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Korean J Physiol Pharmacol 2023;27(4):345-356
https://doi.org/10.4196/kjpp.2023.27.4.345
Author contributions: H.H. and Y.L. performed the experiments. G.L. per-
formed data analysis. J.H. made corrections to the manuscript. F.O. and J.Y.
supervised and coordinated the study. Y.L. and Z.W. wrote the manuscript.
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Copyright © Korean J Physiol Pharmacol, pISSN 1226-4512, eISSN 2093-3827
Original Article
Hydrogen sulfide ameliorates abdominal aorta coarctation-
induced myocardial fibrosis by inhibiting pyroptosis through
regulating eukaryotic translation initiation factor 2αα
phosphorylation and activating PI3K/AKT1 pathway
Yaling Li1,#, Zhixiong Wu1,#, Jiangping Hu1,#, Gongli Liu1, Hongming Hu2, Fan Ouyang1,*, and Jun Yang2,*
1Department of Cardiology, Zhuzhou Central Hospital, Zhuzhou 412000, 2Department of Cardiology, The First Affiliated Hospital of University of South China,
Hengyang 421001, China
ARTICLE INFO
Received August 18, 2022
Revised October 31, 2022
Accepted November 1, 2022
*Correspondence
Jun Yang
E-mail: yangjunincn@163.com
Fan Ouyang
E-mail: 1641261977@163.com
Key Words
Fibrosis
Hydrogen sulfide
Phosphatidylinositol 3-Kinase
Phosphorylation
Pyroptosis
#These authors contributed equally to
this work.
ABSTRACT This study aimed to assess the effects of exogenous hydrogen sulfide (H2S)
on abdominal aorta coarctation (AAC) induced myocardial fibrosis (MF) and autopha-
gy in rats. Forty-four Sprague–Dawley rats were randomly divided into control group,
AAC group, AAC + H2S group, and H2S control group. After a model of rats with AAC
was built surgically, AAC + H2S group and H2S group were injected intraperitoneally
with H2S (100 µmol/kg) daily. The rats in the control group and the AAC group were
injected with the same amount of PBS. We observed that H2S can improve left ven-
tricular function and the deposition of myocardial collagen fibers, inhibit pyroptosis,
down-regulate the expression of P-eif2α in myocardial tissue, and inhibit cell au-
tophagy by activating the phosphatidylinositol 3-kinase (PI3K)/AKT1 signaling path-
way (p < 0.05). In addition, angiotensin II (1 µM) H9c2 cardiomyocytes were injured
in vitro
experiments, and it was also observed that pyroptosis was inhibited after H2S
(400 µmol/kg) intervention, the expression of P-eif2α in cardiomyocytes was signifi-
cantly down-regulated, and the PI3K/AKT1 signaling pathway was activated at the
same time. Therefore, increasing the expression of P-eif2α reverses the activation of
the PI3K/AKT1 signaling pathway by H2S. In conclusion, these findings suggest that
exogenous H2S can ameliorate MF in rats with AAC by inhibiting pyroptosis, and the
mechanism may be associated with inhibiting the phosphorylation of eif2α and acti-
vating the PI3K/AKT1 signaling pathway to inhibit excessive cell autophagy.
INTRODUCTION
As people’s lifestyle has been changing, the incidence rate of
hypertension rises year by year. Statistics suggests that nearly
50% of deaths from cardiovascular disease are associated with
hypertension. Myocardial injury caused by pressure overload that
is accompanied by hypertension shows a close association with
cardiovascular death. Cardiomyocyte hypertrophy and apop-
tosis may occur under the pressure overload, and myocardium
is gradually replaced by fibrosis tissues (e.g., type I and type III
fibrous collagen, elastin, fibronectin, as well as proteoglycan) [1].
Myocardial fibrosis (MF) acts as a vital link during the myocar-
dial remodeling process in hypertension; it is also reported as the
major cause of cardiac failure. Nevertheless, the specific regula-
tory mechanism of MF under the pressure overload remains
unclear. Existing studies have shown that pyroptosis mediates
the formation of “pyroptosis-inflammatory response-fibrosis”
axis-like pathological changes in lung, liver, kidney and other
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Li Y et al
tissue fibrotic diseases, expands the inflammatory cascade, and
aggravates the course of tissue fibrosis [2-4]. Recent studies have
also shown that cardiomyocyte pyroptosis is closely bound up
with the occurrence of cardiovascular diseases, especially MF [5].
Therefore, elaborating the mechanisms regulating cell pyroptosis
and MF will open a new target for the treatment of hypertensive
cardiomyopathy.
Autophagy refers to a physiological process, through which
sirtuin is degraded, and excessive or injured organelles are elimi-
nated following the lysosomal degradation. Some scholars have
demonstrated that autophagy is likely to be activated in myocar-
dial or hypoxic-ischemic injury, and moderate autophagy refers
to a process of self-salvation that helps cells adapt to inflamma-
tion, anoxia, reperfusion injury, as well as other stress responses
[6,7]. Nevertheless, through excessive autophagy, cells will be
damaged, so the apoptosis or death of cells will be promoted [8].
Some existing studies have suggested that phosphatidylinositol
3-kinase (PI3K)/AKT signaling pathway is another classic sig-
naling pathway related to the regulation of autophagy [9]. And
the previous research of the research group also showed that
activating the PI3K/AKT1 signaling pathway can improve MF
[10]. Nevertheless, MF under autophagy-induced stress overload
and the internal regulatory mechanism remain incompletely
studied. Recently, Li
et al
. [11] have shown that DHA can inhibit
pyroptosis by activating the PI3K/AKT1 pathway, which in turn
alleviates ischemia-reperfusion injury. Liu and Tie [12] have also
shown that the new target for the treatment of atherosclerosis
may be closely bound up with the activation of the PI3K/AKT1
pathway to inhibit pyroptosis. Studies have shown that one key
site of translational control is the phosphorylation of eukaryotic
translation initiation factor 2α (ei f2α), which is triggered by four
stress-response kinases, and therefore the expression of eif2α
phosphorylation tends to increase during disease [13]. And it is
gratifying that related studies have shown that eif2α phosphory-
lation is involved in PI3K signaling [14,15]. Therefore, we specu-
lated whether the improvement of abdominal aorta coarctation
(AAC)-induced MF by hydrogen sulfide (H2S) was related to the
activation of PI3K/AKT1 signaling pathway by eif2α phosphory-
lation to inhibitpyroptosis.
H2S, a novel gaseous signaling pathway, has been shown to have
intensive biological effects on the body. Besides, it can dilate blood
vessels as well as control autophagy, anti-inflammation and anti-
apoptosis [16]. However, whether the gaseous signaling molecule
H2S can enhance pressure overload-induced MF and its signal
transduction mechanism has been rarely discussed. Studies have
found that the model of rats with AAC can simulate MF caused by
hypertension under the pressure overload. Therefore, this study
intends to establish a rat model of MF induced by abdominal
aortic constriction and a model of H9c2 cardiomyocyte injury
induced by angiotensin II (Ang II, 1 µM) [17], observe the effect of
H2S on pressure overload-induced MF in rats, and discuss the ef-
fect of eif2α phosphorylation on PI3K/AKT1 signaling pathway.
METHODS
Chemicals and reagents
Sigma-Aldrich provided sodium hydrogen sulfide (NaHS)
(207683-19-0). Wuhan Boster Biological Technology, Ltd. Pro-
teinch provided the antibodies for ATG16L1 (19812-1-AP), Be-
clin1 (11306-1-AP), MMP1 (10371-2-AP), MMP13 (18165-1-AP),
TIMP1 (16644-1-AP), IL-6 (21865-1-AP), ICAM-1 (60299-1-Ig),
Caspase1 (22915-1-AP), Caspase3 (19677-1-AP), CTH (12217-
1-AP). Cell Signaling Technology provided Rabbit polyclonal
P-eif2α (Rabbit mAb#9722), anti-P-PI3K (Rabbit mAb#17366),
anti-P-AKT1 (Rabbit mAb#9018), polyclonal anti-GAPDH (Dan-
foss, Rabbit mAb #5174). Abcam provide IL-1β (ab254360), IL-
18 (ab191860), Cleaved-Caspase3 (ab52072), Beyotime Institute of
Biotechnology provided phenyl methyl sulfonyl fluoride (PMSF;
PB0425), Sigma-Aldrich provided Bicinchoninic Acid (BCA) Pro-
tein Assay kit (B9643), and Beyotime Institute of Biotechnology
provided Enhanced Chemiluminescence Reagent kit (P0018S),
SDS-PAGE Gel Preparation kit and PMSF (P0690).
Establishment of model
We maintained 44 male Sprague–Dawley (SD) rats (slacker
company) weighing 220 ± 30 g in a constant humidity environ-
ment at a steady temperature (24°C ± 3°C), under a 12 h-day-
night cycle and free access to water and feed, 44 experimental rats
were split into 4 groups in a random manner: normal rats admin-
istrated with H2S group (H2S group), AAC and administrated
with H2S group (AAC + H2S group), AAC-treated group (AAC
group), normal group (control group). Following skin prepara-
tion, the rats were anesthetized by intraperitoneal injection of
10% chloral hydrate (350 mg/kg; The rats showed no signs of
peritonitis, pain or discomfort). In the AAC + H2S group and the
AAC group, exposing the abdominal aorta above the renal artery,
carefully separate the abdominal aorta, fix a blunt needle with a
diameter of 7 mm around the abdominal aorta above the renal
artery, then ligate 5 mm above both renal arteries and quickly
withdraw the blunt needle, resulting in lumen stenosis. During
the whole procedure, the rectal temperature was maintained at
36.5°C–37.5°C with a heating lamp. Rats in the control group and
H2S group underwent the same procedure, whereas the abdomi-
nal aorta was not narrowed. All groups were administrated with
penicillin 100,000 units/animal for anti-infection for 3 days once
a week. AAC + H2S group and H2S group underwent intraperi-
toneal injection of NaHS (100 µmol/kg). The rats in the control
group and AAC group were daily administrated with PBS. After
4 weeks, they were weighed and anesthetized with chloral hydrate
(350 mg/kg, intraperitoneal anesthesia), the cervical spine was
dislocated, and the heart was removed and weighed, and then
incubated at –80°C for experiments and all the experiments were
approved by the University Committee on the Use and Care of
Hydrogen sulfide ameliorates abdominal aorta coarctation myocardial fibrosis
Korean J Physiol Pharmacol 2023;27(4):345-356www.kjpp.net
347
Animals of South China University (SYXK2020-0002).
H2S content assay
Myocardial H2S content was assayed by ELISA kit (Beyotime
Institute of Biotechnology). Then add 50 µl of diluted (1:1) stan-
dard to the reaction well, add 50 µl of the test sample to the reac-
tion well, and incubate at 37°C for 1 h. Remove the liquid in the
hole and wash three times. Add 80 µl of affinity streptin-HRP to
each well, gently shake and mix, incubate at 37°C for 30 min, and
wash three times. Add 50 µl each of substrate A and B to each
well, shake gently to mix, and incubate at 37°C for 10 min. Finally,
add 50 µl of stop solution quickly, and measure the result imme-
diately after adding the stop solution.
Masson’s staining
Take the left ventricle of rats was fixed in 10% neutral forma-
lin, paraffin embedded, sections were achieved 4 µm, rigorously
following the instructions Masson staining steps. Subsequently,
different fields of view at 40 × 10 magnification were selected, sec-
tions were placed under a microscope for observation.
Transmission electron microscope (TEM) observation
Four weeks later, the rats were killed in a humanitarian man-
ner, left ventricular apex rapidly small piece of tissue was taken
and placed in 2.5% glutaraldehyde at 4°C fixed 3 h, remove the
glutaraldehyde solution was immersed in PBS at 4°C fixed line
TEM inspection.
Western blotting assay
After taking each group of grinding myocardium cell lysate
and PMSF, harvested supernatant was quantified in a colori-
metrical manner BCA. After being extracted, we heated the
protein to 95°C (10 min) and then underwent denaturing and
glue, electrophoresis processes. Subsequently, it was placed to a
membrane, a hatched anti, Tris buffered saline Tween rinsed,
incubated secondary antibody (goat anti-rabbit, goat anti-mouse),
and lastly obtained by ECL (Beyotime, Institute of Biotechnol-
ogy). Using the Alpha Imager software (Alpha Imager Software)
for each target protein bands for gray scale scanning, respectively,
in comparison with GAPDH gradation value, after statistical
analysis using GraphPad Prism 5 (GraphPad Software).
Cell culture
With obtaining the H9c2 cell line from ATCC, the cells were
cultured in standard DMEM supplemented with 10% calf serum
(Gibco), and during the experiment, the CO2 concentration and
air concentration in the incubator were ensured to be 5% and
95%, respectively, and the temperature was humidified at 37°C.
Cells were randomly seeded onto 6-well dishes when them grew
to a certain density, and treated them with NaHS (400 µM; H2S
dononer) for 30 min before H9c2 cardiomyocyte injury was in-
duced using Ang II (1 µM) [17], and the cell lysate was collected
for analysis after 24 h.
Statistical analysis
At least three independent experiments were repeated (n = 3).
All values are expressed as the mean ± standard error. Student’s
t-test was performed with GraphPad Prism software to evaluate
statistical significance and one-way analysis of variance (ANO-
VA) was used to analyze comparisons among multiple groups.
But when there are two variables, we use two-way ANOVA.
When p < 0.05, it is considered to be statistically significant. The
data are statistically evaluated by variance analysis, and then
Prism is used for Tukey post-test of inter-group comparison.
RESULTS
Variations in rat bodyweight and heart weight/
bodyweight ratio
Four weeks later, 10, 10, 9, and 9 rats survived in the control
group, AAC group, AAC + H2S group, and H2S group, respec-
tively. Table 1 suggests that the mentioned groups showed no sta-
tistically significant differences in bodyweight, heart weight and
heart weight/bodyweight ratio (Table 1).
Table 1. Effects of H
2
S on body weight, heart weight and heart weight/body weight in rats
Groups Body weight (g) Heart weight (mg) Heart weight/body weight (mg/g)
Control 365.62 ± 8.23 966.20 ± 44.34 2.64 ± 0.28
AAC 370.89 ± 7.18 1,115.36 ± 67.12 3.01 ± 0.63
AAC + H2S 375.22 ± 10.43 1,040.69 ± 58.16 2.77 ± 0.86
H2S 361.02 ± 11.63 1,016.97 ± 65.98 2.81 ± 0.35
Values are expressed as means ± SD. H2S, hydrogen sulfide; AAC, abdominal aorta coarctation.
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Li Y et al
Results of rat blood pressure and heart color Doppler
ultrasound examination
The cardiac functions of rats were examined by heart color
Doppler ultrasound examination, while the arterial blood pres-
sure was ascertained by common carotid artery intubation for
each group. According to the results suggested, AAC group and
AAC + H2S group left ventricular ejection fraction and fractional
shortening not statistically significantly different from those of
the control group (p > 0.05). The arterial blood pressures of AAC
group and AAC + H2S group were elevated noticeably compared
with those of the control group (p < 0.05), and after receiving H2S
intervention, AAC + H2S group and AAC group were not statisti-
cally significantly different in arterial blood pressure (p > 0.05).
The above findings indicate that the AAC rat model under pres-
sure overload was successfully established, while the successfully
established model showed no significant loss of cardiac function
during the observation period and no anti-hypertensive effect of
the H2S donor on the AAC rats (Table 2).
Change in the level of myocardial endogenous H2S in
rats
To explore the in the level variation of myocardial endogenous
H2S in rats of each group, the H2S level in myocardial tissue in
rats of all groups was ascertained by ELISA. The results showed
that compared with the results of the control group, the H2S level
in myocardial tissue in AAC group was dropped apparently (p <
0.05), while that in myocardial tissue in AAC + H2S group rose
after exogenous H2S donor was added, which showed a statisti-
cally significant difference (p < 0.05); H2S group and the control
group were not statistically significantly different in H2S level in
myocardial tissue (p > 0.05) (Fig. 1).
The effect of H2S on pressure overload-induced MF
The deposition of cardiac interstitial collagen and interstitial
fibrosis was identified by Masson staining. The Fig. 2 suggests
that collagen fibers in AAC group were blue as observed under a
microscope, myofilaments were arranged in an irregular man-
ner, and collagen fibers increased more obviously than those of
the control group; the deposition of collagen in AAC + H2S group
decreased evidently in amount compared with that in the AAC
group; the deposition of collagen in H2S group remained nearly
unchanged in comparison with the results of the control group
(Fig. 2).
The effect of H2S on MMPs/TIMPs expression
Since MMPs/TIMPs dysregulation displays a close association
with the excessive deposition of collagen in myocardial intersti-
tium, the variation of the expression of MMPs/TIMPs was detect-
ed in this study by Western blotting assay. The result of Western
blotting assay suggested that the expression levels of MMP1 (p
= 0.0031), MMP13 (p = 0.0368) and TIMP1 (p = 0.0352) in AAC
group were significantly up-regulated compared with those of the
control group, whereas those of MMP1 (p = 0.0237), MMP13 (p
= 0.0286) and TIMP1 (p = 0.0383) in myocardial tissue in AAC
+ H2S group declined significantly compared with those of the
AAC group (p < 0.05), there were no statistically significant dif-
ferences in the expression levels of MMP1 (p = 0.4081), MMP13
(p = 0.0517) and TIMP1 (p = 0.1826) between H2S group and the
control group (Fig. 3).
Table 2. Effects of H
2
S on urine protein, blood pressure, left ventricular ejection fraction and fractional shortening in rats
Groups Urine protein (mg/L) Blood pressure (mmHg) Left ventricular ejection
fraction (%) Fractional shortening (%)
Control 7.72 ± 1.16 84.74 ± 7.88 76.66 ± 5.75 45.71 ± 6.39
AAC 6.86 ± 0.77 162.70 ± 11.35* 71.05 ± 6.27 38.78 ± 7.30
AAC + H2S 9.27 ± 0.54 155.31 ± 10.02* 79.94 ± 5.33 42.58 ± 3.95
H2S 10.02 ± 2.16 95.19 ± 9.86 81.42 ± 7.14 41.06 ± 5.77
Values are expressed as mean ± SD. H2S, hydrogen sulfide; AAC, abdominal aorta coarctation. *p < 0.05
vs
. control group.
Fig. 1. The contents hydrogen sulfide (H
2
S) of myocardial in ab-
dominal aorta coarctation (AAC)-induced myocardial fibrosis
treated with control, AAC, AAC + H
2
S, and H
2
S. Values are presented
as mean ± SD. *p < 0.05
vs
. control,
#
p < 0.05
vs
. AAC.
Hydrogen sulfide ameliorates abdominal aorta coarctation myocardial fibrosis
Korean J Physiol Pharmacol 2023;27(4):345-356www.kjpp.net
349
Effect of H2S on the expression of myocardial
pyroptosis-related protein in pressure-overloaded
rats
To observe the effect of H2S on pyroptosis in the myocardial
tissue of rats under the pressure overload, the expression levels of
relevant proteins Caspase1, Caspase3 were detected by Western
blotting assay. Our findings revealed that compared with the
results of the control group, the protein expression levels of Cas-
pase1 (p = 0.0211), Caspase3 (p = 0.0085), in myocardial tissue in
AAC group rose noticeably (p < 0.05), while those of Caspase1
(p = 0.0017), Caspase3 (p = 0.0086) in myocardial tissue in AAC
+ H2S group declined significantly compared with those of the
AAC group. In comparison with the results of the control group,
the protein expression levels of Caspase1 (p = 0.9599), Caspase3
(p = 0.9043), in H2S group were almost unchanged, suggesting no
differences with statistical significance (Fig. 4).
A B
C D
Fig. 2. Upregulation of cardiac pro-
duction of myocardial collagen fibers
in abdominal aorta coarctation (AAC)-
injected rat
in vivo
, this effect can
be antagonized by hydrogen sulfide
(H
2
S). Representative images of Masson
with myocardial collagen fibers in the
heart tissues of AAC-injected rat models
(Blue represents collagen fibers). Images
were acquired at 10 × 40 magnification.
(A) Control group. (B) AAC group. (C)
AAC + H
2
S group. (D) H
2
S group.
A
B C
Fig. 3. Western blotting analysis the
expression of (A) MMP1, (B) MMP13,
(C) TIMP1 in abdominal aorta coarcta-
tion (AAC)-induced myocardial fibro-
sis treated with control, AAC, AAC +
H
2
S, and H
2
S. Values are presented as
mean ± SD. H
2
S, hydrogen sulfide. *p <
0.05
vs
. control,
#
p < 0.05
vs
. AAC,
##
p <
0.01
vs
. AAC.
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Li Y et al
The effect of H2S on the expressions of proteins
related to myocardial inflammation in rats under the
pressure overload
To observe the effect of H2S on inflammation in the myocardial
tissue of rats under the pressure overload, the expression levels of
relevant proteins IL-6, IL1β, IL18, and ICAM-1 were detected by
Western blotting assay. Our findings revealed that compared with
the results of the control group, the protein expression levels of
IL-6 (p = 0.0158), IL1β (p < 0.0001), IL18 (p = 0.0002) and ICAM-
1 (p = 0.0407) in myocardial tissue in AAC group rose noticeably,
while those of IL-6 (p = 0.0174), IL1β (p = 0.0221), IL18 (p = 0.0004)
and ICAM-1 (p = 0.0088) in myocardial tissue in AAC + H2S
group declined significantly compared with those of the AAC
group (p < 0.05); In comparison with the results of the control
group, the protein expression levels of IL-6 (p = 0.0820), IL1β (p =
0.0614), IL18 (p = 0.0990) and ICAM-1 (p = 0.0635) in H2S group
were almost unchanged, suggesting no differences with statistical
significance (Fig. 5).
AB
Fig. 4. Western blotting analysis the
expression of (A) Caspase1, (B) Cas-
pase3 in abdominal aorta coarctation
(AAC)-induced myocardial fibrosis
treated with control, AAC, AAC + H
2
S,
and H
2
S. Values are presented as mean
± SD. H
2
S, hydrogen sulfide. *p < 0.05
vs.
control, **p<0.01
vs
. control,
##
p<0.01
vs
.
AAC.
AB
Fig. 5. Western blotting analysis the
expression of (A) ICAM-1, (B) IL6, IL1ββ,
IL18 in abdominal aorta coarctation
(AAC)-induced myocardial fibrosis
treated with control, AAC, AAC + H
2
S,
and H
2
S. Values are presented as mean
± SD. H
2
S, hydrogen sulfide. *p < 0.05
vs
.
control, ***p < 0.001
vs
. control,
#
p < 0.05
vs
. AAC,
##
p < 0.01
vs
. AAC,
###
p < 0.001.
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Korean J Physiol Pharmacol 2023;27(4):345-356www.kjpp.net
351
The effect of H2S on the ultrastructure of myocardium
in rats under the pressure overload
In this study, we observed the variations in ultrastructure of
myocardium and autophagosomes in rats under the pressure
overload. According to the results, myocardial fibers were closely
arranged, and there were considerable mitochondria in the con-
trol group with no swelling or vacuolation. In comparison with
the results of the control group, myocardial fibers were disorderly
arranged, variations (e.g., mitochondrial swelling and vacuola-
tion) were observed, and more autophagosomes could be ob-
served in the field of vision in the AAC group. As compared with
the results of the AAC group, myocardial fibers were arranged
regularly in AAC + H2S group, mild edema occurred in myocar-
dial fibers with no lytic necrosis, mitochondria were free from
swelling or vacuolation, and less autopahgosomes were observed
in the field of vision. In comparison with the results of the control
group, the ultrastructure of myocardial tissue in H2S group were
almost unchanged (Fig. 6).
The effect of H2S on the expression of autophagy-
related proteins in myocardium of rats under the
pressure overload
To observe the expression levels of autophagy-related proteins
in myocardial tissue of rats under the pressure overload and
the intervention effect of H2S. We used Western-blot method to
detect the expression of P-eif2α, autophagy-related pathways,
autophagy-related proteins. The results indicated that the protein
expression levels of P-eif2α (p = 0.0398), ATG3 (p = 0.0282),
ATG16L1 (p = 0.0112), Beclin1 (p = 0.0109), LC3A/B (p = 0.0430)
in myocardium in AAC group increased significantly than the
control group (Fig. 7), while those of P-PI3K (p = 0.0150), P-AKT1
(p = 0.0126) declined noticeably (Fig. 8)
.
Compared with the re-
sults of the AAC group, the protein expression levels of P-eif2α (p
= 0.0043), ATG3 (p = 0.0393), ATG16L1 (p = 0.0422), Beclin1 (p
= 0.0357), LC3A/B (p = 0.0146) expression levels of these proteins
in myocardial tissue in AAC + H2S group were significantly de-
creased, while those of P-PI3K (p = 0.0443), P-AKT1 (p = 0.0350)
increased noticeably. In comparison with the results of the con-
trol group, the protein expression levels of P-eif2α (p = 0.6695),
ATG3 (p = 0.0571), ATG16L1 (p = 0.3420), Beclin1 (p = 0.3308),
LC3A/B (p = 0.2117), P-PI3K (p = 0.8407), P-AKT1 (p = 0.2335) in
myocardial tissue of H2S group were almost unchanged, suggest-
ing no differences with statistical significance.
H2S inhibits Ang II-induced cardiomyocyte pyroptosis
through down-regulation of eif2αα phosphorylation
As we all know, γ-Cystathionase (CTH, EC: 4.4.1.1) is the key
enzyme for the production of H2S so in our experiments we used
Wertern blotting to detect the expression of CTH (Fig. 9). In or-
Fig. 6. Hydrogen sulfide (H
2
S) inhibi-
tions abdominal aorta coarctation
(AAC)-induced myocardial fibrosis
mitophagy. Representative images of
transmission electron microscopy (black
arrows indicate mitophagy). Transmis-
sion electron micrographs at bars of 0.5
µm. (A) Control group. (B) AAC group. (C)
AAC + H
2
S group. (D) H
2
S group.
AB
CD
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Li Y et al
der to further clarify the internal regulatory mechanism of H2S
intervention and protection of pressure overload cardiomyopathy,
this study used Ang II to induce an
in vitro
experimental model
of H9c2 cardiomyocytes, and Western blotting was used to ex-
plore the effect of H2S on eif2α phosphorylated protein, PI3K/
AKT1 signaling pathway and the expression of pyroptosis-related
proteins. The results showed that compared with the control
group, the expression of P-eif2α (p = 0.0280) in cardiomyocytes
of the Ang II group was increased, the PI3K (p = 0.0483)/AKT1 (p
= 0.0483) autophagy pathway was inhibited, and the pyroptosis-
related proteins Caspase1 (p = 0.0469) and Caspase3 (p = 0.0212)
were also significantly upregulated (Fig. 10). After H2S interven-
AB
C D
Fig. 7. Western blotting analysis the
expression of (A) Atg3, (B) Atg16L1,
(C) Beclin1, (D) LC3A/B in abdominal
aorta coarctation (AAC)-induced myo-
cardial fibrosis treated with control,
AAC, AAC + H
2
S, and H
2
S. Values are
presented as mean ± SD. H
2
S, hydrogen
sulfide. *p < 0.05
vs.
control,
#
p < 0.05
vs
.
AAC.
A
B C
Fig. 8. Western blotting analysis the
expression of (A) P-eif2αα, (B) P-PI3K,
(C) P-AKT1 in abdominal aorta coarc-
tation (AAC)-induced myocardial fi-
brosis treated with control, AAC, AAC
+ H
2
S, and H
2
S. Values are presented as
mean ± SD. H
2
S, hydrogen sulfide. *p <
0.05
vs
. control,
#
p < 0.05
vs
. AAC,
##
p <
0.01
vs
. AAC.
Hydrogen sulfide ameliorates abdominal aorta coarctation myocardial fibrosis
Korean J Physiol Pharmacol 2023;27(4):345-356www.kjpp.net
353
tion, the expression of P-eif2α (p = 0.0084) was down-regulated,
the PI3K (p = 0.0092)/AKT1 (p = 0.0092) autophagy pathway
was activated, and the pyroptosis-related proteins Caspase1 (p =
0.0374) and Caspase3 (p = 0.0360) were also significantly down-
regulated; the expression levels of the above proteins in the car-
diomyocytes of the H2S and control groups did not change sig-
nificantly, the difference was not statistically significant (p > 0.05).
However, in the H2S intervention group, eif2α phosphorylation (p
= 0.0029) activation (BTdCPU: 10 µm [18]) was added, and PI3K
(p = 0.0092)/AKT1 (p = 0.0266) signaling pathway was obviously
inhibited, and the pyroptosis proteins Caspase1 (p = 0.0316) and
Caspase3 (p = 0.0466) were significantly up-regulated (Fig. 11).
DISCUSSION
As people’s lifestyle has been changed, and aging of population
is being accelerated, the incidence rate of hypertension shows
an increase year by year. Also, hypertension is another major
risk factor leading to cardiac failure and cardiovascular death.
Statistics suggested that nearly half of cardiovascular deaths are
clinically associated with hypertension, and cardiac failure is one
of the underlying causes. The four-year mortality rate of cardiac
failure reaches 50%, and pressure overload caused by hyperten-
sion may cause ventricular stiffness and cardiac dysfunction to
enhance, which has been reported to show a close relation to MF
in recent years [19]. Thus, reversing or postponing MF may be
critical to enhance hypertension and prognosis of cardiac failure
[20]. In this study, a model of rats under the pressure overload-
induced MF was successfully built through AAC induced car-
diomyocyte injury model. It was found that the hypertension of
AAC group was obviously higher than that of the control group.
By Masson staining, it was observed that deposition of collagen
fibers obviously increased in myocardial interstitium of rats
with AAC. Also, the results of Western blotting assay suggested
that the expression of MMPs/TIMPs in myocardial tissue in rats
under the pressure overload in AAC group was noticeably dys-
regulated. MMPs act as the major enzyme system that degrades
extracellular matrix components, while TIMPs refer to endog-
AB
CFig. 10. Western blotting analysis the
expression of (A) P-eif2αα, (B) P-PI3K, P-
AKT1, (C) Caspase1, Caspase3 in H9c2
cells treated with control, Ang II, Ang
II + H
2
S, and H
2
S. Values are presented
as mean ± SD. Ang II, angiotensin II; H
2
S,
hydrogen sulfide. *p < 0.05
vs
. control,
***p < 0.001
vs
. control,
#
p < 0.05
vs
. Ang
II,
##
p < 0.01
vs
. Ang II,
###
p < 0.001
vs.
Ang II.
Fig. 9. Western blotting analysis the expression of CTH in H9c2 cells
treated with control, Ang II, Ang II + H
2
S, and PAG. Values are pre-
sented as mean ± SD. CTH, γ-Cystathionase; Ang II, angiotensin II; H
2
S,
hydrogen sulfide; PAG, DL-Propargylglycine. **p < 0.01
vs
. control,
#
p <
0.05
vs.
Ang II,
$
p < 0.05
vs
. Ang II + H
2
S.
354
https://doi.org/10.4196/kjpp.2023.27.4.345Korean J Physiol Pharmacol 2023;27(4):345-356
Li Y et al
enous specific inhibitors of MMPs. The interaction and dynamic
balance between MMPs and TIMPs act as a critical factor to
maintain ECM stability [21], and enhanced activity of MMPs dis-
rupts the dynamic equilibrium of the ECM and causes collagen
fibril deposition, therefore TIMPs, which are inhibitors of MMPs,
increase negatively to achieve dynamic equilibrium [22], which
is also consistent with our experimental results. Studies have also
found that obvious MMPS/TIMPS dysregulation exists in rats
under the pressure overload, and it is associated with excessive
collagen deposition in myocardial interstitium and MF.
Pyrotosis is a kind of pro-inflammatory regulatory necro-
sis mode [23]. There are two main molecular mechanisms of
pyroptosis: classical Caspase-1-dependent and non-Caspase-
1-dependent pathways, both of which can lead to the release of
pro-inf lammatory cytokines IL-1β and IL-18 and expand local or
systemic inflammation. Recent studies have also identified a new
pathway of pyroptosis: activation of Caspase-3 by specific stimuli
(originally Caspase-3 is a substrate for apoptosis), followed by
Caspase-3-induced cleavage of Gasdermin E (GSDME) into C-
terminal fragments (GSDME-CT) and N-terminal fragments
(GSDME-NT), of which GSDME-NT is involved in pyroptosis
[24,25]. Pyroptosis is a highly regulated cell death process, and
in many cases, inhibition of this process by pharmacological or
genetic intervention has a protective effect on the heart. There-
fore, this process is a potential target for therapeutic intervention
to prevent cardiovascular disease, and recent studies have also
confirmed that cardiomyocyte pyroptosis is closely bound up
with the occurrence of cardiovascular diseases, especially MF
[5]. The Qiu team’s research [26] shows that inhibiting autophagy
can reduce pyroptosisand ultimately improve non-alcoholic liver
cirrhosis; this indicates that autophagy activation induces py-
roptosis. Although, the study in Jiang [27] found that autophagy
inhibits pyroptosis, which is contrary to previous reports. Based
on the in-depth analysis of previous autophagy activation can
induce myocardial remodeling after myocardial infarction, our
laboratory believes that moderate autophagy refers to a process of
self-salvation [6,7], excessive autophagy, cells will be damaged, so
the apoptosis or death of cells will be promoted [8], this has been
further explained in our study. We found that in AAC-induced
SD rats, pyroptosis proteins Caspase1, Caspase3, inflammation-
related proteins IL-6, IL-1β, IL-18, ICAM-1, the expression of
autophagy-related proteins ATG3, ATG16L1, LC3A/B and Beclin1
was significantly increased, the PI3K/AKT1 signaling pathway
was inhibited, and the formation of autophagosomes in SD rats
induced by AAC was also observed by electron microscopy, indi-
cating that AAC-induced autophagy in SD rats. After activation,
the induced cell pyroptosis leads to pressure overload and MF,
which may be related to the PI3K/AKT1 signaling pathway.
Eif2α mainly by binding to guanine triphosphate, provides
promoter methionyl transfer RNA (Mett RNAi Met) to the small
ribosomal subunit initiation complex, thereby participating in
protein initiation phase synthesis [28]. Studies have pointed out
that the level of phosphorylation of eif2α in response to various
stresses is a key part of the overall stress response, which is called
the integrated stress response [29,30]. Moreover, some studies
have pointed out that eif2α phosphorylation activation blocks
Fig. 11. Western blotting analysis the
expression of (A) P-eif2αα, (B) P-PI3K, P-
AKT1, (C) Caspase1, Caspase3 in H9c2
cells treated with control, Ang II, Ang II
+ H
2
S, Ang II + H
2
S + BTdCPU, and H
2
S.
Values are presented as mean ± SD. Ang
II, angiotensin II; H
2
S, hydrogen sulfide;
BTdCPU, eif2α phosphorylation activa-
tion. *p < 0.05
vs
. control,
#
p < 0.05
vs
.
Ang II,
##
p < 0.01
vs
. Ang II,
$
p < 0.05
vs
.
Ang II + H
2
S,
$$
p < 0.05
vs
. Ang II + H
2
S.
A
B
C
Hydrogen sulfide ameliorates abdominal aorta coarctation myocardial fibrosis
Korean J Physiol Pharmacol 2023;27(4):345-356www.kjpp.net
355
protein synthesis, and activated P-eif2α protein negatively regu-
lates PI3K activity [14], which is considered a common factor in
the occurrence of many diseases [13,31]. It is well known that
PI3K/AKT1 signaling pathway is a classical autophagy pathway,
and some studies have shown that renal denervation can improve
myocardial remodeling induced by pressure overload, and its
mechanism is related to the inhibition of autophagy. There are
also studies showing that autophagy inhibitor 3-MA can improve
left ventricular hypertrophy induced by pressure overload [32].
All these studies suggest that inhibiting excessive autophagy may
be a key intervention to improve myocardial remodeling under
pressure overload. In this study, it was also observed that the
expression of P-eif2α was significantly increased and the PI3K/
AKT autophagy pathway was inhibited in AAC group, which
was also confirmed in H9c2 cardiomyocytes induced by Ang II.
This study shows that pyrophagy is involved in the mechanism of
pressure overload-induced MF and is associated with the inhibi-
tion of eif2-phosphorylated PI3K/AKT1 autophagy pathway.
H2S refers to the third gaseous signal molecule found after
NO and CO, CSE is the key enzyme of endogenous H2S source
in cardiovascular system [33], CBS and 3MST participate in the
production of endogenous H2S in peripheral vascular tissue [34].
Several studies have reported that H2S have multiple biological
effects on the cardiovascular system and have protecting effect
on the heart. The previous research of the research group also
showed that hydrogen sulfide can improve myocardial remodel-
ing by reducing endoplasmic reticulum stress [35]. Some studies
suggested that H2S can facilitate the process of left ventricular
remodeling in rats with hypertension-induced heart failure [36],
whereas the effect of H2S on AAC-induced MF in rats and the rel-
evant regulatory mechanisms remain unclear. The results show
that the heart function has not significantly decreased in the early
stage of pressure overload, and we speculate that it may be in the
compensatory phase at this time, however, significant changes in
myocardial structure and abnormal molecular signal functions
have been observed at this stage. After intervention with H2S
donor, the deposition of collagen in myocardium in rats in AAC
+ H2S group under the pressure overload was obviously reduced,
while MMPs/TIMPs dysregulation was noticeably improved, at
the same time, it was found that it can significantly inhibit py-
roptosis.
In vitro
experiments have also seen that H2S can inhibit
Ang II-induced pyroptosis. Meanwhile, both
in vitro
and
in vivo
experiments showed that H2S could inhibit eif2α phosphoryla-
tion and activate the PI3K/AKT1 autophagy pathway. In order
to further confirm whether hydrogen sulfide activates the PI3K/
AKT1 autophagy pathway by inhibiting eif2α phosphorylation,
in this study, after adding eif2α phosphorylation activation (BT-
dCPU: 10 µm [18] MCE, Shanghai, China) to the Ang II-induced
cell injury model, it was clearly observed that pyroptosis inhibited
in the H2S treatment group, and the PI3K/AKT1 autophagy path-
way was inhibited, which suggests that hydrogen sulfide activates
the PI3K/AKT1 autophagy pathway to improve cell pyroptosis by
inhibiting eif2α phosphorylation.
In summary, this study found that H2S can activate the PI3K/
AKT1 autophagy pathway by regulating eif2α phosphorylation
expression, thereby inhibiting pyroptosis and improving pres-
sure overload MF, however, more of these regulatory mechanisms
remain to be further investigated. The results of this study are
expected to provide new targets for intervention and treatment
of hypertensive cardiomyopathy, as well as to develop novel
exogenous H2S-releasing drugs to improve the occurrence and
development of MF, and further open up the clinical application
prospects of H2S-releasing drugs for anti-myocardial remodeling.
FUNDING
This study was supported by grant from the National Natural
Science Foundation of China (Grant Nos. 81870230), Hunan
Natural Science Foundation (2022JJ40885).
ACKNOWLEDGEMENTS
None.
CONFLICTS OF INTEREST
The authors declare no conf licts of interest.
REFERENCES
1. Berk BC, Fujiwara K, Lehoux S. ECM remodeling in hypertensive
heart disease.
J Clin Invest.
2007;117:568 -575.
2. Li J, Xue J, Wang D, Dai X, Sun Q, Xiao T, Wu L, Xia H, Mostofa
G, Chen X, Wei Y, Chen F, Quamruzzaman Q, Zhang A, Liu Q.
Regulation of gasdermin D by miR-379-5p is involved in arsenite-
induced activation of hepatic stellate cells and in fibrosis via secre-
tion of IL-1β from human hepatic cells.
Metallomics.
2019;11:483-
495.
3. Chou X, Ding F, Zhang X, Ding X, Gao H, Wu Q. Sirtuin-1 amelio-
rates cadmium-induced endoplasmic reticulum stress and pyropto-
sis through XBP-1s deacetylation in human renal tubular epithelial
cells.
Arc h Toxic ol.
2019;93:965-986.
4. Wree A, Eguchi A, McGeough MD, Pena CA, Johnson CD, Canbay
A, Hoffman HM, Feldstein AE. NLRP3 inf lammasome activation
results in hepatocyte pyroptosis, liver inflammation, and fibrosis in
mice.
Hepatology.
2014;59:898-910.
5. Zhaolin Z, Guohua L, Shiyuan W, Zuo W. Role of pyroptosis in car-
diovascular disease.
Cell Prolif.
2019;52:e12563.
6. Asano J, Sato T, Ichinose S, Kajita M, Onai N, Shimizu S, Ohteki T.
Intrinsic autophagy is required for the maintenance of intestinal
stem cells and for irradiation-induced intestinal regeneration.
Cell
Rep.
2017;20:1050-1060.
356
https://doi.org/10.4196/kjpp.2023.27.4.345Korean J Physiol Pharmacol 2023;27(4):345-356
Li Y et al
7. Terman A, Brunk UT. Oxidative stress, accumulation of biological
‘garbage’, and aging.
Antioxid Redox Signal.
2006;8:197-204.
8. Lo SH, Hsu CT, Niu HS, Niu CS, Cheng JT, Chen ZC. Cryptotan-
shinone inhibits STAT3 signaling to alleviate cardiac fibrosis in type
1-like diabetic rats.
Phytother Res.
2017;31:638-646.
9. Aoki M, Fujishita T. Oncogenic roles of the PI3K/AKT/mTOR axis.
Curr Top Microbiol Immunol.
2017;407:153-189.
10. Xiao T, Luo J, Wu Z, Li F, Zeng O, Yang J. Effects of hydrogen sul-
fide on myocardial fibrosis and PI3K/AKT1-regulated autophagy in
diabetic rats.
Mol Med Rep.
2016;13:1765-1773.
11. Li Z, Zhao F, Cao Y, Zhang J, Shi P, Sun X, Zhang F, Tong L. DHA
attenuates hepatic ischemia reperfusion injury by inhibiting pyrop-
tosis and activating PI3K/Akt pathway.
Eur J Pharmacol.
2018;835:1-
10.
12. Liu Y, Tie L. Apolipoprotein M and sphingosine-1-phosphate com-
plex alleviates TNF-α-induced endothelial cell injury and inflam-
mation through PI3K/AKT signaling pathway.
BMC Cardiovasc
Disord.
2019;19:279.
13. Moon SL, Sonenberg N, Parker R. Neuronal regulation of eIF2α
function in health and neurological disorders.
Trends Mol Med.
2018;24:575-589.
14. Kazemi S, Mounir Z, Baltzis D, Raven JF, Wang S, Krishnamoorthy
JL, Pluquet O, Pelletier J, Koromilas AE. A novel function of eIF2al-
pha kinases as inducers of the phosphoinositide-3 kinase signaling
pathway.
Mol Biol Cell.
2007;18:3635-3644.
15. Koromilas AE, Mounir Z. Control of oncogenesis by eIF2α phos-
phorylation: implications in PTEN and PI3K-Akt signaling and
tumor treatment.
Future Oncol.
2013;9:10 05-1015.
16. Calvert JW, Coetzee WA, Lefer DJ. Novel insights into hydro-
gen sulfide--mediated cytoprotection.
Antioxid Redox Signal.
2010;12:1203-1217.
17. Chen K, Rekep M, Wei W, Wu Q, Xue Q, Li S, Tian J, Yi Q, Zhang
G, Zhang G, Xiao Q, Luo J, Liu Y. Quercetin prevents in vivo and in
vitro myocardial hypertrophy through the proteasome-GSK-3 path-
way.
Cardiovasc Drugs Ther.
2018;32:5-21.
18. Burwick N, Zhang MY, de la Puente P, Azab AK, Hyun TS, Ruiz-
Gutierrez M, Sanchez-Bonilla M, Nakamura T, Delrow JJ, MacKay
VL, Shimamura A. The eIF2-alpha kinase HRI is a novel therapeu-
tic target in multiple myeloma.
Leuk Res.
2017;55:23-32.
19. Bing OH. Hypothesis: apoptosis may be a mechanism for the transi-
tion to heart failure with chronic pressure overload.
J Mol Cell Car-
diol.
1994;26:943-948.
20. Heusch G, Libby P, Gersh B, Yellon D, Böhm M, Lopaschuk G, Opie
L. Cardiovascular remodelling in coronary artery disease and heart
failure.
Lancet.
2014;383:1933-1943.
21. Pei Z, Meng R, Li G, Yan G, Xu C, Zhuang Z, Ren J, Wu Z. Angio-
tensin-(1-7) ameliorates myocardial remodeling and interstitial
fibrosis in spontaneous hypertension: role of MMPs/TIMPs.
Tox icol
Lett.
2010;199:173-181.
22. Nagase H, Visse R, Murphy G. Structure and function of matrix
metalloproteinases and TIMPs.
Cardiovasc Res.
2006;69:562-573.
23. Xu YJ, Zheng L, Hu YW, Wang Q. Pyroptosis and its relationship to
atherosclerosis.
Clin Chim Acta.
2018;476:28-37.
24. Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, dif-
ferences, and crosstalk.
Cell Death Differ.
2019;26:99-114.
25. Beier JI, Banales JM. Pyroptosis: an inflammatory link between
NAFLD and NASH with potential therapeutic implications.
J Hepa-
tol.
2018;68:643-645.
26. Qiu T, Pei P, Yao X, Jiang L, Wei S, Wang Z, Bai J, Yang G, Gao N,
Yang L, Qi S, Yan R, Liu X, Sun X. Taurine attenuates arsenic-in-
duced pyroptosis and nonalcoholic steatohepatitis by inhibiting the
autophagic-inflammasomal pathway.
Cell Death Dis.
2018;9:946.
27. Jiang C, Jiang L, Li Q, Liu X, Zhang T, Dong L, Liu T, Liu L, Hu G,
Sun X, Jiang L. Acrolein induces NLRP3 inflammasome-mediated
pyroptosis and suppresses migration via ROS-dependent autophagy
in vascular endothelial cells.
Toxicology.
2018;410: 26-40.
28. Wek RC. Role of eIF2α kinases in translational control and
adaptation to cellular stress.
Cold Spring Harb Perspect Biol.
2018;10:a032870.
29. Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman
AM. The integrated stress response.
EMBO Rep.
2016;17:1374-1395.
30. Ron D, Walter P. Signal integration in the endoplasmic reticulum
unfolded protein response.
Nat Rev Mol Cell Biol.
2007;8:519-529.
31. Sano R, Reed JC. ER stress-induced cell death mechanisms.
Biochim
Biophys Acta.
2013;1833:3460-3470.
32. Huo JY, Jiang WY, Geng J, Chen C, Zhu L, Chen R, Ge TT, Chang Q,
Jiang ZX, Shan QJ. Renal denervation attenuates pressure overload-
induced cardiac remodelling in rats with biphasic regulation of
autophagy.
Acta Physiol (Oxf ).
2019;226:e13272.
33. Zheng SF, Bao RK, Zhang QJ, Wang SC, Lin HJ. Endogenous hy-
drogen sulfide promotes apoptosis via mitochondrial pathways in
the livers of broilers with selenium deficiency exudative diathesis
disease.
Biol Trace Elem Res.
2018;186:249-257.
34. Prabhudesai S, Koceja C, Dey A, Eisa-Beygi S, Leigh NR, Bhattacha-
rya R, Mukherjee P, Ramchandran R. Corrigendum: Cystathionine
β-synthase is necessary for axis development in vivo.
Front Cell Dev
Biol.
2018;6:121. Erratum for:
Front Cell Dev Biol.
2018;6:14.
35. Long J, Liu M, Liu S, Tang F, Tan W, Xiao T, Chu C, Yang J. H2S
attenuates the myocardial fibrosis in diabetic rats through modulat-
ing PKC-ERK1/2MAPK signaling pathway.
Technol Health Care
.
2019;27(S1):307-316.
36. Predmore BL, Lefer DJ, Gojon G. Hydrogen sulfide in biochemistry
and medicine.
Antioxid Redox Signal.
2012 ;17:119-140.
