Abstract
Background
This study investigated the mechanism of action of Fibraurea recisa
Pierre (FRP) in the treatment of chronic urticaria (CU) using a rat
model and combinatorial analysis of network pharmacology, metabolomics,
and molecular dynamics and dynamics simulation data, providing a
rationale for its clinical use.
Methods
Twenty-four Sprague-Dawley rats were categorized into control, model,
high-dose FRP (40 mg/kg body weight), and low-dose FRP (20 mg/kg body
weight) groups. The CU model was induced by ovalbumin. Ultra-high
performance liquid chromatography-tandem mass spectrometry (UPLC/MS)
was used to estimate the levels of various components in FRP. The rats
in different groups were evaluated for scratching behavior,
histopathological changes in the skin tissues based on
hematoxylin/eosin staining, and the levels of inflammatory factors and
indicators of mast cell degranulation. Metabolomics, network
pharmacology, molecular docking and dynamics simulation, and Western
blotting were used to analyze the mechanism of action of FRP.
Results
We identified 2,206 compounds in FRP based on UPLC/MS data analysis.
Our data showed that the main active components in FRP were palmatine,
jatrorrhizine, and coclaurine. FRP administration significantly reduced
the scratching frequency, pathological characteristics of skin tissues,
levels of inflammatory factors, and the degree of mast cell
degranulation. Based on the combined analysis of metabolomics and
network pharmacology data, phosphatidylinositol 3-kinase (PI3K)-protein
kinase B (Akt) signaling pathway was identified as the key target of
FRP. Molecular docking and molecular dynamics simulation demonstrated
strong and stable binding of Akt with palmatine, jatrorrhizine, and
coclaurine. Western blotting confirmed that FRP increased the levels of
p-Akt and p-PI3K in skin tissue within the CU model.
Conclusion
FRP significantly alleviated the symptoms and pathological changes of
CU by modulating inflammation through upregulation of the PI3K-Akt
signaling pathway.
Keywords: network pharmacology, metabolomics, chronic urticaria,
Fibraurea recisa, inflammation, immunity
Graphical Abstract
[38]graphic file with name FPHAR_fphar-2025-1571819_wc_abs.jpg
1 Introduction
Chronic urticaria (CU) is a common skin allergy with a global
prevalence rate ranging from 0.1% to 3.4%. It is characterized by the
presence of hives and itching for at least 6 weeks ([39]Zhang et al.,
2022). The pathogenesis of CU involves activation of mast cells and
secretion of large amounts of inflammatory mediators such as histamine
(HIS), leukotrienes (LT), and interleukins (IL), which trigger
angioedema (swelling in the deeper layers of the skin) and recruit
various immune cell types to the affected areas ([40]Gimenez-Arnau et
al., 2023). In the initial stages of CU, patients experience sudden
itching and mild skin edema. As the disease progresses, the edema
worsens and the wheals spread and merge. In severe cases, skin
ulceration is observed. This disease negatively impacts the physical
and mental health of the patients. More than half of the patients
cannot recover spontaneously within 1 year. Therefore, there is an
urgent need to discover new and effective interventions for this
disease ([41]He et al., 2021).
Currently, treatment with second-generation antihistamines is the
primary treatment to control or relieve symptoms of CU ([42]Valtellini
et al., 2024). However, most patients do not respond adequately to
conventional dosage of these drugs and higher dosage causes adverse
reactions such as dizziness and drowsiness. In severe cases, patients
may be treated with glucocorticoids. However, long-term use of
glucocorticoids can lead to drug dependence and cause adverse effects,
including centripetal obesity. Moreover, discontinuation of these drugs
increases the risk of symptom rebound and exacerbation ([43]Maurer et
al., 2024).
Traditional Chinese medicine (TCM) is widely used for the clinical
treatment of CU because of its high safety profile and a
multi-component, multi-target approach leading to synergistic effects.
TCM preparation from the dried vine stem of Fibraurea recisa Pierre
(FRP) shows antibacterial and anti-inflammatory properties ([44]Wang H.
Y. et al., 2024). In traditional medicine practices of certain ethnic
minorities, FRP is commonly used to treat skin diseases. In traditional
Dai medicine, FRP is one of the main components in the classic formula
“Yajie Shaba,” which is used for treating various allergies ([45]Liping
et al., 2022). Previous studies have confirmed that palmatine, an
alkaloid in FRP, is effective in alleviating CU because of its
anti-inflammatory and autophagy-regulating effects ([46]Xiao et al.,
2023). Although FRP is a promising drug for treating CU, the mechanisms
of action of its various components have not well established.
Metabolomics and network pharmacology are valuable tools for studying
complex diseases. They both have their own distinct advantages and
disadvantages. Metabolomics is a high-throughput methodology to analyze
the metabolome alterations in diseases, metabolite changes in response
to drug treatment, and drug-disease relationships. However, metabolome
data cannot be used to identify specific targets and mechanisms of drug
action. Network pharmacology uses computational and experimental
methods to identify the active components of drugs, predict disease
targets, analyze drug target functions, and unravel the drug-disease
target relationships. However, limitations in technology, data
availability, and modeling approaches currently impact the accuracy of
prediction results and require further improvements. The combined use
of metabolomics and network pharmacology is a powerful approach for an
in-depth analysis of drug action at the level of metabolites and
systematic analysis of the drug-target interactions at the network
level in human diseases ([47]Shi et al., 2023; [48]Li et al., 2023).
This study performed integrated network pharmacology and metabolomics
analysis to determine the mechanisms by which FRP alleviates CU in a
rat model. Firstly, UPLC/MS was used to determine the chemical
composition of FRP. Then, network pharmacology was used to predict and
analyze potential targets of action of each active component of FRP to
determine their underlying mechanism in alleviating CU. Finally,
metabolomics was used to determine the dynamic changes in the
metabolome after treatment with FRP. Through this systematic research
process, we aimed to determine the effects of FRP on the metabolic
state, core targets, and main signaling pathways in the CU rats, and
identify potential mechanisms by which FRP alleviates CU.
2 Material and methods
2.1 Experimental animals and drugs
24 male Sprague-Dawley rats of specific pathogen-free grade, aged
8 weeks and weighing approximately 200 ± 20 g, were used. These rats
were provided by Changzhou Cavens Experimental Animals Co., Ltd., and
the company’s experimental animal production license number is SCXK
(Su) 2022-0010. The experimental rats were housed in an SPF-grade
environment. The breeding temperature was strictly controlled within
the range of 18°C–25°C, the relative humidity was maintained at
40%–60%, the light and dark periods were each 12 h and alternated, and
the experimental rats had free access to food and water. All operations
and research contents involving experimental animals have been approved
by the Animal Ethics Committee of Yunnan University of Chinese Medicine
(Grant No.: R-062022157).
Loratadine (HY-17043, MCE, Shanghai, China). Ovalbumin (OVA, S7951,
Sigma Aldrich, St. Louis, MO, United States). Palmatine, coclaurine,
jatrorrhizine (AB1049, AB0718, AB0536, Chengdu Alpha Biotechnology Co.,
Ltd.) FRP was purchased from Guangxi Suli Medicinal Materials Co.,
Ltd., and was identified by Yin Zili, associate professor of Yunnan
University of Chinese Medicine. Weigh 480 g of FRP, decoct it with
eight times the volume of distilled water for the first time and six
times the volume of distilled water for the second time, and each
decoction lasts for 30 min. Remove the impurities and retain the
obtained water extract of the medicine for further processing.
2.2 UPLC/MS for the identification of FRP components
100 μL of FRP was added to a centrifuge tube, 100 μL of 70% methanol
(Merch, Darmstadt, Germany) was added, and vortex for 15 min.
12,000 r/min, 4°C, centrifugation for 3 min, pipetting the supernatant,
filtering with microporous filter membrane and then detecting on the
machine. A is ultrapure water, and B is acetonitrile (Merch, Darmstadt,
Germany). The flow rate was 0.35 mL/min, the column temperature was
40°C, and the injection volume was 2 μL. The elution gradient program
was set to: Elution gradient: 0 min 5% B, 95% B in 9 min, and
maintained at 95% for 1 min, 10.00–11.10 min, reduced to 5% B, and
equilibrated at 5% to 14 min. The mass spectrometry conditions were set
to electrospray ion source temperature 550°C; Ion spray voltage:
5,500 V (positive ion mode), 1–4,500 V (negative ion mode); The ion
source gas I, gas II, and curtain gas are set to 50, 60, and 25 psi,
respectively. Based on the self-managed Metware Database, the FRP
component is obtained by using the CV value of <0.5 as the filter
condition.
2.3 Determination of the content of effective components in FRP
Take 2 mL of the FRP, add 25 mL of acetonitrile and 0.1% phosphoric
acid (P816338, Shanghai Macklin Biochemical Technology Co., Ltd.) in a
ratio of 1:1, perform ultrasonic treatment at 37°C for 40 min (power:
250 W, frequency: 360 kHz). Let it cool down to room temperature, make
up the volume to 25 mL with acetonitrile and 0.1% phosphoric acid, and
filter it through a 0.45 μm filter membrane before injecting it into
the machine. According to the literature, the main active components in
FRP are palmatine (AB1049, Chengdu Alpha Biotechnology Co., Ltd.,
Chengdu, China), jatrorrhizine (AB0536, Chengdu Alpha Biotechnology
Co., Ltd., Chengdu, China), and coclaurine (AB0718, Chengdu Alpha
Biotechnology Co., Ltd., Chengdu, China). Therefore, accurately weigh
5 mg of palmatine, jatrorrhizine, and coclaurine reference substances
respectively, place them in a 100 mL volumetric flask, add acetonitrile
and 0.1% phosphoric acid in a ratio of 1:1 to completely dissolve them.
Finally, obtain a solution containing 50 μg of the reference substances
per 1 mL. Shake well and filter it through a 0.45 μm microporous filter
membrane before injecting it into the machine (Agilent 1260 Infinity
III Liquid Chromatography System, Agilent Technologies, Inc., Santa
Clara, United States).
The model of the chromatographic column is Agilent 5 TC-C18
([49]Gimenez-Arnau et al., 2023) 250 × 4.6 mm (article number:
588925-902). Use acetonitrile (A) and 0.1% phosphoric acid aqueous
solution (B) as the mobile phase. The gradient elution program is set
as follows: 0–5 min, 20%–50% A; 5–10 min, 50%–80% A; 10–20 min,
80%–100% A. The flow rate is set at 1 mL/min, the column temperature is
set at 25°C, the detection wavelength is 265 nm, and the injection
volume is 10 μL.
2.4 Group administration and model replication of CSU
SD rats were divided into control group, model group, FRP high-dose
group (FRP-H) and FRP low-dose group (FRP-L). Except for the control
group, other rats were intraperitoneally injected with 1 mg of OVA
(S7951, Sigma Aldrich, St. Louis, MO, United States) suspension on days
0, 2, and 4, and intraperitoneally injected with 2 mg of OVA on day 14.
On days 5–14 after modeling, the control group and the model group were
gavaged with an equal volume of distilled water once a day. The
concentrations of FRP-H and FRP-L groups were 40 mg/kg and 20 mg/kg.
2.5 Rat scratching behavior
Rats exhibit scratching behavior when they feel itching, and we
assessed the relief of itching in rats by scratching behavior. When
10 min after the last intraperitoneal injection of OVA intervention,
the scratching frequency of rats in each group was evaluated within
20 min. The scratching behavior is judged by the rats rubbing the skin
on their backs, scratching their heads and torsos, and rubbing against
each other.
2.6 Histopathological detection of rat skin
The skin tissue of rats in each group was fixed with 4%
paraformaldehyde at 4°C for 12 h, embedded with paraffin wax and cut
into 5 μm thick sections, deparaffinized and hydrated. Stain with
hematoxylin eosin (HE, KGA224, KeyGEN Biotech, NanJing, China) for
5 min at room temperature to study pathological changes, dehydrate with
ethanol gradient, mount with neutral gum, and observe and photograph
under a light microscope with a field of view of 100×. The remaining
slices are stored at room temperature for subsequent experiments.
2.7 Detection of the expression of inflammatory factors in skin tissue
After the blood was taken, the rats were euthanized by cervical
dislocation, and the shaved back skin tissue was obtained for
subsequent experimental analysis. After blood collection, shaved back
skin tissue was obtained for subsequent experimental analysis. The skin
tissue of each group was washed with pre-cooled PBS at 4°C, weighed and
minced, and normal saline was added at a ratio of 1:9 to make skin
tissue homogenate. Centrifuge at 5,000 g for 10 min, take the
supernatant for detection, and detect the expression of IL-4
(SEKR-0004, Solarbio, Beijing, China), IL-6 (SEKR-0005), IL-12
(E-EL-R0064c; Elabscience, TX, United States) and Interferon-γ (IFN-γ,
E-EL-R0009, Elabscience) in skin tissues according to the instructions
of the ELISA kit.
2.8 Mast cell activation and degranulation detection
Toluidine blue staining is commonly used for skin mast cell testing.
The prepared skin tissue wax tablets were soaked in 0.5% toluidine blue
solution (G1032, Servicebio, Wuhan, China) for 30 min at room
temperature and washed with distilled water. Soak in 0.5% glacial
acetic acid solution for 5 s and dehydrate with ethanol and transparent
xylene. It is capsulated with neutral gum. Finally, the light
microscope (Olympus Corporation, Japan) was randomly selected for 100 ×
without repeating, and mast cell counts were taken.
2.9 Mast cell tryptase (MCT) and eosinophil protein X (EPX) assays
After the wax tablets of skin tissue were dewaxed, the antigen was
extracted in 0.01 mol/L citrate, and the antigen was exposed in the
microwave for 20 min 0.03% H[2]O[2] was added to inactivate endogenous
peroxidase for 15 min, and blocked with goat serum blocking solution
for 20 min at room temperature. Subsequently, sections are mixed with
anti-TPSAB1 antibody (13343-1-AP, Proteintech, Wuhan, China) at a 1:50
dilution and with anti-EPX antibody (bs-3881R) (Bioss, Beijing, China)
at a 1:100 dilution and incubated overnight at 4°C. After rewarming at
37°C for 1 h, incubate with secondary antibody and biotin-labeled goat
anti-rabbit IgG for 30 min, then continue with biotin-labeled
streptavidin for 30 min. t is chromogenic with the DAB kit,
counterstained with hematoxylin, and sealed with neutral resin. Hooting
at 200 levels × random optical microscope (Olympus Corporation, Japan)
was not repeated, and the Image-Pro Plus 6.0 software detected the
comprehensive optical density value for each field of view.
2.10 Detection of biochemical markers in serum
Rats in each group gently pressed the sides of the orbit with their
fingers. After protruding the eyeball, use a capillary to enter through
the gap between the eyeball and the corner of the eye. A blood sample
can be collected by gently rotating the capillary and slowly moving it
behind the eyeball. Centrifuge the collected blood sample at
3,000 r/min for 10 min, aspirate 1 mL of supernatant, and follow the
instructions of the ELISA kit. The levels of immunoglobulin E (IgE,
SEKR-0019), LTB4 (CSB-E08035r, Cusabio, Wuhan, China), and HIS
(E-EL-0032c) in serum were detected at a wavelength of 450 nm using a
microplate reader.
2.11 Metabolomics
50 μL of rat plasma in the model group and FPR-H group are removed and
200 μL of methanol solution containing internal standard is added. Hake
for 5 min, centrifuge at 18,000 rpm for 10 min, and take 200 μL of
supernatant. After evaporation by vacuum evaporator, 100 μL of pure
water and 100 μL of methanol are reconstituted. Centrifuge at
18,000 rpm for 10 min, and finally take 80 μL of supernatant for
storage. 5 μL was filtered through a 0.22 μm filter membrane for
LC-QTOF/MS (Sciex TripleTOF 5600+ LC-QTOF/MS, Sciex) for positive and
negative ion detection, and the detection results were compared between
groups, and the results were repeated twice. Liquid phase conditions:
Waters HSS T3 1.8 μm, 2.1 × 100 mm Column with 0.3 mL/min flow rate,
40°C column heater, 5 μL injection volume, Aqueous phase A (ultrapure
water + 0.1% formic acid), B phase organic phase (acetonitrile),
gradient elution program is: 0–1.5 min, 95% A, 5% B; 1.5–2.5 min,
95%–85% A, 5%–15% B; 2.5–6 min, 85%–40% A, 15%–60% B; 6–10 min,40%–5%
A,60%–95% B; 10–12 min,5% A,95% B; 12–12.5 min, 5%–95% A, 95%–5% B;
12.5–15.5 min, 95% A, 5% B. Mass spectrometry conditions: The positive
and negative ion modes were scanned by Turbo V electrospray ionization,
and the parameter was set to the ion spray voltage was 7 kV; Turbine
spray temperature, 550°C; Declustering potential, 70 V; Collision
energy, 30 eV. The TOF/MS has a scanning range of m/z 50–1,200 Da.
2.12 Network pharmacology
The chemical components of FRP detected by UPLC/MS were screened out
for network pharmacology analysis. The screening conditions were set as
high for gastrointestinal absorption, yes for BBB permeability, and at
least two yes for drug similarity, and the active ingredient of FRP was
used as the product if it met the criteria. The Smiles number of the
compound was searched in the Pubchem database, and the target of the
compound was predicted by the Swiss target perdiction database, and the
target with “Probability” greater than 0.1 was selected for subsequent
analysis. With “CSU” as the keyword, the gene name was transformed by
the OMIM, TTD and Genecards databases, and the duplicate values were
deleted as the target of CSU. The intersections of the screened active
ingredient targets and disease targets were made, and the intersection
results were imported into Cytospace 3.9.1 software, and the network
diagram of “FR-active ingredient-target” was drawn, and the main active
ingredients of FRP in the treatment of CSU were screened out according
to the degree value. Import the intersection targets into the String
database, set the minimum interaction score to 0.4, check the hidden
protein name, and output it in TSV (A-B) format. Cytoscape 3.9.1
software was imported, the network diagram was drawn, and the key
targets of Rhubarb vine in the treatment of CSU were screened according
to the degree value. Finally, the intersection targets were introduced
into the micro-biotech data analysis platform for GO enrichment
analysis and KEGG enrichment analysis to find the main pathways for FRP
to intervene in CSU. Using the key targets as receptors and the
components as ligands for molecular docking, select the top three
components and targets ranked by binding energy. Use the Gromacs
program to perform molecular dynamics simulations on the protein-small
molecule complexes to evaluate the dynamic laws of the binding between
ligands and receptors.
2.13 Detection of Akt, p-Akt, P13K, p-P13K protein expression
The skin tissue in each group was washed with pre-cooled PBS, weighed
and shredded. Add 1 mL of RIPA lysate containing PMSF per (Solon, OH,
United States) 100 g of tissue, centrifuge at 12,000 g at 4°C for
5 min, and transfer the supernatant to a pre-chilled EP tube. Protein
quantification was performed according to the BCA kit method. Depending
on the molecular weight of the protein to be measured, SDS-PAGE gels
are prepared. Add an appropriate amount of pre-cooled 1 × of running
buffer, and add the sample and the reference protein to the lane.
Adjust the voltage to bring the destination strip to the predetermined
position. After shearing the PVDF (Millipore, Schwalbach, Germany)
membrane and transferring the membrane at constant pressure, the
membrane was stained with Ponceau red s staining solution for 5 min.
Put it in an antibody incubation box containing TBST, rinse it on a
shaker for 5 min, discard TBST, and add 5% skimmed milk powder prepared
with TBST. Block at room temperature shaker for 1 h, discard the
blocking solution, add diluted primary antibody (Akt antibody dilution
at a ratio of 1:500 (ab8805, Abcam, Cambridge, United Kingdom), p-Akt
at a ratio of 1:1,000 (ab38449, Abcam, Cambridge, United Kingdom), P13K
at a ratio of 1:20,000 (ab191606, Abcam, Cambridge, United Kingdom),
p-P13K (ab182651, Abcam, Cambridge, United Kingdom) at a ratio of
1:500, anti-β-actin-antibody at a ratio of 1:1,000 (ab8229, Abcam,
Cambridge, United Kingdom), and incubate overnight at 4°C. Wash the
membrane five times with TBST for 5 min each time. Continue to add
diluted secondary antibodies (Rabbit Anti-Mouse IgG H&L (HRP) (ab6728,
Abcam, Cambridge, United Kingdom) diluted 1:10,000 and Goat Anti-Rabbit
IgG H&L (HRP) (ab6721, Abcam, Cambridge, United Kingdom) diluted
1:10,000 and incubated for 1 h at room temperature. Wash the membrane
five times with TBST for 5 min each time. After soaking in liquid A and
solution B of the prepared luminescence reagents for 5 min, the
detection was carried out with Tanon 6600 luminescence imaging
workstation (Tanon 6600 Luminous Imaging Workstation, Tanon
Corporation). The optical density value was analyzed by Image Pro Plus
6.0 software, and the relative expression level of the protein was
calculated as the gray value of the target protein/the gray value of
the internal reference protein.
2.14 Data analysis
Data were analyzed and graphed using Graphpad Prism 9 (Version 9.4.0).
All data were presented as means ± SD, and statistical differences
between groups were performed using one-way ANOVA and Tukey’s tests. P
value of less than 0.05 was considered to be significant.
3 Results
3.1 Chemical composition of FRP based on UPLC/MS
The chemical composition of FRP was analyzed by UPLC/MS. The total ion
chromatogram of the FRP sample recorded in both positive and negative
ion modes is shown in [50]Figure 1. Based on the UPLC/MS results, we
identified 2,206 compounds in FRP, including 452 alkaloids, 435 amino
acids and their derivatives, 288 phenolic acids, 146 lignin and
coumarin compounds, 145 organic acids, and 134 terpenoids. The top 1%
of metabolites are in [51]Table 1.
FIGURE 1.
[52]FIGURE 1
[53]Open in a new tab
The total ion chromatogram of the FRP sample in (A) positive and (B)
negative ion mode.
TABLE 1.
The compounds with a content ranking in the top 1%.
Compounds CAS
13,13α-Didehydro-9,10-dimethoxy-2,3-(methylenedioxy)-berbine
1-[(4-hydroxyphenyl)
methyl]-7-methoxy-1,2,3,4-tetrahydroisoquinolin-8-ol
Isocorybulbine 22672-74-8
Corybulbine 518-77-4
Jatrorrhizine 3621-38-3
Coclaurine 486-39-5
3,4,11-trimethoxy-13-methyl-7,8,12b,13-tetrahydro-5h-6-azatetraphen-10-
ol
Tetrahydroprotopapaverine 26193-25-9
Reticuline 485-19-8
Dehassiline 142717-64-4
O-Rnethylstepharinosine
Lauroscholtzine; N-Methyllaurotetanine 2169-44-0
N (6), N (6)-Dimethyl-L-lysine 2259-86-1
Dehydrocrebanine 77784-22-6
Columbamine 3621-36-1
Adenine 73-24-5
Zarzissine 160568-14-9
Vidarabine 5536-17-4
Dehydrocorydalmine 6877-27-6
Adenosine 58-61-7
9-Alpha-Ribofuranosyladenine
9-Arabinosyladenine
Palmatine 3486-67-7
[54]Open in a new tab
3.2 Determination of active components in FRP
Contemporary phytochemical studies have reported that
benzylisoquinoline alkaloids are the principal bioactive constituents
in FRP. Systematic network pharmacology screening data demonstrated
that palmatine, coclaurine and jatrorrhizine were the primary active
alkaloids in FRP. At 265 nm, retention times for the FRP test samples
and coclaurine were 4.568 min and 4.621 min respectively; retention
times for the FRP test samples and jatrorrhizine were 6.925 min and
7.347 min, respectively; retention times for the FRP test samples and
palmatine were 7.471 min and 7.727 min, respectively ([55]Figure 2).
The separation method showed good specificity because the differences
in retention times between the reference compounds and the test samples
were all within ± 0.5 min.
FIGURE 2.
[56]FIGURE 2
[57]Open in a new tab
The liquid chromatograms of (A) the test sample and (B) the mixed
reference substances of Coclaurine, Jatrorrhizine, and Palmatine. (C)
The chemical structures of Coclaurine, (D) Jatrorrhizine, and (E)
Palmatine.
3.3 Effect of FRP on scratching behavior in rats
As shown in [58]Figure 3A, we generated the CU model rats and the
FRP-treated CU model rats. Compared with the control group, rats in the
CU model group and the FRP-treated CU model group showed different
degrees of itching response (P < 0.001). The FRP-H and FRP-L groups
showed significant improvements in scratching latency and time compared
to the CU model group (P < 0.01 and P < 0.05, respectively; [59]Figure
3B).
FIGURE 3.
[60]FIGURE 3
[61]Open in a new tab
(A) The experimental procedure. (B) The effect of FRP on the scratching
frequency of rats. (C) The effect of FRP on the pathological changes of
rat skin tissues. All data are presented as
[MATH: X¯
:MATH]
±SD deviation (n = 6). Compared with the Control group, ^###P < 0.001;
compared with the model group, ^*P < 0.05, ^**P < 0.01.
3.4 Effect of FRP on the histopathological changes in the rat skin tissues
According to the H&E staining results, the skin of rats in the control
group showed normal epidermal structure, including uniform thickness,
neat and orderly arrangement of cells, consistent staining, and normal
hair follicle structure without significant dilation of the
capillaries. In the CU model group, the epidermal structure was
partially absent because of parakeratinization or hyperkeratosis, and
the granular layer was thickened. Furthermore, we observed disordered
collagen fibers, widened fiber bundle distance, dilated blood vessels
and lymphatic vessels, and presence of multiple inflammatory cell
infiltrates around superficial blood vessels. However, FRP intervention
significantly improved CU-related histopathological characteristics
([62]Figure 3C).
3.5 Effect of FRP on the levels of inflammatory cytokines in rats
The levels of IL-4, IL-6, IL-12, and IFN-γ in skin tissue were
significantly higher in the model group compared with the control group
(P < 0.001), but were significantly decreased after FRP intervention (P
< 0.001; [63]Figure 4). These results suggested that FRP treatment
suppressed the release of inflammatory mediators in CU.
FIGURE 4.
[64]FIGURE 4
[65]Open in a new tab
The effects of FRP on the levels of inflammatory factors (A) IL-4, (B)
IL-6, (C) IL-12, and (D) IFN-γ in the skin tissues of rats in each
group. All data are presented as
[MATH: X¯
:MATH]
± SD deviation (n = 6). Compared with the Control group, ^###P < 0.001;
compared with the model group, ^***P < 0.001.
3.6 Effect of FRP on the skin mast cells in rats
In the control group, we observed fewer mast cells that were dispersed
throughout the dermal layer of the skin tissue. The mast cells
exhibited metachromatic purple-stained cytoplasm and occasional
degranulation. In the skin tissue of the model group rats, we observed
significantly higher number of mast cells, dispersed granules, tissue
edema, and widened distance between collagen bundles (P < 0.001).
However, FRP intervention significantly reduced the number of mast
cells, degranulation in mast cells, and vasodilation in the skin
tissues of the CU model rats (P < 0.001, [66]Figures 5A, B).
FIGURE 5.
[67]FIGURE 5
[68]Open in a new tab
(A) Immunohistochemical images of mast cells, MCT, and EPX in rats of
each group. The effects of FRP on (B) the number of mast cells, (C) the
expression of MCT, and (D) the expression of EPX in rats. The effects
of FRP on the levels of (E) IgE, (F) LTB4, and (G) HIS in the serum of
rats. All data are presented as
[MATH: X¯
:MATH]
± SD deviation (n = 6/n = 3). Compared with the Control group, ^###P <
0.001; compared with the model group, ^*P < 0.05, ^**P < 0.01, ^***P <
0.001.
3.7 Effect of FRP on the expression of MCT and EPX
Based on immunohistochemistry, the expression levels of MCT protein
were significantly higher in the skin tissue of the model group rats
compared to the control group rats (P < 0.001). Furthermore, MCT was
located around the interstitial cells and small blood vessels as
brownish-yellow particles. Compared with the model group, the
expression levels of MCT protein were significantly reduced in the skin
tissues of the FRP-H and FRP-L group rats (P < 0.01 and P < 0.05,
respectively; [69]Figures 5A, C). Immunohistochemistry results also
showed that the expression levels of EPX protein were significantly
higher in the skin tissues of the model group rats than in the skin
tissues of the normal group rats (P < 0.001), but were significantly
reduced in the skin tissues of the FRP-H and FRP-L group rats (P <
0.001, P < 0.01, [70]Figures 5A, D).
3.8 Effect of FRP on immune biomarkers in rats
The serum levels of IgE, LTB4, and HIS were significantly higher in the
model group rats compared with the control group rats (P < 0.001), but
were significantly reduced in the FRP-H and FRP-L group rats compared
with the model group rats (P < 0.001, [71]Figures 5E–G).
3.9 Effect of FRP on skin metabolites in rats
The characteristic peaks from the LC-QTOF/MS data were extracted and
preprocessed. We identified 8,200 chromatographic peaks in the positive
ion mode and 8,134 chromatographic peaks in the negative ion mode
([72]Figures 6A, B). This indicated that the instrument was stable and
ready for further analysis The PCA and PLSDA results showed that
samples from the model group and the FRP treatment group exhibited
distinct separation in both positive and negative ion modes. This
suggested significant metabolic differences between the model group and
the FRP-treatment group rats. Then, we screened metabolites using the
OPLS-DA model with VIP value of >1 and P value of <0.05 as threshold
parameters and identified 74 differential metabolites (62 upregulated
and 12 downregulated) in the positive ion mode and 47 differential
metabolites 9 upregulated and 38 downregulated in the negative ion mode
([73]Figures 6C–H).
FIGURE 6.
[74]FIGURE 6
[75]Open in a new tab
(A) Chromatograms of metabolites in positive ion and (B) negative ion
modes. (C) PCA, (D) PLSDA, and (E) volcano plots under the positive ion
mode. (F) PCA, (G) PLSDA, and (H) volcano plots under the negative ion
mode.
In the volcano diagram, differential metabolites in the same cluster
exhibited similar expression patterns and may have similar functions or
participate in the same metabolic processes ([76]Figures 7A, B). The
diagnostic value of differential metabolites was verified by plotting
ROC curves, we selected differential metabolites with AUC values
greater than 0.9 for further analysis. Furthermore, we performed KEGG
pathway analysis of the differential metabolites and identified
enrichment of pyrimidine metabolism, tryptophan metabolism, arginine
biosynthesis, and nicotinate and nicotinamide metabolism ([77]Figures
7C, D).
FIGURE 7.
[78]FIGURE 7
[79]Open in a new tab
(A) Heatmaps of differential metabolites in positive ion and (B)
negative ion modes. (C) Heatmap of differential metabolites and (D)
Metabolites pathway diagram with an ROC value >0.9. The metabolite
levels were indicated with a color code that gradually decreased from
purple to blue. The purple color indicated higher levels, whereas blue
indicated lower levels.
3.10 Results of network pharmacology analysis
We screened FRP chemical components with the top 1% content with
SwissADME and identified 10 active ingredients and 189 corresponding
targets. We also screened the OMIM, TTD, and Genecards databases for
targets corresponding to the disease. After removing duplicates, we
obtained 1,111 disease-specific targets. Subsequently, we generated a
Venn diagram on the micro-biotech data analysis platform by
intersecting 1,111 disease-specific targets with the 189 active
ingredient targets ([80]Figure 8).
FIGURE 8.
[81]FIGURE 8
[82]Open in a new tab
(A) Venn diagram of the target points of differential metabolites of
FRP and disease target points. (B) PPI network diagram of key target
points.
The FRP active ingredients and their targets were imported into the
String database and a “drug-active ingredient-target” network diagram
was constructed using the Cytoscape 3.9.1 software. Based on this
analysis, we identified corybulbine, isocorybulbine, reticuline,
demethyleneberberine, and dehydrocorydalmine as the main active
ingredients of FRP with high degree values ([83]Figure 9).
FIGURE 9.
[84]FIGURE 9
[85]Open in a new tab
Network diagram of FRP-active ingredients-key target points. The
inverted triangle represents the drug, the circles represent components
screened by the drug, and the squares represent intersection targets
that are color coded from blue to orange based on the degree value. The
CytoNCA plug-in was used to analyze the four topological parameters of
nodes, including betweenness centrality (BC), closeness centrality
(CC), eigenvector centrality (EC), and degree centrality (DC). The
orange-red icons were sorted according to the degree value.
Molecular docking was performed between the three active ingredients
(palmatine, jatrorrhizine, and coclaurine) and the core target. The top
three docking structures based on binding energy values were
Akt-jatrorrhizine (−10.4 kcal/mol), Akt-palmatine (−9.6 kcal/mol), and
Akt-coclaurine (−9.2 kcal/mol) ([86]Figure 10). All the three
structures were all less than −7.0 kcal/mol, thereby indicating stable
ligand-receptor binding.
FIGURE 10.
[87]FIGURE 10
[88]Open in a new tab
Molecular docking diagrams of (A) Akt-Coclaurine, (B)
Akt-Jatrorrhizine, (C) Akt-Palmatine, (D) PIK3CA-Coclaurine, (E)
PIK3CA-Jatrorrhizine, (F) PIK3CA-Palmatine, (G) STAT3-Coclaurine, (H)
STAT3-Jatrorrhizine, and (I) STAT3-Palmatine. (J) The binding energies
of the docking complexes in each group.
Molecular dynamics simulation results further confirmed the binding of
Akt with palmatine, jatrorrhizine, and coclaurine. The Root Mean Square
Deviation (RMSD) curve demonstrated that the protein conformation
fluctuated during the simulation, but did not break. This indicated
that the binding of these three compounds of FRP to Akt was strong and
generated a protein structure with good stability ([89]Figure 11A). The
Root Mean Square Fluctuation (RMSF) data suggested that the three
components did not significantly alter the stability of the binding
region of Akt while binding to the small molecules. The RMSF values for
most sites in Akt were 0.2 nm, thereby indicating that the structure of
the protein-small molecule binding region was stable ([90]Figure 11B).
The radius of gyration curve represents the density of the overall
protein structure. Jatrorrhizine, coclaurine, and palmatine showed
stable gyration radii with the Akt protein from the beginning to the
end of the simulation. This suggested that binding of the three
compounds did not affect stability of the protein conformation
([91]Figure 11C). The number of hydrogen bonds between Akt and the
three compounds are indicative of their binding strength. Palmatine
showed the highest hydrogen bond density and strength with Akt,
followed by coclaurine and jatrorrhizine ([92]Figure 11D). Furthermore,
we also detected the exposure degree of the binding receptor to the
surrounding solvent molecules during the simulation. Our data showed
that the interaction between the solvent and the surface area of the
protein-small molecule complex was stable throughout the simulation
process ([93]Figure 11E). The binding of jatrorrhizine and coclaurine
molecules to the Akt protein was stable from the beginning to the end
of the simulation and the complex structure remained unchanged. The
binding of a palmatine molecule to the Akt protein was stable for the
first 40 ns, but the stability decreased slowly in the next 60 ns.
Overall, the complex remained stable. This suggested reduction in the
solvent accessible surface area of the structure. The binding free
energy can be used to determine the variability and stability of the
ligand-protein binding mode. The Molecular Mechanics Poisson-Boltzmann
Surface Area (MMPBSA) binding free energies of the coclaurine,
jatrorrhizine, and palmatine molecules were −56.865, −64.71, and
−135.327 kJ/mol respectively ([94]Figure 11F).
FIGURE 11.
[95]FIGURE 11
[96]Open in a new tab
(A) The curve diagram of RMSD changes of the docking complex. (B) The
RMSF analysis diagram of the docking complex. (C) The Rg analysis
diagram of the docking complex. (D) The analysis diagram of the number
of hydrogen bonds in the docking complex. (E) The curve diagram of the
time changes of the solvent and surface area of the docking complex.
(F) The binding free energy of the docking complex calculated by
MMGBSA.
Gene Ontology (GO) and KEGG pathway enrichment analysis of the target
proteins was performed using the micro-bioinformation analysis
platform. GO analysis included identification of enriched biological
processes (BP), molecular functions (MF), and cell components. The top
enriched biological processes were cell chemotaxis, regulation of MAP
kinase activity, and positive regulation of protein serine/threonine
kinase activity. The top enriched cell components were membrane raft,
membrane microdomain, and membrane region. The top enriched molecular
functions were protein tyrosine kinase activity and protein
serine/threonine kinase activity ([97]Figures 12A–C). The top enriched
KEGG pathways were Pl3K-Akt signaling pathway, proteoglycans in cancer,
endocrine resistance, pancreatic cancer, and breast cancer ([98]Figure
12D).
FIGURE 12.
[99]FIGURE 12
[100]Open in a new tab
Network pharmacology (A) BP, (B) CC, (C) MF, and (D) KEGG enrichment
pathway diagrams.
3.11 Combined analysis of network pharmacology and metabolomics
We used the Cytoscape 3.9.1 software and Swiss Target Prediction to
predict the potential targets of 58 differential metabolites that were
identified by metabolomics. Subsequently, we intersected the 45 targets
screened by network pharmacology with the 727 targets related to
metabolites and identified 34 common target proteins ([101]Figure 13A).
These common target proteins were evaluated using the Cytoscape 3.9.1
software. Based on the degree value, the top target proteins were MMP9,
PTGS2, MTOR, ERBB2, CDC42, PIK3R1, and PIK3CA. KEGG pathway analysis of
these common target proteins showed that the top enriched pathway were
PI3K-Akt signaling pathway, Kaposi sarcoma-associated herpesvirus
infection, proteoglycans in cancer, pancreatic cancer, and endocrine
resistance ([102]Figures 13B, C).
FIGURE 13.
[103]FIGURE 13
[104]Open in a new tab
(A) Target Venn diagram, (B) PPI network diagram of key target points,
and (C) KEGG enrichment pathway diagram of the combined analysis.
3.12 Effects of FRP on the expression of Akt, p-Akt, PI3K, and p-P13K in rats
Based on Western blotting, the expression levels of p-Akt and p-P13K
were significantly decreased in the model group compared with the
control group (P < 0.001). However, the expression levels of p-Akt and
p-P13K were significantly increased in the FRP treatment groups
compared with the model group (P < 0.001, P < 0.01, P < 0.05,
[105]Figure 14).
FIGURE 14.
[106]FIGURE 14
[107]Open in a new tab
(A) Protein band diagrams of each group in the PI3K-Akt pathway. The
effects of FRP on the protein expressions of (B) Akt, (C) p-Akt, (D)
PI3K, and (E) p-PI3K in rats. All data are presented as
[MATH: X¯
:MATH]
± SD deviation (n = 3). Compared with the Control group, ^###P < 0.001;
compared with the model group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.
4 Discussion
Recurrent occurrence of itchy wheals or angioedema on the skin surface
are the main clinical manifestations of CU. The repeated occurrence of
itchy symptoms every day significantly reduces the quality of life in
patients with CU ([108]Goncalo et al., 2021; [109]Akca and Tuncer Kara,
2020). FRP is an ethnic medicine that has been used for hundreds of
years to clinically treat allergic skin diseases, but its mechanism of
action was unclear. Numerous studies have demonstrated that the total
alkaloids and other active components of FRP play a significant role in
the treatment of Alzheimer’s disease, improvement of hyperglycemia,
regulation of cellular apoptosis, and modulation of inflammatory
responses. In acute toxicity tests using gavage administration in mice,
the total alkaloids of FRP were both safe and effective, thereby
indicating significant potential for further clinical development. This
study quantitatively evaluated the scratching behavior of CU model and
FRP treated CU model rats to determine the efficacy of FRP in
alleviating the main symptoms of CU. FRP treatment significantly
relieved reduced itching symptoms in the CU model rats. Furthermore, we
performed histopathological analysis of skin tissues by H&E staining.
The rats were sensitized and challenged with OVA to induce CU-like
allergic reactions. H&E staining results showed that low- or high-dose
FRP treatment alleviated pathological morphology of skin tissues in the
CU model rats to varying degrees.
CU is highly related with IgE-mediated mast cell degranulation and
release of histamine (HIS). IgE is an antibody type that binds to mast
cells and stimulates them to degranulate and release HIS and other
inflammatory mediators upon encountering an allergen ([110]Kolkhir et
al., 2024). After patients with CU are first exposed to allergens, the
humoral immune response of the body is activated, leading to the
production of IgE antibodies. IgE specifically binds to high-affinity
receptors on the surface of the mast cells, thereby sensitizing them
for future encounter with the same allergen ([111]Orzan et al., 2022;
[112]Kolkhir et al., 2022). When the same allergen invades sensitized
individuals, it rapidly stimulates effector immune cells to undergo
degranulation and triggers release of pro-inflammatory mediators such
as HIS and LTB4. HIS acts on the blood vessels of the skin and mucous
membranes throughout the body and increases blood vessel permeability
and capillary dilation. This leads to formation of wheals and induces
clinical symptoms of CU. LTB4, a lipid mediator derived from
arachidonic acid, is closely related with the onset of CU ([113]Zeng et
al., 2024; [114]Lu et al., 2024). Mast cells recruit effector T cells
to the site of inflammation by releasing LTB4. This helps sustain and
amplify the inflammatory response. Therefore, LTB4 is a key factor that
links activation of mast cells and T cells. It plays an important role
in the pathological process of CU and is a major focus of CU treatment
research.
Dermal mast cells are main effector cells involved in both wheal
formation and sensory nerve stimulation in CU. When mast cells
degranulate, they release several pro-inflammatory mediators, including
MCT, which are crucial for the development and sustenance of allergic
reactions ([115]Puxeddu et al., 2024; [116]Metz et al., 2024).
Eosinophils are also regarded as pro-inflammatory cells in the immune
response related to CU and release a variety of inflammatory factors
and toxic substances. EPX is a toxic protein primarily secreted by
eosinophils and is often used as a specific marker for eosinophil
activity. The inflammatory response in CU leads to significantly
increased levels of MCT and EPX ([117]Elieh-Ali-Komi et al., 2023;
[118]Sanchez et al., 2021). Our study results showed that mast cell
degranulation was significantly reduced in the CU model rats after FRP
intervention. Furthermore, the levels of IgE, HIS, LTB4, EPX, and MCT
were significantly higher in the skin tissues of model group rats than
those in the control group, but were significantly reduced after FRP
intervention. These findings suggested that FRP effectively reduced
mast cell degranulation and the release of HIS mediated by IgE.
IL-4, a signature cytokine of the Th2 lymphocytes, promotes
proliferation and differentiation of mast cells, and stimulates mast
cells to release HIS, a key mediator in the formation of wheals. IL-4
also increases the expression and signal transduction of high-affinity
IgE receptor cells. Furthermore, IL-4 regulates IgE expression levels
by inducing B cells to switch from IgG synthesis to IgE production,
thereby increasing the total serum IgE levels in the body ([119]Wu et
al., 2022; [120]Aranda et al., 2023). This induces Th2
lymphocyte-dominated inflammatory response, which is at the core of CU
pathogenesis and activation of mast cells. Activated mast cells secrete
a variety of inflammatory factors, including IL-4, which further
exacerbate CU and the associated vicious cycle ([121]McSweeney et al.,
2022). IL-6 activates T cells and exacerbates the inflammatory response
in the body ([122]Korn and Hiltensperger, 2021).
IL-12 is a key cytokine that promotes polarization of T cells into the
Th1 phenotype. It inhibits secretion of IgE by the B cells. A low
concentration of IL-12 often reflects weakened cellular immune
response. IL-12 secretion is related to the production of IFN-γ, a
representative cytokine of Th1 lymphocytes. IL-12 exerts antagonistic
effect on IL-4, a Th2 lymphocyte-related cytokine. IFN-γ inhibits the
activity of Th2 cells, promotes differentiation of Th cells into Th1
cells, and blocks the synthesis of IgE ([123]Peng et al., 2021). This
study found that FRP treatment effectively restored the normal balance
between Th1 and Th2 cells by reducing the levels of pro-inflammatory
factors such as IL-4 and IL-6, and increasing the levels of
anti-inflammatory factors such as IL-12 and IFN-γ, thereby alleviating
the inflammatory response caused by CU.
The metabolomics study identified 58 differential metabolites related
to CU and FRP-treated CU model group. Pyroglutamic acid, a natural
amino acid derivative, inhibits inflammatory response and improves
intestinal health in mice. The state of intestinal health is closely
related with the occurrence and development of allergic diseases.
Genkwanin, an antioxidant, exerts anti-inflammatory effects by
inhibiting inflammatory mediators and related signaling pathways, and
protects cells against oxidative stress damage ([124]Gang et al.,
2018). Indomethacin, a non-steroidal anti-inflammatory drug,
demonstrates antipyretic and anti-inflammatory effects. Cholic acid
reduces inflammation by inhibiting the NLRP3 inflammasome ([125]Rashid
et al., 2021). Piperazine derivatives are highly effective histamine H1
receptor antagonists and are a valuable therapeutic option for treating
CU. The deficiency of argininosuccinate lyase leads to the accumulation
of argininosuccinic acid, which is a key factor in altering substances
that trigger inflammation. Carbazole derivatives demonstrate
significant antibacterial and anti-inflammatory effects. L-Tryptophan
plays an important role in regulating inflammatory response in a
variety of human chronic inflammatory diseases ([126]Zeng et al.,
2024). Isoliquiritigenin inhibits inflammation by suppressing the NF-κB
signaling pathway. Piperine is associated with antioxidant activity and
reduces inflammation and pain ([127]Choi et al., 2020).
Activation of the PI3K-AKt signaling pathway regulates pyrimidine
metabolism, improves mitochondrial morphology, reduces oxidative stress
damage, and mitigates the inflammatory response ([128]Shang et al.,
2021). Tryptophan, an essential amino acid for the human body, plays a
key role in several physiological processes and signal transduction.
Tryptophan metabolic pathway regulates a variety of physiological
functions, including inflammation. Arginine biosynthesis plays a key
role during allergic reactions ([129]Sun et al., 2023). Inflammatory
mediators such as HIS, platelet-activating factor, and LT released by
the mast cells, stimulate endothelial cells to convert arginine into
NO, thereby exacerbating the allergic reaction. Niacin and nicotinamide
metabolism regulate inflammation and immune functions ([130]Gonzalez et
al., 2023). The changes in the activity and/or levels of these
compounds and the functional characteristics of the enriched pathways
suggest that the intervention effect of FRP on CU is related to its
inflammatory effects.
Metabolomics data can be used to identify metabolome changes induced by
FRP treatment that are associated with improving CU, but cannot
comprehensively explain the underlying regulatory mechanism. Network
pharmacology can be used for in-depth investigation of the mechanism of
action of FRP and overcome the drawback of metabolomics. By combining
FRP metabolomics and analysis of drug characteristics, we identified
jatrorrhizin, coclaurine, and palmatine, all of which are
berberine-like compounds, as active ingredients of FRP. These compounds
are associated with significant analgesic, anti-inflammatory, and
anti-hepatitis effects ([131]Hong et al., 2023). Jatrorrhizine, a key
component in FRP, demonstrates significant anti-inflammatory effects.
Coclaurine demonstrates good binding energy in several molecular
docking studies with various cellular proteins and participates in
several cellular processes, including cell differentiation, growth,
movement, and apoptosis ([132]Hu et al., 2024). Palmatine is an
effective immunomodulatory compound that enhances innate immune
response by activating key immune genes. The anti-inflammatory effects
of palmatine have been confirmed in several studies. For example,
palmatine exhibits synergistic anti-inflammatory effects in LPS-induced
microglia and significantly inhibits the production of TNF-α and iNOS.
Preliminary studies by our research group have shown that palmatine
significantly alleviates CU by regulating inflammation ([133]Wang H. et
al., 2024). Jatrorrhizin, coclaurine, and palmatine are all associated
with anti-inflammatory properties and are potential active components
in FRP involved in the alleviation of CU. Therefore, we further
investigated the mechanisms by which these components in FRP alleviate
CU.
The combined analysis of network pharmacology and metabolomics data
showed that FRP alleviated CU through its regulatory effects on the
PI3K/Akt signaling pathway and targets such as STAT3, ERBB2, MMP9,
PTGS2, PIK3R1, PIK3CA, and MTOR. High expression of STAT3 in mast cells
triggers inflammatory reactions. Plasma levels of MMP9 are
significantly increased in the CU patients ([134]Bartko et al., 2024).
PTGS2 is a potential regulator of immune responses and inflammation in
CU. PIK3R1, a regulatory subunit of PI3K, mediates inflammation and
indirectly regulates its own expression by inhibiting the production of
IL-6. PIK3CA inhibition is associated with anti-inflammatory effects
([135]Du et al., 2024). MTOR plays a key role in recognizing
nutritional signals and modulating cell growth and proliferation.
Inhibition of mTOR reduces the levels of inflammatory cytokines. ERBB2
activates the PI3K/Akt signaling pathway and inhibits the expression of
inflammatory factors. Our data showed that FRP suppressed CU by
modulating the expression of these key targets through regulation of
the PI3K/Akt signaling pathway. PI3K/Akt signaling pathway regulates
multiple biological processes such as cell division, proliferation,
apoptosis, and metabolism, and plays an important role in the
inflammatory response ([136]Hu et al., 2023). Several studies have
shown that the PI3K/Akt signaling pathway is involved in relieving the
symptoms of CU by negatively regulating the production of IgE, thereby
suppressing the activation and degranulation of mast cells. Molecular
docking and molecular dynamics simulation results showed stable binding
of Akt to the three active components of FRP, namely, palmatine,
jatrorrhizine, and coclaurine. Furthermore, we verified that the
expression levels of p-PI3K and p-Akt were significantly higher in the
FRP group than in the model group. This strongly suggested that FRP
inhibited the activation of mast cells and susbequent release of
inflammatory mediators by regulating the PI3K/Akt signaling pathway.
5 Conclusion
Based on integrated analysis of metabolomics and network pharmacology
data, and evaluation of the expression of immune-related indicators, we
observed that FRP effectively alleviated the clinical symptoms of CU by
reducing inflammation through modulation of the PI3K/Akt signaling
pathway. Higher concentration of FRP demonstrated increased activation
of the PI3K/Akt signaling pathway and significant downregulation of
inflammatory factors. Concurrently, the frequency of scratching was
reduced. These findings suggested a positive correlation between FRP
concentration, AKT pathway modulation, and downstream biological
effects. Based on our results in this study, we plan to conduct
clinical trials to further obtain more comprehensive and reliable data
regarding the efficacy and safety of FRP to develop robust evidence for
supporting its broad application for the clinical management of
urticaria.
Acknowledgments