Abstract
Accumulating evidence suggests that Astragaloside IV (AS-IV) improves
cardiac function and protects the cardiovascular system. However, the
molecular targets involved remain ambiguous. In this work, we report
research suggesting that AS-IV can antagonize arrhythmias and reduce
the cardiac damage induced by aconitine in zebrafish. Zebrafish have
certain benefits with respect to studying the effect of drugs on
cardiovascular disease. The possible mechanisms involved are analyzed,
and hub gene targets are predicted. First, a model of cardiac damage
induced by aconitine was created, and then a safe drug concentration of
AS-IV was screened, and the appropriate drug dose gradient was selected
within a safe drug concentration range. Second, we confirmed the
protective effect of AS-IV in the cardiovascular system by observing
changes in zebrafish heart rates and the cardiac and vascular
structure. Third, we aimed to demonstrate the antagonistic mechanism of
AS-IV on heart rate and cardiac damage induced by aconitine in
zebrafish, with differentially expressed genes (DEGs) detected by RNA
sequencing. The DEGs were then further analyzed by bioinformatic
techniques, such as function enrichment analysis, protein-protein
interaction network, and DNA-microRNA networks, for example. Next, we
predicted the hub genes of the cardiac protective effects of AS-IV.
Finally, we validated these genes in different transcriptome sequence
datasets of cardiac damage. Thus, we conclude that miR-26b-5p/ATF3/JUN
are key targets of AS-IV and play an important role in maintaining
cardiac homeostasis and regulating cardiac remodeling.
Keywords: astragaloside IV, aconitine, zebrafish, cardiac damage,
bioinformatics analysis, RNA sequencing
Introduction
Cardiovascular disease (CVD) may be considered a series of pathological
conditions involving the heart and circulatory system at all levels. In
recent years, CVD has become a major threat because of its high
morbidity, disability, and mortality ([35]Benjamin et al., 2018).
However, the molecular mechanisms of CVD have not been clearly
elucidated. A better understanding of the factors that result in the
occurrence and development of CVD could reveal potential therapeutic
targets for the disease.
Radix Astragali (Astragalus mongholicus Bunge) is an important herb
that has been used in the clinic in China for more than 2,000 years.
Radix Astragali is known as Huangqi in Traditional Chinese Medicine,
and its protective effect on the cardiovascular system (including a
cardiotonic effect, cardiomyocyte protection, and blood pressure
regulation), has become the focus of fundamental and clinical research
([36]Ren et al., 2013; [37]Li et al., 2017a; [38]Zang et al., 2020).
Astragaloside IV (AS-IV), a key bioactive component of Radix Astragali,
has attracted increasing attention over recent years due to its
potential therapeutic benefits in terms of improving cardiac function
and protecting the myocardium ([39]Zhao et al., 2012). The study of its
pharmacological effects and mechanism of cardiovascular protection will
contribute to the further development and utilization of AS-IV.
The latest reports show that AS-IV could alleviate heart failure by
promoting angiogenesis through the Janus kinase/signal transducers and
activators of transcription (JAK/STAT) 3 pathway ([40]Sui et al.,
2019), as well as alleviating doxorubicin-induced cardiomyopathy by
inhibiting nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase-derived oxidative stress ([41]Lin et al., 2019), and protecting
cardiac function after myocardial infarction by regulating the
phosphatase and tensin homolog/phosphoinositide 3-kinase/protein kinase
B (PTEN/PI3K/Akt) signaling pathway ([42]Cheng et al., 2019). These
findings indicate that further in-depth studies on the mechanism of
AS-IV in the treatment of heart disease are of great significance and
provide the groundwork for clinical interventions at a later stage. In
contrast to the above experiments, we used zebrafish as a model and
utilized high-throughput sequencing and bioinformatic technology to
screen and verify the expression of hub genes.
The zebrafish is one of the most important model vertebrates currently
used in the field of drug screening and toxicity assessment in CVD
([43]Asnani and Peterson, 2014), for several reasons. First, zebrafish
embryos and young fish have transparent bodies, meaning that their
hearts can be clearly imaged under the microscope, while their heart
rates and blood circulation are also clearly visible and easy to
observe. Second, zebrafish share similar physiological and biochemical
characteristics with mammals, with a genome that is up to 87% similar
to the human genome, and can therefore be used to study the molecular
mechanism of human diseases or in high-throughput drug screening
([44]Howe et al., 2013). Third, cardiac fluorescence transgenic
(cmlc2:GFP) zebrafish emit green fluorescence under a fluorescence
microscope, which improves the accuracy and convenience with which
changes in zebrafish heart morphology and structure may be observed
([45]Gut et al., 2017). When studying drug interventions for CVD,
changes in the structure of zebrafish hearts can be observed at
different time periods.
As transcriptome sequencing technology has developed and matured,
high−throughput platforms have become widely applied in gene expression
analysis. In particular, RNA sequencing (RNA-seq) has been widely used
in the diagnosis and treatment of CVD ([46]Conesa et al., 2016).
Screening the hub genes from the large amount of genetic data generated
by RNA-seq relies on bioinformatic technology, which solves biological
problems using several methods including applied mathematics,
informatics, statistics, and computer science ([47]Li et al., 2018).
Given the importance of microRNA (miRNA) as a regulatory molecule in
gene regulatory networks, further confirmation and functional analysis
of the miRNAs involved in cardiac homeostasis will provide more
effective therapeutic targets for the treatment of CVD.
The aim of present study was to verify whether AS-IV could protect
against the cardiac damage induced by aconitine, and to explore the
molecular mechanisms of its cardiac protective effect. First, we
discuss the antagonistic effects of AS-IV at different concentrations
on aconitine-induced cardiac damage in zebrafish by observing changes
in heart rate, cardiac morphology, and the distance between the sinus
venous and bulbus arteriosus (SV-BA). Second, we discuss the use of
high−throughput sequencing technology to detect the differentially
expressed genes (DEGs) of AS-IV in heart protection, followed by data
mining, processing, and bioinformatics analysis to determine potential
signaling pathways and gene targets. Finally, we validate these genes
in different transcriptome sequence datasets of cardiac damage in the
Gene Expression Omnibus (GEO) database. Thus, we present the hub genes
associated with AS-IV intervention in aconitine-induced cardiac damage
in zebrafish. The specific workflow followed is represented in
[48]Figure 1 .
Figure 1.
[49]Figure 1
[50]Open in a new tab
Workflow for AS-IV intervention in aconitine-induced cardiac damage in
zebrafish.
Materials and Methods
Zebrafish
Zebrafish, transgenic (cmlc2: GFP) Tübingen (TU) strain, belonging to
the Danio family of Cyprinidae, were provided by the Institute of
Pediatrics, Children's Hospital of Fudan University. The heart of a
young zebrafish glows green under a fluorescence microscope, which
facilitates the observation of zebrafish hearts and the measurement of
related indicators. Adult zebrafish were bred in a water temperature of
28°C, at a pH of 7.0 to 7.5, and conductivity of 400–600 μs/cm.
Zebrafish were fed three times a day, and exposed to fluorescent light
for 14 h and to darkness for 10 h ([51]Westerfield, 1994). Embryos were
carefully collected at 10-min intervals to ensure precise developmental
timing within a group. Embryos were submerged in culture medium with
cautious and placed in an incubator at 28°C on a 14 h light, 10 h dark
cycle. All procedures were approved by the Children's Hospital of Fudan
University.
Drug Configuration
AS-IV (99.24%) and aconitine (98.01%) were purchased from Chengdu Must
Bio-Technology Limited, preserved at 4°C in a sealed environment, and
protected from light. Dimethyl sulfoxide (DMSO) was used to facilitate
dissolution. Ten milligrams AS-IV and 10 mg aconitine were mixed into
100 μl DMSO to form the mother liquor, which was diluted before use
according to the required concentration of zebrafish culture fluid.
Groups and Experimental Scheme
Model of Cardiac Damage Induced by Aconitine
At 48 hpf, the embryos were divided into a control group and an
aconitine group, with 20 embryos in each group. The embryos were then
placed into six wells, with an aconitine concentration of 15mg/L
([52]Fang et al., 2012). At 72 hpf, the embryos were returned into
ordinary culture medium and placed in an incubator at 28°C in a
six-well plate, with each well containing 4 mL of liquid. Next,
relevant indicators, including heart rate and SV-BA distance, were
observed and recorded. The experiment was repeated twice.
Safe Concentration Screening of AS-IV
The collected embryos (6 hpf) placed in a 12-well plate, and each well
contains 20 embryos and 4 ml culture medium with a different drug
concentration. They were then placed in incubators, and the culture
medium was changed every 24 h, three times in total. At 24 hpf, 48 hpf,
72 hpf, and 96 hpf, the number of zebrafish deaths, changes in heart
morphology, and blood cell accumulation were observed and counted under
a microscope.
Intervention of AS-IV
The 48 hpf-embryos were divided into five groups, with 20 embryos in
each group, as follows: control group, aconitine group, and AS-IV
10/25/40 mg/L groups. In the aconitine group, embryos were placed in 15
mg/L aconitine medium. In the three AS-IV groups, 10, 25, and 40 mg/L
AS-IV solution, respectively, was added into 15 mg/L aconitine medium.
The remaining procedures were as described above.
Zebrafish Development Observation and Recording
All embryos were reared at 28°C and heart rates (beats per minute, bpm)
were measured at room temperature [53](Supplementary Material, Video 1)
. Prior to the measurements, each dish was removed from the incubator
and placed under the microscope light for 4 min at room temperature,
allowing the embryos to acclimatize to the light and eliminate the
effect of any startle response. The embryos were then anesthetized with
tricaine ([54]Wilson et al., 2009).
The embryos were subsequently adjusted to a side-lying position with
cautious use of a needle. Ten embryos were collected in each treatment
group. The heart beats of the embryos were recorded by counting beats
per minute on the live video, as previously described ([55]Keßler
et al., 2015). The blood enters the atrium via the SV, and leaves the
ventricle via the BA. During normal development, the atrium and
ventricle overlap from the side view. However, if the development
process is blocked, the position of the atrium and ventricle changes,
and the distance between the SV and BA also changes accordingly.
Therefore, the SV-BA distance can quantitatively reflect the degree of
influence of drugs on the heart of zebrafish ([56]Du et al., 2015).
SV-BA distance was measured by Image J
([57]http://www.rsb.info.nih.gov/ij/), as previously described.
Data Analysis
All data were plotted using GraphPad Prism 7.0. Data were expressed as
mean ± standard error of the mean (SEM), and the statistical software
SPSS24.0 was used for statistical analysis. The t-test was used for
comparison between the two groups, and analysis of variance (ANOVA) was
used for comparison between the means of multiple groups. A P-value
≤0.05 was considered statistically significant.
RNA Extraction and RNA-Seq Library Construction
This experiment was divided into in two groups including Aconitine
group and Aconitine/AS-IV group, in order to obtain the DEGs of AS-IV
on aconitine-induced cardiac damage. The effect of aconitine on gene
expression was eliminated accordingly.
Total RNA extraction was performed with TRIZOL reagents from Invitrogen
following the manufacturer's instructions. RNA library construction was
then performed by BGI Co., Ltd, Shenzhen, China
([58]http://www.genomics.cn/). An Agilent 2100 Bioanalyzer was used to
detect RNA concentration, RNA Integrity Number (RIN) value, 28S/18S,
and fragment size to determine the integrity of RNA, and the purity of
RNA as detected using an ultraviolet spectrophotometer NanoDrop
(OD260/280) ([59]Qian et al., 2014).
Bioinformatic Analysis of RNA-Seq Data
First, raw data from Illumina HiSeq sequencing were filtered, and clean
reads were matched to the reference sequence. Based on the comparison
results, differential splicing gene detection, single nucleotide
polymorphisms (SNP), Indel detection, fusion gene detection, and other
analyses were performed. Quantitative analysis was conducted on the
known genes, and differential expression analysis was conducted
according to the expression number of genes in different sample groups.
Second, for experiments without biological replication, we used the
PossionDis algorithm to perform differential gene detection, and
screened |log2(FoldChange)|>1&qvalue<0.001 as the DEGs. According to
the result of DEGs, the heatmaps function
([60]https://CRAN.R-project.org/package=pheatmap) in R software was
used to performed clustering analysis.
Third, the DEGs were analyzed by a series of bioinformatics analyses
such as Gene Ontology (GO) ([61]Qian et al., 2014) function analysis
([62]http://www.geneontology.org/), Kyoto Encyclopedia of Genes and
Genomes (KEGG) ([63]Kanehisa et al., 2019) signal pathway analysis
([64]https://www.kegg.jp/), protein-protein interaction (PPI)
([65]Szklarczyk et al., 2019) network prediction
([66]http://string-db.org), and DNA-microRNA ([67]Sticht et al., 2018)
network analysis ([68]http://mirwalk.umm.uni-heidelberg.de/). Finally,
the obtained gene target network was imported into Cytoscape software
([69]https://cytoscape.org/) for visual editing. CytoHubba ([70]Chin
et al., 2014) was employed to investigate node composition and pick out
hub nodes with high degree of connectivity in the network.
Validation of Hub Gene Targets
The expression of hub genes was verified in different transcriptomes.
We retrieved the RNA-seq and miRNA-seq datasets from the GEO database
([71]https://www.ncbi.nlm.nih.gov/geo/). Then RNA-seq series matrix
files were submitted to Biojupies ([72]http://biojupies.cloud), which
was a web-automated generation application in the cloud. The samples in
the dataset were divided into control group and the treatment group.
All procedures followed the methods described in a previous paper
([73]Torre et al., 2018). The limma package ([74]Ritchie et al., 2015)
in R software ([75]http://www.bioconductor.org/packages/) was used for
differential expression analysis of miRNA-seq microarray data.
The Sybyl X-2.0 (Tripos, St. Louis, MO, USA) ([76]Jain, 2003) was
employed to validate the compound-target association in our study. The
compound was retrieved from ZINC database
([77]http://zinc15.docking.org). The 3D (three dimensional) structures
of target proteins were obtained from the RCSB-PDB database
([78]http://www.rcsb.org/) and Uniprot ([79]https://www.uniprot.org/)
database. The proteins and ligands were preprocessed by the docking
suite tool to remove water molecules, protonate 3D hydrogenation, fix
termini treatment, and extract ligand substructure ([80]Ragunathan
et al., 2018). The docking mode was automatic and standard.
Results
Model of Cardiac Damage Induced by Aconitine
A previous study has shown that at 72 hpf, the half maximal effective
concentration (EC50) of the cardiac toxicity of aconitine to zebrafish
embryos is 14.49 mg/L ([81]Fang et al., 2012). The cardiac toxicity of
aconitine is dose- and time-dependent. Our results show that the heart
rates of the aconitine 15 mg/L group increased significantly compared
with the control group ([82] Table 1 , [83]Figure 2A ) (P* < 0.01).
Over time, some zebrafish manifested ventricular arrest or an irregular
heartbeat. We also found that aconitine 15 mg/L caused statistically
significant prolongation of the SV-BA distance ([84] Table 1 ,
[85]Figure 2B ) (P**<0.01). [86]Figure 2C reveals that aconitine 15
mg/L caused the ventricle of the zebrafish to shrink, reduced
pericardial edema, and led to accumulation of blood cells in the sinus
veins and the dorsal aorta (yellow line), suggesting that aconitine can
result in damage to the heart.
Table 1.
Heart rates and SV-BA distance of zebrafish in the control and
aconitine group at 72 hpf (n = 10).
Groups Number Heart rate (bpm)
[MATH: (x¯±s) :MATH]
SV-BA (μm)
[MATH: (x¯±s) :MATH]
P* P**
Aconitine 10 207.2 ± 5.619* 207.2 ± 5.619** <0.0001 <0.0001
Control 10 124.3 ± 2.119* 126.5 ± 3.988**
[87]Open in a new tab
*Compared with the control group, heart rates of the aconitine 15 mg/L
group increased significantly.
**Compared with the control group, the SB-BA distance of the aconitine
15 mg/L group increased significantly.
Figure 2.
[88]Figure 2
[89]Open in a new tab
(A) Heart rates of zebrafish in aconitine group at 72 hpf (n = 10). (B)
SV-BA distance, comparing control group with aconitine group (n = 10).
(C) Effect of aconitine on the morphology of zebrafish heart.
Intervention of AS-IV
This experiment was divided into two parts. The first part was the
screening of the safe dose range of AS-IV. The second part was the
measurement of change in heart rate, SV-BA distance, and cardiac
morphological features of zebrafish after AS-IV intervention.
In the first part, the testing concentrations of AS-IV were 25, 50, 75,
and 100 mg/L ([90] Figure 3A ). A dose of 50 mg/L AS-IV saw the
beginning of abnormal tail development of the zebrafish ([91] Figure 3B
), with no significant change in pericardial structure. Therefore, we
selected 10, 25, and 40 mg/L as a reasonable concentration gradient for
the later interventions.
Figure 3.
[92]Figure 3
[93]Open in a new tab
(A) Effects of different doses of AS-IV on mortality, and development
of the heart and other parts of the zebrafish. (B) At 50 mg/L AS-IV,
abnormal tail vascular development and tail malformation starts to
become apparent. (C) Comparison of heart rates of zebrafish in each
group (histogram). (D) Comparison of distance SV-BA in each group
(trend graph). (E) Changes in heart morphology of zebrafish in each
group. (a) Control group, (b) Aconitine group, (c) AS-IV 10 mg/L group,
(d) AS-IV 25 mg/L group, (e) AS-IV 40 mg/L group.
In terms of the second part of the experiment, there were no
statistically significant differences in heart rate between the AS-IV
10 mg/L group and the aconitine group (P* > 0.05). There was, however,
a statistically significant difference between the AS-IV 25 and 40 mg/L
groups compared with the aconitine group (P** < 0.01, P*** < 0.01)
([94] Table 2 , [95]Figure 3C ). In addition, there was no
statistically significant difference in SV-BA distance between the
AS-IV 10 mg/L and AS-IV 25 mg/L groups compared with the aconitine
group (P* > 0.05, P** > 0.05). In contrast, there was a statistically
difference between the AS-IV 40 mg/L group compared with the aconitine
group (P*** < 0.05) ([96] Table 2 , [97]Figure 3D ).
Table 2.
Heart rates and SV-BA distance of zebrafish in each group (n = 10).
Groups Number Heart rates (bpm)
[MATH: (x¯±s) :MATH]
SV-BA (μm)
[MATH: (x¯±s) :MATH]
Control 10 152.4 ± 2.0 147.1 ± 6.1
Aconitine 10 204.4 ± 6.6 178.5 ± 4.9
AS-IV 10 mg/L^* 10 190.0 ± 3.6 171.8 ± 5.1
AS-IV 25 mg/L^** 10 176.8 ± 4.9 166.6 ± 5.5
AS-IV 40 mg/L^*** 10 172.5 ± 3.2 157.4 ± 8.2
F – 25.100 4.159
P – 0.000 0.006
[98]Open in a new tab
Compared with aconitine group, the heart rate of AS-IV 10mg/L is no
statistically significant differences (P*>0.05), however, the heart
rate of AS-IV 25mg/L and 40mg/L groups is significantly decreased.
(P**<0.01, P***<0.01).Compared with aconitine group, the SV-BA of AS-IV
10mg/L and AS-IV 25mg/L groups is no statistically significant
differences (P*>0.05, P**>0.05), however, the SV-BA of AS-IV 40mg/L
group is decreased. (P***<0.05).
At 72 hpf, we can see that the pericardium of the zebrafish in the
aconitine 15 mg/L group is clearly enlarged. [99]Figure 3E shows that
the pericardium boundary is close to the yolk sac. However, the extent
of pericardial edema is reduced in the AS-IV groups (yellow line).
Therefore, we can conclude that the cardioprotective effective AS-IV is
dose-dependent, and in this experiment 40 mg/L AS-IV is the appropriate
concentration for the intervention of aconitine-induced cardiac damage
in zebrafish.
Mapping of RNA-Seq Reads to Zebrafish Genome
In this section, the Illumina HiSeq platform was used to obtain the
transcriptome sequence data of the AS-IV intervention in an
aconitine-induced cardiac-damaged model of zebrafish. Clean reads
([100] Table 3 ) obtained through filtration were statistically
analyzed, including data volume statistics, base content distribution,
and base mass distribution statistics. The utilization rate of the
transcriptome data was directly reflected by the percentage of total
mapped reads ([101] Table 4 ) in total reads (>70%). We conclude,
therefore, that the error rate in sequencing bases was low, and that
the sequencing data are credible.
Table 3.
Statistics for clean reads.
Clean reads Clean reads^1 (M) Clean bases^2 (G) Q20 (%) Q30 (%) GC (%)
Read length (bp)
Aconitine_
AS-IV 40.0979M 6.0147G 98.37 95.63 48.55 150
Aconitine 40.1863M 6.0279G 98.35 95.57 48.67 150
[102]Open in a new tab
^1Clean reads (M); the number of clean reads obtained after filtration
and in units of M.
^2Clean bases (G): the number of clean reads obtained after filtration
and in units of G.
Table 4.
Statistics for mapped reads.
Mapped reads Total reads[103] ^1 Total mapped[104] ^2 Reads map to+
Reads map to− Splice mapped
Aconitine_
AS-IV 40097856 32334002
(80.64%) 16182534
(40.36%) 16151468
(40.28%) 17374239
(43.33%)
Aconitine 40186304 32830290
(81.70%) 16409274
(40.83%) 16421016
(40.86%) 17253601
(42.93%)
[105]Open in a new tab
^1
Total reads: the number and percentage of reads mapped to the genome.
^2
Total mapped: the percentage of total mapped reads in total reads
should be more than 70%.
Differential Expression Analysis
Under different experimental conditions, genes with significant
differences in expression level are called DEGs. The results of DEGs
showed that a total of 938 genes were obtained [106](Supplementary
Material, Table 1) of which 752 were up-regulated and 186 were
down-regulated ([107] Figure 4A ). The differences of gene expression
levels and the statistical significance in the two comparison samples
are rapidly examined by the Volcano Plot. The differential expression
between aconitine/AS-IV group and aconitine group in is shown as a
volcano plot in the [108]Figure 4B .
Figure 4.
[109]Figure 4
[110]Open in a new tab
(A) Statistical chart of the number of DEGs. The horizontal axis
represents comparison of different samples, the vertical axis
represents the number of DEGs, red represents up-regulation, blue
represents down-regulation. (B) Volcano Plot of DEGs. Each dot in the
volcano plot of DEGs represents a gene. The red dots in the plot
represent up-regulated DEGs, the blue dots represent down-regulated
DEGs, and the black dots represent non-differentially expressed genes.
The dotted line means the threshold lines of FDR and fold change (C)
Biological Process diagram of DEGs. X axis represents -log10 (p-value).
Y axis represents enrichment names. (D) Cellular component diagram of
DEGs. (E) Molecular function diagram of DEGs. (F) Diagram of KEGG
pathway.
Gene Ontology and KEGG Pathway Enrichment Analysis of DEGs
Gene ontology is the international standard classification system of
gene function. Through the GO enrichment analysis of the differentially
enriched genes, their GO classifications and gene functions can be
found. The GO annotation system contains three main branches: namely, a
biological process, a molecular function, and a cellular component
([111] Figures 4C–E ). The GO enrichment analysis of the DEGs was
performed, and the results of top five items in three main branches
includes: biological process contains regulation of transcription from
RNA polymerase II promoter, regulation of cell death, response to cAMP,
positive regulation of cell differentiation, response to radiation. The
items that relate to molecular function are RNA polymerase II core
promoter proximal region sequence-specific DNA binding,
sequence-specific DNA binding, RNA polymerase II core promoter proximal
region sequence-specific binding, sequence-specific DNA binding,
structural constituent of eye lens. The items that relates to cellular
component are transcription factor complex M band, integral component
of plasma membrane, muscle myosin complex, intracellular.
Similar to the GO enrichment principle, KEGG pathway significance
enrichment can determine the most important biochemical metabolic
pathways and signal transduction pathways involved in DEGs ([112]
Figure 4F ). A scatter diagram is a graphical presentation of the
results of the KEGG enrichment analysis. The top five of these are
mitogen activated protein kinase (MAPK) signaling pathway, toll-like
receptor signaling pathway, Janus kinase-signal transducers and
activators of transcription (JAK-STAT) signaling pathway, insulin
signaling pathway, and endocytosis. The enrichment degree of MAPK
signaling pathway is much higher other signaling pathways, so it is
considered as a key signaling pathway in cardioprotective effect of
AS-IV.
Network Analysis From Protein-Protein Interaction Data
First, we inputted DEGs into a STRING database to obtain a
protein-protein interaction (PPI) network [113](Supplementary Material,
Data Sheet 1) . The network was then imported into Cytoscape software,
and core genes were extracted using the Cytohubba. The top 20 genes
were screened out, of which the top five were FOS, JUN, JUNBA, JUNBB,
and ATF3. From these genes, we can see that JUN, JUNBA, and JUNBB are
all immune response complexes, and that ATF3 is of particular
significance as an upstream regulator ([114] Figure 5A ). The
log2(FoldChange) value (Aconitine vs Aconitine/AS-IV) of ATF3 and JUN
is 2.168 and 1.122, respectively.
Figure 5.
[115]Figure 5
[116]Open in a new tab
(A) Hub genes identified by Cytoscape network analysis. The shade of
the color indicates the degree of the gene. The list on the right is
the result of the top 20 genes. (B) Protein-protein interaction data
network of ATF3. Red, up-regulation; blue, down-regulation. Color depth
and size of the dots were proportional to the fold change and
correlated degree, respectively; thickness of the line between the two
nodes indicated the connection strength positively. (C) DNA-microRNA
network of ATF3. The shade of the color indicates the degree of the
genes. The list on the right is the result of the top 5 genes.
Second, we took ATF3 as the center, screened out the genes interacting
with it, and then constructed an ATF3 gene regulatory network, which
demonstrated the upstream and downstream regulatory relationship of
ATF3 more intuitively. These genes included immune response complexes
(FOS, JUN, MMP9), cytokine signal inhibitors (SOCS3A, SOCS3B), and
genes involved in regulating the cell cycle (MAPK10, TRAF5, TRAF6)
([117] Figure 5B ).
It is known that miRNA plays an important role in regulating cardiac
homeostasis; therefore, to further study the miRNA related to ATF3, we
constructed a DNA-microRNA network based on FOS, JUN, JUNBA, JUNBB, and
ATF3, and screened the top miRNA using the Cytohubba plug-in. The
results show that the miRNA with the highest core gene degree was
miR-26b-5p, and that JUN could also be regulated. Therefore, we believe
that the hub genes might be miR-26b-5p/ATF3/JUN ([118] Figure 5C ).
Validation of Hub Targets miR-26b-5p/ATF3/JUN
The end stage of many CVDs such as heart failure, myocardial
infarction, the heart is prone to myocardial fibrosis, cardiac
enlargement, and other cardiac damage symptoms ([119]Tzahor and Poss,
2017; [120]Yuan and Braun, 2017). There is currently no known effective
prophylaxis for myocardial damage. The present study demonstrates that
miR-26b-5p/ATF3/JUN may be the potential targets of cardiac protection
through RNA-seq and a series of bioinformatics analyses.
First, RNA-seq dataset ([121]GSE108157) of left ventricle samples in 11
patients with end-stage heart failure was analyzed by Biojupies to
obtain the DEGs. We found by differential expression analysis that
there were 298 overlapping DEGs in zebrafish and human samples.
Interestingly, the top five genes (FOS, JUN, JUNBA, JUNBB, and ATF3)
screened in the zebrafish were all included in these DEGs. Furthermore,
ATF3 and JUN are up-regulated in zebrafish, while in patients they are
down-regulated ([122] Table 5 ). Furthermore, the RNA-seq matrix of
myocardial infarction tissue in 20 mice was screened from the GEO
database ([123]GSE23294), then the differential expression analysis was
performed to further verify the expression of ATF3 and JUN. The results
suggested that ATF3 and JUN were both downregulated in heart damage
tissue of mouse and human ([124] Table 5 ). Finally, miRNA-seq
transcriptomic dataset with 48 heart failure patients and 32 healthy
donors ([125]GSE136547) was analyzed to evaluate the expression of
miR-26b-5p. Compared with healthy persons, miR-26b-5p was upregulated
in the heart failure patients ([126] Table 5 ). The results of this
experiment partly confirm that miR-26b-5p/ATF3/JUN may play a
considerable role in cardiac protection.
Table 5.
Hub genes expression in different transcriptome sequence datasets.
Genes Sample type Log2FC Regulation
atf3 Zebrafish 2.168584 Up
jun Zebrafish 1.122770 Up
ATF3 Mouse −0.092700 Down
JUN Mouse −0.234537 Down
ATF3 Human −0.583325 Down
JUN Human −0.350524 Down
miRNA-26b-5p Human 0.118979 Up
[127]Open in a new tab
With the aim to improving the accuracy of the results, virtual
screening was further performed to assess the binding affinity between
AS-IV and ATF3/JUN. SYBYL X 2.0 molecular docking experiment was
exploited to elaborate the potential relationship between protein and
compound based on the Cscore. When Cscore value <3, it means that the
binding affinity is weak. The formation of the hydrogen bond was of
vital importance for any stable interaction between compounds and
proteins. The information of Cscore and hydrogen bond were shown in
[128]Table 6 . It is concluded that AS-IV has a stable interaction with
ATF3/JUN.
Table 6.
Molecular docking results of AS-IV and ATF3/JUN.
Compound Structure Cscore (Value) Hydrogen bond (Number)
JUN ATF3 JUN ATF3
AS-IV graphic file with name fphar-11-00957-g006.jpg 4 5 2 3
[129]Open in a new tab
Discussion
Our study demonstrated that AS-IV can improve the manifestations of
aconitine-induced cardiac damage, such as increased heart rate,
pericardial edema, and increased SV-BA interval induced by aconitine in
zebrafish. Other questions addressed by this study include: Do
different concentrations of AS-IV affect the hearts of zebrafish? What
is the safe concentration range of AS-IV? Further work indicated at an
AS-IV concentration of 50 mg/L, abnormal tail development in the
zebrafish was first observed, with the abnormality most commonly being
a warped tail. No pericardial edema or other cardiac damage was
observed in zebrafish at different concentrations of AS-IV. Within the
safe drug concentration range, we finally determined that the
appropriate concentration of AS-IV for aconitine-induced cardiac damage
is 40 mg/L. Through statistical analysis, we find that AS-IV at
different concentrations could reduce the increase in heart rate
induced by aconitine, and reduce the degree of pericardial edema and
SV-BA spacing in a dose-dependent manner. Therefore, our data suggest
that AS-IV has a protective effect on cardiac damage induced by
aconitine in zebrafish.
The DEGs of AS-IV intervention in aconitine-induced cardiac damage were
identified. Of a total of 938 genes, 752 were up-regulated and 186 were
down-regulated, according to high-throughput sequencing technology. To
further understand these DEGs, a PPI network of DEGs was constructed to
screen out the top 20 core genes, among which the top five genes were
FOS, JUN, JUNBA, JUNBB, and ATF3. Among these five genes, FOS, JUN,
JUNBA, and JUNBB were immune response factors, while ATF3, as the
upstream regulatory gene, was of particular significance. In order to
further study the genes with a specific interaction with ATF3, 29
important proteins from the PPI network were predicted which were
closely related to ATF3, including proteins involved in the immune
response (JUN, JUNB, JUND, FOS, etc.) Given that miRNA is an important
regulatory molecule in the gene regulatory network, we further
constructed a DNA-microRNA interaction network, among which miR-26b-5p
had the highest binding degree. Therefore, miR-26b-5p/ATF3/JUN became
the hub genes in this study.
To obtain reliable evidence regarding the relationship between the
expression of miR-26b-5p/ATF3/JUN and cardiac damage-related diseases,
we further verify the expression of miR-26b-5p/ATF3/JUN in human and
mouse cardiac damage tissue in the GEO database. By analyzing and
comparing the DEGs, we find that the expression of miR-26b-5p/ATF3/JUN
in cardiac damage specimens is just opposite to the regulation of AS-IV
cardioprotective effect in this study. Furthermore, molecular docking
experiment was performed to elaborate the potential relationship and
the hydrogen bond formation between AS-IV and ATF3/JUN. Therefore, it
can be considered that AS-IV may play a role in cardiac protection by
regulating miR-26b-5p/ATF3/JUN.
Although it is well recognized that ATF3 protein represents one of the
53 basic leucine zipper (b-Zip) transcription factors in humans, and
that the transcriptional activation or repression activity of ATF3 is
located at both the N- or C-terminal region, the detailed mechanism of
this activity remains elusive ([130]Hai and Hartman, 2001; [131]Hartman
et al., 2004). As the “hub” of the cell adaptive response, ATF3 can
activate or inhibit target gene transcription when it binds with
different structures to form homologous or heterologous dimers
([132]Hai et al., 2010; [133]Zhou et al., 2014). This may be why ATF3
plays a dual role in heart disease. Recent studies demonstrate that
activation of ATF3 expression improves CVD. Up-regulation of ATF3 in
cardiac fibroblasts is a compensatory mechanism for self-protection
during hypertensive ventricular remodeling and heart failure. To
further explore the mechanism of the cardioprotective effect of ATF3,
research shows that ATF3 inhibits the target gene Map2K3 to mediate
cardioprotection, and further inhibits the p38-TGF-β signaling pathway
([134]Li et al., 2017b). Another study also reveals that ATF3
expression in cardiomyocytes can improve cardiac function. This process
is achieved by dampening the inflammatory responses and inhibiting the
expression of extracellular matrix (ECM) remodeling genes, and can also
control peripheral glucose tolerance to preserve homeostasis in the
heart ([135]Kalfon et al., 2017).
On the contrary, [136]Zhou et al. (2011) first reported that ATF3
deficiency promotes myocardial hypertrophy and fibrosis in heart
failure caused by excessive stress load, suggesting that ATF3 has a
cardioprotective function. Research by Heng Lin indicates that ATF3
therapy, or the use of the ATF3-inducer, tert-Butylhydroquinone,
protects against pressure-overload heart failure by inhibiting
Beclin-1-associated detrimental autophagic activity ([137]Lin et al.,
2014). In a word, many phenomena cannot simply be explained by
up-regulation or down-regulation; they are more like the concept of Yin
and Yang in Chinese philosophical theory, in which the ultimate goal is
to pursue a balance between Yin and Yang. The result of the present
study will provide a novel evidence for the cardioprotective effect of
ATF3 expression.
JUN is a family of protein kinases in the MAPK signal transduction
cascade. It is activated by double phosphorylation of tyrosine and
threonine residues, enters the nucleus, regulates the expression of
specific genes, and participates in the regulation of cell apoptosis
and other physiological activities ([138]Kyriakis et al., 1994;
[139]Huang et al., 2004; [140]Sun et al., 2019). Conversely, c−JUN has
also been demonstrated to have anti−apoptotic properties. He et al.
found that up-regulation of miR-138 can reduce hypoxia-induced
apoptosis via up-regulating the MLK3/JNK/c-Jun signaling pathway in
cardiac muscle samples of patients with congenital heart disease
([141]He et al., 2013). Renata Windak et al. ([142]Windak et al., 2013)
investigated the function of c-Jun, and the result suggested that it
can prevent stress-imposed maladaptive remodeling of the heart in mice.
One possible explanation for these experiments is that the role of
c−Jun in promoting survival and apoptosis may be the adaptation of
myocardial tissue to chronic hypoxia; that is, some mechanism of
self-protection and self-repair.
Collectively, miRNA-26b also plays an important role in the
cardiovascular system. In terms of clinical studies, different
expressions of three miRNAs (miR-26b-5p, miR-660-5p, and miR-320a) were
observed in patients with myocardial infarction and healthy individuals
([143]Jakob et al., 2017). In vitro study demonstrated that miR-26b-5p
regulates cell proliferation to suppresses angiogenesis in
hepatocellular carcinoma. Vivo studies have shown similar results. The
latest research ([144]Qi et al., 2020) demonstrated that downregulation
of miR-26b-5p expression can facilitate exercise-induced physiological
cardiac hypertrophy by augmenting autophagy in rats. [145]Han et al.
(2012) underscore the functional relevance of miR-26b-5p in regulating
gene expression during cardiac hypertrophy.
Conclusion
In summary, we have provided evidence that AS-IV shows antagonistic
action against arrhythmia induced by aconitine in zebrafish, and could
also improve structural damage in zebrafish hearts, such as reducing
the SV-BA distance and relieving pericardial edema, in order to play a
protective role in the heart. Mechanistically, via the RNA-seq and
bioinformatics analysis of DEGs in zebrafish, it is found that
miR-26b-5p/ATF3/JUN and MAPK signaling pathway were identified as hub
gene targets and key signaling pathway, respectively. The present study
represents new evidence and a novel therapeutic approach of
cardioprotective effect of AS-IV on zebrafish. However, although
zebrafish have certain benefits in studying heart disease, mammals or
human studies are necessary to explore the pharmacological mechanisms
of AS-IV and aconitine-induced cardiac damage at a deeper level.
Systematic relationship between hub gene targets is required to discuss
in further vitro and vivo experiments.
Data Availability Statement
The datasets [[146]GSE108157/ [147]GSE23294/[148]GSE136547] for this
study can be found in the GEO database
[[149]https://www.ncbi.nlm.nih.gov/geo/].
Author Contributions
All authors contributed to the article and approved the submitted
version. MW and YS have contributed equally to this work.
Funding
This study was supported by the National Natural Science Foundation of
China (8177141253), key specialty cultivation project of TCM
(2018-2020), and Shanghai University of Traditional Chinese Medicine
(project code:2019LK022).
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.
Acknowledgments