Abstract
Successful recovery from hepatectomy is partially contingent upon the
rate of residual liver regeneration. The traditional Chinese medicines
known as Periplaneta americana extracts (PAEs) positively influence
wound healing by promoting tissue repair. However, the effect of PAEs
on liver regeneration is unknown. We used a mouse liver regeneration
model after 70% partial hepatectomy (PH) and a hepatocyte culture to
determine whether PAEs can promote liver regeneration as effectively as
skin regeneration and establish their modes of action. L02 cells were
divided into serum-starved control (NC) and three PAEs (serum
starvation + 0.1 mg/ml, 0.5 mg/ml, or 1 mg/ml PAEs) groups. L02 cell
proliferation was assessed at 24 h, 48 h, and 72 h by CCK-8 assay.
Forty male C57 mice were randomly divided into control (NC), normal
saline (NS), PAEs400 (400 mg/kg/d), and PAEs800 (800 mg/kg/d) groups (n
= 10 per group). The NS and both PAEs groups were administered normal
saline and PAEs, respectively, by gavage for 10 days. Two hours after
the tenth gavage, the NS and both PAEs groups were subjected to 70% PH
and the residual liver was harvested after 48 h. The hepatic
regeneration rate was evaluated and hepatocyte proliferation was
estimated by immunohistochemical (IHC) staining for Ki-67. Twelve DEG
libraries (three samples per group) were prepared and sequencing was
performed in an Illumina HiSeq 2000 (Mus_musculus) at the Beijing
Genomics Institute. The genes expressed in the liver tissues and their
expression profiles were analyzed by bioinformatics. KEGG was used to
annotate, enrich, and analyze the pathways. PAEs promoted hepatocyte
proliferation in vitro and in vivo and accelerated mouse liver
regeneration after 70% PH. The screening criteria were fold change (FC)
≥ 2 and q-value < 0.001. We identified 1,092 known DEGs in PAEs400 and
PAEs800. Of these, 153 were categorized in cellular processes. The KEGG
analysis revealed that the aforementioned DEGs participated in several
signaling pathways closely associated with cell proliferation including
PI3K-Akt, MAPK, Apelin, Wnt, FoxO, mTOR, Ras, VEGF, ErbB, Hippo, and
AMPK. It was concluded that PAEs can effectively improve liver
regeneration via the synergistic activation of different signaling
pathways.
Keywords: bioinformatics, liver regeneration, Periplaneta americana
extracts, proliferation, signaling pathways
Introduction
The liver performs and regulates numerous physiological functions and
has the ability to regenerate. Parenchymal cells or hepatocytes
constitute ~80% of the liver tissue and execute most of the
physiological functions of this organ. The balance of the liver
consists of non-parenchymal endothelial, stellate, and Kupffer cells as
well as lymphocytes ([59]Taub, 2004). Successful recovery after partial
hepatectomy (PH) depends mainly on rapid residual liver regeneration
and liver function recovery. However, few clinically available drugs
significantly enhance hepatocyte proliferation or liver regeneration.
Therefore, the quest for new drugs or therapeutic targets that can
improve liver regeneration is of great theoretical and practical
importance.
Several animal models have been designed to evaluate liver
regeneration. In 1931, Higgins and Anderson proposed a rat model of 2/3
hepatectomy ([60]Nevzorova et al., 2015). It continues to be popular to
this day as it involves the excision of intact liver lobes and does not
damage the residual liver. In contrast, other models using toxicants
such as carbon tetrachloride to induce injury to the residual liver
([61]Mao et al., 2014). Though the excised lobes never regenerate, the
residual liver tissue restores the original liver mass within ~1 week
after surgery ([62]Taub, 2004). The PH liver regeneration model is used
to synchronize and evaluate cell cycle events and signal transduction
in vivo ([63]Fausto et al., 2006). Certain in vitro methods also
synchronize mammalian cell cultures and are used to study regulatory
mechanisms of cell cycle progression and cell proliferation. These
include serum starvation/deprivation, chemically-induced cell cycle
arrest, mitotic shake-off, counterflow centrifugal elutriation, and
newer live cell methods. Each of these methods has inherent
disadvantages. Nevertheless, serum starvation is simple, reversible,
reliable, and generally applicable to mammalian cells ([64]Langan et
al., 2017) including hepatocytes.
Liver regeneration is extremely complex and involves multiple factors
and pathways. Parenchymal and non-parenchymal liver cells proliferate
to replace lost hepatic tissue. However, hepatocytes proliferate first
([65]Michalopoulos and DeFrances, 2005). Liver regeneration
approximately entails the priming phase (early period after PH;
quiescent hepatocytes transition from G0 to G1), the proliferation
phase (progression phase; DNA synthesis and hepatocyte proliferation),
and the termination phase (hepatocyte proliferation stops as soon as
the liver mass is restored) ([66]Fausto, 2000; [67]Tao et al., 2017).
The earliest signals initiating the regenerative response remain to be
elucidated. However, it is known that IL-6 (interleukin-6) and TNF-α
(tumor necrosis factor-α) participate in the events that transform
hepatocytes from G0 to G1 in the priming phase and this process is
mediated by the NF-κB, JAk/STAT, and MAPK signaling pathways ([68]Taub,
2004; [69]Michalopoulos and DeFrances, 2005; [70]Mao et al., 2014;
[71]Tao et al., 2017). Complete mitogens and auxiliary mitogens are
involved in the proliferation phase. The former activates secondary or
delayed gene responses, stimulate DNA replication, and induce
hepatocyte proliferation via the JAk/STAT, PI3K/AKT, mTOR, ERK, and
MAPK signaling pathways. Complete mitogens include TGF-α (transforming
growth factor-α), HGF (hepatocyte growth factor), EGF (epidermal growth
factor), and EGFR (epidermal growth factor receptor) ([72]Taub, 2004).
The latter may partially contribute to regeneration by accelerating or
magnifying the effects of complete mitogens even though they are not
mitogenic in hepatocytes. Auxiliary mitogens include VEGF (vascular
endothelial growth factor), IGF (insulin-like growth factor), Bas (bile
acids), NE (norepinephrine), and estrogen ([73]Tao et al., 2017). The
mechanisms of the termination phase are poorly understood. TGF-β1
(transforming growth factor-β1) is the best known antiproliferative
factor ([74]Tao et al., 2017). Despite extensive investigation, the
exact mechanisms of liver regeneration have not yet been clarified.
Certain animal products have been used in traditional Chinese medicine
(TCM). The ancient Chinese science of disease treatment has been
advocated for thousands of years. Periplaneta americana (PA), the
American cockroach, has a long history of application for the treatment
of various injuries ([75]Alves and Alves, 2011; [76]Zhu et al., 2018).
Over the past few years, physiological and pharmacological studies have
demonstrated that TCM powder and extracts of Periplaneta americana
(PAEs) ([77]Zeng et al., 2019) have tissue repair ([78]Yang et al.,
2015; [79]Li et al., 2016; [80]Zhu et al., 2018), antitumor ([81]Luo et
al., 2014; [82]Zhao et al., 2017), antibacterial ([83]Basseri et al.,
2016; [84]Ali et al., 2017), antiviral ([85]Li and Hu, 2003),
antifungal ([86]Yun et al., 2017), antifibrotic ([87]Li D. et al.,
2018), antiosteoporotic ([88]Huang et al., 2017),
cardiomyocyte-protecting ([89]Li J. et al., 2019), and
immunity-enhancing ([90]Zeng et al., 2019) efficacy. Animal medicines
have complex ingredients and it is difficult to isolate and identify
effective components. Many researchers are actively engaged in the
analysis and identification of ingredients of PAEs and have made some
progresses. It has been reported that ([91]Lv N. et al., 2017; [92]Li
et al., 2020) PAEs contain polysaccharides, peptides, nucleosides,
polyols, steroids, terpenes, alkaloids, flavonoids, and isocoumarins,
and the effective constituents of the extracts may include
polysaccharides, peptides, nucleosides. However, PAEs have not been
fully explored in clinical applications as their active constituents
have not been adequately purified and their molecular mechanisms are
not fully understood. At present, only a few representative clinical
PAEs prescriptions such as “Kangfuxin solution”, “Xinmailong
injection”, “Ganlong capsule” among others are used mainly for the
treatment of cutaneous lesions ([93]Li L. J. et al., 2019), chronic
heart failure ([94]Lu et al., 2018), alimentary canal diseases ([95]Li
Q. J. et al., 2018), and chronic hepatitis B ([96]Li and Hu, 2003).
PAEs stimulate healing and inhibit hepatic fibrosis progression.
However, it remained uncertain whether PAEs would promote liver
regeneration as effectively as skin regeneration. Here, we used a mouse
liver regeneration model after 70% PH and a hepatocyte culture to
determine the beneficial effects of PAEs in liver regeneration and
establish their modes of action.
Materials and Methods
Preparation of Periplaneta americana Extracts (PAEs)
The original PAEs extractum was supplied by Tengchong Pharmaceutical
Co. Ltd., Baoshan, Yunnan, China. The extraction protocol of PEAs
followed the patent ([97]Li and Hu, 2003) involving Ganlong capsule, a
medicine for the treatment of chronic hepatitis B clinically in China.
The main ingredients are sticky sugar amino acid. Briefly, dried adult
Periplaneta americana was coarsely crushed and the powder was soaked in
95% ethanol (1:4 w/v) for 24 h and extracted by heat reflux extraction
at 80°C for 8 h. After oil-water separation, the aqueous phase was
filtered and evaporated under reduced pressure down to the original
PAEs extractum with ≤ 15% moisture content. This condensate was stored
at -20°C until subsequent use. For intragastric administration or cell
culture preparation, the material was diluted in 0.9% (w/v) saline or
double-distilled water, respectively.
HPLC Analysis
Chromatographic analysis was performed on a Waters E2695 series HPLC
system (Waters, MA, USA) equipped with an ultraviolet detector 2998,
using an Agilent C18 SB-AQ (4.6mm×150mm, 5μm) at a column temperature
of 25°C. The flow rate and injection volume were 1 ml/min and 20 µl,
respectively. The methanol–water (2:98) system was employed as the
mobile phase for quantitative determination of multiple standard
compounds, i.e., cytosine, uracil, cytidine, uridine, inosine, and
guanosine. All these standard compounds were dissolved together by 3%
methanol to form a mixed standard solution (10 μg/ml). The optimized
detection wavelength was 254 nm.
The inosine standard compound (C14328000) was purchased from Dr.
Ehrenstorfe Co., LTD, Germany. The other standard compounds, cytosine
(CDAA-281383), uracil (CDAA-28071620), cytidine (CDAA-281384), uridine
(CDAA-280717), and guanosine (CDAA-280791), were purchased from ANPEL
Laboratory Technologies Co., LTD, Shanghai.
Cell Culture
The normal hepatocyte cell line L02 was obtained from the Tumor
Institute of the Third Affiliated Hospital of Kunming Medical
University, Kunming, Yunnan, China. The L02 cells were cultured in
Gibco RPMI 1640 Medium without HEPES and supplemented with 10% fetal
bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100
U/ml penicillin, and 100 g/ml streptomycin (Invitrogen, Carlsbad, CA,
USA) in 5% CO[2] at 37°C.
Cell Counting Kit-8 (CCK-8) Assay
L02 cell proliferation was measured with a CCK-8 kit (Dojindo
Laboratories, Kumamoto, Japan). Briefly, cells in the logarithmic
growth phase were seeded in 96-well plates at a density of 5×10^3/well.
Each well contained 100 μl culture medium. After 24 h, the cells were
incubated in 100 μl culture medium without FBS for 24 h and divided
into the serum-starved control group (NC) and the serum starvation +
0.1, 0.5, 1, 25, or 50 mg/ml PAEs groups. After 24, 48, and 72 h, the
CCK-8 kit reagents were added to the culture plates in the dark. The
cells were incubated under 5% CO[2] at 37 °C for 40 min. The
absorbances were read at 450 nm in an automated plate reader (BioTek).
Each experiment was repeated at least in triplicate. Based on the
instructions of the CCK-8 kit, the cell proliferation ratio was
calculated as follows:
[MATH:
Cell prolifer
ation r
atio =
(APAE<
/mi>s−Ab
)/(Ac−Ab) :MATH]
(1)
where A[PAEs], A[c], and A[b] represent the average absorbances of the
PAEs group, the control group, and the blank (culture medium without
FBS), respectively.
70% Partial Hepatectomy (PH) of C57 Mice
The present study was approved by the Medical Ethics Committee of
Kunming Medical University (Kunming, Yunnan Province, China). All
experimental procedures were performed in accordance with protocols
approved by the Institutional Animal Care and Utilization Committee.
Healthy 8–14-week male C57 mice weighing 20–24 g were obtained from the
Experimental Animal Center of Kunming Medical University. All
experimental mice were maintained under standard general anesthesia
with intraperitoneal (IP) 3% (v/v) chloral hydrate. The dosage was 1.2
ml/100 g BW. A midline laparotomy was performed and the left lateral
and left and right median lobes were ligated and resected. Here, 70% of
the mouse liver was surgically removed under sterile conditions
([98]Nevzorova et al., 2015). The mortality rate was < 10%. The main
cause of death was bleeding caused by ligation line falling off after
70% PH.
PAEs Administration
To evaluate the effect of PAEs on liver regeneration, male C57 mice
were randomly divided into the nonspecific control (NC), normal saline
(NS), and low- and high-dose PAEs groups (n = 10 per group).
The PAEs oral dose of adult was no more than 18 g/d in the patent
([99]Li and Hu, 2003) involving Ganlong capsule, a medicine for the
treatment of chronic hepatitis B clinically in China. According to the
body surface area (BSA) normalization method, a human equivalent dose
(HED) was calculated as follows ([100]Reagan-Shaw et al., 2008):
[MATH: HED (mg/kg)=Animal dose (mg/kg)×Ani
mal K
mHuman
Km :MATH]
(2)
where the K[m] factor is 3 for a mouse and 37 for a human. The dose of
mice was no more than about 3.7 g/kg/d because oral dose of 60 kg adult
was no more than 18 g/d. In the present liver regeneration experiment
and in reference to previous studies by others ([101]Zhang et al.,
2013; [102]Li, 2016; [103]Li et al., 2016; [104]Chang et al., 2017;
[105]Tang et al., 2018), gavage administration was adopted, in which
low- and high-dose PAEs groups were set as PAEs400 (400 mg/kg/d) and
PAEs800 (800 mg/kg/d), respectively.
By gavage, the NS mice were administered with normal saline and the
PAEs mice were administered with 400 or 800 mg/kg/d for 10 days. At 2 h
after the 10th gavage, 70% PH was performed on the NS and PAEs mice. At
48 h after 70% PH, all mice were euthanized by CO[2] inhalation. Liver
tissue samples of all mice including the NC were collected immediately.
Hepatic Regeneration Rate (HRR) Measurement
After 70% PH, the hepatic regeneration rate (HRR) of the residual liver
including the right and caudate lobes was calculated as follows:
[MATH: HRR=[Wc−(Wa−Wb)]/[Wa−Wb]×100 :MATH]
(3)
[MATH:
Wa=Wb<
/mi>/70%
:MATH]
(4)
where W[a] is the calculated initial weight of the whole mouse liver at
the start of the 70% PH, and W[b] and W[c] are the actual weights of
the surgically excised liver tissue and the residual liver tissue at
the time of death, respectively. This original weight was calculated as
a fixed proportion of the liver weight according to [106]Y. Nevzorova
et al. (2015) and [107]M. Meier et al. (2016).
Immunohistochemical (IHC) Staining
The right lobe was split into two parts. One section was immediately
frozen in liquid nitrogen for later sequencing while the other part was
fixed in 10% (v/v) neutral formaldehyde for immunohistochemical (IHC)
staining. The segments were fixed overnight, embedded in paraffin, and
cut into 4-µm-thick sections (Leica RM2235). Hepatocyte proliferation
was estimated by IHC staining for the nuclear antigen Ki-67. This
antigen is preferentially expressed during all active cell cycle phases
(G1, S, G2, and M) but not in G0 ([108]Scholzen and Gerdes, 2000).
Anti-Ki-67 Rabbit pAb (WL01384a) was provided by Wanleibio Co. Ltd.,
Shenyang, China. Immunohistochemical staining was performed using the
following protocol included with the kit: primary antibody: 1:100, 4°C,
overnight; secondary antibody-biotin: 1:150, 37°C, 1 h; and
streptavidin-HRP: 1:200, 37°C, 30 min. The Ki-67-positive hepatocytes
(N[P]) and the total hepatocytes (N[T]) were counted in random fields
at ×400 magnification. The positive rate (%) of Ki-67 expression was
used to enumerate the proliferating hepatocytes in the IHC staining
sections and was calculated as follows:
[MATH:
Positiv
e rate
(%)=NP/NT×1
00 :MATH]
(5)
RNA Sample Preparation, cDNA Library Preparation, and Illumina Sequencing
Nine samples per group were randomly selected. Three samples were
randomly mixed and three mixed samples were obtained per group. Total
RNA was extracted from each mixed sample with TRIzol reagent
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. Twelve DGE libraries (NC, NS, PAEs400, and PAEs800) were
processed in an Illumina gene expression sample prep kit (Illumina, San
Diego, CA, USA). After cDNA library quality and quantity control,
Illumina sequencing was performed in an Illumina HiSeq 2000
(Mus_musculus) at the Beijing Genomics Institute (BGI), Shenzhen,
China). The SRA data have been uploaded to NCBI (BioProject:
PRJNA635249).
Mapping DEGs to the Mus_Musculus Genome
Raw reads were prepared as follows to create clean reads ([109]Cock et
al., 2010). Raw reads with adapters and unknown bases (>5%) were
removed. Low-quality reads with quality values < 10 for >20% of their
bases were filtered out. Clean high-quality tags were mapped to the
reference genome with HISAT ([110]Kim et al., 2015) and to the
reference sequences with Bowtie2 ([111]Langmead and Salzberg, 2012).
Based on the mapping results, the RSEM analysis ([112]Li and Dewey,
2011) was executed to quantify the gene expression level and obtain a
read count of each gene in each sample. The gene expression levels were
calculated by the fragments per kilobase per million reads (FPKM)
method ([113]Mortazavi et al., 2008).
Functional Analysis of Differentially Expressed Genes (DEGs)
A differential expression analysis between the treatment and control
was implemented using a rigorous algorithm. The threshold P-value was
determined by the false discovery rate (FDR) method in multiple tests
([114]Wang et al., 2010). FDR < 0.001 and absolute value of the log2
ratio ≥ 1 were set as the detection thresholds for genes with
significant differential expression. To identify significantly enriched
terms, the Mus_musculus (mm10) genome was used as the background to
identify GO terms enriched within the DEG dataset via a hypergeometric
test using a corrected P-value (≤ 0.05) as a threshold. A Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
was performed to identify significantly enriched pathways within the
DEG datasets. It was compared with the genome database via a
hypergeometric test using a corrected P-value (≤ 0.05) as a threshold.
Data and Statistical Analysis
The data were compiled in a spreadsheet in MS Office Excel 2010.
Statistical analyses were performed by running t-tests on independent
samples to identify significant differences. P < 0.05 was considered
statistically significant.
Results
HPLC Quantitative Analysis
The amounts of six physiological small molecules, i.e., cytosine,
uracil, cytidine, uridine, inosine, and guanosine, in PAEs were
determined by HPLC. These nucleosides and nucleobases were deemed to be
the potential active components in PAEs in the study of intestinal
barrier improvement and anti-inflammation effects ([115]Ma et al.,
2018). As shown in [116]Figure S1A, these six compounds in mixed
standard substances were analyzed, with a satisfied degree of
separation and methodological investigation being obtained.
[117]Figures S1B, C provide clear evidence that the concentrations of
these six compounds in PAEs sample could be determined. Accordingly,
based on the external standard method, the amounts of these six
compounds, i.e., cytosine, uracil, cytidine, uridine, inosine, and
guanosine, in PAEs were calculated and the results were 5.91, 6.12,
7.63, 5.97, 3.26, and 1.89 mg/g, respectively. In addition, two unknown
substances were found in our study.
Hepatocyte Proliferation In Vitro and In Vivo
In vitro, PAEs at low dose (1 mg/ml) was found to accelerate moderate
L02 cell (in Gibco RPMI 1640 Medium without HEPES and supplemented with
10% FBS) proliferation. On the other hand, virtually all cells were
killed by PAEs at high doses (25 or 50mg/ml). The result was submitted
as supplementary material ([118]Figure S2). The CCK-8 assay showed that
different PAEs concentrations (0.1, 0.5, or 1 mg/ml) accelerated L02
cell proliferation under serum starvation conditions ([119]Figures 1A,
B).
Figure 1.
[120]Figure 1
[121]Open in a new tab
PAEs efficacy in hepatocyte proliferation both in vitro and in vivo
relative to control. (A) Representative images of CCK-8 color reactions
in 96-well plates at 48 and 72 h after treatment with different PAEs
concentrations. (B) Differences in L02 cell proliferation ratios
between PAEs treatment group (0.5 mg/ml) and NC group were significant
at 48 and 72 h according to Student’s t-test (*P < 0.05). (C) Ki-67
expression levels in residual liver cell nuclei and cytoplasm at 48 h
after 70% PH. (E) Representative images of residual liver at 48 h after
70% PH. Each group included nine mice. (D, F) Differences between PAEs
and NS groups were significant according to Student’s t-test (*P <
0.05; **P < 0.01).
According to the report of [122]Wang et al. (2011), abnormal
manifestations of the test animals were not observed during the acute
toxicity experiment of PAEs. The estimated LD50 was >2000 mg/kg PAEs in
normal ICR mice. According to the report of [123]Zhou (2008), LD50 was
> 10 g/kg TCM powder of Periplaneta americana in normal female and male
KM mice, and the toxicity grade of Periplaneta Americana was
practically non-toxic. In our study, abnormal manifestations were not
observed in C57 mice who were administered PAEs with 400 or 800 mg/kg.
By immunohistochemical staining, Ki-67 was localized in the hepatocyte
nuclei of the PAEs800 group. All postoperative groups presented with
higher Ki-67 expression than the NC group ([124]Figure 1C). The PAEs800
group had a significantly higher Ki-67 expression than that in the NS
(P < 0.05) and PAEs400 (P < 0.01) groups 48 h after 70% PH ([125]Figure
1D).
Hepatic Regeneration Rate (HRR)
The HRR was significantly higher in the PAEs groups than the NS group
48 h after 70% PH (P < 0.05 or P < 0.01). However, the difference in
HRR between the PAEs400 and PAEs800 groups ([126]Figures 1E, F) was not
statistically significant (P > 0.05).
Quantitative Identification of Differentially Expressed Genes (DEGs) 48 h
After 70% PH
Analysis of the gene expression spectrum detected 11,986 transcripts
including 9,266 coding and 2,720 noncoding transcripts. The expression
levels of various genes increased or decreased by ≥ 2× (q-value <
0.001) at 48 h after 70% PH ([127]Figure 2A). The PAEs groups presented
with significantly fewer DEGs but more downregulated genes than the NS
group.
Figure 2.
[128]Figure 2
[129]Open in a new tab
Quantitative identification of differentially expressed genes (DEGs)
48 h after 70% PH. (A) Number of DEGs increased or decreased by ≥ 2×
(q-value < 0.001) in various comparison groups. (B) Venn diagram of
DEGs between PAEs400/NS and PAEs800/NS. (C) Numbers of known DEGs in
various elements of Venn diagram.
A Venn diagram analysis identified 1,182 DEGs that were commonly
expressed among the PAEs and NS groups. Moreover, 408 and 1,077 DEGs
were specifically expressed in the PAEs400 and PAEs800 groups,
respectively ([130]Figure 2B). However, as we focused on the possible
effects of the PAEs after 70% PH, we concentrated on the intersection
of the 1,182 DEGs. A sequence analysis showed 1,092 reported and 90
unreported genes at the intersection. Of the former, 1,057 were
downregulated and only 35 were upregulated in the PAEs400 group while
1,055 reported genes were downregulated and only 37 reported genes were
upregulated in the PAEs800 group ([131]Figure 2C).
Bioinformatics Analysis of Differentially Expressed Genes (DEGs)
Gene Ontology (GO) was used to annotate and enrich the DEGs and a Kyoto
Encyclopedia of Genes and Genomes (KEGG) analysis was used to annotate,
enrich, and analyze the signaling pathways. The KEGG pathway analysis
functionally classified the 1,092 reported genes expressed in both the
PAEs400 and PAEs800 treatment groups. They were categorized by the
online BGI according to cellular processes, environmental information
processing, genetic information processing, human diseases, metabolism,
and organismal systems. [132]Figure 3A shows that 153 known genes were
separated into the cellular process category. Except for Pdgfrl (kinase
insert domain protein receptor), the expression levels of 152 elements
in the NS and PAEs groups were significantly higher than those of the
NC group. However, the PAEs groups displayed intermediate expression
levels. To facilitate the visualization and interpretation of gene
expression from these data, we used GeneMaths LOG10(FPKM+1) to rank the
DEGs according to their expression patterns and display them in concise
and intelligible graphical format ([133]Figure 3B). A KEGG pathway
enrichment analysis identified the enriched pathways by a two-tailed
Fisher’s exact test and assessed the enrichment of the 153 DEGs against
all identified elements. Enrichment level 2 was restricted to “Cell
Growth and Death” and “Signal Transduction” ([134]Figure 3C). Pathways
with corrected P-values ≤ 0.05 were considered statistically
significant. The KEGG pathway enrichment analysis indicated that 81
DEGs participated in the cell cycle, apoptosis, and necroptosis
processes and/or were involved in the signaling pathways related to
cell growth or death (PI3K-Akt, MAPK, Apelin, Wnt, FoxO, mTOR, Ras,
VEGF, ErbB, Hippo, and AMPK) ([135]Figures 3D, E, [136]Table 1). Of the
81 DEGs, 34 participated in the liver regeneration process via ≥ 2
signaling pathways. Akt3 was implicated in all of the aforementioned
signaling pathways except for Wnt and Hippo.
Figure 3.
[137]Figure 3
[138]Open in a new tab
KEGG analysis of DEGs. (A) Annotation and classification by KEGG
analysis. (B) Expression of 153 DEGs in the “Cellular Processes”
category. (C) Top 20 pathways. Enrichment of 153 DEGs by KEGG pathway
enrichment analysis against all identified elements. Level 2 restricted
to “Cell Growth and Death” and “Signal Transduction”. Among them, 26
DEGs enriched in PI3K-AKT signaling pathway, and 21 in MAPK signaling
pathway. Only seven DEGs enriched in VEGF signaling pathway, but rich
ratio was the highest. This analysis result indicated an extremely
complex molecular mechanism in the process by which PEAs can promote
liver regeneration. (D) DEG expression levels in pathways. Legend shown
in [139]Table 1. (E) PAEs accelerate hepatocyte proliferation and
promote liver regeneration via complex networks. DEG-pathway network
constructed by linking 153 DEGs (blue triangles), associated signaling
pathways (yellow round rectangles), and cellular processes (green
ellipses).
Table 1.
Genes altered by ≥ 2× between the PAEs intervention and NS groups
according to the enrichment results.
GeneSymbol Definition Graphic Symbol in [140]Figure 3D Number Map ID
//Map name
1 Adcy2 adenylate cyclase 2 [EC:4.6.1.1] graphic file with name
fphar-11-01174-g005.jpg 26 ko04151
//PI3K-Akt signaling pathway
2 Adcy3 adenylate cyclase 3 [EC:4.6.1.1]
3 Akt3 RAC serine/threonine-protein kinase [EC:2.7.11.1]
4 Bmpr1b bone morphogenetic protein receptor type-1B [EC:2.7.11.30]
5 Bub1 checkpoint serine/threonine-protein kinase [EC:2.7.11.1]
6 Camk2a calcium/calmodulin-dependent protein kinase (CaM kinase) II
[EC:2.7.11.17]
7 Ccnb1 G2/mitotic-specific cyclin-B1
8 Cd36 CD36 antigen
9 Cdc14a cell division cycle 14 [EC:3.1.3.16 3.1.3.48]
10 Cdc25b M-phase inducer phosphatase 2 [EC:3.1.3.48]
11 Cdc25c M-phase inducer phosphatase 3 [EC:3.1.3.48] graphic file with
name fphar-11-01174-g006.jpg 20 ko04010
//MAPK signaling pathway
12 Cidea DNA fragmentation factor, 45 kD, alpha subunit
13 Cidec DNA fragmentation factor, 45 kD, alpha subunit
14 Ctsk cathepsin K [EC:3.4.22.38]
15 Dbf4 activator of S phase kinase
16 Ddit4l DNA-damage-inducible transcript 4
17 Dpt Notch 1
18 Egf epidermal growth factor
19 Fgf13 fibroblast growth factor graphic file with name
fphar-11-01174-g007.jpg 13 ko04371
//Apelin signaling pathway
20 Fgf7 fibroblast growth factor
21 Flnc filamin
22 Flt1 FMS-like tyrosine kinase 1 [EC:2.7.10.1]
23 Fos proto-oncogene protein c-fos
24 Fzd2 frizzled 2
25 Fzd9 frizzled 9/10
26 Fzd10 frizzled 9/10 graphic file with name fphar-11-01174-g008.jpg
12 ko04310
//Wnt signaling pathway
27 Grm1 metabotropic glutamate receptor 1
28 Hspa1l heat shock 70 kDa protein 1/2/6/8
29 Igf1r insulin-like growth factor 1 receptor [EC:2.7.10.1]
30 Itga7 integrin alpha 7
31 Itga11 integrin alpha 11
32 Itgb6 integrin beta 6
33 Itgbl1 tenascin graphic file with name fphar-11-01174-g009.jpg 11
ko04068
//FoxO signaling pathway
34 Lama2 laminin, alpha 1/2
35 Lama4 laminin, alpha 4
36 Lamb2 laminin, beta 2
37 Lrrc2 erbb2-interacting protein
38 Map3k7cl mitogen-activated protein kinase kinase kinase 7
[EC:2.7.11.25]
39 Mapk12 p38 MAP kinase [EC:2.7.11.24] graphic file with name
fphar-11-01174-g010.jpg 12 ko04150
//mTOR signaling pathway
40 Myl2 myosin regulatory light chain 2
41 Mylk2 myosin-light-chain kinase [EC:2.7.11.18]
42 Mylk4 myosin-light-chain kinase [EC:2.7.11.18]
43 Napb alpha-soluble NSF attachment protein
44 Nexn myosin-light-chain kinase [EC:2.7.11.18]
45 Nfatc2 nuclear factor of activated T-cells, cytoplasmic 2 graphic
file with name fphar-11-01174-g011.jpg 15 ko04014
//Ras signaling pathway
46 Nfatc4 nuclear factor of activated T-cells, cytoplasmic 4
47 Nlrc3 NACHT, LRR, and PYD domains-containing protein 3
48 Nos1 nitric-oxide synthase, brain [EC:1.14.13.39]
49 Pdgfb platelet-derived growth factor subunit B
50 Pdgfrl kinase insert domain protein receptor [EC:2.7.10.1]
51 Peg10 poly [ADP-ribose] polymerase [EC:2.4.2.30]
52 Pgf placenta growth factor graphic file with name
fphar-11-01174-g012.jpg 7 ko04370
//VEGF signaling pathway
53 Pik3r6 phosphoinositide-3-kinase regulatory subunit alpha/beta/delta
54 Pla2g4e cytosolic phospholipase A2 [EC:3.1.1.4]
55 Plcb4 phosphatidylinositol phospholipase C, beta [EC:3.1.4.11]
56 Pm20d2 translation initiation factor 2 subunit 3
57 Prkab2 5’-AMP-activated protein kinase, regulatory beta subunit
graphic file with name fphar-11-01174-g013.jpg 8 ko04012
//ErbB signaling pathway
58 Prkag3 5’-AMP-activated protein kinase, regulatory gamma subunit
59 Pvalb atrophin-1 interacting protein 1
60 Pygm glycogen phosphorylase [EC:2.4.1.1]
61 Pygo1 SHC-transforming protein 1
62 Rasgrf1 Ras-specific guanine nucleotide- releasing factor 1 graphic
file with name fphar-11-01174-g014.jpg 9 ko04390
//Hippo signaling pathway
63 Rps6ka2 ribosomal protein S6 kinase alpha-1/2/3/6 [EC:2.7.11.1]
64 Rragd Ras-related GTP-binding protein C/D
65 Shc2 SHC-transforming protein 2
66 Slc25a31 solute carrier family 25 (mitochondrial adenine nucleotide
translocator), member 4/5/6/31
67 Slc25a4 solute carrier family 25 (mitochondrial adenine nucleotide
translocator), member 4/5/6/31 graphic file with name
fphar-11-01174-g015.jpg 7 ko04152
//AMPK signaling pathway
68 Stac3 signal transducing adaptor molecule
69 Tgfb3 transforming growth factor beta-3
70 Thbs2 thrombospondin 2/3/4/5 graphic file with name
fphar-11-01174-g016.jpg 8 ko04110
//Cell cycle
71 Thbs3 thrombospondin 2/3/4/5
72 Thbs4 thrombospondin 2/3/4/5
73 Tnxb tenascin
74 Ttc9 peptidyl-prolyl isomerase D [EC:5.2.1.8]
75 Ttk serine/threonine-protein kinase TTK/MPS1 [EC:2.7.12.1] graphic
file with name fphar-11-01174-g017.jpg 9 ko04210
//Apoptosis
76 Vit collagen, type VI, alpha
77 Wnt9a wingless-type MMTV integration site family, member 9
78 Wnt11 wingless-type MMTV integration site family, member 11
79 Xirp1 methyl CpG binding protein 2 graphic file with name
fphar-11-01174-g018.jpg 10 ko04217
//Necroptosis
80 Zcchc5 poly [ADP-ribose] polymerase [EC:2.4.2.30]
81 3632451
O06Rik high mobility group protein B1
[141]Open in a new tab
Discussion
Periplaneta americana (PA) is a common source of animal medicine that
has a long history of use in TCM for the treatment of wounds and burns
([142]Alves and Alves, 2011; [143]Zhu et al., 2018). Over the past two
decades, PAEs have been tested for the promotion of gastric and
duodenal ulcer healing ([144]Lu et al., 2019), treatment of hepatic
fibrosis ([145]Li D. et al., 2018), inhibition of tumor growth
([146]Zhao et al., 2017), stimulation of skin wound healing ([147]Yang
et al., 2015; [148]Song et al., 2017), and induction of skin fibroblast
migration ([149]Li L. J. et al., 2019). The active constituents in PAEs
with wound healing efficacy have been screened ([150]Li Q. J. et al.,
2018). The abovementioned studies, however, did not provide evidence
for the healing potential of PAEs. To the best of our knowledge, the
present study is the first to report on the liver regeneration efficacy
of PAEs involving the synergy of multiple signaling pathways.
The present results have shown that PAEs promote hepatocyte
proliferation both in vitro and in vivo and accelerate mouse liver
regeneration after 70% partial hepatectomy. That PAEs can accelerate
liver regeneration by affecting hepatocyte proliferation is evident by
the increase in incidence of Ki-67 immunohistochemical stained
hepatocyte nuclei. The DEGs bioinformatics analysis revealed that PAEs
influence the expression levels of different genes after PH. On closer
analysis, some of the DEGs participated in cellular processes and
signaling pathways associated with cell growth or death. The cellular
processes included cell cycle, apoptosis, and necroptosis. The
signaling pathways included PI3K-Akt, MAPK, Apelin, Wnt, FoxO, mTOR,
Ras, VEGF, ErbB, Hippo, and AMPK ([151]Figure 3E).
The murine 70% PH model is not associated with tissue injury or
inflammation because the liver lobes are removed intact. Of note, the
expression levels of the DEGs related to cell death were lower in the
PAEs groups than the NS group. It stands to reason therefore that PAEs
may protect hepatocytes by inhibiting cell death after liver injury.
They may promote post-PH liver regeneration by inducing the
proliferation of existing mature hepatocytes without activating the
progenitor cells ([152]Mao et al., 2014). Though the PAEs accelerated
liver regeneration, the expression levels of the DEGs related to cell
growth were lower in the PAEs group than the NS group. It is suggested
that they may be concerned more with the process of liver regeneration
after 70% PH.
After 70% PH, most of the hepatocytes in the residual lobes undergo one
or two proliferative processes ([153]Michalopoulos and DeFrances,
2005). In rats, the first peak in hepatocyte DNA replication occurs
after ~24 h. A second smaller peak is observed between 36–48 h and
there is a maximum rate of cell division at 36 h after PH. In contrast,
there is species variation in peak DNA replication following PH; in
mice, the peak was 12‑16 h later compared to rats ([154]Mao et al.,
2014). [155]Nevzorova et al. (2015) suggested that for studies on the
progression phase, the mice should be sacrificed at 36 (S-phase onset),
40, 48 (peak of DNA synthesis), and 60 h (termination of cell cycle
activity) after PH. Here, we collected the liver samples at 48 h after
liver resection. A peak in DNA replication and significant upregulation
of the DEGs related to cell growth occurred in NS at this time point.
There were comparatively higher HRR and relatively lower expression
levels of the DEGs associated with cell cycle and proliferation in the
PAEs groups. Hence, PAEs accelerate the initial hepatocyte
proliferation process and hasten the arrival of the cells to the second
proliferative event. PAEs improved L02 cell proliferation in vitro;
thus, they possess liver regeneration potential.
Previous studies had explored the effects of single factors and
pathways on liver regeneration. However, more recent reports
demonstrated that numerous signaling pathways are involved in this
process and their interactions are complex. [156]Gupta et al. (2019)
found that ALR (augmenter of liver regeneration, a protein that was
identified to specifically support liver regeneration) induced
miRNA-26a expression, upregulated the p-Akt/cyclin D1 pathway, and
promoted hepatic cell proliferation. [157]Lai et al. (2015) found that
EGR-1 (early growth response 1 gene)-induced GGPPS plays an important
role in post-PH liver regeneration via RAS/MAPK signaling. [158]Ye et
al. (2018) reported that Diwu Yanggan capsules can improve the liver
regeneration microenvironment by regulating the Ras/Raf/Mek/Erk
signaling pathway and regeneration-related factors. It has been
reported in knockout mice or use of inhibitors that single-pathway
disruption does not block but causes delay in regeneration. This
suggests that a complex pathway network is vital for optimal liver
regeneration and the generation of adequate hepatic mass
([159]Michalopoulos, 2007; [160]Mao et al., 2014). In an IL-6^-/- mouse
model, liver regeneration was delayed ([161]Cressman et al., 1996).
Gene expression and hepatocyte proliferation can be corrected with a
preoperative IL-6 injection. After PH, Tnfr1^-/- mice presented with
multiple liver regeneration defects whereas Tnf^-/- mice underwent
normal liver regeneration. Thus, other ligands may bind to TNFr1
([162]Yamada et al., 1997; [163]Hayashi et al., 2005). In practice,
pathophysiological processes are seldom induced by isolated changes in
single molecular effectors. Indeed, physiological and pathological
modulations are nearly always the result of the complex interactions of
multiple signaling pathways.
PAEs are prepared from Periplaneta americana and contain various
biologically active ingredients including polysaccharides, peptides,
nucleosides, polyols, steroids, terpenes, alkaloids, flavonoids, and
isocoumarins. The effective constituents may be attributed to
polysaccharides, peptides, nucleosides, among others ([164]Lv N. et
al., 2017; [165]Li et al., 2020). Therefore, PAEs may have multiple
molecular targets and biochemical properties. In our study, the main
ingredients of PAEs are sticky sugar amino acid, based on the patent
([166]Li and Hu, 2003) involving Ganlong capsule; however, they are
still not considered a natural pure compound drug. The efficacy of PAEs
at accelerating liver regeneration after 70% PH was mediated by the
synergistic effect of numerous targets and pathways associated with
cell growth and death. These pathways can form complex interconnecting
networks. In fact, certain DEGs participated in ≥ 2 pathways
([167]Figure 4). This discovery corroborates the synergy of action
theory of multicomponent therapeutics TCM postulated by [168]Lv C. et
al. (2017). In the latter, the gene expression profiles of four active
constituents in Danshen were analyzed. In this connection, molecular
analysis of the expressions of the key signaling members by qPCR and
western-blotting using L02 cells culture in vitro along with animal
experimentation is clearly desirable. This would help unravel the major
pathways and explore the mechanism that guide the enhancement of liver
regeneration by the active ingredients of PAEs.
Figure 4.
Figure 4
[169]Open in a new tab
PAEs accelerate liver regeneration via a complex network after 70% PH
in mice.
Though the liver can recover following hepatectomy, liver failure will
nonetheless occur if the tissue loss is very extensive. This response
is known as the Small-for-Size-Syndrome (SFSS). [170]Tschuor et al.
(2016) reported that deficient liver regeneration is the principal
cause of SFSS. Successful regeneration may lead to recovery from acute
liver failure. However, no regenerative strategies for existing acute
liver failure have been established ([171]Kojima et al., 2020). It is
unequivocal from the present results that PAEs administered prior to
surgery could enhance the postoperative liver regeneration capacity.
Hence, PAEs might have clinical potential for the treatment of
post-hepatectomy liver failure.
The previous study in mice showed that the toxicity grade of
Periplaneta Americana was practically non-toxic ([172]Zhou, 2008). In
our liver regeneration experiment, the mortality rate was <10%. The
main cause of death was bleeding caused by ligation line falling off
after 70% PH. Abnormal manifestations were not observed in mice who
were administered PAEs. Based on the BSA normalization method
([173]Reagan-Shaw et al., 2008), human equivalent dose of PAEs (about
3.9 g/d), which was converted from the high dose (800 mg/kg) of mice,
was less than 1/4 of the safe dose (18 g/d) ([174]Li and Hu, 2003). In
the experiment, the doses of PAEs used were safe and effective. This
indicates that PAEs are safe and indeed have the potential for clinical
application.
Conclusions
To the best of our knowledge, the present study is the first to
demonstrate that preoperative PAEs gavage increased postoperative liver
regeneration capacity in mice. In the present liver regeneration
experiment, the doses of PAEs used were safe and effective. This
indicates that PAEs are safe and indeed have the potential for clinical
application. More importantly, we show here that PAEs improved liver
regeneration via a complex network of targets and signaling pathways.
Though the present therapeutic approach using PAEs has practical merit,
significance, and potential, the molecular targets, active
constituents, and modes of action have not yet been elucidated. This
will certainly be the scope of our future study.
Data Availability Statement
The sequencing data has been deposited into the Sequence Read Archive
(accession: PRJNA635249).
Ethics Statement
The animal study was reviewed and approved by Medical Ethics Committee
of Kunming Medical University.
Author Contributions
XZ, XW, and KW designed the project and helped analyze the data and
finalize the manuscript. YZ, MZ, and DZ performed most of the
experiments, participated in discussions and data analysis, and
prepared the first draft of the manuscript. YR and LS helped analyze
the main data and revise the manuscript. ZM, JZ, and CX conducted
certain experiments and maintained the experimental mice. ZY, ZQ, and
RX performed tissue paraffin embedding, sectioning, and
immunohistochemical (IHC) staining. SL, QK, HZ, SZ, and LL assisted
with animal surgery and tissue sample excision.
Funding
The present study was supported in part by the National Natural
Sciences Foundation of China (Grant No. 81760430) and the Yunnan
Provincial Department of Education (Grants No. 2020J0137 and 2018Y040).
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