Abstract
Banxia Houpu decoction (BXHPD) has been used to treat depression in
clinical practice for centuries. However, the pharmacological
mechanisms of BXHPD still remain unclear. Network Pharmacology (NP)
approach was used to explore the potential molecular mechanisms of
BXHPD in treating depression. Potential active compounds of BXHPD were
obtained from the Traditional Chinese Medicine Systems Pharmacology
Database and Analysis Platform Database. STRING database was used to
build a interaction network between the active compounds and target
genes associated with depression. The topological features of nodes
were visualized and calculated. Significant pathways and biological
functions were identified using Gene Ontology and Kyoto Encyclopedia of
Genes and Genomes analyses. A total of 44 active compounds were
obtained from BXHPD, and 121 potential target genes were considered to
be therapeutically relevant. Pathway analysis indicated that MAPK
signaling pathway, ErbB signaling pathway, HIF-1 signaling pathway and
PI3K-Akt pathway were significant pathways in depression. They were
mainly involved in promoting nerve growth and nutrition and alleviating
neuroinflammatory conditions. The result provided some potential ways
for modern medicine in the treatment of depression.
Background
Depression is a widespread chronic mental illness characterized by low
mood, sadness, and insomnia. Depression affects physical health
[[36]1]. The world health organization predicts that depression will be
the leading cause of death of burden among all health diseases by 2030
[[37]2].
Current therapies, including selective 5-serotonin reuptake inhibitors,
serotonin-noradrenergic reuptake inhibitors and tricyclic
antidepressants [[38]3], can alleviate some major symptoms of
depression. But these medications trigger a series of serious side
effects like anxiety, gastrointestinal discomfort, drug resistance,
withdrawal response and so on [[39]4]. Therefore, it is necessary to
develop safe, effective new drugs and therapies with lower side
effects. Studies have shown that the use of complementary and
alternative medicine (CAM) for the treatment of depression is common
[[40]5, [41]6]. As the important part of the CAM, traditional Chinese
medicine (TCM) reported positive results for managing depression: less
adverse reactions than other antidepressants and no significant
differences from medication [[42]7, [43]8].
Banxia Houpu decoction (BXHPD) is a famous Chinese medicine
prescription, which firstly recorded in Jingui Yaolue (Synopsis of
Prescriptions of the Golden Chamber) in Eastern Han Dynasty of Chinese
history (25AD–220AD). BXHPD was 5 herbs, which included Pinellia
rhizome (PR, Banxia), Magnolia officinalis cortex (MOC, Houpu), Poria
(PO, Fuling), ginger rhizome (GR, Shengjiang), and Perilla folium (PF,
Zisu). In the theory of TCM, BXHPD could promote chi, eliminate
stagnation, calm the adverse chi and dissolve phlegm. BXHPD was widely
used in the treatment of depression and achieved effective results
[[44]9, [45]10]. Zheng Q et al reported that the effective rate of
BXHPD combined with western medicine was 94.5%, and the cure rate was
55.2%, compared to 81.1% and 22.9% treated with western medicine alone
in the treatment of depression [[46]10].
Some previous studies predicted the molecular mechanism on depression
of BXHPD, which mainly included activation of the inflammatory response
system [[47]11], monoamine hypothesis [[48]12], Hypothalamic Pituitary
Adrenal (HPA) axis dysfunction [[49]13], low expression of
brain-derived neurotrophic factor (BDNF) [[50]14] and so on. The
biological mechanism in holistic manner are still unknown. The main
problems are as follows: (1) On account of the complex composition, the
biological effects on depression of BXHPD were not unified. BXHPD
included many active compounds, most of which were different in
therapeutic mechanism. For instance, Baicalein [[51]15] and Luteolin
[[52]16] were related to the suppression of inflammation about nerve
through the regulation of pathways or the expression level of
inflammatory related factors. Beta-sitosterol might affect the content
of neurotransmitter [[53]17]. (2) The previous studies mainly focused
on the gene targets or biological functions rather than multiple
pathways: Anti-depression on BXHPD proved to elevate brain
5-hydroxytryptamine (5-HT) levels, attenuate abnormalities in
dopaminergic system functions [[54]18], ameliorate the damages of lipid
peroxidation [[55]19], and adjust the amino acid metabolism and energy
metabolism [[56]20].
In order to explore details of the mechanism and relevant pathways, it
is essential to elucidate the molecular and biological basis of TCM
preparations and NP approaches have been proven to be a powerful
approach [[57]21, [58]22]. The holistic philosophy of TCM shares
similar characteristic of NP. TCM network pharmacology approach was
established by "network target, multi-components" mode, which predicted
the target profiles, revealed drug-gene-disease co-module associations,
and interpreted the combinatorial rules and network regulation effects
of herbal formulae [[59]23]. Herein we focused on the following issues:
(1) Which active ingredients were involved in the regulation of
depression? (2) Which active ingredients and proteins regulated the
target to achieve the biological activity? (3) What pathways or
biological processes did the active ingredients regulate? The main
contribution of our work was to clarify the potential mechanism of
BXHPD in the treatment of depression using NP. The workflow of the
experimental procedures was showed in [60]Fig 1.
Fig 1. The workflow of the experimental procedures.
[61]Fig 1
[62]Open in a new tab
Methods
Identification of active compounds
The main chemical constituents of BXHPD were collected from the
Traditional Chinese Medicine Systems Pharmacology Database and Analysis
Platform Database (TCMSP) ([63]http://tcmspw.com/tcmsp.php/) [[64]24].
TCMSP database was a network pharmacological database of Chinese herbal
medicines and it provided pharmacokinetic information for each compound
[[65]25], such as drug similarity (DL) and oral bioavailability (OB).
Compounds with good drug-like were selected according to their
characteristics (including absorption, distribution, metabolism and
excretion [ADME]) [[66]25].
OB and DL were important indicators to evaluate ADME [[67]26]
characteristics. OB represented the correlation of effective compounds.
OB ≥30% meant that the effective compounds had a strong correlation. DL
of new molecules was assessed using the following formula [[68]27]:
[MATH:
f(A,B)=A⋅B|A|2<
/mn>+|B|2−A⋅B :MATH]
. "A" is the molecular descriptor of BXHPD based on Dragon software
([69]http:www.talete.mi.it/products/dragon_description.htm). "B"
represents the average descriptor of all drugs based on The Drug Bank
database [[70]28]. The Dragon software calculates the average of all
descriptors and the active molecules with DL ≥ 0.18 were selected.
Therefore, compounds with OB ≥ 30% and DL ≥ 0.18 were selected for
further study. Pinellia ternata, Magnolia officinalis, Poria cocos,
Ginger and Perilla frutescens were used separately as search terms to
gain the pharmacokinetic information of active compounds.
Screening of potential targets
An important step after screening active compounds was to identify
their molecular targets for triggering biological effects [[71]29].
UniProt ([72]https://www.uniprot.org/) is a protein database with
abundant information and extensive resources [[73]30]. The protein
targets retrieved from TCMSP database were put into Uniprot, and the
corresponding gene names were extracted from UniProt KB. We restricted
species to human beings and converted them into official gene names.
Gene Cards ([74]https://www.genecards.org/) is a searchable and
comprehensive database which automatically integrates data from about
125 web-based sources of genes, including genomes, transcriptomes and
proteomics [[75]31]. Gene Cards database is applied to search disease
targets for depression. Potential antidepressant targets of BXHPD were
obtained by intersecting the targets of active ingredients and
potential disease-related targets.
Construction and topological analysis of PPI network
STRING ([76]https://string-db.org/cgi/input.pl) was used for expanding
protein-protein interaction (PPI) network data [[77]32]. The gene names
of potential targets converted from UniProt were put into STRING to
download interactive information table of PPI. Cytoscape V3.7.1 was
used to visualize PPI and complete topological analysis [[78]33]. Core
genes of BXHPD for anti-depression were obtained meeting degree ≥ 38
(two times of median of all nodes).
"Degree", "Betweenness" and "Closeness" were used to assess the
topological importance of the selected core genes [[79]34]. "Degrees"
is defined as the number of links to a node that reflects the node's
interaction with another node. Nodes with an extremely high level of
degree tend to be critical in interaction networks. "Betweenness" is
defined as the number of closest associations. "Closeness" indicates
the sum of the nodes distance from all other nodes.
Biological function enrichment and metabolic pathway acquisition
The Gene Ontology (GO) is a database that functionally marks genes and
proteins as three main terms: biological process (BP), cellular
component (CC) and molecular function (MF) [[80]35]. The function of
genes can be defined and described in many ways through CC, BP and MF
[[81]36]. Kyoto Encyclopedia of Genes and Genomes (KEGG) database is
used to determine advanced functions and biological correlation of a
large number of genes [[82]37]. KEGG pathways infer the relationships
between proteins and the biological functional annotation of proteins.
David 6.8 is used for GO and KEGG pathway analysis of the core
antidepressant targets of BXHPD [[83]38]. The corresponding data were
obtained by using human genes as the range and P ≤ 0.05 as the
screening value. The relevant biological processes or related pathways
with the largest number of corresponding target points were selected to
draw bar charts.
Construction of a compound-target-pathway network
The active compounds, core targets and signal pathways were utilized to
construct a compound-target-pathway (C-T-P) network. In this C-T-P
network, "nodes" of different colors represented the protein targets,
compounds, signal pathways or diseases. "Edge" indicated the
interaction of compound targets, compound pathways, or diseases. The
networks were constructed by using Cytoscape v3.7.1.
Results
Active compounds of BXHPD
In the TCMSP database, 13 kinds of compounds from PR, 15 from PO, 5
from GR and 14 from MOC were included. After removed 5 duplicates from
49 compounds, 44 compounds were further studied ([84]Table 1).
Table 1. Basic information of active compounds of BXHPD.
MOL ID MOL NAME OB% DL TARGETS NUMBER
MOL000006 luteolin 36.16 0.25 55
MOL002714 baicalein 33.52 0.21 34
MOL000358 beta-sitosterol 36.91 0.75 34
MOL000449 Stigmasterol 43.83 0.76 29
MOL002670 Cavidine 35.64 0.81 26
MOL005970 Eucalyptol 60.62 0.32 24
MOL002773 beta-carotene 37.18 0.58 23
MOL000296 hederagenin 36.91 0.75 22
MOL000519 coniferin 31.11 0.32 21
MOL000492 (+)-catechin 54.83 0.24 7
MOL001749 ZINC03860434 43.59 0.35 4
MOL005980 Neohesperidin 57.44 0.27 4
MOL006957 (3S,6S)-3-(benzyl)-6-(4-hydroxybenzyl)piperazine-2,5-quinone
46.89 0.27 3
MOL006129 6-methylgingediacetate2 48.73 0.32 3
MOL000953 CLR 37.87 0.68 3
MOL001755 24-Ethylcholest-4-en-3-one 36.08 0.76 2
MOL005030 gondoic acid 30.7 0.2 2
MOL006936 10,13-eicosadienoic 39.99 0.2 2
MOL003578 Cycloartenol 38.69 0.78 2
MOL006967 beta-D-Ribofuranoside, xanthine-9 44.72 0.21 2
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In the 44 compounds, 12 compounds had no protein targets. A total of
159 protein targets for 32 compounds were obtained from TCMSP. After
inputting the 159 protein targets into the Uniprot, we found that 11
protein targets could not found the corresponding genes. The 11 protein
targets above were removed. Adding up to 148 gene targets and 32
compounds were preserved for further researches.
The construction of compounds-target network
In order to identify the relationship between TCM and the corresponding
targets [[86]39], a compounds-target network was built to show the
correspondence of active BXHPD compounds and targets ([87]Fig 2). The
compounds with the most amount of targets contained luteolin (55),
baicalein (34), beta-sitosterol (34) and stigmasterol (29).
Fig 2. Compounds-target network.
[88]Fig 2
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Pink hexagons represent compounds and blue circles represent targets.
Acquisition of core targets
From Gene Cards database, 8,959 genes were related to depression. After
intersecting 148 potential active targets with 8,959 depression related
genes, 121 potential drug target genes were obtained. The PPI network
was built by STRING ([90]Fig 3). The PPI network consisted of 120 nodes
and 1476 edges. After the visual topological analysis by CytoNCA (an
application in Cytoscape), 24 core targets were obtained ([91]Table 2).
Among the 24 core targets, ALB has the greatest degree (71), followed
by FOS (65), IL6 (63) and EGFR (60).
Fig 3. PPI network.
[92]Fig 3
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Table 2. Core targets of BXHPD.
Gene symbol Degree Gene symbol Degree
ALB 71 CCND1 49
FOS 65 ERBB2 47
IL6 63 ESR1 47
EGFR 60 APP 45
VEGFA 60 CAT 44
TNF 59 BCL2L1 44
MAPK1 57 MMP2 43
JUN 57 CYCS 43
CASP3 57 CASP8 43
MYC 55 PPARG 42
MMP9 54 CTNNB1 41
PTGS2 52 AR 41
[94]Open in a new tab
GO and KEGG pathways analysis for core genes
GO enrichment analysis and KEGG pathways analysis were performed on the
23 target genes by David ([95]Fig 4). Pathways with P-value<0.05 were
considered as significant pathways.
Fig 4. GO and KEGG functional analysis.
[96]Fig 4
[97]Open in a new tab
(A) GO biological process terms. (B) GO cellular component terms. (C)
GO molecular function terms. (D) KEGG pathway.
GO enrichment analysis contains three broad categories: BP, CC and MF.
BP terms mainly contained response to estradiol, response to drug,
positive regulation of transcription, DNA-templated, positive
regulation of smooth muscle cell proliferation and positive regulation
of transcription from RNA polymerase II promoter. CC terms mainly
contained cytosol, membrane raft, protein complex, and extracellular
space and nucleus. MF terms mainly contained identical protein binding,
enzyme binding, transcription factor binding and protein binding. KEGG
pathways mainly contained MAPK signaling pathway, PI3K-Akt signaling
pathway, Oxytocin signaling pathway, ErbB signaling pathway and HIF-1
signaling pathway.
KEGG enrichment analysis
KEGG Enrichment Analysis reveals the possible biological processes of
pathway by analyzing biological processes, which unlocks the molecular
roles of targets in the treatment of depression. There were more than
20 pathways associated with depression. In these pathways, MAPK
signaling pathway ([98]Fig 5A) and PI3K-Akt signaling pathway ([99]Fig
5B) were most closely related to depression.
Fig 5. KEGG enrichment diagram.
[100]Fig 5
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(A) MAPK signaling pathway. (B) PI3K-AKT signaling pathway. Red
asterisks represent core targets.
One of the MAPK signaling pathway: ERK, part of the MAPK pathway in the
prefrontal cortex and hippocampal played an important role in
antidepressant processing [[102]40]. ERK activates Elk-1, Sapla and
c-Myc through phosphorylation. Elk-1, Sapla and c-Myc combine to form
SRF, SRF activates c-fos through DNA, c-fos produces proliferation
differentiation function, via DNA activation. The PI3K-Akt signaling
pathway was one of the important pathways of depression. GF activates
RTK on cell membrane, RTK activates IRS1, IRS1 activates PI3K(Class
IA), PI3K activates AKT through PIP3, AKT inhibits BAD by
phosphorylation, BAD ultimately affects Cell survival by inhibiting
Bcl-xL and Bcl-2.
Component-target-pathway network
Compound-Target-Pathway networks of BXHPD were shown in [103]Fig 6.
There were 9 compounds, 24 targets and 17 pathways. The nodes represent
drug compounds, active components and targets. The edges represent
their relationship. Through the network, we could find that some
pathways had high enrichment in the network such as PI3k-Akt pathway,
which suggested that these pathways may play a key role in therapeutic
effects of BXHPD.
Fig 6. Compound-target-pathway networks.
[104]Fig 6
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Green nodes stand for chemical compounds in BXHPD; Red nodes stand for
known target genes from chemical compounds and depression; Blue nodes
stand for the main biological pathway; Red lines stand for the
relationship between compounds and target genes; Brown lines stand for
the relationship between target genes and biological pathway.
Discussion
Through the screening of active compounds, Luteolin (53 targets),
Baicalein (34 targets), Beta-sitosterol (34 targets), Stigmasterol (29
targets) and Cavidine (26 targets) might have primary effects for
antidepression in BXHPD. (1) As an active flavonoid derived from
astragalus root, baicalein could alleviate the development of
depressive symptoms by up-regulating the level of dopamine in the
hippocampus and the level of BDNF [[106]41]. Additionally, it
stimulated the activity of Erks, a member of MAPK family [[107]42], and
inhibited the down-regulation of nuclear factor-kappa B (NF-κB) pathway
[[108]43, [109]44], such as IL-6 and TNF-α, to inhibit
neuroinflammation and to protect nerves. (2) Luteolin was also a
flavonoid from a variety of plants that prevented nerve cell death
[[110]45] and suppressed the levels of inflammatory factors such as
IL-6 and TNF-α to reduce nerve damage [[111]46]. Not only the activity
of Acetylcholinesterase and antioxidant enzymes could be improved, but
the formation of nitric oxide could be done by luteolin to achieve
antioxidant and anti-inflammatory effects [[112]16]. (3) Cavidine was
an isoquinoline alkaloid from Corydalis impatiens, which was an
anti-inflammatory substance that inhibited NF-κB pathway related
pro-inflammatory substances such as IL-6 and TNF-α [[113]47, [114]48].
Cavidine might be related to the suppression of inflammation about
nerve. (4) Zhao et al. found that beta-sitosterol affected the content
of dopamine and 5-HT, which might be a potential drug ingredient target
[[115]49]. (5) Stigmasterol was famous for the treatment of depression
in TCM, which might regulate the level of nerve steroids to control
depressive symptoms [[116]50].
Our results showed that the important pathways of BXHPD mainly
contained PI3k-Akt signaling pathway, MAPK signaling pathway, Oxytocin
signaling pathway, ErbB signaling pathway and HIF-1 signaling pathway.
These pathways are linked to the core genes chiefly including FOS, IL6,
TNF-α, Bcl-2, c-Jun, ERK, and EGF gene in our research in the treatment
of depression. Those results were supported by lots of studies. (1)
PI3k-Akt pathway phosphorylates the downstream factor Forkhead box O3a
and inhibits neurotoxic apoptosis conducted by Corticosterone (CORT),
which is the effective antidepressant process [[117]51, [118]52]. In
addition, PI3k-Akt pathway controlled BDNF (such as IL-6, TNF-α) to
reverse depression-like behavior in a neurotrophic and neuroprotective
way [[119]53, [120]54]. PI3k-Akt pathway was related to neuroplasticity
and could achieve anti-depression effect by enhancing the formation of
synapses and the extension of axon dendrites [[121]55, [122]56].
PI3k-Akt pathway also adjusted downstream molecular to achieve
antidepressant effects, such as Bcl-2 [[123]57] and c-Jun [[124]58],
both of which appeared in core targets of our research ([125]Table 2).
(2) MAPK pathway was a well-known pathway related to neuronal
proliferation and differentiation among depression [[126]59]. ERK, an
important gene of MAPK pathway in the prefrontal cortex and hippocampal
played an important role in antidepressant processing, such as
increasing the expression of BDNF [[127]40] and promoting
growth-related microtubulin in the hippocampus [[128]60]. Patel et al.
showed that MAPK pathway could promote the formation of nitric oxide
(NO), thus improving neuroplasticity and inhibiting apoptosis ([129]Fig
4A) [[130]61]. (3) Oxytocin signaling pathway affected nerve excitation
transmission [[131]62] through the modulation of 5-HT [[132]63] and
interaction with gamma-aminobutyric acid [[133]64]. Wang et al. found
that Oxytocin signaling pathway could reverse depression by
down-regulating CORT and by affecting the HPA axis [[134]65]. Oxytocin
improved depression symptom by down-regulating c-fos protein and by
inhibiting the ERK pathway as well [[135]66]. (4) The ErbB pathway was
also associated with depression by adjusting neuregulin and affecting
downstream Akt and ERK signaling pathways [[136]67, [137]68]. EGF and
VEGF were parts of the ErbB family and were associated with nerve
growth and nutrition [[138]69]. (5) HIF-1 signaling pathway was also
relevant to depression in our study. In the pathway, Hypoxia-inducible
factor (HIF-1) was a main regulator in hypoxia response and played a
role in energy supply in depression through neurotransmitter
transmission [[139]50]. (6) Other pathways in our result ([140]Fig 4B)
were indirectly linked to depression, in terms of nerve growth and
inflammation, such as FoxO signaling pathway [[141]70, [142]71],
Jak-STAT signaling pathway [[143]72, [144]73], prolactin signaling
pathway [[145]74, [146]75] and TGF-beta signaling pathway [[147]76,
[148]77].
Our research had some limitations. First, it was uncertain whether
Chinese medicine was absorbed in the intestinal tract as the active
compounds in our research. Second, core genes of specific pathways
related to downstream products were not objectively presented in our
results. Third, whether the role of pathways in our study was
up-regulated or down-regulated was unclear. Fourth, our study used the
network construction method to speculate the potential anti-depression
targets of BXHPD, which still needs to be further verified by
biological experiments.
Conclusions
BXHPD can effectively alleviate the symptoms of depression through the
molecular mechanisms predicted by NP. NP analysis demonstrates that
there were multi-scale curative activity in regulating depression
related biological processes. Pathway enrichment analysis indicated
that MAPK signaling pathway, ErbB pathway, HIF-1 pathway and PI3K-Akt
pathway were significant pathways in depression. They mainly were
involved in promoting nerve growth and nutrition and alleviating
neuroinflammatory conditions. This study provided some potential ways
for modern medicine in the treatment of depression.
Supporting information
S1 File
(RAR)
[149]Click here for additional data file.^ (105.1KB, rar)
Acknowledgments