Abstract
Fu-you formula (FY), a Traditional Chinese Medicine (TCM) formula
composed of 12 herbs, as an in-hospital preparation, has been used
treat to precocious puberty (PP) for decades. However, the lack of
phytochemical characterization and mechanism of FY remains the main
limitation for its spreading. In this study, we analyze the components
and mechanisms of FY in PP, based on the integrated pharmacology.
Investigated main constituents, targets, pathways of FY by using an
integrative pharmacology, and recognized main constituents by
HPLC-MS/MS. Then, observed the levels of Follicle-stimulating hormone
(FSH), luteinizing hormone (LH), and estrogen (E[2]) in danazol-induced
PP in Sprague–Dawley (SD) rats. Lastly, retrospective study analyzed
the clinical data of 575 patients who were diagnosed PP, treated by the
FY, and followed-up in our hospital from 2014–2020. The result that
total of 116 important candidate targets were selected based on
pharmacological analysis. Selected the top 10 values key targets such
as the estrogen receptor alpha (ESR1) and insulin-like growth factor 1
(IGF1), were localized and the related gene functions were determined.
Gene functions were associated with biological regulation, a cellular
process, or signaling pathway, such as the Estrogen signaling pathway,
MAPK signaling pathway and PI3K-Akt signaling pathway. By recognizing
the five compounds related to the ESR1 and IGF1, which are Quercetin,
kaempferol, Luteolin, Apigenin, and Emodin. The results of the
molecular docking study further showed that the flavonoids had a strong
binding affinity for ESR1 and IGF1 after docking into the crystal
structure. The results showed that the FY could effectively reduce
E[2], LH, and FSH levels in SD rats. Furthermore, the results of the
retrospective analysis of medical records showed that the FY could
remarkably reduce E[2] levels in girls with PP.
Keywords: integrated pharmacology, precocious puberty, Chinese
medicine, protein-protein interaction network, Fuyou formula
Introduction
Precocious puberty (PP) is a common endocrine disorder among children.
It occurs before the age of eight years in girls and before the age of
9 years in boys. In recent years, the annual incidence of this
condition has been on the rise, and the incidence among girls is 5–10
times that among boys ([44]Chinese Society of Pediatric Endocrinology
and Metabolism (CSPEM), 2015). At present, early initiation of the
gonadal axis is believed to be the cause of PP. Therefore, modern
medicine holds the view that the administration of a gonadotropin
releasing hormone antagonist (GnRHa) in the treatment of PP is the most
effective method. However, clinical results show that the long-term use
of a GnRHa has inhibitory effects on growth and the thyroid in
children. In addition, some children require simultaneous treatment
with growth hormone or even thyroxine. Clinical studies on the FY as a
treatment for girls with PP at our hospital have shown that it can
control the early symptoms, and effectively reduce estrogen levels and
bone age ([45]Liu et al., 2009; [46]Pan et al., 2019). At present, the
literature comprises mostly clinical reports and observations of
curative effects. However, in-depth research on the effective
components, key targets, and mechanisms of action of the FY are still
lacking. Integrative pharmacology could enhance our comprehension and
facilitate the prediction of potential targets, pathways, and effects,
which might provide clues for the design of subsequent research
studies. In the present study, we used an integrative pharmacological
approach to understand the systemic, organ-related, and molecular
effects of the FY. The components and mechanisms of the FY in the
treatment of PP were preliminarily analyzed and explored. The TCM
integrated pharmacology platform was used and a TCM-component-network
target-disease multi-level network was considered as the underlying
framework.
Materials and Methods
Materials and Reagents
Danazol was obtained from the A&D Technology Corporation (Beijing,
China). Leuprorelin acetate microspheres for injection were purchased
from Livzon (Zhuhai, China). FSH, LH, and E[2] ELISA kits were obtained
from Cloud-Clone Corp (Wuhan, China). The TCM standards Quercetin
(serial number: 100081–201610, purity: 99.90%), Luteolin (serial
number: 111520–202006, purity: 94.40%), kaempferol (serial number:
110861–202013, purity: 93.20%) and Emodin (serial number:
110756–201913, purity: 96.0%) were purchased from the National
Institutes for Food and Drug Control (Beijing, China). Apigenin (serial
number: [47]B20981–20 mg, purity: 98.00%) was purchased from the
Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
Plant Materials and Fu-you Formula Preparation
The FY was an in-hospital preparation (Approval number: Z20053679). It
comprised a mixture of [48]Prunella vulgaris L. (Xiakucao); Carapax
Trionycis (Cubiejia); Gentiana scabra Bunge (Longdan); Chrysanthemum
morifolium (Ramat.) Hemsl (Juhua); Lycium chinense Mill (Digupi);
Alisma plantago-aquatica L (Zexie); Scrophularia ningpoensis Hemsl
(Xuanshen); Paeonia suffruticosa Andrews (Mudanpi); Rehmannia glutinosa
(Gaertn.) DC (Shengdihuang); Hordeum vulgare L (Maiya); Concha oetreae
(Muli); Thalluslaminariae (Kunbu)
(1.5:1:0.6:0.6:1:1:1.5:0.6:1.2:2:3:1). All herbs were purchased from
the Beijing Bencao Fangyuan Pharmaceutical Group Co. Ltd. and the FY
was prepared by the Preparation Center of the Beijing Children's
Hospital (Lot number: 20201202).
Construction of the Compound-Target and Disease-Target Database
To identify the corresponding targets of the 12 active ingredients of
the FY, several approaches combining chemometric methods, information
integration, and data mining were implemented. First, all active
compounds were submitted to the TCM-IP platform
([49]http://www.tcmip.cn/TCMIP/index.php/Home/Index/index.html) [50]Xu
et al. (2019), as well as the TCMSP ([51]http://tcmspw.com/tcmsp.php)
[52]Ru et al. (2014), and TCMID
([53]http://119.3.41.228:8000/tcmid/search/) [54]Huang et al. (2018) to
mine compound-target interactions. The biological targets of the active
ingredients were obtained from the STITCH ([55]http://stitch.embl.de/)
[56]Szklarczyk et al. (2016); SwissTarget
([57]http://www.swisstargetprediction.ch/) [58]Gfeller et al. (2014);
CTD ([59]http://tcmspw.com/index.php) [60]Yan et al. (2019); and SymMap
([61]https://www.symmap.org/) [62]Wu et al. (2019) databases. Known
therapeutic targets for PP were obtained from the DrugBank
([63]http://www.drugbank.ca/) [64]Wishart et al. (2018); Online
Mendelian Inheritance in Man (OMIM) ([65]http://www.omim.org)
[66]Hamosh et al. (2005); and DisGeNET
([67]https://www.disgenet.org/home/) [68]Janet et al. (2020) databases.
Protein-Protein Interaction Network Construction
The protein-protein interaction (PPI) data were imported from the
STRING
([69]https://string-db.org/cgi/input.pl?sessionId=rEkaDRgfV0vC&input_pa
ge_show_search=on) PPI databases. An interactive network for the
candidate drug targets and known PP-related targets of the FY was
constructed based on their interaction data and was visualized using
the Cytoscape software ([70]Shannon et al., 2003). Interactions between
the targets of the traditional Chinese medicine components of the FY
and the targets related to PP were determined. Furthermore, the gene
interaction network of the Chinese medicine components of the FY and PP
was established. The degree centrality (DC) equal to two times the
median value, was applied as the core for selection of the network
nodes (hubs node). Thus, the median of node connectivity, closeness
centrality, and betweenness centrality were the key values that
determined the selection of nodes. Nodes that met three values
simultaneously were selected as the candidate key targets of the FY in
the treatment of PP.
Gene Oncology Enrichment and Pathway Analysis
We performed gene ontology (GO) analysis of the non-repetitive putative
targets of the FY using the database for Annotation, Visualization, and
Integrated Discovery (DAVID) to gain insights into their involvement in
two different categories namely, biological process and molecular
function ([71]Sherman and Lempicki, 2009). Tissue enrichment analysis
was performed using the FunRich software ([72]http://www.funrich.org)
([73]Pathan et al., 2015). We then performed Kyoto Encyclopedia of
Genes and Genomes (KEGG) signaling pathway enrichment analysis of the
candidate targets of the FY after topological analysis. A P-value <
0.05 was considered significant, and the enriched GO terms were
identified using the hypergeometric test. A bubble chart was plotted
using the OmicShare tools, a free online platform for data analysis
([74]www.omicshare.com/tools).
Chemical Components Analysis
Characterization of main chemical components in FY was assayed by
HPLC-MS/MS (AB SCIEX QTRAP 5500). Chromatographic separation was
performed on a Hypersil Gold C18 column (150 × 2.1 mm, 5 μm) (Thermo
Scientific), with column temperature set at 40°C. The mobile phase was
solution A, 2 mM ammonium acetate in water containing 0.4‰ formic acid,
and solution B, methanol. Gradient elution program was: 0–1.5 min,
60–10% A; 1.5–3.5 min, 10% A; 3.5–3.51 min, 10–60% A; 3.51–6.0 min, 60%
A. The flow rate of mobile phase was 0.4 ml/min. The mass spectrometer
was operated in negative ion mode with a needle potential of -4,500 V;
the source temperature was set at 500°C. Nitrogen was used as the
sheath gas and auxiliary gas at pressures of 50 and 40 psi. Multiple
reactions monitoring (MRM) mode was used to identify the five compounds
by monitoring their transitions from the molecular ions to product
ions. The proper amounts of standard substance were weighed and
dissolved in methanol-water (1:1, v/v) to prepare standard solutions at
1 μg/ml. Meanwhile, 10 μL of FY was mixed with 1 ml of methanol-water
(1:1, v/v) by vortexing for 10 min, then centrifuge for 15 min at
15,000 rpm. The supernatant fluid was used as sample solution. The
chromatograms of standard solution and sample solution were used to
compounds matching.
In Silico Molecular Docking
In silico molecular docking studies of bio-active peptides or chemical
drug molecules that exert their action by binding with specific
receptors provides evidence on binding conformation, pattern and
affinity. To identify the binding ability of active constituents with
PP related targets, the crystal structures of ESR1 (PDB code: 6VIG) and
IGF-1 (PDB code: 1IMX) were obtained from RCSB Protein Data Bank
([75]http://www.rcsb.org/), and three main compounds structure of
Quercetin, Apigenin and Luteolin were obtained from PubChem
([76]https://pubchem.ncbi.nlm.nih.gov/) to establish molecular docking
model with Discovery Studio 4.5. The CDOCKER module of Dock Ligands in
Discovery Studio 4.5 was used to do the docking. The kinetic method was
used to randomly search the small molecule conformation, and then the
simulated annealing method was used to optimize each conformation in
the receptor active site region, so as to make the docking results more
accurate.
Animals
At postnatal day (PND) 3, female Sprague-Dawley rats and their mothers
were obtained from SPF Biotechnology Co., Ltd. (Beijing, license no:
SYXK (Beijing) 2016–0038). The rats were housed in the laboratory
animal room and maintained at 24 ± 2°C, with 42 ± 5% humidity on a 12-h
light/dark cycle (lights on from 07:30 to 19:30) in a
specific-pathogen-free animal room. The animals were supplied food and
water ad libitum and acclimated for three days before the start of the
experiments. All animal experiments were performed in strict compliance
with Chinese guidelines, including the standards for Laboratory Animals
(GB14925–2001), and the Guideline on the Humane Treatment of Laboratory
Animals (MOST 2006a). All animal procedures were approved by the
Beijing Administration Office for Laboratory Animals.
Animal Grouping and Drug Administration
The animals were randomly divided into four groups: the control group,
model group, positive control (leuprorelin) group, and FY group. At PND
5, the rats in the three experimental groups were given a single
subcutaneous injection of 300 µg/25 µL danazol (ethylene glycol:ethanol
= 1:1, v/v). The rats in the control group were given a subcutaneous
injection of 25 µL of glycol/ethanol ([77]Morishita et al., 1993; Ju et
al., 2019). The rats in the positive control (leuprorelin) group were
subcutaneously injected with 100 μg/kg leuprorelin. The rats in the FY
group were given a solution formulated with dry ointment powder, by
intragastric administration every day. The rats in the control and
model groups were given the same amount of normal saline. The rats that
exhibited vaginal opening were sacrificed at diestrus after a complete
estrous cycle. The remaining rats were sacrificed at the same time
point. All rats were anesthetized with an intraperitoneal injection of
2% pentobarbital sodium. Blood samples were collected from the
abdominal aorta before sacrifice. Blood serum was separated by
centrifugation (3,500 rpm, 20 min, 4°C) and preserved at −80°C for
further analysis of serum hormone levels.
Drug dosage: The Fy dose was calculated according to the clinical
dosage administered to 6-year-old girls. According to the following
formula:
[MATH: dB=dAdA
×RB/RA×(WA/WB
)1/3 :MATH]
with d [B] representing the animal/human body weight dose, d [A]
representing the known human/animal body weight dose, W [A] and W [B]
representing known human and animal weights, respectively, and R [A]
and R [B] representing known human/animal body shape coefficients,
respectively. Every two days, the animals were weight, and the dose was
recalculated.
Serum Hormone Level Detection
After anesthesia, blood was collected from the abdominal aorta, and the
serum was centrifuged at 4°C and stored at −20°C until further
analysis. The serum concentrations of FSH, LH, and E[2] were measured
using ELISA kits, according to the manufacturers’ instructions. The
ELISA kits, which employ a competitive inhibition enzyme immunoassay
technique, were purchased from Cloud-Clone Corp (Wuhan, China).
Retrospective Analysis of Cases
Children with PP, treated with the FY at the outpatient department of
our hospital from 2014 to 2020 were also evaluated. The inclusion
criteria were as follows: 1) continuous use of the FY for 1 year; and
2) evaluation of sex hormone levels every 6 months. The exclusion
criteria were as follows: 1) the presence of other endocrine diseases;
2) the presence of ovarian cysts. This retrospective study was approved
by the Medical Ethics Committee of Beijing Children’s Hospital, Capital
Medical University.
Statistical Analysis
All results were presented as the mean ± SD. Differences were analyzed
using one-way ANOVA. The data were further analyzed and plotted using
the SPSS 19.0 software (IBM SPSS Software, New York, United States).
Differences were considered statistically significant at p < 0.05.
Results
Formula Analysis of Fu-you Formula
[78]Prunella vulgaris L.and Carapax Trionycis act on the liver and
relieves congestion, nourishes yin and clears heat; Gentiana scabra
Bunge, Chrysanthemum morifolium (Ramat.) Hemsl, Lycium chinense Mill,
Alisma plantago-aquatica L, Scrophularia ningpoensis Hemsl., Paeonia
suffruticosa Andrews, Rehmannia glutinosa (Gaertn.) DC, which clear
heat and removes dampness, nourishes yin, and cools the blood; Hordeum
vulgare L, Concha oetreae and Thalluslaminariae act on the liver and
relieves congestion, used as an adjuvant. All herbs combined act on the
liver, clear congestion, nourish yin, and clear heat, can reduce the
size of nodules in the breast, eliminate vaginal secretions, and
dissipate scrofula, and reduce sputum production. The TCM composition
are listed in [79]Table 1.
TABLE 1.
Composition of the FY.
Chinese name Scientific name Family Lot No Place of origin Parts of
plant used
Xia Ku Cao [80]Prunella vulgaris L. Lamiaceae 20201010 Jiangsu, China
Dried erial parts
Cu Bie Jia Carapax Trionycis Trionyxsinensis Wiegmann 20201018 Hubei,
China Carapace
Long Dan Gentiana scabra Bunge Gentianaceae 20200927 Yunan, China Dried
roots and rhizomes
Ju Hua Chrysanthemum morifolium (Ramat.) Hemsl Compositae 20201027
Anhui, China Capitulum
Di Gu Pi Lycium chinense Mill Solanaceae 20201105 Hebei, China Dried
root bark
Ze Xie Alisma plantago-aquatica L Alismataceae 20201126 Fujian, China
Dried tuber
Xuan Shen Scrophularia ningpoensis Hemsl Scrophulariaceae 20201019
Zhejiang, China Dried root tuber
Mu Dan Pi Paeonia suffruticosa Andrews Paeoniaceae 20201123 Anhui,
China Dried root bark
Sheng Di Huang Rehmannia glutinosa (Gaertn.) DC Plantaginaceae 20201104
Henan, China Dried root tuber
Mai Ya Hordeum vulgare L Triticum 20201030 Hebei, China Dried ripe
fruit
Mu Li Concha oetreae Ostrea 20200917 Guangdong, China Shell
Kun Bu Thalluslaminariae Laminaria 20200922 Fujian, China Dried lobes
[81]Open in a new tab
Chemical Composition and Prediction Target Analysis
Four hundred and thirteen chemical components were detected from 12 TCM
in the FY. Furthermore, 37,468 predicted drug targets were obtained.
The predicted target information and the characterization of each herb
were derived from data on the TCM-component-targets, as shown in
[82]Table 2. Analysis of the common targets among the predicted
targets, yielded a total of 4,741 predicted targets, among the 12 TCM.
No common intersection targets were detected among the 12 TCM. 97
targets were detected between the two key herbs, which each had common
targets with other herbs, as shown in [83]Table 3.
TABLE 2.
Basic data on the components and targets of traditional Chinese
medicines.
TCM Number of components Number of targets
[84]Prunella vulgaris L. 39 10,189
Carapax Trionycis 16 374
Gentiana scabra Bunge 45 1289
Chrysanthemum morifolium (Ramat.) Hemsl 101 11,384
Lycium chinense Mill 22 1358
Alisma plantago-aquatica L 26 795
Scrophularia ningpoensis Hemsl 25 564
Paeonia suffruticosa Andrews 36 9072
Rehmannia glutinosa (Gaertn.) DC 49 1777
Hordeum vulgare L 32 496
Concha oetreae 10 56
Thalluslaminariae 12 114
[85]Open in a new tab
TABLE 3.
Number of common targets.
TCM [86]Prunella vulgaris L. Carapax Trionycis Gentiana scabra Bunge
Chrysanthemum morifolium (Ramat.) Hemsl Lycium chinense Mill Alisma
plantago-aquatica L Scrophularia ningpoensis Hemsl Paeonia suffruticosa
Andrews Rehmannia glutinosa (Gaertn.) DC Hordeum vulgare L Concha
oetreae Thalluslaminariae
[87]Prunella vulgaris L. — 97 358 3975 417 262 168 3991 293 181 25 61
Carapax Trionycis 97 — 48 95 58 37 30 88 43 41 7 16
[88]Open in a new tab
Construction and Analysis of Compound-Target Network of Fu-you Formula
After removing redundant targets, 4,741 targets obtained from 12 herbs
intersected with 166 disease targets to obtain 79 mapped-genes. We then
explored the predicted therapeutic targets of the FY, using multiple
online databases as previously described. A network of potential
targets of the compounds in the FY was then constructed using the
Cytoscape software, as shown in [89]Figure 1A. Based on the 1,224 core
nodes obtained, 116 key candidate targets for the treatment of PP girls
were screened out, 75 were direct targets and 41 were predicted
targets. The degree values were determined for the top 10 hub genes,
two of the most important targets are ESR1 and IGF1. After further
analysis, there were 96 chemical components acting on 10 hub genes in
the formula, and 42 chemical components acting on ESR1 and IGF1, which
the five components most widely distributed in medicinal materials were
Luteolin, Quercetin, Apigenin, Kaempferol and Emodin. The interaction
relationships between targets were determined and a network map of the
hub targets for the treatment of PP in girls was constructed
([90]Figure 1B).
FIGURE 1.
FIGURE 1
[91]Open in a new tab
Compound-target interaction network and preliminary gene ontology (GO)
analysis of drug targets. (A) Compound-key target network of the FY.
(B) Compound-hub target network of the FY. (C) GO analysis of drug
targets classified into three categories: biological process, molecular
function, and cellular component. (D) Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway analysis of core targets of the FY in the
treatment of PP. (E) Chinese medicinal materials-core component-key
target-main pathways.
Gene Oncology and Kyoto Encyclopedia of Genes and Genomes Enrichment Analysis
of Hub Targets for the Fu-you Formula in Precocious Puberty
Based on the results of GO and KEGG pathway analyses, the enriched
pathways were determined. A total of 2,462 hub genes were identified
based on the GO analysis, which were associated with the target genes
or proteins of cells (cellular component), molecular functions
(function), or biological processes (in process). Gene function
information is presented in [92]Figure 1C. A total of 133 KEGG pathways
were enriched, which were associated with key candidate targets. The
top 20 pathways were sorted by P-value, as shown in [93]Figure 1D.
Construction and Analysis of the Multi-Layer Network Correlation Diagram of
Traditional Chinese Medicine-Core Component-Hub Target-Main Pathways in the
Treatment of PP With the Fu-you Formula
The KEGG pathways of the top 30 key candidate targets, based on their
P-values were selected to construct the multi-level network association
diagram of the “traditional Chinese medicine-core component-key
target-main pathways” for the FY in the treatment of PP in girls
([94]Figure 1E). The P-values were used to sort 30 KEGG pathways, which
included 10 hub genes.
Phytochemical Characterization of Fu-you Formula
To identify the main constituents of FY, we analyzed the FY using
HPLC-MS/MS. Five compounds were recognized from FY as shown in
[95]Table 4. The standard solution and the sample solution were
analyzed by the HPLC-MS/MS method upper to identify the five
constituents of FY. Accroding to the chromatograms ([96]Figure 2), the
consistent chromatographic peaks of the five compounds could be
recognized in standard solution and sample solution, including of
Luteolin, Quercetin, Apigenin, Kaempferol and Emodin. Therefore, it is
convincing that FY contains these five constituents.
TABLE 4.
MRM transitions for identify of the target compounds.
Analyte Precursorion (m/z) Production (m/z) DP EP CE CXP
Luteolin 284.8 132.8 −150 −2 −40 −40
Quercetin 300.9 150.8 −120 −2 −27 −40
Apigenin 269.0 116.8 −150 −2 −40 −40
Kaempferol 284.9 93.0 −180 −2 −40 −40
Emodin 268.9 224.8 −150 −2 −36 −40
[97]Open in a new tab
FIGURE 2.
[98]FIGURE 2
[99]Open in a new tab
Chromatograms of HPLC-MS/MS of FY. [(I) Standard solution; (II) Sample
solution of FY; Retention time of Kaempferol and Emodin were 3.28 and
4.18 min, respectively].
Molecular Docking
To further validate the potential targets possessing good affinity to
the ingredients, molecular docking was performed for 3 high content
ingredients with the 2 high relevance degree proteins. The docking
results of the 3 flavonoids with the target proteins ESR1 and IGF1 are
shown in [100]Table 5 and [101]Figure 3. As shown in the results, all
the active compounds have favorable binding energy (<0 Kcal/moL) with
their relative potential target proteins, and 3 flavonoids interact
with ESR1 more stronger, which adds chips to the reliability of the
virtual screening results.
TABLE 5.
Molecular docking results of target and active compounds.
Active compounds Binding energy (Kcal/moL)
IGF1 ESR1
Quercetin −9.81275 −41.9021
Apigenin −16.8225 −43.4102
Luteolin −13.2934 −40.1986
[102]Open in a new tab
FIGURE 3.
[103]FIGURE 3
[104]Open in a new tab
(A) Active binding sites of compounds with IGF1. (B) Active binding
sites of compounds with ESR1. (C) Molecular docking patterns of
compounds and IGF1. (D) Molecular docking patterns of compounds and
ESR1.
Serum Test Results
Compared with the normal control group, E[2], LH, and FSH levels in the
model group were significantly increased (p < 0.01), indicating that
the model of PP was successfully established. After treatment with the
FY, the E[2] and LH levels in rats with PP were significantly reduced
compared with the model group (p < 0.01); and FSH levels were
significantly reduced (p < 0.05), compared with the model group.
However, in the leuprorelin group, only the LH levels showed a
reduction (p < 0.05) ([105]Figure 4A and [106]Figure 4B).
FIGURE 4.
FIGURE 4
[107]Open in a new tab
Effects of the FY on sex hormone levels in PP. (A) Serum estrogen
(E[2]) levels among all groups of rats. (B) Serum luteinizing hormone
(LH) and follicle-stimulating hormone (FSH) levels among all groups of
rats. (C) Serum E[2] levels among all groups of children. (D) Serum LH
and FSH were detected among all groups of children.
Retrospective Analysis of Cases
Retrospective analysis showed that 575 children who met the inclusion
criteria were included. It showed that E[2], LH, and FSH levels were
significantly reduced after 12 months treatments (p < 0.01).
Significant differences were noted in E[2] levels between groups (p <
0.01). The FSH levels were significantly lower at 12 months after
treatment compared with 6 months after treatment (p < 0.05)
([108]Figures 4C,D).
Discussion
The results of the analysis of the common targets of the FY revealed a
total of 97 targets for [109]Prunella vulgaris L. and Carapax
Trionycis, which had common targets with other drugs. These findings
suggest that these herbs had close synergistic effects with the other
herbs. The following herbs: Gentiana scabra Bunge, Chrysanthemum
morifolium (Ramat.) Hemsl., Lycium chinense Mill, Alisma
plantago-aquatica L, Scrophularia ningpoensis Hemsl, Paeonia
suffruticosa Andrews and Rehmannia glutinosa (Gaertn.) DC had a total
of 51 targets. These findings indicate that these 7 herbs in the
prescription also had synergistic effects. Furthermore, no common
targets were detected for Hordeum vulgare L, Concha Etreae, and
Thalluslaminariae, indicating that these 3 herbs are not key active
ingredients in the formulation. Our previous study about data mining
showed that the most frequently used herbs were Anemarrhena
asphodeloides Bunge, Rehmannia glutinosa (Gaertn.) DC, Phellodendron
chinense C.K.Schneid, Paeonia suffruticosa Andrews, and Prunella
Vulgaris L. Common medicinal were cold and bitter, mostly attributed to
the liver and kidney. The core of the herbs based on Zhibai Dihuangwan,
and there were 4 kinds of herbs with FY.
At present, GnRHa is recommended to treat CPP, but not incompleteness
precocious puberty in the relevant guidelines. The premature thelarche
is the most common type of incomplete precocious puberty, 14–23% of
which will develop to CPP ([110]Zhu et al., 2008). So intervene as
early as possible is the present clinical needs to solve the problem.
Literature shows that Zhibai Dihuang Pill and Dabuyin Pill can treat
precocious puberty ([111]Liu and Wang, 2018; [112]Wang and Zhao, 2019;
[113]Wang et al., 2020), but there is no indication for this in their
instructions, so it belongs to off-label drug use.
The above-mentioned number of common targets of each herb is consistent
with compatibility principles of the formulation. We identified main
chemical constituents of FY using HPLC-MS/MS and confirmed that the
main constituents related to the key targets in FY are Quercetin,
kaempferol, Luteolin, Apigenin and Emodin. Analysis of the formulation
reveals that the core components are mainly flavonoids, as well as
kaempferol and quercetin, which are all phytoestrogens. Modern
pharmacological studies have shown that phytoestrogens can make two-way
adjustments, as they are similar to endogenous estrogen in structure
and function. When the level of estrogen in the body is lower than the
normal level, it can play an estrogen-like role, which can prevent and
cure women's menopausal syndrome, prostate cancer, osteoporosis and
cardiovascular diseases. On the other hand, when the level of estrogen
in the body is higher than the normal level. such as breast
hyperplasia, uterine fibroids and other diseases, it can produce
estrogen antagonism and effectively weaken the response of target cells
to estrogen ([114]Chen et al., 2017; [115]Cai and Zhanf, 2020).
Among the top 10 core targets selected, ESR1, IGF1 and other direct
therapeutic targets are reportedly related to the onset and development
of PP ([116]Ye et al., 2011; [117]Yang and Zhao, 2013; [118]Wang et
al., 2016), and are important targets in the treatment of PP. We
further analyzed and clarified the core targets of biological function,
gene function, and signal pathways. The results showed that the key
components of the FY alone or combined were associated with
transcription factor binding, transcription factor regulation,
biological and cellular processes, such as GO or biological
process-related gene/protein molecular function, and the estrogen
signaling pathway, MAPK signaling pathway, and PI3K-Akt signaling
pathways. These pathways mediate hormones that act on target tissues to
achieve endocrine regulation. The E[2] hormone binds with ESR1 to form
a hormone-receptor complex that activates the estrogen signaling
pathway. The MAPK signaling pathway and PI3K-Akt signaling pathway
regulate the secretion of GnRH, LH, and FSH, as well as metabolic
processes associated with bones. In addition, the growth
hormone-insulin-like growth factor-1 (GH-IGF1) is the most important
neuroendocrine factor associated with growth and development. Excessive
IGF1 levels can inhibit GH secretion, and thereby inhibiting the growth
of articular cartilage and epiphyseal cartilage, and retard growth in
children ([119]Su et al., 2017).
Treatment with GnRH analogues, such as Leuproline, which act by
downregulating pituitary GnRH receptors ([120]Carel et al., 2009),
represent the standard of care for the treatment of CPP. The integrated
pharmacological results suggest that the mechanism of the FY in the
treatment of PP may include: competitive binding of E[2] with ESR1and
reduction in serum IGF1 concentrations. At the same time, animal
experiments showed that the FY could reduce E[2], LH, and FSH levels in
rats with PP. Retrospective analysis of the medical records also showed
that the FY could control the early symptoms of PP, and effectively
inhibit E[2] levels. The results of the animal experiments and
retrospective analysis of medical records have confirmed the
feasibility of studies of the mechanism of action of Chinese herbal
compounds by integrative pharmacological methods.
In addition, the complex key targets of the FY and predictions based on
the KEGG pathway analysis results show that the treatment may have
effects on ovarian steroidogenesis, prostate and breast cancer, and
other signaling pathways. These results are consistent with our
previous clinical studies, which have shown that the effects of complex
mixtures in girls with PP and ovarian cysts may be a more favorable
intervention. Mixtures such as the FY may diminish ovarian cysts,
regulate character development, improve liver function and qi
stagnation, alleviate yin deficiencies and heat symptoms, reduce levels
of E[2], and retard bone aging and maturation. The present findings
also provide a novel basis for the clinical application, and further
research and development of the FY.
However, this study has some limitations that are worth mentioning.
First, the reliability of the effects of FY against PP depends on
database, so biological verification is necessary to evaluate the
reliability of bioinformatics analysis in vitro, in vivo and in silico.
Secondly, quantitative analysis of the synergistic effect of the main
compounds should be investigated in the future.
Conclusion
In conclusion, we combined methods of big data discovery with
biological validation to study the mechanism of actions of the FY in PP
at the systemic level. We used the TCM-IP database for the treatment of
PP and considered various components and molecular mechanisms in the
preliminary analysis. We determined that the FY acts through multiple
component interactions with targets. The mixture also exerts its
effects through multiple pathways involved in the regulation of PP, and
may thus play a role in treatment of the condition. Whether other
pathways or mechanisms predicted in this network pharmacological
approach also contribute to the beneficial effects of the FY requires
further investigation.
Acknowledgments