Abstract
Dried ginger-aconite decoction (DAD) is a traditional Chinese medicine
(TCM) formula that has been extensively used in the treatment of
myocardial ischemia reperfusion injury (MI/RI). However, its specific
mechanism against MI/RI has not been reported yet. Therefore, this
paper studies the potential active components and mechanism of DAD
against MI/RI based on network pharmacology and experimental
verification. Sixteen active components of DAD were screened according
to oral bioavailability and drug similarity indices. Through Cytoscape
3.7.0, a component-target network diagram was drawn, and potential
active components of DAD against MI/RI were determined. Protein-protein
interaction (PPI) and compound-target-pathway (C-T-P) networks were
established through the software to discover the biological processes,
core targets and core pathways of DAD against MI/RI. High Performance
Liquid Chromatography (HPLC) analysis identified the presence of
potentially active core components for network pharmacological
prediction in DAD. It was found that DAD might have played a
therapeutic role in anti-MI/RI by activating the PI3K/Akt/GSK-3β
signaling pathway in order to reduce mitochondrial hypoxia injury and
myocardial cell apoptosis. The network pharmacological prediction was
validated by Hypoxia/reoxygenation(H/R) model in vitro and ligation
model of the ligation of the left anterior descending branch in vivo.
It was verified that DAD had activated PI3K/AKT/GSK-3β to reduce
myocardial apoptosis and play a therapeutic function in MI/RI.
Keywords: dried ginger-aconite decoction, myocardial ischemia
reperfusion injury, network pharmacology, energy metabolism, tcm
Introduction
Myocardial ischemia-reperfusion injury (MI/RI) denotes the further
destruction of the cardiac structure, and the further aggravation of
metabolic dysfunction or even irreversible damage of the myocardial
cell, following the restoration of blood supply of ischemic and anoxic
myocardial tissue, which mainly involves re-expansion of myocardial
infarction area and life-threatening arrhythmia ([42]Lee et al., 2002;
[43]Raedschelders et al., 2012; [44]Inoue, 2016). A common clinical
cardiovascular disease, it has since developed into a killer ailment
with high morbidity and mortality ([45]Hausenloy and Yellon, 2013;
[46]Heusch, 2017). It typically occurs among the middle-aged and
elderly population; however, as social competition becomes increasingly
fierce, pressure on the youth has also increased, as they are also
prone to develop myocardial ischemic diseases ([47]Ingram et al., 2013;
[48]Han et al., 2018). Some studies indicate that 18 million people die
of cardiovascular diseases every year globally, of which MI/RI
incidence accounts for around 50% ([49]Wei, 2017). MI/RI pathogenesis
involves the interaction of multiple mechanisms, including
vasoconstrictor release, non-reperfusion, deep inflammatory response,
apoptosis and necrosis ([50]Chen et al., 2020; [51]Li et al., 2020a;
[52]Samiotis et al., 2021). Albeit not quite effective, the current
treatment methods for MI/RI are percutaneous coronary intervention and
the use of related thrombolytic drugs; nevertheless, MI/RI still has a
high mortality rate worldwide. Therefore, research and attention on the
mechanism of MI/RI have a considerable significance for its prevention
and treatment.
GRAPHICAL ABSTRACT.
[53]GRAPHICAL ABSTRACT
[54]Open in a new tab
The graphical abstract of this study.
Traditional Chinese medicine (TCM) plays an indispensable role in the
prevention and treatment of MI/RI. It spans a long history, including
Yi Qi Huoxue decoction, Gualou Xiebai Baijiu decoction and Si Ni
decoction ([55]Deng et al., 2017; [56]Zheng and Bao, 2017; [57]Gao et
al., 2019). It has been extensively used in MI/RI treatment, and dried
ginger-aconite decoction (DAD), which comprises two kinds of Chinese
herbal medicines, is one such medicine. Composed of aconite and dried
ginger, DAD is recorded in the Treatize on Febrile and Miscellaneous
Disease. Considering the efficacy of Yang for resuscitation, DAD is
used to clinically treat ischemic heart diseases ([58]Xu, 1986).
Previous studies have indicated that DAD has a protective effect on the
hearts of rats with MI/RI, and such effect is closely associated with
its antioxidant and apoptosis effect ([59]Shi et al., 2014). However,
its bioactive compounds and their pharmacological mechanisms are still
relatively unclear.
Network pharmacology integrates biological systems and
multi-directional pharmacological approaches, incorporates biological
networks and drug action networks, transcends the constraints of
single-target beliefs, and begins from multi-target research strategies
in order to achieve a comprehensive network analysis of drug effects
([60]Xu et al., 2014; [61]Chen et al., 2017; [62]Zhang et al., 2018a).
It is a significant approach to study the mechanisms of the
multi-components, multi-targets and multi-pathways of TCM ([63]Hopkins,
2008; [64]Li and Zhang, 2013). The varied components of DAD are
complex. Previous studies have determined that DAD can treat MI/RI by
reducing the apoptosis of cardiomyocytes; however, the exact mechanism
remains vague. Therefore, a comprehensive method is applied in this
study to illustrate the molecular mechanisms of DAD. Network
pharmacology is used to predict the active components and mechanisms of
DAD in MI/RI treatment. HPLC is applied to determine whether DAD
contains certain components for network pharmacological prediction.
Afterward, in vivo and in vitro experiments are conducted to validate
its mechanism on network pharmacological prediction. A graphical
abstract of this study is presented in Graphical Abstract.
Materials and Methods
Materials
Aconitum abietetorum W.T.Wang and L.Q.Li (No. 51078020190334YC) and
Zingiber officinale Roscoe (No. 51078020191020YC) were obtained from
Jiangyou City, Sichuan Province, China. The geographical location of
Jiangyou is within 31°32′26′′−32°19′18′′ north and
104°31′35′′−105°17′30′′ east. Material authentication for TCM
identification was carried out by Professor Gang Zhang of Shaanxi
University of Chinese Medicine. The samples were deposited at the
Herbal Medicine Museum of the same university.
Fetal bovine serum (FBS) was purchased from BI (United States).
Phosphate buffer saline (PBS) and Dulbecco’s modified Eagle medium
(DMEM) were procured from Gibco (United States). Penicillinstreptomycin
mixture and Cell Counting Kit-8 (CCK-8) from Shanghai Biyuntian Co.,
Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) and trypsin were also
procured from Gibco (United States). The assay kits for malondialdehyde
(MDA), superoxide dismutase (SOD)apoptosis, atpase, creatine kinase
(CK), mitochondrial permeability transition pore (MPTP), lactate
dehydrogenase (LDH) and glutathione peroxidase (GSH-PS) were all
obtained from Boster Biological Technology Co., Ltd. (Wuhan, China).
Cyt-C, β-action, GADPH, Casp9, PI3K, AKT, Bax, Bcl-2 and
phosphorylated(P)-AKT, GSK3β were also purchased from Boster Biological
Technology Co., Ltd. (Wuhan, China). 6-gingerol (202,003), aconitine
(No. A0608), mesaconitine (A0196) and hypaconitine (A0609) were all
purchased from Chengdu Munst Biotechnology Co., Ltd. Standard purity
was set as more than 98%. Methanol and triethylamine were purchased
from Shaanxi Weitong Chemical Co., Ltd.
Dried ginger-aconite decoction Preparation
Aconite and dried ginger were mixed at a 1:1 ratio. They were soaked in
water for 0.5 h, then were boiled twice for 1 h each time. The
filtrates were collected via gauzes, combined and concentrated to
1 g/ml to obtain the extract. For this study, 100 g of aconite and
100 g of dried ginger were prepared. Both components were completely
immersed in water for 0.5 h. Then, 1.6 L water was added, letting the
mixture boil for 1 h twice. The extract was then collected, filtered
with gauze, and concentrated to 200 ml. The supernatant was obtained
after centrifugation of the solution at 3,000 r/min, sterilized with
0.22 μm aqueous microporous membrane, and sealed.
Network Pharmacology
Screening of Dried ginger-aconite decoction Active Components and Collection
of Targets
The chemical constituents of aconite and dried ginger were examined
from the Traditional Chinese Medicine Integrated Database (TCMSP,
[65]https://tcmspw.com/tcmsp.php) and the Comparative Toxicogenomic
Database (CTD, [66]http://ctdbase.org/), with aconite and dried ginger
as the keywords. Active components of DAD were screened via oral
bioavailability (OB) and drug-like quality (DL) ([67]Cao et al., 2018),
with DL ≥ 0.18 and OB ≥ 30% as the thresholds.
The primary compounds of aconite and dried ginger are alkaloids and
volatile oil, both of which are irreplaceable and have good
pharmacological activity. Thus, the following nine compounds were
supplemented: deoxyaconitine, aconitine, hypaconitine, mesaconitin,
6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, and zingerone. The
targets of all active compounds were obtained and imported into the
Universal Protein (UniProt) database ([68]https://www.uniprot.org/) to
standardize their names.
Predicting Targets of DAD Against MI/RI
With “myocardial ischemia-reperfusion injury” as the keywords, MI/RI
targets in the disgenet database ([69]https://www.disgenet.org/)
limited to “Homo sapiens” were obtained. The interactions of the DAD
and MI/RI targets were considered as the potential therapeutic targets.
The protein-protein interaction (PPI) of the common targets was
accomplished in the string database ([70]https://string-db.org/); the
parameter organism was set to Homo sapiens, while the other basic
settings were set as default. Using the Cytoscape 3.7.0 software,
compound-target (C-T) and PPI were constructed.
Pathway and Functional Enrichment Analysis
The database for Annotation, Visualization and Integrated Discovery
(DAVID) v6.8 ([71]www.david.ncifcrf.gov/) provides a comprehensive set
of functional annotation tools for researchers to understand the
biological meanings behind extensive lists of genes. It was employed to
undertake pathway enrichment analyses using the Gene Ontology (GO) and
the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Pathway
terms with p < 0.05 were deemed significant. Using the Cytoscape 3.7.0
software, compound-target-pathway (C-T-P) was constructed.
In vitro Experiment
HPLC Method for Component Analysis
DAD was filtered through a 0.22 μm nylon membrane prior to HPLC
analysis. An HPLC System (Thermo, United States) was used to separate
the components of DAD. All components were separated by Waters Bridge
C18 (4.6 mm × 150 mm, 5 μm) and a C18 guard. Flowrate was set at 1.0
mL, min^−1. The column temperature was 30°C. The wavelength was set at
237 nm. The mobile phases were (A) methanol and (B) triethylamine
aqueous solution, with gradient elution of 0–15 min (A: B = 30:70),
15–40 min (A:B = 65:35) and baseline (A:B = 30:70).
Grouping and Modeling
Rat myocardial cells (H9C2) were purchased from Wuhan Punosai Life
Science and Technology Co., Ltd. (Wuhan, China). The cells were
cultured in DMEM with 10% FBS, 100 U/ml penicillin and 100 μl/ml
streptomycin. They were maintained inside a humidified incubator with
95% air and 5% CO[2] at 37°C. They were subjected to experimental
procedures when they reached an 80% confluence level of population.
They were classified into five groups: control group,
Hypoxia/reoxygenation(H/R) group, DAD low-dose group (0.125 mg/ml), DAD
medium-dose group (0.25 mg/ml), and DAD high-dose group (0.5 mg/ml).
For all experiments, the cells were rendered quiescent by serum
starvation for 24 h before treatment. Following pretreatment with DAD
at varied doses for 24 h, the cells for all groups–except for the
control group and the H/R group–were incubated in DMEM and glucose-free
DMEM, respectively and then placed inside a hypoxia chamber (Stem Cell
Technologies, San Diego, CA, United States). The chamber was flushed
with 95% (v/v) N[2] and 5% (v/v) CO[2] at a flowrate of 15 L/min for 10
min, and maintained at 37°C to induce hypoxia injury. After hypoxia for
12 h, reoxygenation was conducted by replacing the medium to DMEM that
contained 4.5 mM glucose (pH 7.4) and by subsequent incubation in a
CO[2] incubator (5% (v/v) CO[2], 95% (v/v) air) for 2 h ([72]Wang et
al., 2018).
Survival Rate of H9C2 Cells
CCK8 assay was applied to determine the influence of DAD on the
survival rate of H9C2 cells that were damaged by oxygen. The cells were
briefly seeded onto 96-well plates and then cultured until they
adhered. Afterward, the cells were treated with DAD at varied
concentrations (0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml). Model group and
control group were given DMEM (without glucose) and DMEM, respectively.
Model according to the above method. Afterward, 10 μl of CCK-8 was
added, and the mixture was incubated for another 2 h. Absorbance was
recorded at 450 nm, and the experiments were performed in parallel in
triplicate.
Detection of Apoptosis Rate
The H/R damaged cells in each group were digested with 0.25% trypsin
and centrifuged at 1,500 r/min for 5 min. The supernatant was discarded
and the cells were collected. The collected cells were then resuspended
with PBS (pH 7.2), washed with PBS twice, and centrifuged at
1,500 r/min for 5 min, before the supernatant was discarded. The
precipitated cells were resuspended with 500 μl of binding buffer, then
5 μl of annexin V-FITC and 5 μl of PI staining solution was added.
After mixing, the cells were incubated at room temperature in the dark
for 5–15 min. Finally, the apoptosis for each group was detected by
flow cytometry (NovoCyte 452180529501, Thermo, United States).
Biochemical Testing
After H/R injury, the cells in each group were obtained. According to
the manufacturer’s protocols, the SOD, MDA, Na^+-K^+-ATP and
Ca^2+-Mg^2+-ATP levels were detected by their respective commercial
kits.
In vivo Experiment
Establishment and Grouping of MI/RI in Rat Models
Sixty Sprague-Dawley male rats were purchased from Chengdu Da Shuo
Experimental Co., Ltd. (Sichuan, China). They were housed in a
specific-pathogen-free (SPF) environment. The rats in DAD low-dose, DAD
medium-dose and DAD high-dose groups were orally administered with
1.4 g/kg, 2.8 g/kg and 5.6 g/kg DAD once daily, respectively. Those in
the positive control group were orally administered with 0.09 g/kg per
day of compound danshen dripping pills (CDDP), and those in the model,
and sham group were orally administered with the same volume of 0.9%
NaCl. A week later, all rats were operated, with the sham group only
opening the chest without ligation. Left thoracotomy and
pericardiectomy, followed by left anterior descending coronary artery
ligation, were performed. After 40 min of ischemia, the ligature was
opened for reperfusion for 2 h. The serum and heart tissue samples were
prepared for future experiment. All animal experiments were performed
in accordance with the Animal Care and Use Committee of the Institute
of Materia Medica, China (No. TCM-2019–194,040-E08).
Detection of Myocardial Infarction Area in MI/RI Rats
Prior to the experiment, 2% TTC was placed in a 37°C thermostat for
0.5 h. Four rats were randomly selected from each group. Their hearts
were removed, flushed with PBS, and rapidly frozen at −20°C. The
specimens were uniformly cut into 1 mm slices under the line of
ligature and placed in a 37°C, 2% TTC solution to dye for 20°min, and
then fixed with 10% formaldehyde solution. Ultimately, the myocardial
infarction area was white and the non-infarction area was red. The
infarct area was calculated using ImageJ software (Media Cybernetics,
Inc., Rockville, MD, United States). The applied equation was as
follows,
[MATH:
Infarction Range=Inf
arction RangeLeft Ventricular Area
×100%. :MATH]
Immunohistochemical Staining
Immediately after reperfusion, the heart was removed and rinsed in
precooled saline. The myocardium from the anterior wall of the left
ventricle was removed. The heart was then fixed with precooled 4%
paraformaldehyde and rinsed with water for 12 h. The specimens were
dehydrated afterward. They were then immersed in xylene, and
hematoxylin-eosin (H-E) staining was conducted after routine
paraffin-embedded staining. Then, the slices were sealed with
conventional resin, and the pathological changes in the myocardium were
observed under an optical microscope (Olympus BX 41, Japan).
Myocardial Tissue Apoptosis Detection
After MI/RI modeling, the heart tissue was removed. The tissue sections
were washed in a phosphate buffer solution, and fixed in a 4%
paraformaldehyde solution. They were then cut into paraffin sections
with a thickness of 4 μm, and proteinase K was added. After a strict
color rendering according to the kit instructions, five visual fields
were randomly selected for shooting, and the color images of ten
independent fields were randomly captured and digitized. The cells with
clear nuclear markers were defined as TUNEL positive. Image J software
was used for recording, and the apoptosis rate was calculated. The
applied equation was as follows:
[MATH:
Apoptosis index=Numbe
r of TUNEL positive cellsTotal number of
cardiomyocytes×100%. :MATH]
Biochemical Testing
After reperfusion, the rats were intraperitoneally anesthetized using
chloral hydrate (30 mg/kg), and the blood samples were obtained from
the abdominal aorta. The samples were left standing at room temperature
for 30 min and then centrifuged at 3,000 r/min for 15 min. The serum
was collected and stored at −80°C until used. Based on the
manufacturer’s protocols, the GSH-Sp, MDA, CK and LDH levels were
detected by the respective commercial kits.
Detection of MPTP Open Holes in Myocardial Tissues
The fresh myocardial tissue just removed was rinsed with PBS; the
excess water on the surface of the myocardial tissue was absorbed using
a filter paper. The proper part of the entire heart tissue was taken;
its mass was accurately measured, and the tissue homogenate was
prepared using a mass-to-volume ratio of 1:9. The entire operation
needed to be conducted in an ice bath. Finally, the tissue homogenate
was centrifuged for 3,500 r/min for 10 min. The supernatant was
collected and stored at −80°C for later use. The openness of the MPTP
holes in the homogenate was determined according to the kit
instructions.
Western Blot Analysis (in vivo and in vitro)
The myocardial tissue and the H9C2 cells were lyzed by RIPA buffer
(Shanghai Weiao Biological Technology Co., Ltd., China) containing
cocktail protease inhibitors (1:100) and a protein phosphatase
inhibitor (1:50) for 30 min on ice. The protein concentration in the
supernatants was determined by BCA assay (Shanghai Weiao Biological
Technology Co., Ltd., China). Protein samples were loaded with 10%
SDS-polyacrylamide gel (Shanghai Weiao Biological Technology Co., Ltd.
China), and then electrophoretically transferred onto PVDF (Millipore.
Billerica, MA, United States). The membranes were blotted with 5%
fat-free milk in a TBST buffer for 2 h at room temperature and then
incubated at 4°C overnight with the following primary antibodies:
anti-Caspase-9 (1:600), anti-Bax (1:500), anti-Bcl-2 (1:500),
anti-Cyt-c (1:500), anti-PI3K (1:500), anti-Akt (1:1,000), anti-p-Akt
(1:1,000), anti-p-GSK-3β (1:1,000), and anti-GAPDH (1:1,000). The
membrane was rinsed thrice on the second day and then incubated with
HRP-conjugated secondary antibodies for 1 h at room temperature. The
blots were imaged under an enhanced chemiluminescence (ECL) system. The
target band molecular weights and the net optical density were analyzed
using the AlphaEase FC software (Alpha Innotech, United States).
Statistical Analysis
All data were expressed as mean ± standard deviation (SD). GraphPad
Prism 7 software was employed to ascertain statistically significant
differences. The differences among multiple groups were assessed using
one-way analysis of variance (ANOVA). The difference between the means
was considered statistically significant when p < 0.05.
Results
Network Pharmacology
DAD Active Compounds and Target Screening
From aconite and dried ginger, 16 compounds ([73]Table 1) were
retrieved from the TCMSP database, and 171 targets were retrieved from
the TCMSP and CTD databases ([74]Figure 1A). A total of 966 targets of
MI/RI were obtained from the DisGeNet databases. A total of 80 targets
([75]Table 2) were obtained through the intersection of the 966 MI/RI
targets and the 171 putative targets of aconite and dried ginger. These
80 mutual targets were identified as potential therapeutic targets for
DAD against MI/RI ([76]Figure 1B). The C-T network included 187 nodes
(16 for potential bioactive components and 171 for protein targets).
Among the bioactive components, aconitine (DAD, degree = 48), 6-ginger
(DAD, degree = 31) and mesaconitine (DAD, degree = 25), hypaconitine
(DAD, degree = 24) exhibited the greatest correlation with MI/RI. These
could be the key components of DAD against MI/RI.
TABLE 1.
Information on the 16 active compounds in the DAD.
Herbal name TCMSP ID Compound OB DL
Aconite MOL002395 Deoxyandrographolide 56.3 0.31
Aconite MOL002398 Karanjin 69.56 0.34
Aconite MOL002424 aconitine 7.87 0.23
Aconite MOL000538 hypaconitine 31.39 0.26
Aconite MOL002089 mesaconitin 8.7 0.25
Aconite MOL002388 Delphin_qt 57.76 0.28
Aconite MOL002392 Deltoin 46.69 0.37
Dried ginger MOL002467 6-gingerol 35.64 0.16
Dried ginger MOL002459 10-gingerol 19.14 0.28
Dried ginger MOL002495 6-shogaol 31 0.14
Dried ginger MOL002516 zingerone 25.23 0.05
Dried ginger MOL000359 sitosterol 36.91 0.75
Dried ginger MOL002464 1-Monolinolein 37.18 0.3
Dried ginger MOL002501
[(1S)-3-[(E)-but-2-enyl]-2-methyl-4-oxo-1-cyclopent-2-enyl]
(1R,3R)-3-[(E)-3-methoxy-2-methyl-3-oxoprop-1-enyl]-2,2-dimethylcyclopr
opane-1-carboxylate 62.86 0.3
Dried ginger MOL002514 Sexangularetin 35.64 0.16
Dried ginger MOL000358 beta-sitosterol 36.91 0.75
[77]Open in a new tab
FIGURE 1.
[78]FIGURE 1
[79]Open in a new tab
The networks of dried ginger and aconite decoction anti-MI/RI. (A) The
compound-target network of DAD. The red nodes represent active
compounds and the green nodes represent targets. The target surrounding
the active components are proportional to their degree. (B) Overlap of
DAD and MI/RI targets. The blue circles represent DAD targets and the
yellow circles represent MI/RI targets. The shaded area is the target
of DAD anti-MI/RI. (C) The protein-protein interaction network of
protein targets obtained from STRING database and constructed by
Cytoscape. The colors of the nodes are illustrated from blue to yellow
to orange in descending order of degree values.
TABLE 2.
Targes information of DAD anti-MI/RI.
Target name Full name of the target Uniprot ID
MAPK3 Mitogen-activated protein kinase 3 [80]P27361
CYP2C9 Cytochrome P450 2C9 [81]P11712
CYP2C8 Cytochrome P450 2C8 [82]P10632
CYP3A4 Cytochrome P450 3A4 [83]P08684
ARNTL Aryl hydrocarbon receptor nuclear translocator-like protein 1
[84]O00327
CD36 Platelet glycoprotein 4 [85]P16671
GATA4 Transcription factor GATA-4 [86]P43694
ITGA2B Integrin alpha-IIb [87]P08514
ITGB3 Integrin beta-3 [88]P05106
MTOR Serine/threonine-protein kinase mtor [89]P42345
OLR1 Ox-LDL receptor 1 [90]P78380
S100B Protein S100-B [91]P04271
TNF Tumor necrosis factor [92]P01375
BAX Apoptosis regulator BAX [93]Q07812
BCL2 Apoptosis regulator Bcl-2 [94]P10415
BDNF BDNF [95]P23560
CASP3 Caspase-3 [96]P42574
MAPK1 Mitogen-activated protein kinase 1 [97]P28482
CHAT SH2 domain-containing protein 3C [98]Q8N5H7
CHRNA5 Neuronal acetylcholine receptor subunit alpha-5 [99]P30532
IL1B Interleukin-1 beta [100]P01584
IL6 Interleukin-6 [101]P05231
NFKB1 Nuclear factor NF-kappa-B p105 subunit [102]P19838
TP53 Cellular tumor antigen p53 [103]P04637
TRPA1 Transient receptor potential cation channel subfamily a member 1
[104]O75762
ABCB1 ATP-dependent translocase ABCB1 [105]P08183
CYP1A2 Cytochrome P450 1A2, EC 1.14.14.1 [106]P05177
GSK3B Glycogen synthase kinase-3 beta, GSK-3 beta [107]P49841
CCND1 G1/S-specific cyclin-D1 [108]P24385
PPARG PPAR-gamma [109]P37231
PTGS2 Prostaglandin G/H synthase 2 [110]P35354
BIRC5 Baculoviral IAP repeat-containing protein 5 [111]O15392
GDF15 Growth/differentiation factor 15 [112]Q99988
CASP8 Caspase-8 [113]Q14790
NOS2 Nitric oxide synthase, inducible [114]P35228
CAT Catalase [115]P04040
MMP2 72 kDa type IV collagenases [116]P08253
ADIPOQ Adiponectin [117]Q15848
MMP9 Matrix metalloproteinase-9 [118]P14780
MPO Myeloperoxidase [119]P05164
PARP1 Poly [ADP-ribose] polymerase 1 [120]P09874
SOD1 Superoxide dismutase [Cu-Zn] [121]P00441
SOD2 Superoxide dismutase [Mn], mitochondrial [122]P04179
AKT1 RAC-alpha serine/threonine-protein kinase [123]P31749
BECN1 Beclin-1 [124]Q14457
FAS Tumor necrosis factor receptor superfamily member 6 [125]P25445
FN1 Fibronectin [126]P02751
GHRL Appetite-regulating hormone [127]Q9UBU3
HIF1A Hypoxia-inducible factor 1-alpha [128]Q16665
NFKBIA NF-kappa-B inhibitor alpha [129]P25963
PRKCE Protein kinase C epsilon type [130]Q02156
RPS6KB1 Ribosomal protein S6 kinase beta-1 [131]P23443
NFE2L2 Nuclear factor erythroid 2-related factor2 [132]Q16236
HMOX1 Heme oxygenase 1 [133]P09601
DDIT3 DNA damage-inducible transcript 3 protein [134]P35638
GCLC Glutamate--cysteine ligase catalytic subunit [135]P48506
FABP4 Fatty acid-binding protein [136]P15090
GCLM Glutamate--cysteine ligase regulatory subunit [137]P48507
BCL2L1 Bcl-2-like protein 1 [138]Q07817
HMGCR 3-hydroxy-3-methylglutaryl-coenzyme a reductase [139]P00347
IFNG Interferon gamma [140]P01579
LCAT Phosphatidylcholine-sterol acyltransferase [141]P04180
LPL Lipoprotein lipas [142]P06858
TGFB1 Transforming growth factor beta-1 proprotein [143]P01137
TLR4 Toll-like receptor 4 [144]O00206
PTGS1 Prostaglandin G/H synthase 1 [145]P23219
PIK3CG PI3K-gamma [146]P48736
F2 Prothrombin [147]P00734
SCN5A Sodium channel protein type 5 subunit alpha [148]Q14524
F10 Coagulation factor X [149]P00742
ACHE Acetylcholinesterase, AChE, EC 3.1.1.7 [150]P22303
ADRB2 Beta-2 adrenergic receptor [151]P07550
DPP4 Dipeptidyl peptidase 4 [152]P27487
ESR1 Estrogen receptor [153]P03372
NR3C2 Mineralocorticoid receptor [154]P08235
CHRM3 Muscarinic acetylcholine receptor M3 [155]P20309
PDE3A cGMP-inhibited 3′,5′-cyclic phosphodiesterase A [156]Q14432
HTR2A 5-hydroxytryptamine receptor 2A [157]P28223
SLC6A4 Sodium-dependent serotonin transporter [158]P31645
PON1 Serum paraoxonase/arylesterase 1 [159]P27169
[160]Open in a new tab
PPI Network Analysis
To examine the potential interactions of the 80 targets, String 11.0
database was used to build a PPI network. The minimum combined score
between the targets was set as the medium confidence (0.400). The PPI
network of the potential target was saved as a TSV file and then
entered into Cytoscape 3.7.0 for visualization ([161]Figure 1C). In the
PPI network, targets with high degrees played a significant role in
central correlation. The top 5 targets, which were ranked in terms of
degree value, were acquired as the core targets. These targets were
AKT1 (degree = 47), IL6 (degree = 41), TNF (degree = 38), MAPK3 (degree
= 36) and TP53 (degree = 30).
GO Enrichment Analysis
The biological function of DAD against MI/RI was identified by GO
enrichment of the 80 potential therapeutic targets. A total of 158 GO
items were obtained from the GO enrichment analysis of 80 potential
therapeutic targets, including 118 biological processes (BP), 22 cell
components (CC) and 18 molecular functions (MF) (p < 0.05). To realize
a brief demonstration, only the top 10 significant GO entries were
selected for further analysis. The top ten analyses for BP, CC and MF
were selected respectively ([162]Figure 2A), which indicated that DAD
might regulate cell apoptosis, inflammation and mitochondrial energy
metabolism to exert its therapeutic effects against MI/RI.
FIGURE 2.
[163]FIGURE 2
[164]Open in a new tab
(A) The gene ontology (GO) enrichment analysis for key targets. (B) The
KEGG pathway enrichment analysis of key targets. (C) The
compound-target-pathway network constructed by Cytoscape. The blue
nodes represent active components in DAD, the red nodes represent
putative targets, the green nodes represent the signaling pathways.
Node’s size is proportional to their degree. (D) PI3K/ Akt signaling
pathway network (PI3K/Akt signaling pathway network is derived from
[165]https://www.cellsignal.cn/pathways/pathways-akt-signaling)
Pathway Enrichment
To examine the potential pathways of DAD on MI/RI, a pathway enrichment
of the 80 potential therapeutic targets was conducted. The top 20
significantly enriched pathways are presented in [166]Figure 2B. Among
the potential pathways, PI3K/AKT signaling was the most prominently
enriched based on the gene numbers. To further clarify and elucidate
the molecular mechanism of DAD treatment on MI/RI, a C-T-P network
diagram was drawn based on the top 20 signaling pathways, as well as
the targets and compounds involved ([167]Figure 2C). After integrating
drug target predictions, pathway and function enrichments, and network
analyses, AKT1, PIK3G, MAPK3, MAPK1, NFKB, TNF, NFKBA, MTOR, GSK3β and
TP53 were identified. These targets were highly associated with
apoptosis and inflammation. Likewise, they were considered as the key
targets of DAD against MI/RI. Interestingly, of the aforementioned
targets, only GSK3β was downstream of the PI3K/AKT signaling pathway
([168]Figure 2D). Thus, it was speculated that the anti-MI/RI effect of
DAD might be associated with its regulation of apoptosis and
mitochondrial energy metabolism by targeting PI3K/AKT/GSK3β signaling
pathways with their relevant activators.
In vitro Experiments
HPLC Analysis
Network pharmacology predicted that aconitine, 6-ginger, mesaconitine
and hypaconitine in DAD were the potential active components of
anti-MI/RI in DAD. The phytochemical composition of DAD was assessed
using HPLC. As shown in [169]Supplementary Figure S1, DAD contained
aconitine, 6-ginger, mesaconitine.
H9C2 Cells’ Survival Test Results
The effects of DAD were initially assessed based on the cell viability
of the H9C2 cells damaged by H/R. It was found that the exposure of
H9C2 cells to H/R injury had led to a decrease in cells (p < 0.01).
Compared to the control group, the survival rate of the H/R group was
only 58%. When the H9C2 cells were pretreated with 0.125–0.5 mg/ml DAD,
cell viability was significantly restored (p < 0.01). DAD (0.25 mg/ml)
had the greatest effect on cell survival rate, which increased by 25%
(p < 0.01), compared to the H/R group ([170]Figure 3A). These data
suggest that DAD pretreatment may provide protection against
H/R-induced cardiomyocyte injury.
FIGURE 3.
[171]FIGURE 3
[172]Open in a new tab
Effects of dried ginger-aconite decoction (DAD) on survival rate and
biochemical parameters of H9C2 cells damaged by H/R. (A) Effect on the
survival rate of H9C2 cells. (B) Effect on oxidative stress factors
MDA, SOD. (C) The effect on the activity of ATPase. Data were presented
as mean standard deviation (SD). #p < 0.05, ##p < 0.01 vs. control
group. *p < 0.05, **p < 0.01 vs. H/R group.
Results of Biochemical Testing
The outcome of cardiomyocyte hypoxia was insufficient oxygen as
required by the mitochondria, which would lead to mitochondrial damage,
reduced ATP production and aggravated oxidative damage of the
cardiomyocytes. SOD is an oxygen free radical scavenger in human body
([173]Khatua et al., 2012; [174]Ling et al., 2019). The final product
of oxidative damage is MDA, which can damage the mitochondria. The
change in MDA can reflect the degree of oxidative damage of the cells
([175]Mao et al., 2008; [176]Radmanesh et al., 2017). The enzymatic
activities of Na^+-K^+-ATP and Ca^2+-Mg^2+-ATP–indirectly reflect
Changes in the amount of ATP ([177]Zhu et al., 2019). After
pretreatment with varied DAD doses, the MDA levels of H9C2 cells
damaged by H/R could be reduced to varied degrees, as well as increased
the activities of SOD, Na^+-K^+-ATP and Ca^2+-Mg^2+-ATP. In the
administration group ([178]Figure 3B-[179]C), DAD (0.25 mg/mg)
manifested the best therapeutic effect (p < 0.05). These results imply
that the protective effect of DAD on H/R-damaged H9C2 is related to the
mitochondria.
Effect of DAD on the Apoptosis Rate of H9C2 Cells With H/R Injury
As discussed, ischemia and hypoxia aggravate the oxidative damage of
cardiomyocytes and eventually induce the apoptosis of cardiomyocytes.
H/R injury significantly increased the apoptosis rate of the H9C2
cells, which increased by 35% compared to the control group (p < 0.01).
after DAD preconditioning. The apoptosis rate of the H9C2 cells damaged
by H/R significantly decreased, while the apoptosis rate of the DAD
group (0.25 mg/kg) decreased by 18% (p < 0.01) compared to the H/R
group ([180]Figure 4A).
FIGURE 4.
[181]FIGURE 4
[182]Open in a new tab
Effects of dried ginger-aconite decoction (DAD) on apoptosis rate and
Bax/Bcl-2 expression of H9C2 cells after H/R injury. (A) The effect on
apoptosis rate. (B) The effect on the expression of Bax/Bcl-2. Data
were presented as mean standard deviation (SD) of three independent
experiments. #p < 0.05, ##p < 0.01 vs. control group. *p < 0.05, **p <
0.01 vs. H/R group.
Western Blot Analysis
Network pharmacological analysis implied that the molecular mechanism
of the anti-MI/RI effect of DAD might be associated with apoptosis. The
mammalian BCL-2 family member Bcl-2 was an anti-apoptotic protein,
while Bax protein induced apoptosis by enhancing cytochrome c (Cyt-C)
release from the mitochondria ([183]Aamazadeh et al., 2020; [184]Lin et
al., 2020). Therefore, the two targets of Bax and Bcl2 (Bax and Bcl2
belonged to the targets of DAD in anti-MI/RI) were validated in vitro.
Compared to the control group, the expression of Bax had significantly
increased and the expression of Bcl-2 had significantly decreased after
the H9C2 cells were damaged by H/R (p < 0.01). Compared to the H/R
group, the expression of Bax in the H9C2 cells damaged by H/R had
significantly decreased, while the expression of BCL2 had significantly
increased when the H9C2 cells were pretreated by DAD (p < 0.01). In the
administration group ([185]Figure 4B), DAD (0.25 mg/mg) manifested the
best therapeutic effect. In vitro studies were found consistent with
network pharmacology, with DAD being shown to resist MI/RI by reducing
myocardial cell apoptosis. In vitro studies were found consistent with
network pharmacology, with DAD being shown to resist MI/RI by reducing
myocardial cell apoptosis.
In vivo Experiments
Results of ECG and Myocardial Infarction Area in MI/RI Rats
The electrocardiogram test results ([186]Figure 5A) of rats presented
that the ST segment was elevated after reperfusion for each group
compared to the sham operation group, indicating that the model had
been successfully established. Compared to the sham group, the MI/RI
group had significantly increased the infarct size (45%) of the
myocardial tissue ([187]Figure 5B), (p < 0.01). Compared to the MI/RI
group, the infarct size of the myocardial tissue for each
administration group had significantly reduced. The lowest infarct size
was 9.2% in the CDDP (positive) group. Among the three DAD groups (p <
0.01), the MI area of rats in the DAD (2.8 g/kg) medium dose group was
the lowest (19.3%) (p < 0.01). Meanwhile, although the high dose of DAD
(5.6 g/kg) did not manifest a significant reduction in infarct size, a
protective trend of infarct size reduction could be perceived.
FIGURE 5.
[188]FIGURE 5
[189]Open in a new tab
Changes of electrocardiogram in MI/RI rats and effects dried
ginger-aconite decoction (DAD) on myocardial infarction area in MI/RI
rats. (A) After modeling, ECG changes in Sham group, MI/RI group, CDDP
group and DAD (1.4 g/kg, 2.8 g/kg, 5.6 g/kg) groups. (B) Effects on
infarction range reduction of I/R injured rats. Data were presented as
mean standard deviation (SD). ^# p < 0.05, ^## p < 0.01 vs. sham group.
*p < 0.05, **p < 0.01 vs. MI/RI group.
Histopathological Examinations
The degree of myocardial injury could be determined by
histopathological examinations ([190]Zhou et al., 2020). In the sham
group, the myocardial tissue was intact, with a clear texture and
regular arrangement of the myocardial fibers, and without apparent cell
swelling and fracture; the nuclei material was evenly distributed,
without apparent pathological changes. In contrast, in the MI/RI group,
the texture of the myocardial tissue was blurred, the shape of the
myocardial fiber was disordered, the myocardial tissue was faulted, the
interstitium was severely swollen, the nuclear morphology was changed,
and some of the nuclei had disappeared. DAD treatment (1.4, 2.8 and
1.4 g/kg groups) partially attenuated the myocardial tissue
histopathological damages, with the greatest improvement realized in
the 2.8 g/kg group ([191]Figure 6).
FIGURE 6.
[192]FIGURE 6
[193]Open in a new tab
Representative myocardial tissue histopathological sections on the
effects of dried ginger-aconite decoction on myocardial infarction size
in MI/RI rats (200×). Myocardial tissue injury was assessed by
hematoxylin-eosin (H&E) staining.
Effect of DAD on Myocardial Cell Apoptosis in MI/RI Rats
TUNEL assay was applied to evaluate the effects of DAD on the apoptosis
of myocardial tissue cells in MI/RI rats. Compared to that of the sham
operation group, the apoptosis rate (70%) of the MI/RI group had
significantly increased (p < 0.01). DAD (1.4, 2.8 and 5.6 g/kg)
treatment had significantly mitigated the increased percentage of
apoptotic cells compared to the model group (p < 0.01). Among the three
DAD groups, the 2.8 g/kg group exhibited the lowest apoptosis rate
(45%). Meanwhile, although a high dose of DAD (5.6 g/kg) did not
significantly reduce the apoptosis rate, a decreasing trend in such
rate was observed ([194]Figure 7).
FIGURE 7.
[195]FIGURE 7
[196]Open in a new tab
Effects of dried ginger-aconite decoction (DAD) on apoptosis of
myocardial cells in MI/RI rats. Apoptosis of cardiomyocytes (green) and
DAPI (nuclei Blue). Representative images were shown, and the scale bar
indicated 20 µm. The percent of apoptosis cells were calculated for
statistical analysis. Data were presented as mean standard deviation
(SD). ^# p < 0.05, ^## p < 0.01 vs. sham group. *p < 0.05, **p < 0.01
vs. MI/RI group.
Results of Biochemical Testing
Myocardial enzymes are vital indicators in the clinical detection of
heart health ([197]Radhiga et al., 2012; [198]Xiang-Qian et al., 2019).
The activity of the LDH and CK enzyme sharply increased after
myocardial injury. The expressions of LDH and CK significantly
increased in the MI/RI group (p < 0.01), suggesting that serious heart
damage might occur. After treatment, the activities of CK and LDH
decreased in each dose group of DADs (1.4, 2.8 and 5.6 g/kg), while the
activities of the 2.8 g/kg group had significantly decreased (p < 0.01)
([199]Figure 8A). Oxidative stress injury is a key mechanism of I/R
injury. Under ischemia and hypoxia conditions, the mitochondria of
cardiomyocytes are damaged, the permeability of the mitochondria
membrane is transformed ([200]Figure 8B), and reactive oxygen species
are released into the cytoplasm through the damaged mitochondria. SOD
and GSH-Px are known as free-radical scavengers in vivo. Remarkably,
after MI/RI, mitochondrial swelling degree and MDA had increased
alongside a decreased GSH-Px activity. After treatment with varied DAD
(1.4, 2.8 and 5.6 g/kg), the degree of mitochondrial swelling and the
degree of elevated MDA level among MI/RI rats were reduced, while the
activity of GSH-Px, SOD were restored ([201]Figure 8C). Among the three
DAD groups, DAD (2.8 g/kg) manifested the best therapeutic effect (p <
0.05).
FIGURE 8.
[202]FIGURE 8
[203]Open in a new tab
Effects of dried ginger-aconite decoction (DAD) on biochemical indices
and MPTP conversion pores in MI/RI rats. (A) Effects on activities of
myocardial enzymes CK, and LDH. (B) Effects on mitochondrial
transformation pore MPTP. (C) Effect on oxidative stress factors MDA,
and GSH-Px. Data were presented as mean standard deviation (SD). #p <
0.05, ##p < 0.01 vs. sham group. *p < 0.05, **p < 0.01 vs. MI/RI group.
Promotion of PI3K/AKT/GSK-3β by DAD
As a unique molecular target in the mitochondria, Cyt-C can activate
apoptosis factors such as CASP9 and can lead to the apoptosis of
damaged myocardium ([204]Gao et al., 2016). Considering that the
anti-MI/RI mechanism of DAD has been shown to be associated with
apoptosis in vitro, the network pharmacologically predicted pathway and
the related mitochondrial targets in vivo were further validated,
namely the PI3K/AKT/GSK-3β pathway and the mitochondrial targets–Cyt-C
and CASP9. Western blot analysis presented that the expression of
PI3K/AKT/GSK-3β was inhibited (p < 0.05), and that the expression of
Cyt-C and CASP9 was significantly increased in the MI/RI group compared
to the sham group (p < 0.05). After DAD intervention, the expression of
PI3K/AKT/GSK-3β was significantly activated, and the expressions of
Cyt-C and CASP9 were significantly decreased in the DAD groups compared
to the MI/RI group ([205]Figure 9). Among the three DAD groups, DAD
(2.8 g/kg) manifested the best therapeutic effect (p < 0.05).
FIGURE 9.
[206]FIGURE 9
[207]Open in a new tab
Experimental validation of key signaling pathways and mitochondrial key
targets in vivo. Dried ginger-aconite decoction(DAD) activates the
PI3k/Akt/GSK-3β signaling pathway and inhibits the expression of Cyt-C
and CASP9. Data were presented as mean standard deviation (SD) of three
independent experiments. ^# p < 0.05, ^## p < 0.01 vs. sham group. *p <
0.05, **p < 0.01 vs. MI/RI group.
Discussion
MI/RI is not only a primary cause of death among patients with
cardiovascular and cerebrovascular diseases, but can also seriously
affect the prognosis of patients with ischemic heart diseases. While
the effects of DAD against MI/RI have been demonstrated, its exact
mechanism is vague. In this case, pharmacological approaches are
adopted to explore relevant molecular pharmacological mechanisms and
validate them empirically.
Sixteen active components and 171 targets of DAD were obtained using OB
and DL parameters as significant evaluation indices and supplements of
significant components. A higher degree of the compound in the C-T
network denotes a greater significance. This study found that the
degree values of aconitine, 6-ginger, hypaconitine and mesaconitine
were among the top. These could be the key components of DAD against
MI/RI. The HPLC method was used to determine the above components in
DAD that were predicted by network pharmacology. We found that DAD
contained aconitine, 6-ginger, mesaconitine, which was consistent with
the results of network pharmacology.
Through network pharmacology, it was found that BP that was highly
correlated with DAD anti-MI/RI was the negative regulation of the
apoptotic process, the lipopolysaccharide-mediated signaling pathways,
the regulation of mitochondrial membrane potential, the inherent
apoptotic signaling pathway as a response to DNA damage, the external
apoptotic signaling pathway lacking ligand, and release of cytochrome C
from mitochondria. An analysis of the C-T-P network revealed that DAD
anti-MI/RI acted on multiple targets and signaling pathways. The core
targets of the active compounds in DAD were determined, namely AKT1,
PIK3G, MAPK3, MAPK1, NFKB, TNF, NFKBA, MTOR, GSK3β and TP53. As with
BP, these targets were associated with apoptosis and inflammation.
Various studies have likewise confirmed that apoptosis is the key
mechanism of anti-MI/RI ([208]Zhang et al., 2018b; [209]Li et al.,
2020b). Apoptosis plays a vital function in MI/RI prognosis. Studies
have determined that the inhibition of myocardial cell apoptosis during
MI/RI can mitigate the enlargement of the infarct area and can
effectively protect cardiac functions ([210]Geng et al., 2020).
Significantly, PI3K/AKT/GSK-3β, an apoptotically-related signaling
pathway, has the highest anti-MI/RI correlation in DAD ([211]Chen et
al., 2017). Therefore, DAD may play an anti-MI/RI function by
inhibiting myocardial apoptosis through the PI3K/AKT/GSK-3β signaling
pathway. To further validate this hypothesis, in vivo and in vitro
experiments are conducted to validate its mechanism on network
pharmacological prediction.
Mitochondria is the energy factory of the cells and is also the site of
ATP synthesis ([212]Wang et al., 2020). On a physiological level, a
stable mitochondria provides ATP to the body; when it is damaged (i.e.,
by hypoxia injury), it can produce superoxides and reactive oxygen
species, leading to adverse stimuli like calcium overload and oxidative
stress, and further inducing apoptosis and necrosis in cells
([213]Latini et al., 2015). The abnormal openness of MPTP, as a key
regulator of mitochondrial functions, can induce mitochondrial
structure disorders, which influence mitochondrial functions and
eventually result to cell apoptosis. Under normal physiological
conditions, MPTP remains closed, while Ca^2+ overload and excessive
oxidative stress can induce it to open ([214]Tait and Green, 2010).
Cyt-C generally exists in the space between the inner and outer
membranes of the mitochondria, and cannot cross the outer membrane to
reach the cytoplasm under physiological conditions ([215]Joseph and
Levine, 2015). When the MPTP is abnormally open and causes damage to
the mitochondrial membrane structure, Cyt-C is released from the
mitochondria into the cytoplasm and acts as a vital pro-apoptotic
factor. It binds to the apoptotic protease activator 1 in the
synergistic role of deoxyadenosine triphosphate. Caspase-9 is
activated, eventually leading to apoptosis ([216]Mace et al., 2014).
Interestingly, in vitro studies have depicted that DAD reduces
apoptosis and increases ATPase activity in H/R-damaged H9C2 cells.
Meanwhile, in vivo studies have presented that DAD can reduce
myocardial injury in MI/RI rats, with the rate of apoptosis of
myocardial cells, the oxidative damage, the degree of mitochondrial
MPTP opening, and the expressions of Cyt-C and CASP9 likewise all
reduced. Therefore, as predicted by network pharmacological analysis,
it was verified that DAD could reduce cardiomyocyte apoptosis both in
vivo and in vitro.
The PI3K/AKT/GSK-3β signaling pathway is a fundamental pathway in
MI/RI. Phosphatidylinositol 3-kinase (PI3K, a lipid kinase) can
specifically catalyze the phosphorylation of the phosphatidylinositol-3
hydroxyl group ([217]Stokes and Condliffe, 2018). It phosphorylates
PIP2 to produce PIP3 firstly ([218]Zhang et al., 2017), and then
activates AKT ([219]Chen et al., 2014). Activated AKT can yield a
series of phosphorylation cascade reactions and can regulate
significant downstream effector molecules such as Glycogen synthase
kinase-3β (GSK-3β) to exert their biological functions ([220]Ya-Fei et
al., 2010). GSK-3β is a serine/threonine kinase ([221]Sun et al., 2011;
[222]Barré and Perkins, 2014), and is the most extensively studied
downstream target of AKT. It can promote cardiomyocyte apoptosis
through an intrinsic mitochondrial pathway ([223]Yan et al., 2011);
meanwhile, phosphorylated GSK3β has no biological activity, which can
reduce myocardial cell apoptosis. ([224]Jun et al., 2011). The
PI3K/AKT/GSK-3β signaling pathway plays a vital function in the growth,
survival, apoptosis and proliferation of cells. Recent studies have
presented that the activation of this signaling pathway can reduce body
damage caused by hypoxia ([225]Kaneko et al., 2016; [226]Li et al.,
2018; [227]Jing et al., 2019). Interestingly, in vivo studies have
indicated that DAD can activate the expression of the said signaling
pathway.
The innovation of this study involves the prediction of active
components, BP and mechanism of action of DAD against MI/RI using
network pharmacology. This study has demonstrated that DAD plays an
anti-MI/RI role by activating PI3K/AKT/GSK3β to reduce cardiomyocyte
apoptosis. Nevertheless, the limitations of this study should be
acknowledged. Firstly, DAD at its highest concentration has either no
or minimal effect against MI/RI. In the dose-setting process, the
clinical equivalent dose was selected as the medium-dose group. In
[228]Figures 3, [229]5–[230]9, dose dependence was not found, which
might be because the concentration gradient established was not large
enough. Future studies may focus on the study of the “dose-effect”
relationship of DAD in regulating SOD and MDA, as well as other
indices. Moreover, most TCMs can play multiple therapeutic roles, and
network pharmacology can predict DAD anti-MI/RI by inflammation
relevant signaling pathways. Thus, further studies may explore
inflammation-related signaling pathways and regulators. In addition,
the active compounds neutralized in DAD have been identified by network
pharmacology. However, the compounds that exert therapeutic effects are
still unknown and deserve further study. Overall, the aforementioned
limitations should continue to be studied in order to clarify the
therapeutic mechanisms of DAD.
Conclusion
In this study, a comprehensive strategy that involved network
pharmacological analysis, HPLC technology and experimental verification
was adopted to determine the potential active components and molecular
mechanisms of DAD against MI/RI. Based on the TCMSP database and on
core compounds, 16 active compounds of DAD were obtained. The presence
of four of these components was identified in DAD by HPLC, in which the
components were potential therapeutic ingredients as predicted by
network pharmacology. Through the analysis of BP, hub targets and hub
signaling pathways and experimental verification, it was concluded that
DAD could play an anti-MI/RI role by inhibiting myocardial apoptosis
via PI3K/AKT/GSK3β. The experimental results were consistent with the
network pharmacological predictions. Relatively, this study evidently
clarified the anti-MI/RI mechanism of DAD, which could provide a
certain basis for future studies on DAD.
Data Availablility Statement
The original contributions presented in the study are included in the
article/[231]Supplementary Material, further inquiries can be directed
to the corresponding author.
Ethics Statement
All animal experiments were carried out in accordance with the Animal
Care and Use Committee of the Institute of Materia Medica, China (No.
TCM-2019- 040-E08).
Author Contributions
FX, G-JD, and Y-YW performed the experiments, analyzed the data and
wrote the manuscript. ML, BW, and P-FW designed the study. FG, LC, and
A-PL revised the article. All authors have read and agreed to the final
version of the manuscript.
Funding
This research was financially supported by National Natural Science
Foundation of China, China, (81373988); Project of science and
Technology Department of Shaanxi Province (2017JM8080); Project of
Shaanxi Provincial Administration of traditional Chinese Medicine
(JCPT007); Project of Education Department of Shaanxi Province
(20JC012); and Subject Innovation Team of Shaanxi University of Chinese
Medicine, China, (2019-QN02). The person in charge of the project is
ML.
Conflict of Interest
The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.
Supplementary Material
The Supplementary Material for this article can be found online at:
[232]https://www.frontiersin.org/articles/10.3389/fphar.2021.609702/ful
l#supplementary-material.
[233]Click here for additional data file.^ (11.7MB, zip)
[234]Click here for additional data file.^ (171.1KB, pdf)
References