shaoyao-Gancao
decoction (SGD) on proteins with significant changes in the dorsal root
ganglion (DRG) in rats and to explore the role of the Semaphorin 3G
(Sema3G) protein in the DRG and its downstream factors, interleukin-6
(IL-6) and CC-motif chemokine ligand 2(CCL2), in the treatment of
chronic inflammatory pain (CIP).
Methods
We created a CIP rat model using 100 μL of complete Freund's adjuvant
(CFA) that was injected into the left posterior plantar of rats. Then,
we administered SGD intragastrically. We tested the animals for
behavioral changes and protein expression levels in DRG pre- and
post-drug intervention.
Results
Rats in the SGD group showed significantly increased paw withdrawal
threshold (PWT), paw withdrawal latency (PWL), and relative expression
levels of the Sema3G protein in the DRG (all P < 0.05), while the
relative mRNA expression levels of IL-6 and CCL2 in the DRG of the rats
were significantly decreased (P < 0.05) when compared with the model
group.
Conclusion
In this study, we found that Shaoyao-Gancao decoction was effective in
improving the PWT and PWL of rats with CIP. It reduced CIP by
upregulating the expression of Sema3G in the DRG and inhibiting the
relative mRNA expression levels of IL-6 and CCL2.
Keywords: CCL2, Chronic inflammatory pain, Dorsal root ganglion,
sema3G, IL-6, Shaoyao-Gancao decoction
1. Introduction
Chronic inflammatory pain (CIP) refers to the pathological pain caused
by the increased excitability of nociceptors in response to the release
of pain-causing chemicals. This can change the physiological
environment of injured tissue when inflammation occurs in the body. The
condition manifests mainly as hyperalgesia and allodynia.
Shaoyao-Gancao Decoction (SGD) is a traditional Chinese medicine
preparation used mainly in the treatment of abdominal pain and acute
spasms of the legs and feet, and it was first documented in the
Treatise on Febrile Diseases. [[35]1] Pain disorders and
musculoskeletal/joint diseases are treated with SGD due to its strong
analgesic effect [[36]2]. It is one of the most commonly used oral
analgesics in East Asia [[37]3].
Modern pharmacological studies have confirmed that SGD has
anti-spasmodic, analgesic, anti-inflammatory, and anti-allergenic
properties and is used in a variety of treatments for cough and asthma
relief, neuromuscular blocking, and improving immune regulation
[[38]4]. Experimental studies on the anti-inflammatory and analgesic
mechanisms of SGD demonstrated that it could relieve the pain symptoms
of cervical spondylosis in animal models and reduce the expression
levels of the inflammatory factors interleukin-1β (IL-1β),
interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α)
[[39]5,[40]6]. Additionally, SGD was found to be effective in
inhibiting the synthesis and release of microglial inflammasomes in the
spinal cord anterior horn, thereby reducing the expression of
inflammatory factors [[41]7]. SGD has been shown to intervene in CIP,
although the mechanism by which it does so in the dorsal root ganglion
(DRG) is not well understood.
The DRG is the gathering point for first-order neurons involved in
afferent sensation. It receives and integrates sensory information from
the peripheral nerve endings and transmits it to the spinal dorsal
horn. Chronic inflammatory pain affects DRG functions, resulting in
changes such as protein degradation and abnormal cytokine release
[[42][8], [43][9], [44][10]].
In this study, we used whole-protein four-dimensional (4D) label-free
quantitative proteomics to detect proteins with significant changes in
the DRG as a result of treating CIP with SGD. We followed this with a
validation analysis of the differential proteins to offer insights into
the analgesic mechanism of SGD. We found that 1,4-alpha-glucan
branching enzyme 1 (Gbe1), Myosin regulatory light chain 2 (Myl2), and
Semaphorin 3G (Sema3G) were the common differentially expressed
proteins. Gbe1 and Myl2 have not been reported previously to be
associated with pain or inflammation, whereas Sema3G has been shown to
be associated with sleep deprivation and remifentanil-induced
hyperalgesia [[45]11], with demonstrated inhibitory effects on
inflammatory factors and chemokines in lipopolysaccharide (LPS)-induced
inflammation models in vitro [[46]12]. Therefore, for the present
study, we selected Sema3G to investigate whether SGD could play an
anti-inflammatory and analgesic role in CIP via the regulation of
Sema3G.
Abnormal cytokine release in the DRG is thought to cause chronic
inflammatory pain. The deficiency of Sema3G in glomerular podocytes has
been shown in a previous study [[47]13] to enhance the expression of
inflammatory cytokines, including IL-6 and CC-motif chemokine ligand
2(CCL2). Studies in animal models of CIP found that local inflammation
and pain induced the upregulation of IL-6 and CCL2 in the DRG
[[48]9,[49]14,[50]15]. Therefore, in the current study, we selected
IL-6 and CCL2 in the DRG as downstream cytokines.
We can summarize the key theoretical foundations of this study as
follows: (1) The occurrence and progression of CIP are closely
associated with protein changes in the DRG. (2) SGD has a clear
analgesic effect, but its mechanism of inflammation regulation in the
DRG requires further investigation.
Based on the above theoretical basis, we hypothesized that SGD is
likely to improve CIP by regulating the expression levels of Sema3G,
IL-6, and CCL2 in the DRG.
2. Materials and methods
2.1. Animals
We purchased a total of 36 specific-pathogen-free (SPF) male
Sprague-Dawley (SD) rats, aged 6–8 weeks and weighing 200 ± 20 g, from
the Shanghai SLAC Animal Liability Co., Ltd. (License No. SCXK
[Shanghai] 2012–0001). These were raised in the laboratory animal
research center at the Fujian University of Traditional Chinese
Medicine (License No. SYXK [Fujian] 2020–0002). The feeding environment
was maintained at an appropriate temperature and humidity with a 12-h
alternating light and dark cycle. The experimental procedure was
conducted in strict accordance with the regulations for the use of
experimental animals in China and the animal management system of our
institution.
The rats were fed a breeding diet for lab rats and mice (manufactured
by Beijing HFK Bioscience Co., Ltd., license No. SCXK [Beijing]
2019–0008). The nutritional components of this feed (calculated per
kilogram of feed) are shown in [51]Table 1. The animals did not receive
any additional food supplements.
Table 1.
Nutritional components of the breeding diet (Calculated per kilogram of
feed).
Nutritional composition Content
Crude protein ≥200 g
Crude fat ≥40 g
Moisture ≤100 g
Coarse gray powder ≤80 g
Coarse fiber ≤100 g
Calcium 10–18 g
Phosphorus 6–12 g
Lysine acid ≥13.2 g
Methionine + Cystine ≥7.8 g
Vitamin E ≥120IU
[52]Open in a new tab
2.2. Drugs and reagents
We used the following drugs and reagents: a bicinchoninic acid (BCA)
protein concentration assay kit and a sodium dodecyl
sulphate-polyacrylamide gel electrophoresis preparation kit (Wuhan
Boster Biological Technology Co., Ltd., China); protein markers,
Western antibody diluent, and a hyper-sensitivity
electrochemiluminescence kit (Shanghai Beyotime Biotechnology Co.,
Ltd., China); polyvinylidene fluoride (PVDF) membrane (Millipore, USA);
Sema3G primary antibody (Abcam, UK); glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antibody and goat anti-rabbit secondary antibody
(Proteintech, USA); GelstainRedTM nucleic acid dye (Suzhou UElandy
Biotechnology Co., Ltd., China); DNA extraction reagent and a total RNA
extraction kit (Beijing Solarbio Science & Technology Co., Ltd.,
China); and Sema3G recombinant adenovirus and blank control recombinant
adenovirus (Shenzhen Brain Case Biotechnology Co., Ltd., China).
2.3. Instruments
The following instruments were used in this study: a von Frey Pain
Threshold Detector (North Medical, USA), a thermal radiation
dolorimeter (Chengdu Techman Software Co., Ltd., China), an automatic
microplate reader (BioTek, USA), a high-speed refrigerated centrifuge
(Eppendorf, Germany), a thermostatic water bath (Tanon Science &
Technology Co., Ltd., Shanghai, China), an electrophoresis apparatus, a
chemiluminescence imaging system (Bio-Rad, USA), pipettes (Eppendorf,
Germany), a water purifier (Thermo Fisher Scientific, USA), a Milli-Q
ultra-purified water system (Millipore, USA), a DNA amplification
instrument (model 9600) (PE Company, USA), a timsTOF Pro mass
spectrometer (Bruker) and a nanoElute ultra-performance liquid
chromatography (UPLC) system (Bruker).
3. Methods
3.1. Groups
The research study consisted of the following two experiments:
Experiment 1
We randomly numbered a total of 18 SPF male SD rats and divided them
into the following three groups using a random number table: the
control group (n = 6), the model group (n = 6), and the SGD group
(n = 6). The rats in the control group were injected with 100 μL of
normal saline at the left posterior plantar during modeling, while
the rats in the model and SGD groups were injected with 100 μL of
complete Freund's adjuvant (CFA) at the left posterior plantar.
Experiment 2
We randomly numbered another set of 18 SPF male SD rats, and using a
random number table, we divided them into three groups: the control
group (n = 6), the model + negative control group (n = 6), and the
model + virus overexpression group (n = 6). The rats in the control
group were fed normally before modeling and did not receive any
intrathecal injections. Four weeks before modeling, the rats in the
model + negative control group were injected intrathecally with a
blank control recombinant adenovirus (rAAV-hSyn-EGFP), while the
rats in the model + virus overexpression group received intrathecal
injections of the rAAV-hSyn-Sema3g-P2A-EGFP virus. During modeling,
rats in the control group were injected with 100 μL of normal saline
at the left posterior plantar, while the rats in the
model + negative control group and the model + virus overexpression
group were injected with 100 μL of CFA at the left posterior
plantar.
3.2. Modeling
All the experimental rats were fasted for 12 h before modeling, with
free access to water. The rats were anesthetized with an
intraperitoneal injection of 30 mg/kg of 3 % pentobarbital sodium by
weight. After the rats were fully anesthetized, 100 μL of CFA was
slowly injected subcutaneously at the left posterior plantar using a
microsyringe. After modeling, the animals were kept at room temperature
(25 °C) to awaken. We closely monitored the body temperature,
respiration, heartbeat, and other vital signs of the rats throughout
the modeling process. After waking, the rats were placed in cages for
subsequent observation.
3.3. Administration
In [53]experiment 1, the rats in the SGD group were administered SGD
intragastrically 24 h after the completion of modeling. The decoction
was prepared as per the original prescription in the Treatise on
Febrile Diseases [[54]1] (Paeonia alba radix [12 g], honey-fried
licorice [12 g]), and the dosage for the rats was calculated according
to the daily clinically effective dose for 60-kg adults. The following
formula was used: daily dose/rat = dose/kg (human body
weight) × conversion factor 6.17 × body weight of each rat (the dose of
SGD in adults was 0.4 g/kg [body weight]) [[55]16]. The dose for rats
with a standard weight of 150 g was calculated as
0.4 g/kg × 6.17 = 2.468 g/kg. Based on the standard, for rats with a
body weight of 250 g, the dosage of SGD was calculated to be about
2.068 g/kg.
After determining the dosage, SGD of the corresponding concentration
was prepared based on the intragastric volume of the rats. In this
experiment, we used granules (purchased from the traditional Chinese
medicine pharmacy of Fujian Rehabilitation Hospital). The rats in the
model group were given the same dose of normal saline by gavage every
day, while the rats in the control group were catched at the same time
every day without other intervention measures. The above interventions
were implemented once a day for seven consecutive days.
3.4. Injection of the virus
[56]Experiment 2 involved injecting a virus directly into the
intrathecal space. The rats in the model + negative control group were
injected intrathecally with a blank control recombinant adenovirus
(rAAV-hSyn-EGFP) (AAV/9, virus titer 5.43 E + 12 vg/mL) 28 days before
modeling, while the rats in the model + virus overexpression group
received intrathecal injections of the Sema3G recombinant adenovirus
(rAAV-hSyn-rSema3g-P2A-EGFP) (AAV/9, virus titer 2.89 E+12 vg/ml), as
shown in [57]Fig. 1.
Fig. 1.
[58]Fig. 1
[59]Open in a new tab
rAAV-hSyn-rSema3g-P2A-EGFP-WPREs map.
The primer sequence was as follows:
679-F1: TGCCTGAGAGCGCAGGTCGACGCCACCATGGCCCGCTGCTCC, 679-R1:
AGCAGGCTGAAGTTAGTAGCTGTGGCTTCTACCTCCCGGG, 679-F2:
CCCGGGAGGTAGAAGCCACAGCTACTAACTTCAGCCTGCT, 679-R2:
GGTTGATTATCTCGAGAATTCTCACTTGTACAGCTCGTC.
After anesthesia with isoflurane inhalation, the spinous space between
L5-L6 lumbar vertebrae was exposed, and percutaneous lumbar puncture
was performed with 25-μl microsamplers. The hind limb flutter or
lateral swing of the rat tail indicated a successful puncture ([60]Fig.
2).
Fig. 2.
[61]Fig. 2
[62]Open in a new tab
Successful insertion of the intrathecal catheter.
3.5. Paw withdrawal threshold test
The rats underwent a paw withdrawal test (PWT) of the left hindlimb
before modeling and on days 1, 3, 5, and 7 after modeling. The test was
performed between 8:00 a.m. and 12:00 p.m. Constant ambient temperature
and humidity levels were maintained (23 ± 2 °C and 50 ± 2 %,
respectively). The measurement was done as per the up-down method
[[63]17], and licking, lifting, moving, or foot withdrawal actions by
the rats during the stimulation were considered positive reactions.
After each stimulation, the next stimulation began once the rats had
settled down. The mean value obtained from three measurements was
recorded as the test result.
3.6. Paw withdrawal latency test
We checked the pain response time of the left hindlimb of the rats
before modeling and on days 3, 5, and 7 after modeling. The measurement
environment was similar to that for the measurement of the PWT.
Following the Hargreaves method [[64]18], radiation stopped
automatically when the rats began to withdraw their feet, and the time
was recorded. The model side foot of each rat was measured three times
with an interval of at least 5 min and then averaged.
3.7. Whole-protein 4D label-free quantitative proteomics
* (1)
We followed the following procedure for protein extraction: The DRG
tissues of the rats were collected, weighed, and added to an
appropriate amount of lysate. Then they were mixed, sonicated, and
left at a low temperature for 30 min. The supernatant was removed,
centrifuged, and cryopreserved for later use. We used a BCA kit to
determine the protein concentrations.
* (2)
Trypsin digestion was performed as follows: The same amount of
protein was taken from each sample for enzymolysis, and we adjusted
the volume for consistency using lysate. Then trichloroacetic acid
(TCA) was added to achieve a final concentration of 20 %.
Subsequently, the solution was precipitated for 2 h at 4 °C and
centrifuged. The supernatant was discarded, and the precipitate was
washed two to three times with pre-cooled acetone. After the
precipitate dried, tetraethyl ammonium bromide (TEAB) at a final
concentration of 200 mM was added. The precipitate was dispersed by
sonication, trypsin was added, and enzymolysis was performed
overnight. Dithiothreitol was added to achieve a final
concentration of 5 mM, and reduction was carried out at 56 °C for
30 min. Then, iodoacetamide was added to achieve a final
concentration of 11 mM. The solution was incubated for 15 min at
room temperature in a dark place. Finally, the peptide solution was
collected to obtain enzymolysis samples.
* (3)
Liquid chromatography-mass spectrometry was performed as follows:
Peptide fragments were dissolved in liquid chromatography mobile
phase A and separated using a nanoElute UPLC system. The peptide
fragments separated by the UPLC system were injected into a
capillary ion source for ionization and analyzed using a timsTOF
Pro mass spectrometer.
* (4)
We performed the following database searches: We retrieved the
secondary mass spectrometry data of this experiment using the
MaxQuant (V1.6.15.0) software package. The database was
RattUS_norvegicus_10116_PR_20201214.FASTA (29,940 sequences), in
which a negative database and a common contamination database were
added to calculate the false positive rate and eliminate the
influence of contaminated proteins on the identification results.
* (5)
We used the following databases for the bioinformatics analysis:
UniPROt-GOA for Gene Ontology (GO) protein annotation, WoLF PSORT
for the subcellular localization annotation of differential
proteins, and the Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway for annotation, enrichment analysis of protein pathways,
and classification of pathways.
3.8. Western blotting
The DRG tissues of the rats were collected, weighed, and added to an
appropriate amount of lysate. Then they were mixed, sonicated, and left
at a low temperature for 30 min before centrifugation. Next, the
supernatant was removed and cryopreserved for later use. We used a BCA
kit to determine the protein concentrations. After sample loading at 50
μg/well, gel stacking with 10 % polypropylene gel, electrophoresis at
40 V, separation with separating glue at 100 V, and glue cutting, the
target protein and internal reference protein on the corresponding
molecular weight band were transferred to a PVDF membrane at 300 mA.
The proteins were blocked with 5 % skimmed milk for 2 h, and Sema3G
antibody (1:500) and GAPDH antibody (1:5000) were added.
After incubating at 4 °C overnight, the bands were removed and washed
with Tris-buffered saline (TBS) + Tween Buffer (TBST) three times
(5 min each time). After the primary antibody reaction, secondary
antibodies (Sema3G 1:8000 and GAPDH 1:8000) were added for a 2-h
incubation at room temperature. After the secondary antibody reaction,
the membrane was washed with TBST three times (5 min each time). An
Image-LAB image analysis system was used to mark the gray values of the
target and reference proteins, and the expression level of each index
was expressed as the comparison value of the gray values of the target
protein band and the reference GAPDH band.
3.9. Real-time fluorescent quantitative polymerase chain reaction
The complete DRGs were quickly removed from the anesthetized rats, and
the excess liquid on the surface was rinsed with iced normal saline.
The ganglia were placed in a centrifuge tube and stored in a liquid
nitrogen tank. The DRG tissues were removed from the EP tubes for the
measurement of RNA concentrations on Super NANO. We used the miRNA
all-in-one TM reverse-transcription quantitative polymerase chain
reaction (RT-qPCR) miRNA detection kit for RT reactions and PCR
amplification reactions, done under the following reaction conditions:
DNA removal: 42 °C for 2 min; RT reaction: 15 min at 50 °C and 2 min at
85 °C. The PCR amplification reaction included pre-degeneration at
95 °C for 5 min, 40 cycles of amplification at 94 °C for 10 s, and
annealing and extension at 60 °C for 30 s.
We used a Bio-Rad RT-qPCR instrument for detection and analysis. The
primer sequence was as follows: IL-6:
GACTTCCAGCCAGTTGCCTT/CTGGTCTGTTGTGGGTGGTAT; CCL2:
GCATCAACCCTAAGGACTTCAG/TTCTCTGTCATACTGGTCACTTCT; and GAPDH:
GATGCTGGTGCTGAGTATGRCG/GTGGTGCAGGATGCATTGCTCTGA.
4. Statistical analysis
We used SPSS 23.0 (IBM) statistical software for data analysis.
Measurement data of normal distribution were expressed as
mean ± standard deviation (
[MATH: χ‾ :MATH]
± s). The pain behavior data were analyzed using a repeated analysis of
variance (ANOVA). If P > 0.05 in Mauchly's test of sphericity, we
performed a repeated ANOVA. If P < 0.05 in Mauchly's test of
sphericity, we used a multivariate ANOVA or a degrees-of-freedom
correction test.
The statistical analysis of the proteomic analysis of [65]experiment 1
yielded the differentially expressed proteins. In this part of the
experiment, we established three sets of biological replicates, and an
ANOVA statistical test was performed. Proteins with fold changes >0.001
or <1000 and P < 0.001 were defined as significantly differentially
expressed proteins. We used a one-way ANOVA for comparisons between
multiple groups, the least-significant difference method for pairwise
comparisons between groups in cases of homogeneity of variance, and the
Games-Howell post-hoc test in cases of heterogeneity of variance. We
used a non-parametric test for non-normally distributed data. A P value
of <0.05 indicated a statistically significant difference.
5. Results
5.1. Effect of Shaoyao-Gancao decoction on the pain behavior of rats with CIP
The rats were tested for the left lateral plantar PWT before modeling
(day 0), one day after modeling (before intervention), and on days 3,
5, and 7 after modeling. We found that the PWT of the rats in all three
groups before modeling was above 9 g, and there were no statistically
significant differences between the groups (P > 0.05). At 1 day
post-modeling, the PWT of the rats in the model group and the SGD group
was significantly decreased compared with that in the control group
(P < 0.05). At 3–7 days after the procedure, the PWT of the rats in the
model group was significantly decreased compared with that in the blank
group (P < 0.05), and it remained below 2 g, indicating that the rats
were not self-cured and the model was relatively stable. At 7 days
post-modeling, the PWT of the rats in the SGD group was significantly
increased compared with that in the model group (P < 0.05), as shown in
[66]Fig. 3.
Fig. 3.
[67]Fig. 3
[68]Open in a new tab
A. Comparison of the left lateral plantar paw withdrawal threshold of
the rats in each group (
[MATH: χ‾ :MATH]
± s) B. Comparison of the left lateral plantar paw withdrawal latency
of the rats in each group (
[MATH: χ‾ :MATH]
± s)
Note: Compared with the control group, *P < 0.05. Note: Compared with
the model group, ^# < 0.05.
We tested the rats for left lateral plantar PWL before modeling and on
days 3, 5, and 7 after modeling (i.e., after intervention). The PWL of
the rats in the three groups before modeling was about 7 s, and the
differences between the groups were not statistically significant
(P > 0.05). After CIP modeling, the PWL of the rats in the model group
and the SGD group was significantly decreased (P < 0.05). On days 3 and
5 after intervention, the PWL of the rats in the SGD group increased
and was significantly higher than that of the rats in the model group
(P < 0.05), as shown in [69]Fig. 3.
5.2. Effect of Shaoyao-Gancao decoction on the proteomics of DRG in rats with
CIP
On day 8 after the operation, we analyzed the total protein expression
of the DRG of the rats in each group using whole-protein 4D label-free
quantitative proteomics. We found that compared with the control group,
the expression levels of a total of 17 proteins were significantly
increased in the model group, while in the DRG of the rats in the model
group, 52 proteins had significantly decreased expression. Compared
with the model group, the expression levels of a total of 465 proteins
were significantly decreased, while 243 proteins were significantly
increased in the rats of the SGD group, as shown in [70]Fig. 4. With
the comparison between the model group and the control group (See
[71]Fig. 5A) and the SGD group and the model group (see [72]Fig. 5B),
we plotted a differential protein volcano map based on the differential
proteins and the protein change degree obtained from the proteomics
results.
Fig. 4.
[73]Fig. 4
[74]Open in a new tab
Number of differential proteins in the dorsal root ganglion of the rats
in each group. Note: Red indicates the number of proteins with
upregulated expression, and green indicates the number of proteins with
downregulated expression.
Fig. 5.
[75]Fig. 5
[76]Open in a new tab
Volcano plot of differential proteins. Note: The X axis represents the
value of protein change degree after Log2 conversion, and the Y axis
represents the P value of a t-test of significant difference after
Log10 conversion. The red dot represents a protein with upregulated
expression, and the blue dot represents a protein with significantly
downregulated expression. A: Model group vs. control group (number of
proteins) B: Shaoyao-Gancao decoction group vs. model group (number of
proteins).
The GO analysis included biological processes (BP), cellular
compartments (CC), and molecular functions (MF). The BP results of this
experiment revealed that the differential proteins among the three
groups were mainly involved in cellular processes, biological
regulation, metabolic processes, and stimulus response. The CC results
showed that the differentially expressed proteins were mainly from
cells, intracellular proteins, and protein-containing complexes. The MF
results revealed that most of the differential proteins exhibited
binding activity, catalytic activity, and structural molecular
activity, as shown in [77]Fig. 6. [78]Fig. 6A shows the GO annotation
entries of the differential proteins in the model group compared with
the control group, and [79]Fig. 6B shows the GO annotation entries of
the differential proteins in the SGD group compared with the model
group.
Fig. 6.
[80]Fig. 6
[81]Open in a new tab
Gene Ontology (GO) protein annotation. Note: [82]Fig. 6A shows the GO
annotation entries of the differential proteins in the model group
compared with the control group. [83]Fig. 6B shows the GO annotation
entries of the differential proteins in the SGD group compared with the
model group. A: Model group vs. blank group B: Shaoyao-Gancao decoction
group vs. model group.
The subcellular localization annotation of the differential proteins in
the model and control groups was mainly in the nucleus and
extracellular regions, as shown in [84]Fig. 7A. Compared with the model
group, the subcellular localization of differential proteins in the SGD
group was mainly in the nucleus and cytoplasm, as shown in [85]Fig. 7B.
Fig. 7.
[86]Fig. 7
[87]Open in a new tab
Subcellular localization annotation. Note: [88]Fig. 7A shows the
subcellular localization annotation of differential proteins in the
model group compared with the control group. [89]Fig. 7B shows the
subcellular localization annotation of different proteins in the SGD
group compared with the model group.
The KEGG pathway enrichment analysis showed that, compared with the
control group, the upregulated or downregulated proteins in the DRG in
the model group were involved mainly in the transforming growth
factor-beta (TGF-beta) signaling pathway and the sphingolipid signaling
pathway (see [90]Fig. 8A). Compared with the model group, the
upregulated or downregulated proteins in the DRG in the SGD group were
involved mainly in the adipocytokine signaling pathway (see [91]Fig.
8B), the metabolism of xenobiotics by cytochrome P450, chemical
carcinogenesis, porphyrin and chlorophyll metabolism, Th1 and Th2 cell
differentiation, drug metabolism by cytochrome P450, and other related
pathways, as shown in [92]Fig. 9.
Fig. 8.
[93]Fig. 8
[94]Open in a new tab
Bubble diagram of functional enrichment analysis of differential
proteins. Note: [95]Fig. 8A shows the functional enrichment
distribution of differential proteins in the model group compared with
the control group. [96]Fig. 8B shows the functional enrichment
distribution of differential proteins in the SGD group compared with
the model group.
Fig. 9.
[97]Fig. 9
[98]Open in a new tab
Cluster analysis heat map of Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway enrichment.
5.3. Effect of Shaoyao-Gancao decoction on the relative expression level of
Sema3G in the DRG of rats with CIP
To further verify the differential protein Sema3G in the proteomics
results, we conducted a quantitative analysis using Western blotting.
The results showed that in comparison with the control group, the
relative protein expression level of Sema3G in the DRG of the rats in
the model group decreased (P < 0.05), while in comparison with the
model group, the relative protein expression level of Sema3G in the DRG
of the rats in the SGD group increased (P < 0.05), as shown in [99]Fig.
10, [100]Fig. 11, respectively.
Fig. 10.
[101]Fig. 10
[102]Open in a new tab
Relative protein expression of sema3g in the dorsal root ganglion of
the rats in each group.
Fig. 11.
Fig. 11
[103]Open in a new tab
Comparison of relative protein expression levels of Sema3G in the
dorsal root ganglion of the rats in each group (
[MATH: χ‾ :MATH]
± s). Note: a. Compared with the blank group, ****P < 0.05. Note: b.
Compared with the model group, ***P < 0.05.
5.4. Effect of Shaoyao-Gancao decoction on the relative mRNA expression
levels of IL-6 and CCL2 in the DRG of rats with CIP
We reviewed the literature to further explore the anti-inflammatory and
analgesic mechanisms of SGD and found that a Sema3G deficiency could
enhance the expression of inflammatory cytokines, including IL-6 and
CCL2, in glomerular podocytes. Therefore, we used real-time PCR to
detect the mRNA expression levels of IL-6 and CCL2 in the DRG.
The results showed that in the model group, the relative mRNA
expression level of IL-6 in the DRG of the rats was significantly
increased (P < 0.05) when compared with the blank group. In the SGD
group, the relative mRNA expression level of IL-6 in the DRG of the
rats was significantly decreased (P < 0.05) when compared with the
model group, as shown in [104]Fig. 12A.
Fig. 12.
[105]Fig. 12
[106]Open in a new tab
Comparison of the Relative mRNA Expression Levels of IL-6 and CCL2 in
the Dorsal Root Ganglion of the Rats in Each Group. Note: a. Compared
with the control group, *P < 0.05. Note: b. Compared with the model
group, **P < 0.05.
In the model group, when compared with the control group, the relative
mRNA expression level of CCL2 in the DRG of the rats was significantly
increased (P < 0.05). Finally, compared with the model group, the
relative mRNA expression level of CCL2 in the DRG of the rats in the
SGD group was significantly decreased (P < 0.05), as shown in [107]Fig.
12B.
5.5. Effect of intrathecal injection of Sema3G overexpressing virus on the
pain behavior of rats with CIP
We treated rats with intrathecal injections of a recombinant adenovirus
overexpressing Sema3G (rAAV-hSyn-rSema3g-P2A-EGFP) or a blank control
recombinant adenovirus (rAAV-hSyn-EGFP) to further determine whether
the upregulation of Sema3G in the DRG could improve the PWT and PWL
after CIP was induced. We tested the rats in each group for the left
lateral plantar PWT and PWL before modeling (day 0) and at days 1, 3,
5, and 7 after modeling.
Rats in the three groups had a PWT of >9 g before modeling, and the
differences between the groups were not statistically significant
(P > 0.05). On day 1 after modeling, rats in the model + negative
control group and the model + virus overexpression group showed
significantly decreased PWT when compared with the control group
(P < 0.05). On day 7 after modeling, the PWT of the rats in the
model + virus overexpression group was significantly increased compared
with that in the model + negative control group (P < 0.05), as shown in
[108]Fig. 13A.
Fig. 13.
[109]Fig. 13
[110]Open in a new tab
A. Comparison of left lateral plantar paw withdrawal threshold of the
rats in each group (
[MATH: χ‾ :MATH]
± s) B.Comparison of left lateral plantar paw withdrawal latency of the
rats in each group (
[MATH: χ‾ :MATH]
± s). Note: Compared with the control group, *P < 0.05. Note: Compared
with the model + negative control group, ^#P < 0.05.
In all three groups, the PWL of the rats was >7 s before modeling, and
the differences were not statistically significant (P > 0.05). After
CIP modeling, the PWL of the rats in the model + negative control group
and the model + virus overexpression group was significantly decreased
(P < 0.05). On days 3, 5, and 7 after modeling, the rats in the
model + virus overexpression group showed increased PWL, which was
significantly higher than in the model + negative control group
(P < 0.05), as shown in [111]Fig. 13B.
5.6. Effect of intrathecal injection of Sema3G overexpressing virus on the
relative mRNA expression levels of IL-6 and CCL2 in the DRG of rats with CIP
To further investigate whether the intrathecal injection of the Sema3G
overexpressing virus could inhibit the relative mRNA expression levels
of IL-6 and CCL2 in the DRG, we used RT-PCR to detect the relative mRNA
expression levels of IL-6 and CCL2 in the DRG on day 7 after modeling
(i.e., 35 days after the intrathecal injection of the Sema3G virus).
The experimental results revealed that the DRG of the rats in the
model + negative control group had significantly increased mRNA levels
of IL-6 when compared with the control group (P < 0.05). In the
model + virus overexpression group, compared with the model group, the
relative mRNA expression level of IL-6 in the DRG of the rats was
significantly decreased (P < 0.05), as shown in [112]Fig. 14A.
Fig. 14.
[113]Fig. 14
[114]Open in a new tab
Comparison of the relative mRNA expression levels of IL-6 and CCL2 in
the dorsal root ganglion of the rats in each group. Note: a. Compared
with the control group, ***P < 0.05. Note: b. Compared with the
model + negative control group, ****P < 0.05.
In comparison with the control group, the relative mRNA expression
level of CCL2 in the DRG of the rats in the model + negative control
group was significantly increased (P < 0.05). Finally, compared with
the model group, the relative mRNA expression level of CCL2 in the DRG
of the rats in the model + virus overexpression group was significantly
decreased (P < 0.05), as shown in [115]Fig. 14B.
6. Discussion
Shaoyao-Gancao decoction, a standard prescription commonly used in the
clinical treatment of pain-related diseases, is one of the most
commonly used oral analgesics in East Asia. Hence, understanding its
analgesic mechanism assumes much significance.
In this formulation, the ratio of P. alba radix and licorice root is
closely associated with its anti-inflammatory and analgesic effects.
Zhu et al. [[116]19] noted that vinegar-baked P. alba radix and
honey-fried licorice (1:1) showed the best anti-inflammatory and
analgesic effects, while vinegar-baked P. alba radix and honey-fried
licorice (6:1) had only a weak anti-inflammatory effect and no obvious
analgesic efficacy. Based on our earlier clinical research experience
and the experimental exploration of the research group, we used P. alba
radix and honey-fried licorice in a ratio of 1:1 in this experiment.
First described in the Treatise on Febrile Diseases [[117]1], SGD is an
excellent prescription for relieving spasms and pain [[118]20].
Researchers have used metabolomic analyses to confirm the analgesic
effect of paeonol, benzoic acid, paeonol A, paeonolactone C, and other
substances in SGD on neuralgia. Liu et al. demonstrated that the active
substance in SGD could alleviate trigeminal neuralgia by acting on
interleukin-1β, mitogen-activated protein kinase (MAPK) 8, MAPK1, CCL2,
and other targets [[119]21]. A study by Zhu et al. found that SGD plays
a role in osteoarthritis by regulating cell cycles, apoptosis, drug
metabolism, inflammation, and immunity [[120]22].
SGD has shown clear clinical efficacy in the treatment of CIP. In the
treatment of lumbar and leg pain associated with lumbar disc
herniation, Wu et al. [[121]23] found that SGD + balanced acupuncture
was superior to only oral celecoxib capsules; moreover, patients’
visual analog scale (VAS) scores and Japanese Orthopedic Association
(JOA) scores improved, and the levels of IL-6 and CRP were reduced. In
another study on 60 patients with neck pain, SGD showed a good
analgesic effect; in the early stage, the pain was significantly
relieved after treatment, with a significantly lower VAS score than in
a control group [[122]24].
The analgesic effect of SGD has been demonstrated in several basic
research studies. For instance, in rats with CFA-induced arthritis, Sui
et al. found that it alleviated pain by inhibiting the transient
receptor potential vanillin receptor 1 (TRPV1) function in DRG neurons
[[123]25]. Yun et al. reported that SGD alleviated visceral
hyperalgesia following inflammation by inactivating TRPV1 and
inhibiting the synthesis of 5-hydroxytryptamine [[124]26].
The above studies elucidate, to some extent, the anti-inflammatory and
analgesic mechanisms of SGD. In a series of studies on the
anti-inflammatory and analgesic effects of SGD, our research group
found that SGD could alleviate pain by regulating the expression of
miR-146a and miR-155 in the cervical intervertebral disc and neck
muscle tissue, thereby inhibiting the downstream inflammatory signaling
pathways and reducing the release of TNF-α, IL-1β, IL-6, and other
inflammatory factors [[125]6]. Furthermore, in cervical spondylosis
model rabbits, SGD could also reduce the local inflammatory response by
inhibiting the release of inflammatory factors from the NF-κB signaling
pathway [[126]27,[127]28].
The DRG is also sensory ganglion tissue. When stimulus signals from the
nerve endings in the limbs are transmitted to neurons in the DRG and
the information is integrated in this area, nociceptive sensory neurons
project to the dorsal horn of the spinal cord and send signals to
secondary neurons, which are projected to the advanced pain center in
the hypothalamus and cortex [[128]29].
Numerous studies have found that CIP can lead to changes in the
expression of both proteins and cytokines in the DRG. Liu et al.
[[129]30,[130]31] found that the Nogo-A protein was significantly
increased in the DRG of rats with CIP. The expression levels of CCL2
and its receptor (CCR2) in the DRG were significantly increased in CIP
model rats, and CCL2 improved the excitability of nociceptive cells in
the DRG. Chen et al. reported that the expression level of p38 MAPK was
increased in the DRG of rats with CIP [[131]32]. Niu et al.
[[132]33,[133]34] found that CFA injection could upregulate the mRNA
expression of IL-6, IL-17, TNF-α, and CCL2 in the ipsilateral DRG. In
addition, IL-1β mRNA and COX-2 mRNA and their proteins in the DRG
[[134]35] were found to induce the increased expression of CCL2/CCR2,
CCL3, CCL21, and endothelial chemokine (C-X-C motif) ligand 1
(CXCL1)/CXCR2.
We used the proteomics technique in the present study to analyze
changes in the expression of numerous proteins in the DRG. Rats in the
model group showed decreased expression in 52 proteins when compared
with the control group, and in the SGD group, there were 212 proteins
with increased expression in comparison with the model group. There
were three overlapping proteins, for which the encoding genes were
Gbe1, Myl2, and Sema3G, respectively. We did not find any relevant
reports on the occurrence and development of pain associated with the
Gbe1 and Myl2 genes in our literature review. One study suggested that
inhibiting the expression level of Sema3G in the spinal dorsal horn of
rats could effectively improve mechanical hyperalgesia and thermal
hypersensitivity induced by sleep deprivation and remifentanil and
shorten the pain recovery time [[135]36]. However, as there were
studies linking Sema3G and inflammatory factors, we selected the Sema3G
gene for verification and analysis in this study.
In the proteomic analysis, the Sema3G protein in the DRG was
downregulated in the model group when compared with the control group,
while it was upregulated in the SGD group in comparison with the model
group. The Western blotting results also confirmed the results of the
proteomics analysis, i.e., the relative expression level of the Sema3G
protein in the DRG of the rats was downregulated in the model group,
while its relative expression level in the rats in the SGD group was
upregulated as compared to the model group. Thus, Sema3G expression in
the DRG was downregulated during the occurrence and progression of CIP,
despite SGD upregulating its expression. Therefore, we can speculate
that Sema3G can contribute to the analgesic action of the preparation.
This result provides the experimental groundwork for constructing and
verifying adenoviruses that overexpress Sema3G.
The Sema3G protein is a member of the Semaphorin family. There is
growing evidence that Semaphorin plays an important role in
morphological changes and homeostasis across multiple systems, with
implications for several biological processes such as cell metastasis
and cytokine release [[136]37]. In addition, human genomic analysis has
revealed that Semaphorin and its receptors are predisposing or
pathogenic genes in schizophrenia, cancer, and degenerative diseases
[[137]38,[138]39].
A study [[139]40] revealed that Sema3A and Sema3E were mainly
associated with poor tumor prognosis, while Sema3G was generally
associated with a tumor survival advantage. The remaining Sema3 genes
showed survival advantages or disadvantages depending on the type of
cancer. In addition, all Sema3 genes were significantly associated with
immune infiltration subtypes and correlated with stromal cell
infiltration levels and tumor cell stem cells to varying degrees. Other
recent studies have shown that Sema3A can regulate the degeneration of
lumbar intervertebral discs by inhibiting inflammation, antagonizing
the activity of vascular endothelial growth factor, and suppressing
angiogenesis and the growth and development of vascular endothelial
cells [[140]41].
Abnormal vascular hyperplasia is closely associated with the occurrence
and development of a variety of diseases. Class-3 Semaphorin family
proteins show promise in innovative treatments of diseases via the
regulation of blood vessels, nerve ingrowth, and the inflammatory
response.
Semaphorin 3G, a new signaling protein in the class-3 Semaphorin
family, is a secreted protein of Semaphorin that localizes on
chromosome 14 [[141]42]. Exploring the role of the Sema3G protein is a
promising direction in disease research [[142]43]. Previous studies
have shown that Sema3G supplementation promoted both the formation of
healthy vascular networks and the degeneration of diseased vessels
during vascular remodeling, suggesting that it has an important
protective role in ischemic retinopathy [[143]44,[144]45]. Other
studies found that peripheral Sema3G regulates adipocyte
differentiation, which is associated with obesity [[145]46]. Gene chip
analysis has demonstrated that the expression of Sema3G increases by
more than fourfold during adipogenesis. In a pathological state, Sema3G
can exert a powerful anti-tumor effect by promoting anti-invasive
activity in tumor cells and reducing tumor-associated vascular density.
Semaphorin 3G has also been identified as an important prognostic
marker for adult glioma [[146]47]. A recent study found that the Sema3G
protein, secreted by cerebrovascular endothelial cells, increases
synaptic density and enhances synaptic transmission by acting on
Neuropilin-2/PlexinA4 receptors on hippocampal pyramidal neurons.
Cerebral vascular endothelium-derived Sema3G knockdown leads to
cognitive decline [[147]48]. Furthermore, the Sema3G lentiviral vector
can effectively overexpress endogenous Sema3G proteins in human
pancreatic cancer cells, thereby inhibiting cell proliferation,
invasion, and migration [[148]49]. In an analysis of the in vivo and in
vitro functions of Sema3G in the kidney [[149]13], the Sema3G protein,
secreted by glomerular podocytes, was found to protect podocytes from
inflammatory kidney disease and diabetic nephropathy. The deficiency of
Sema3G in podocytes increases the release of inflammatory cytokines and
chemokines such as IL-6 and CCL2.
In summary, Sema3G has an anti-inflammatory function as it can inhibit
the expression of inflammatory factors and chemokines in glomerular
podocytes. Research on the Sema3G protein has benefited many disorders
such as retinopathy, obesity, glioma, cognitive dysfunction, and
diabetic nephropathy [[150]13,[151][44], [152][45], [153][46],
[154][47]].
In the present study, after the plantar injection of CFA, we found that
the relative mRNA expression levels of IL-6 and CCL2 in the DRG of the
rats in the model group were significantly higher than those in the
control group. This was consistent with our observations of obvious
redness and swelling of the foot on the modeling side and the PWT and
PWL values showing a significant downward trend. In the DRG samples
obtained from the rats seven days after the administration of SGD, the
relative mRNA expression levels of IL-6 and CCL2 were significantly
decreased compared with those of the model group. This was consistent
with the downward trend in the PWT and PWL values of the rats in the
SGD group. These findings indicate that SGD reduced the pain threshold
in the rats with CIP by downregulating the relative mRNA expression
levels of IL-6 and CCL2 in the DRG, demonstrating the possible
analgesic mechanism of the preparation.
Pain and cytokines are closely connected. The most commonly tested
biomarker for chronic inflammation is IL-6, which is involved in the
initiation and regulation of inflammation [[155]50]. Increased levels
of IL-6 can be seen in animal models of neuropathic pain, while
decreases in IL-6 levels can inhibit the development of paralgesia. The
expression of IL-6 is enhanced in response to environmental stress
factors such as infection and tissue damage, and this triggers alarm
signals and activates the host's defense mechanisms against stress.
Specifically, CCL2 can induce the aggregation of monocytes at the sites
of inflammation, traumatic infection, toxin exposure, and ischemia
[[156]51].
In addition to its recognized proinflammatory effect, many studies have
suggested that CCL2 is associated with pain [[157]52,[158]53] due to
its role as a neuron-microglial signaling factor in the development of
neuropathic pain. In the case of peripheral chronic inflammation, CCL2
synthesized and released by DRG neurons directly excites nociceptive
neurons via autocrine and/or paracrine processes. In a classic CIP
model, CFA-induced paw swelling in rats caused an increased expression
of CCL2/CCR2 and IL-6 in the ipsilateral DRG. However, treating rats
with CFA-induced inflammatory pain with tripterine significantly
downregulated the relative mRNA expression levels of IL-6 and CCL2 in
the DRG and alleviated pain symptoms [[159]35].
In the present study, our RT-PCR results were consistent with the above
findings, indicating that the pain symptoms of rats with CIP were
alleviated by the downregulation of the relative mRNA expression levels
of IL-6 and CCL2.
To promote complete expression, in the present study, we intrathecally
injected adenovirus at L4/5 four weeks before modeling. In
[160]experiment 2, the local injection of rAAV-hSyn-rSema3g-P2A-EGFP
into the DRG at L4/5 improved the PWT and PWL in the rats with CIP. The
PWT in the model + virus overexpression group was significantly
increased at 35 days after the injection of the virus when compared
with the model + negative control group, and the PWL was significantly
increased on days 31, 33, and 35 after injection. This was similar to
the efficacy observed in the rats with CIP who were treated with SGD in
[161]experiment 1. This indicated that the overexpression of the Sema3G
protein with rAAV-hSyn-rSema3g-P2A-EGFP relieved pain, and the optimal
treatment effect was seen 31 days after the intrathecal virus
injection. Thus, the intrathecal injection of
rAAV-hSyn-rSema3g-P2A-EGFP alleviated inflammatory pain symptoms caused
by the plantar injection of CFA.
Our results also indicated that the intrathecal injection of the blank
control virus did not affect the relative mRNA expression levels of
IL-6 and CCL2, i.e., the relative mRNA expression levels of IL-6 and
CCL2 in the DRG of the rats in the model + negative control group were
significantly higher than those in the control group after CIP
modeling. The intrathecal injection of rAAV-hSyn-rSema3g-P2A-EGFP
effectively inhibited the relative mRNA expression levels of IL-6 and
CCL2 in the DRG. This was similar to a result in the experiment in
which the relative mRNA expression levels of IL-6 and CCL2 revealed a
consistent downward trend in the DRG of the rats with CIP after the
administration of SGD.
Therefore, it is reasonable to speculate that intrathecally injecting
rAAV-hSyn-rSema3g-P2A-EGFP might alleviate CIP by inhibiting the
relative mRNA expression levels of IL-6 and CCL2. The knockdown of
Sema3G expression in the DRG by the rAAV-hSyn-rSema3g-P2A-EGFP virus
might play a similar role to that of SGD.
The limitations of the study are as follows: First, we did not explore
the specific binding mechanism of Sema3G and its ligand Npn2 in detail.
Sema3G and Npn2 are receptor-ligand relationships with high binding
affinity. We did not study whether SGD affects the binding processes of
Sema3G and Npn2 or the molecular mechanisms underlying this. Besides,
due to time and resource limitations in this study, we did not verify
the differential proteins Gbe1 and Myl2, but we discussed the
correlation between Gbe1 and Myl2 and the occurrence and development of
chronic inflammatory pain. In order to obtain insights into the
analgesic mechanism of SGD, the influence of Gbe1 and Myl2 on chronic
inflammatory pain needs to be further considered. As a newly discovered
protein, Sema3G has broad prospects in disease research. The current
exploration of Sema3G is mainly in the field of oncology and cancer. In
order to give play to the greater reference value of basic research in
clinical practice, the possible regulatory mechanism of Sema3G in the
treatment of cancer pain by SGD needs to be subsequently considered.
In conclusion, in this study, we found that SGD improved the PWT and
PWL in rats with CIP. The decoction improved CIP by upregulating the
expression of Sema3G in the DRG and inhibiting the relative mRNA
expression levels of both IL-6 and CCL2.
Ethics approval and consent to participate
This study was conducted with approval from the Ethics Committee of
Fujian University of Traditional Chinese Medicine. All procedures
performed in this study involving human participants were in accordance
with ethical standards of institutional and/or national research
committee and in compliance with the Declaration of Helsinki. Each
enrolled patient signed an informed consent form to use their samples
and records for scientific research.
Consent for publication
Not applicable.
Data availability
The data associated with this study has not been deposited into a
publicly available repository.
The data will be made available on request.
Funding
This work was supported by the National Key Research and Development
Program of China (Grant Nos.: 2022YFC2009700, 2022YFC2009701,
2022YFC2009704).
Authors’ contributions
Rong Lin: Analysis and interpretation of data, drafting the article,
final approval of the version submitted. Jungang Gu: Acquisition of
data, critical revision of the manuscript for intellectual content,
final approval of the version submitted. Zhifu Wang: Conception and
design of the research, critical revision of the manuscript for
intellectual content, final approval of the version submitted. Xiaoxia
Zeng: Acquisition of data, critical revision of the manuscript for
intellectual content, final approval of the version submitted. Hongwei
Xiao: Acquisition of data, critical revision of the manuscript for
intellectual content, final approval of the version submitted. Jincheng
Chen: Analysis and interpretation of data, critical revision of the
manuscript for intellectual content, final approval of the version
submitted. Jian He: Conception and design of the research, analysis and
interpretation of data, critical revision of the manuscript for
intellectual content, final approval of the version submitted.
CRediT authorship contribution statement
Rong Lin: Writing - original draft, Formal analysis. Jun-Gang Gu:
Writing - review & editing, Data curation. Zhi-Fu Wang: Writing -
review & editing, Conceptualization. Xiao-Xia Zeng: Writing - review &
editing, Data curation. Hong-Wei Xiao: Writing - review & editing, Data
curation. Jin-Cheng Chen: Writing - review & editing, Formal analysis.
Jian He: Writing - review & editing, Funding acquisition, Formal
analysis, Conceptualization.
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.
Acknowledgements